Clinical trial NCT01086540
Conditions: Systemic Sclerosis-Associated PAH
Keywords: Pulmonary Arterial Hypertension (PAH) Autoimmune Disease Systemic Scleroderma
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)
Start date: 2011-06-24; Completion date: 2019-12-15
Enrollment: 57 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH) is a serious, life-threatening manifestation of systemic sclerosis (SSc), an autoimmune disease of the connective tissue characterized by scarring (fibrosis) and atrophy of the skin, joints and tendons, skeletal muscles, and internal organs, and immunological disturbances. One-year survival for patients with SSc-PAH ranges from 50-81%. There is currently no cure for SSc-PAH and treatment is limited to vasodilator therapy used in all forms of PAH. In recent studies, immunotherapy was shown to be effective in treating SSc-interstitial lung disease, another serious, life-threatening manifestation of SSc. In addition, there are compelling pre-clinical data and anecdotal clinical reports that suggest modulation of the immune system may be an effective strategy for treating SSc-PAH. To test this approach, this trial will determine if rituximab, an immunotherapy, has a marked beneficial effect on clinical disease progression, with minimal toxicity, in patients with SSc-PAH when compared to placebo.

Detailed description:
This prospective, double-blind, placebo-controlled, multi-center, randomized trial will evaluate the effect of rituximab on disease progression in subjects with SSc-PAH receiving concurrent stable-dose standard medical therapy with a prostanoid, endothelin receptor antagonist, and/or phosphodiesterase 5 (PDE-5) inhibitor. The study will focus on assessment of clinical response and safety measures longitudinally. In addition, the effects of treatment with rituximab on the underlying immune mechanisms associated with B-cell dysregulation and pathogenic autoantibody response in this disease will be investigated. 1000 mg of rituximab or placebo will be administered as two IV infusions given two weeks apart. Clinical assessments and sample collection will occur at monthly visits through Week 48. If a participant has not recovered B cells by Week 48, B cell studies will be conducted quarterly until reconstitution is documented or for 2 years after initial treatment.

This trial will include a sub-study, entitled "Right Ventricular Response to Rituximab in Systemic Sclerosis-Associated Pulmonary Arterial Hypertension - A Magnetic Resonance Imaging Sub-study" (RESTORE Sub-study). The objective of the RESTORE sub-study is to evaluate the therapeutic effect of rituximab on the right ventricle of patients with SSc-PAH. Changes in right ventricular end diastolic volume index (RVEDVI) and stroke volume (SV) determined by cardiac MRI will be used as surrogates of right ventricle function and prognosis. Enrollment for the RESTORE sub-study will parallel that of main trial. Twenty patients from each treatment arm, distributed among all participating sites, will be recruited for this sub-study. Each patient will be studied at baseline (i.e. prior to initiation of study drug) and after 24 weeks or at time of discontinuation. In addition to the data collection and testing specified in the main trial, participants in RESTORE will undergo comprehensive cardiac MRI evaluation. All main trial study inclusion and exclusion criteria apply, as well as additional exclusion criteria that pertain only to the RESTORE sub-study: 1) known hypersensitivity to Gadolinium; 2) inability to tolerate or cooperate with MRI; 3) morbid obesity; and 4) presence of metallic objects or pacemakers.

Arms: arm 1: Rituximab (1000 mg) will be administered as 2 intravenous infusions given 2 weeks apart.

Concurrent stable-dose Pulmonary Arterial Hypertension (PAH) medical therapy will be continued/managed as per standard of care (PAH SOC). arm 2: Placebo will be administered as 2 intravenous infusions given 2 weeks apart.

Concurrent stable-dose Pulmonary Arterial Hypertension (PAH) medical therapy will be continued/managed as per standard of care (PAH SOC).
Interventions: intervention 1: Rituximab intervention 2: Placebo intervention 3: CMRI intervention 4: prednisone intervention 5: methylprednisolone intervention 6: diphenhydramine intervention 7: acetaminophen
Intervention descriptions: intervention 1: Participants receive rituximab intravenous (IV) infusions, 1000 mg each, 14 days apart (Day 0 and Week 2).

Rituximab is supplied as a sterile, clear, colorless, preservative-free liquid concentrate for intravenous (IV) administration in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials, which must be diluted before administration

Standard rituximab pre-medications will be provided in preparation for the rituximab infusions. intervention 2: Participants receive placebo intravenous (IV) infusions 14 days apart (Day 0 and Week 2).

Standard pre-medications will be provided in preparation for the infusions. intervention 3: Up to 20 participants from each treatment arm will be assessed by CMRI at Baseline and at Week 24. intervention 4: Prednisone dose of 40 mg (or equivalent) by mouth administered the night before and the morning of each study drug infusion. intervention 5: Methylprednisolone (or equivalent corticosteroid) administered intravenously 30 minutes prior to each study drug infusion. intervention 6: Diphenhydramine 50 mg (or equivalent) administered by mouth approximately thirty to sixty minutes prior to each study drug infusion.

Dose may be repeated every four hours, as needed. intervention 7: Acetaminophen 650 mg (or equivalent) administered by mouth approximately thirty to sixty minutes prior to each study drug infusion.

Dose may be repeated every four hours, as needed.

Protocol documents:
Prot_001.pdf: 
 
 
 
C O N F I D E N T I A L 
 
 
 
Autoimmunity Centers of Excellence 
 
 
 
Protocol # ASC01 
 
 
A Randomized, Double-blind, Placebo-Controlled, Phase II Multicenter 
Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment of 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension (SSc-PAH) 
 
Short Title:  Rituximab for Treatment of SSc-PAH 
 
 
 
BB-IND 105,863 
 
 
Version 8.0, 24 April 2017 
 
 
 
 
IND Sponsor:  Division of Allergy, Immunology, and Transplantation (DAIT) 
 
National Institute of Allergy and Infectious Diseases (NIAID) 
 
National Institutes of Health (NIH) 
 
 
 
 
 
 
 
Confidentiality Statement 
The information contained within this document is not to be disclosed in any way without the prior permission of 
the Protocol Chair(s), or the Division of Allergy, Immunology and Transplantation; the National Institute of 
Allergy and Infectious Diseases; and the National Institutes of Health. 
 


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Protocol Chairs: 
 
Mark Nicolls, MD 
Chief, Division of Pulmonary and Critical 
Care Medicine,  
Stanford University School of Medicine  
Medical Service (111P) 
VA Palo Alto Health Care System 
3801 Miranda Avenue   
Palo Alto, CA 94304 
Phone: 650-493-5000 ext. 69289 
Fax: 650-849-1942 
E-mail: mnicolls@stanford.edu 
 
David B. Badesch, MD 
Professor of Medicine 
University of Colorado Health Sciences 
Center 
Leprino Building, Room 536 
12401 E. 17th Avenue 
Aurora, CO 80045       
Phone: 720-848-6567 
Fax: 720-848-0480 
E-mail: David.Badesch@ucdenver.edu 
 
 
Protocol Co-Chairs: 
 
Lorinda Chung, MD 
Associate Professor of Medicine 
Stanford University School of Medicine 
Medical Service 
VA Palo Alto Health Care System 
3801 Miranda Ave. 
Palo Alto, CA 94305 
Phone: 650-493-5000 ext 62042 
Fax: 650-849-1213 
Email: shauwei@stanford.edu 
Robyn Domsic, MD 
Assistant Professor of Medicine 
Division of Rheumatology and Clinical 
Immunology 
University of Pittsburgh 
S724 Biomedical Science Tower 
3500 Terrace St. 
Pittsburgh, PA 15261  
Phone: 412-383-8000 
Fax: 412-648-9643 
Email: rtd4@pitt.edu  
 
 
Aryeh Fischer, MD 
Associate Professor of Medicine 
National Jewish Health 
University of Colorado School of Medicine 
1400 Jackson Street G07 
Denver, CO 80206 
Phone: 303-398-1703 
Fax: 303-398-1040 
Email: fischera@njhealth.org  
Roham Zamanian, MD 
Assistant Professor of Medicine 
Director, Adult Pulmonary Hypertension 
Clinical Service 
Vera Moulton Wall Center for Pulmonary 
Vascular Disease 
Division of Pulmonary & Critical Care 
Medicine  
Stanford University School of Medicine 
300 Pasteur Dr. Room H3143 
Stanford, CA 94305 
Phone: 650-725-5495 
Fax: 650-725-5489 
Email: Zamanian@stanford.edu 
 
 


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Protocol Consultant: 
 
Thomas A. Medsger, Jr., MD 
Professor of Medicine 
Division of Rheumatology and Clinical Immunology 
University of Pittsburgh School of Medicine 
BST South 
Room 720 
3500 Terrace Street 
Pittsburgh, PA 15261 
Phone: 412-383-8765 
Fax: 412-648-8894 
E-mail: Medsger@dom.pitt.edu 


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DAIT, NIAID, NIH 
 
Medical Monitor: Ellen Goldmuntz, MD, PhD 
Division of Allergy, Immunology, and Transplantation   
National Institute of Allergy and Infectious Diseases 
National Institutes of Health   
5601 Fishers Lane, Room #7D50 
Rockville, MD 20852 
Phone: 240-627-3502 
E-mail: egoldmuntz@niaid.nih.gov 
 
Project Manager: Beverly Welch, RN, MSN 
Division of Allergy, Immunology, and Transplantation   
National Institute of Allergy and Infectious Diseases 
National Institutes of Health   
5601 Fishers Lane, Room #7D38 
Rockville, MD 20852 
Phone: 240-627-3484 
E-mail: bwelch@niaid.nih.gov 
 
Regulatory Officer: 
Maria-Concetta Veri, PhD 
Division of Allergy, Immunology, and Transplantation  
National Institute of Allergy and Infectious Diseases 
National Institutes of Health  
5601 Fishers Lane, Room 7B48 
Bethesda, MD  20892-6601 
Phone: 240-627-3572 
E-mail: maria.veri@nih.gov 
 
 
 


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SACCC – RhoFED, Inc. 
 
Statistician: Lynette Keyes-Elstein, DrPH 
Rho Federal Systems Division, Inc. 
6330 Quadrangle Drive 
Suite 500 
Chapel Hill, NC 27517 
Phone:  919-595-6410 
Fax:  919-287-3039 
E-mail: lynette_keyes-elstein@RhoWorld.com 
 
Study Coordinator: Carla D’Aveta, CCRP 
Rho Federal Systems Division, Inc. 
6330 Quadrangle Drive 
Suite 500 
Chapel Hill, NC 27517 
Phone: 919-595-6307 
Fax: 919-287-3039 
E-mail: Carla_D’Aveta@RhoWorld.com


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INVESTIGATOR SIGNATURE PAGE 
 
Protocol Title:    
A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for 
the Treatment of Systemic Sclerosis-Associated Pulmonary Arterial 
Hypertension (SSc-PAH) 
 
Protocol Number:   Protocol # ASC01 
 
Protocol Version:   Version 8.0 (24 April 2017) 
 
Study Sponsor: 
Division of Allergy, Immunology, and Transplantation (DAIT) 
 
 
 
National Institute of Allergy and Infectious Diseases 
 
 
 
5601 Fishers Lane 
Rockville, MD 20852 
 
Please print, sign, and date at the indicated location below. A copy should be kept for your 
records and the original signature page sent to the SACCC.   
 
I confirm that I have read the above protocol in the latest version. I understand it, and I will 
work according to the principles of Good Clinical Practice (GCP) as described in the United 
States Code of Federal Regulations (CFR) – 21 CFR Parts 45, 50, 56, and 312, and the 
International Conference on Harmonization (ICH) document “Guidance for Industry: E6 
Good Clinical Practice: Consolidated Guidance” dated April 1996. Further, I will conduct the 
study in keeping with local, legal, and regulatory requirements.  
 
As a Principal Investigator on this protocol, I agree to conduct “A Randomized, Double-
blind, Placebo-Controlled, Phase II Multicenter Trial of a Monoclonal Antibody to CD20 
(Rituximab) for the Treatment of Systemic Sclerosis-Associated Pulmonary Arterial 
Hypertension (SSc-PAH)” by the criteria written in the protocol and understand that no 
changes can be made to this protocol without written permission of the DAIT/NIAID. 
 
 
 
 
Principal Investigator (Print)  
Principal Investigator Signature 
 
 
Date 
 


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PROTOCOL SYNOPSIS 
 
Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II Multicenter Trial of a 
Monoclonal Antibody to CD20 (Rituximab) for the Treatment of Systemic Sclerosis-Associated Pulmonary 
Arterial Hypertension (SSc-PAH) 
ACE Protocol Number:  ASC01  
Protocol Chair(s):  Mark Nicolls, MD; David Badesch, MD 
IND Holder: DAIT/NIAID/NIH 
Primary Objective: The primary objective of this study is to compare patients with SSc-PAH treated with 
rituximab to those on placebo for change in exercise capacity, as determined by six minute walk distance 
(6MWD). 
Secondary Efficacy Objectives:  
 
To compare patients treated with rituximab to those on placebo for change in exercise capacity, as 
determined by 6MWD, at time points other than 24 weeks. 
 
To compare patients treated with rituximab to those on placebo for change in PVR as assessed by 
right heart catheterization. 
 
To compare patients treated with rituximab to those on placebo for time to clinical worsening, defined 
as first occurrence of: 
a. 
death,  
b. hospitalization for SSc-PAH, 
c. 
lung transplantation,  
d. atrial septostomy,  
e. 
addition of other PAH therapeutic agents, or   
f. 
worsening of 6MWD by > 20% and a decrease in functional class. 
 
To compare patients treated with rituximab to those on placebo who changed or added other PAH 
therapeutic agents. 
 
To compare patients treated with rituximab to those on placebo for change in quality of life as 
determined by the short form 36 (SF-36) and the disability index of the Scleroderma Health 
Assessment Questionnaire (SHAQ). 
 
To compare patients treated with rituximab to those on placebo for change in number of new digital 
ulcers. 
 
To compare patients treated with rituximab to those on placebo for change in severity of Raynaud 
phenomenon as measured by the VAS scale of the SHAQ. 
 
To compare patients treated with rituximab to those on placebo for change in DLCO and room air 
oxygen saturation at rest.  
 
A secondary objective of this study is to determine whether the effects on clinical disease progression are 
paralleled by changes in selected biomarkers.   
 
Secondary Safety Objectives: 
 
To assess the safety and tolerability of rituximab for the treatment of SSc-PAH in patients receiving 
stable background medical treatment with prostanoid, endothelin receptor antagonist, PDE-5 inhibitor 
therapy, and/or guanylate cyclase stimulators. 
 
Mechanistic Study Objectives: 
 
CD19 Studies 
 To determine whether the extent and/or duration of CD19+ B cell depletion correlates with 
treatment response as measured by exercise capacity (6MWD),  PVR (right heart 
catheterization), and quality of life (SF-36 and SHAQ). 
 To monitor and correlate the reconstitution patterns of B cell subpopulations with treatment 
response 
 
To determine if the biomarkers anti-U1 RNP, anti-U3 RNP, anti-B23, anti-cardiolipin, anti-Th/To, 
anti-CENP-B, RF, and other autoantibodies, and quantitative immunoglobulin levels, including IgG 
subclasses, correlate with treatment response as measured by PVR (right heart catheterization), 


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Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II Multicenter Trial of a 
Monoclonal Antibody to CD20 (Rituximab) for the Treatment of Systemic Sclerosis-Associated Pulmonary 
Arterial Hypertension (SSc-PAH) 
exercise capacity (6MWD), and quality of life (SF-36 and SHAQ). 
 
To determine if the biomarkers BNP/NT-proBNP, serum Na+, IL-1, IL-6, and other cytokines 
correlate with treatment response as measured by PVR (right heart catheterization), exercise capacity 
(6MWD), and quality of life (SF-36 and SHAQ).   
 
To determine if B cell clones are present in SSc-PAH and what impact rituximab may have on these 
clones. 
 
To determine if B cell clones present in SSc-PAH are correlated with clinical outcomes. 
 
Exploratory Objectives: 
 
To evaluate agreement between PVR calculated by site-based standards and a standardized Fick-
based calculation of PVR. 
 
To assess sensitivity of the secondary efficacy analysis to standardization of the PVR calculation. 
 
To assess the utility of a standardized derived pulmonary artery capacitance as a primary efficacy 
endpoint. 
 
To evaluate the relationship between 6MWD, PVR, and other clinical measures. 
Study Arms:   
Subjects will be randomized in a 1:1 ratio to receive either: 
 
2 infusions of rituximab, 1000 mg each, given 2 weeks apart, or 
 
2 infusions of placebo given 2 weeks apart 
Study Design:  This is a prospective, double-blind, placebo-controlled, multi-center, randomized trial to 
evaluate the effect of rituximab on disease progression in subjects with SSc-PAH receiving concurrent stable-
dose standard medical therapy with a prostanoid, endothelin receptor antagonist, phosphodiesterase 5 (PDE-5) 
inhibitor, and/or guanylate cyclase stimulators. The study will focus on assessment of clinical response and 
safety measures longitudinally. In addition, the effects of treatment with rituximab on the underlying immune 
mechanisms associated with B-cell dysregulation and pathogenic autoantibody response in this disease will be 
investigated.   
 
Subjects with SSc-PAH with elevated mean pulmonary artery pressure as measured by right heart 
catheterization will be enrolled. The diagnosis of SSc-PAH should be confirmed   by a rheumatologist 
experienced in the diagnosis and treatment of systemic sclerosis in conjunction with a pulmonologist or 
cardiologist specializing in management of PAH. Both specialists will be part of the study team at each site.  
 
Rituximab/placebo will be administered as two IV infusions of 1000 mg each, given two weeks apart at Day 0 
and Week 2. All subjects will receive 40mg of prednisone orally the night before and morning of each 
infusion, as well as 100 mg methylprednisolone or equivalent corticosteroid intravenously thirty minutes prior 
to each infusion, with diphenhydramine and acetaminophen orally thirty to sixty minutes prior to each infusion 
of rituximab or placebo. Subjects will remain on their baseline standard medical regimen.  
 
Sixty eligible subjects will be accrued. Each potential study subject will provide written informed consent prior 
to screening procedures. All inclusion and exclusion criteria (Section 4, Selection of Subjects) must be met at 
time of randomization prior to receipt of the first dose of rituximab/placebo (Day 0, Treatment Initiation).  
 
Clinical assessments and sample collection will occur regularly through Week 48 with telephone follow-up 
conducted intermittently. If a subject has not recovered B cells by Week 48, B cell studies will be conducted 
quarterly until recovery is documented or for 2 years after initial treatment (whichever occurs first). Recovery 
is defined as the time point at which B cells recover to within 10% of baseline levels; if a baseline value is not 
available then recovery is defined as a B cell level of at least 90% of the lower limit of normal. During this 
quarterly monitoring period of B cell recovery, AEs and SAEs will be assessed, providing the subject has not 
withdrawn consent, to capture any infectious event ≥ Grade 3 using the National Cancer Institute—Common 
Terminology Criteria for Adverse Events (NCI-CTCAE). No additional study-related data will be collected. 
 
The primary efficacy endpoint is the change in 6MWD from baseline to 24 weeks after treatment initiation. 
Hemodynamic measures will be assessed at baseline and Week 24, contributing to the understanding of the 
relationship between PVR and clinical endpoints. Time to clinical worsening will be assessed as a secondary 
outcome measure through Week 48, contributing to the understanding of the relationship between PVR and 


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Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II Multicenter Trial of a 
Monoclonal Antibody to CD20 (Rituximab) for the Treatment of Systemic Sclerosis-Associated Pulmonary 
Arterial Hypertension (SSc-PAH) 
clinical endpoints. Initiation of new therapy due to disease worsening prior to Week 24 will prompt an 
endpoint visit and right heart catheterization prior to initiation of the new therapy. B cell recovery for all will 
be monitored by serial assessments of B cell counts as determined by flow cytometry. 
 
The total duration of the study is anticipated to be approximately 8 years.  
Study Population:  Any individual 18 to 75 years of age, who has been diagnosed with SSc-PAH within the 
past 5 years (as defined by a clinical diagnosis of SSc with a mPAP ≥ 25 mmHg), a NYHA Functional Class of 
II, III, or IV, a mean PVR of > 3 Wood units, and who meets all entry criteria is eligible for randomization.  
Primary Efficacy Endpoint: Change in exercise capacity, assessed by 6MWD, from baseline to 24 weeks 
after treatment initiation. 
 
Secondary Efficacy Endpoints: 
Secondary efficacy endpoints include the following clinical endpoints: 
1. Change in PVR measured by right heart catheterization, from baseline to 24 weeks after treatment 
initiation. 
2. Exercise capacity, as determined by 6MWD, assessed longitudinally over the 48 weeks following 
treatment initiation.   
3. Assessment of time to clinical worsening, censored at 48 weeks, defined as first occurrence of: 
a. 
death,  
b. hospitalization for SSc-PAH,  
c. 
lung transplantation,  
d. atrial septostomy,  
e. 
addition of other PAH therapeutic agents, or  
f. 
worsening of 6MWD by > 20% and a decrease in functional class. 
4. Percentage of subjects who changed or added PAH therapeutic agents at 24 and 48 weeks. 
5. Change in quality of life as determined by the SF-36 and disability index of the SHAQ from baseline 
to 24 and 48 weeks after treatment initiation. 
6. Number of new digital ulcers assessed longitudinally over both the 24 and 48 weeks following 
treatment initiation. 
7. Severity of Raynaud phenomenon, as measured by the VAS scale of the SHAQ, assessed 
longitudinally over both the 24 and 48 weeks following treatment initiation. 
8. DLCO and oxygen saturation at rest on room air assessed longitudinally over both the 24 and 48 
weeks following treatment initiation. 
9. Change in PVR from baseline to 24 weeks after treatment initiation as a percentage of the baseline 
value. 
 
Secondary efficacy endpoints also include the following mechanistic endpoints: 
1. CD19 Studies 
a. 
CD19+ B cells (absolute number and percentage of gated lymphocytes) at time of B cell 
depletion and longitudinally over the 48 weeks following treatment initiation, where B cell 
depletion is defined as <5 CD19+ cells/μL. 
b. Time to repopulation of CD19+ B cells, defined as the time point at which B cells recover to 
within 10% of baseline levels; if a baseline value is not available, then recovery is defined as 
a B cell level of at least 90% of the lower limit of normal. 
c. 
Reconstitution pattern of B cell subpopulations assessed longitudinally over the 48 weeks 
following treatment initiation. 
2. Level of anti-U1 RNP, anti-B23, anti-cardiolipin, anti-CENP-B, RF autoantibodies, and 
immunoglobulins, including IgG subclasses, measured by ELISA and assessed longitudinally over the 
48 weeks following treatment initiation. 
3. Presence or absence of anti-U3 RNP, anti-Th/To, and other autoantibodies assessed longitudinally 
over the 48 weeks following treatment initiation. 
4. Level of BNP/NT-proBNP, serum Na+, IL-1, IL-6, and other cytokines assessed longitudinally over 
the 48 weeks following treatment initiation. 
5. Presence or absence of B cell clones assessed at baseline and longitudinally over the 48 weeks 


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Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II Multicenter Trial of a 
Monoclonal Antibody to CD20 (Rituximab) for the Treatment of Systemic Sclerosis-Associated Pulmonary 
Arterial Hypertension (SSc-PAH) 
following treatment initiation. 
 
Secondary Safety Endpoints: 
1. All NCI-CTCAE Grade 3-5 AEs, which are defined as possibly, probably, or definitely related to 
rituximab infusion.  
2. All NCI-CTCAE Grade 3-5 adverse events. 
3. All infection related events. 
4. Treatment-related mortality, defined as death occurring at any time after randomization and possibly, 
probably, or definitely resulting from treatment given in the study. Study investigators will make an 
initial determination of the cause of death of any subject and if uncertainty remains, a final decision 
will be made by an independent blinded panel after reviewing all clinical and, if available, autopsy 
data. 
5. All-cause mortality, defined as any death occurring at any time after randomization. 
6. CD19+ B cell levels post treatment (note that analyses will be performed regularly per Table 6.1 
through 48 weeks and quarterly thereafter as documented in Section 3.1, Description of Study 
Design). 
7. Peripheral blood cell counts, liver function, and creatinine levels after treatment. 
 
Exploratory Endpoints: 
Exploratory analysis of standardization of the PVR calculation will be based on PVR calculated from a 
standardized Fick calculation of cardiac output, which is a function of hemoglobin, arterial and mixed-venous 
saturations, and oxygen consumption according to the La-Farge and Miettinen formula. The cardiac output 
calculation is a function of assumed oxygen consumption according to the La-Farge calculation of values 
normalized for age, gender, heart rate. Calculation of a standardized Fick cardiac output will also allow for 
determination of the pulmonary artery capacitance (PACf). See Appendix E, Exploratory Analysis: 
Standardized Fick-based Pulmonary Vascular Resistance and Pulmonary Artery Capacitance for details. 
Additionally, an exploratory analysis of the relationship between change in PVR and change in 6MWD will be 
addressed. Relationships with other clinical endpoints, like change in NTproBNP, will also be explored. 
Sample Size:  The study will include 30 subjects treated with rituximab and 30 subjects treated with placebo.  
Data Analyses:  The primary efficacy analysis of change in 6MWD from baseline to 24 weeks will be 
performed on the intention-to-treat (ITT) population. and will be tested using a repeated measures random 
effects model to model change from baseline in 6MWD as the primary outcome and treatment as the primary 
predictor, controlling for visit day, limitations by musculoskeletal disease, baseline levels of 6MWD, and a 
treatment by visit day interaction.  A random slope and intercept will be fit for each subject.  An unstructured 
covariance matrix will be assumed for the random effects. Additional covariates will be considered for this 
model as part of secondary analysis. 
 
Secondary analyses will include repeating the primary analysis in the per protocol population, subgroup 
analyses of the primary outcome, safety analyses, exploratory analyses of the secondary endpoints, and 
exploratory analyses of the mechanistic endpoints. 
 


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Lay Summary: 
 
Sixty eligible adults between the ages of 18 and 75 who have been diagnosed with SSc-PAH 
within the past 5 years and meet all entry criteria will be enrolled in this study. Participants 
will receive 2 infusions (1000 mg) of either rituximab or placebo 14 days apart.   
 
This study will compare patients treated with rituximab to those on placebo for change in six 
minute walk distance (6MWD). The secondary objectives of this study are to compare 
treatment groups for other measures of clinical disease progression and to determine whether 
the effects on clinical disease progression are paralleled by changes in selected biomarkers. 
Additionally, the safety and tolerability of rituximab for the treatment of SSc-PAH in patients 
receiving stable background medical treatment with prostanoid, endothelin receptor 
antagonist, PDE-5 inhibitor, and/or guanylate cyclase stimulators therapy will be assessed.   
 


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FLOW DIAGRAM OF PROTOCOL 
 
Visit 1 
 
 
Screening  
Day -28 to -1 
 
Visit 2 
Baseline 
Day -7 to 0 
 
 
  
 
 
 
 
 
Visit 3 
Day 0 
 
 
 
  
 
 
 
Visit 4 
Week 2 
 
 
 
 
 
 
Assessments 5-10  
Weeks 4-24 
 
 
 
 
 
 
Assessments 11-16  
Weeks 28-48 
 
 
 
 
 
B Cell Recovery 
 
 
 
 
 
Assessment 17 
Week 104 
 
Screening:  Eligibility determined per assessments listed in Table 6.1. 
Baseline Evaluations: Baseline and Day 0 assessments (Table 6.1) may 
be combined into 1 visit as scheduling dictates so long as they are 
completed within 7 days of the first infusion 
1st Rituximab Infusion 
Subjects receive 1000 mg IV 
rituximab. 
 Pretreated with corticosteroids, 
diphenhydramine, and 
acetaminophen.
Clinical and disease assessments (including exercise capacity [6MWD]), 
questionnaires, and laboratory assessments will be completed per Table 6.1. 
 
The primary endpoint, change in exercise capacity, assessed by 6MWD, at Visit 10 
(Week 24). A key secondary endpoint is change in pulmonary vascular resistance 
by right heart catheterization.
For subjects who have not withdrawn consent and who have received any 
rituximab and B cells have not recovered, follow-up clinical care must include 
visits at least quarterly to monitor recovery of B cells.   
 
Quarterly visits will continue until B cell recovery or for 2 years after first 
infusion (whichever occurs first).
Randomize 60 subjects 
1st Placebo Infusion 
Subjects receive IV placebo.  
Pretreated with corticosteroids, 
diphenhydramine, and 
acetaminophen. 
2nd Rituximab Infusion 
Subjects receive 1000 mg IV 
rituximab.  
Pretreated with corticosteroids,  
diphenhydramine, and 
acetaminophen.
2nd Placebo Infusion 
Subjects receive IV placebo.  
Pretreated with corticosteroids,  
diphenhydramine, and 
acetaminophen. 
Clinical and disease assessments, questionnaires, and laboratory assessments will 
be completed per Table 6.1. 
 
The end of study visit will occur at Visit 16 (Week 48). 
Two year post randomization assessment to be completed on vital status and 
treatment satisfaction for all subjects.  


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ABBREVIATIONS 
6MWD 
6-Minute Walk Distance 
6MWT 
6-Minute Walk Test 
ACE 
Autoimmunity Centers of Excellence 
ADCC 
Antibody-dependent Cellular Cytotoxicity  
ADCT 
Autoimmune Diseases Clinical Trials 
AE 
Adverse event 
AECA 
Anti-Endothelial Cell Antibody 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
ANAs 
Serum Antinuclear Antibodies 
ANCOVA 
Analysis of Covariance 
Anti-B23 
Antibody to the B23 antigen 
Anti-CENP-B 
Antibody to the CENP-B (centromere) antigen 
Anti-Th/To 
Antibody to the Th/To antigen 
APAH 
Associated Pulmonary Arterial Hypertension 
APGAR 
Appearance, Pulse, Grimace, Activity, and Respiration 
AST 
Aspartate Aminotransferase 
ATG 
Anti-thymocyte Globulin 
BMPR2 
Bone Morphogenetic Protein Receptor Type 2 
BNP 
Brain Natriuretic Peptide 
BP 
Blood Pressure 
BUN 
Blood Urea Nitrogen 
C 
Celsius 
CBC 
Complete Blood Count 
CFR 
Code of Federal Regulations 
CHF 
Congestive Heart Failure 
CI 
Cardiac Index 
CMRI 
Cardiac Magnetic Resonance Imaging  
CO2 
Carbon Dioxide 
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTGF 
Connective Tissue Growth Factor 
CXR 
Chest X-ray 
CY 
Cyclophosphamide 
DAIT 
Division of Allergy, Immunology, and Transplantation 
DE 
Delayed Enhancement 
DHHS 
Department of Health and Human Services 
DLCO 
Carbon Monoxide Diffusing Capacity 
DSMB 
Data and Safety Monitoring Board 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
ERA 
Endothelin Receptor Antagonist 
ET-1 
Endothelin-1 
F 
Fahrenheit 
Fc 
Fragment, crystallizable 


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FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
G-CSF 
Granulocyte Colony-Stimulating Factor 
HACA 
Human Anti-chimera Antibody 
HBV 
Hepatitis B Virus 
HCG 
Human Chorionic Gonadotropin 
HCV 
Hepatitis C Virus 
Hg 
Mercury 
HIMC 
Human Immune Monitoring Center 
HIV 
Human Immunodeficiency Virus 
HHV8 
Human Herpesvirus-8 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IFN 
Interferon 
Ig 
Immunoglobulin  
IgG 
Immunoglobulin G 
IJ 
Internal Jugular 
IL 
Interleukin 
IND 
Investigational New Drug Application 
IPAH 
Idiopathic Pulmonary Arterial Hypertension 
IR 
Immune Reconstitution 
IRB 
Institutional Review Board 
ITT 
Intention-to-Treat or Intent-to-Treat 
ITP 
Idiopathic Thrombocytopenic Purpura 
IUD 
Intrauterine Device 
IV 
Intravenous 
IVIG 
Intravenous Immunoglobulin 
kD 
Kilo Dalton 
LOCF 
Last Observation Carried Forward 
LON 
Late Onset Neutropenia 
LPS 
Lipopolysaccharide 
LV 
Left Ventricle 
MAP 
Mean Arterial Pressure 
MCP 
Monocyte Chemotactic Protein 
MI 
Myocardial Infarction 
MID 
Minimally Important Difference 
MOP 
Manual of Procedures 
mPAP 
Mean Pulmonary Arterial Pressure 
MRI 
Magnetic Resonance Imaging 
MTX 
Methotrexate 
Na+ 
Sodium 
NCI 
National Cancer Institute 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NS 
Normal Saline 
NT-proBNP 
N-Terminal Pro Brain Natriuretic Peptide 


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NYHA 
New York Heart Association 
OHRP 
Office of Human Research Protection 
PA 
Posteroanterior 
PAAT 
Pulmonary Arterial Acceleration Time 
PACf 
Fick-based calculation of Pulmonary Artery Capacitance  
PAH 
Pulmonary Arterial Hypertension 
PAP 
Pulmonary Arterial Pressure 
PASP 
Pulmonary Arterial Systolic Pressure 
PCR 
Polymerase Chain Reaction 
PCWP 
Pulmonary Capillary Wedge Pressure 
PDE-5 
Phosphodiesterase 5  
PE 
Physical Examination 
PFTs 
Pulmonary Function Tests 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
PL 
Placebo 
PP 
Per Protocol 
PPD 
Purified Protein Derivative 
PML 
Progressive Multifocal Leukoencephalopathy 
PVR 
Pulmonary Vascular Resistance 
PVRf 
Fick-based calculation of Pulmonary Vascular Resistance 
qPCR 
Quantitative Polymerase Chain Reaction 
RA 
Rheumatoid Arthritis 
RHC 
Right Heart Catheterization  
RNA 
Ribonucleic Acid 
RNP 
Ribonucleoprotein 
RV 
Right Ventricle 
RVEDVI 
Right Ventricular End Diastolic Volume Index (mL/m2) 
SACCC 
Statistical and Clinical Coordinating Center 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SBP 
Systolic Blood Pressure 
SD 
Sprague Dawley 
SF-36 
Short Form 36 
SGOT 
Serum Glutamic-Oxaloacetic Transaminase 
SGPT 
Serum Glutamic-Pyruvic Transaminase 
SHAQ 
Scleroderma Health Assessment Questionnaire 
SLE 
Systemic Lupus Erythematosus 
SP 
Safety Population 
SSc 
Systemic Sclerosis 
SSc-PAH 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension 
SUHX 
SU5416-hypoxia 
SV 
Stroke Volume 
TB 
Tuberculosis 
TGF 
Transforming Growth Factor 
Th 
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TLC 
Total Lung Capacity 
TLS 
Tumor Lysis Syndrome 
TNF 
Tumor Necrosis Factor 
VAS 
Visual Analog Scale 
TTE 
Transthoracic Echocardiography 
WHO 
World Health Organization 


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TABLE OF CONTENTS 
INVESTIGATOR SIGNATURE PAGE................................................................................6 
PROTOCOL SYNOPSIS ........................................................................................................7 
FLOW DIAGRAM OF PROTOCOL ..................................................................................12 
ABBREVIATIONS ................................................................................................................13 
TABLE OF CONTENTS ......................................................................................................17 
1  BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ....................22 
 
SYSTEMIC SCLEROSIS – GENERAL DISEASE CONCEPTS ..........................................22 
1.1
 
Current Treatment for Systemic Sclerosis-Associated PAH ............................25 
1.1.1
 
SUMMARY OF PRE-CLINICAL AND CLINICAL STUDIES ............................................26 
1.2
 
Pre-Clinical Studies .........................................................................................26 
1.2.1
 
Clinical Studies ................................................................................................30 
1.2.2
 
RITUXIMAB/TREATMENT BACKGROUND.................................................................31 
1.3
 
Current Licensing of Rituximab .......................................................................31 
1.3.1
 
Other Diseases in Which Rituximab Use Has Been Described .......................32 
1.3.2
 
KNOWN AND POTENTIAL RISKS OF RITUXIMAB TREATMENT ..................................33 
1.4
 
RATIONALE FOR STUDY ..........................................................................................38 
1.5
 
Rationale for the Entry Criteria .......................................................................39 
1.5.1
 
Rationale for Mechanistic Studies ...................................................................41 
1.5.2
2  STUDY OBJECTIVES AND PURPOSE ......................................................................42 
 
PRIMARY OBJECTIVE ..............................................................................................42 
2.1
 
SECONDARY OBJECTIVES ........................................................................................42 
2.2
 
EXPLORATORY OBJECTIVES ....................................................................................43 
2.3
3  STUDY DESIGN ..............................................................................................................44 
 
DESCRIPTION OF STUDY DESIGN .............................................................................44 
3.1
 
Stratification, Randomization, and Masking ...................................................45 
3.1.1
 
DESCRIPTION OF ENDPOINTS ...................................................................................46 
3.2
 
Primary Efficacy Endpoint ..............................................................................46 
3.2.1
 
Secondary Efficacy Endpoints .........................................................................46 
3.2.2
 
Secondary Safety Endpoints .............................................................................47 
3.2.3
 
Exploratory Endpoints .....................................................................................47 
3.2.4
 
SUBJECT DISPOSITION .............................................................................................48 
3.3
 
Definition of Subject Completion .....................................................................48 
3.3.1
 
Discontinuation of Protocol-Specified Treatment Requirements ....................48 
3.3.2
 Follow-up Requirements for All Treated Subjects ......................................49 
3.3.2.1
 
Subject Withdrawal from the Study..................................................................49 
3.3.3
 Procedures for Subject Withdrawal ............................................................50 
3.3.3.1


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Subject Replacement ........................................................................................50 
3.3.4
4  SELECTION OF SUBJECTS ........................................................................................50 
 
INCLUSION CRITERIA ..............................................................................................50 
4.1
 
EXCLUSION CRITERIA .............................................................................................50 
4.2
 
Coenrollment Guidelines .................................................................................53 
4.2.1
5  TREATMENT OF SUBJECTS ......................................................................................53 
 
DESCRIPTION OF RITUXIMAB ..................................................................................53 
5.1
 
Product Description .........................................................................................53 
5.1.1
 
Packaging and Labeling of Study Product ......................................................53 
5.1.2
 
Storage and Handling of Study Product ..........................................................53 
5.1.3
 
DOSAGE REGIMEN ..................................................................................................54 
5.2
 
ADMINISTRATION OF RITUXIMAB ...........................................................................54 
5.3
 
Medications to Hold Prior to Study Drug Administration...............................57 
5.3.1
 
Preparation for Administration .......................................................................57 
5.3.2
 
Pre-medication for Rituximab Infusion ...........................................................57 
5.3.3
 
Administration..................................................................................................57 
5.3.4
 Guidance for NYHA Class II or III Subjects ...............................................58 
5.3.4.1
 Guidance for NYHA Class IV Subjects .......................................................59 
5.3.4.2
 
Criteria for Permanent Discontinuation of Rituximab Infusion ......................59 
5.3.5
 
Criteria for Withholding Rituximab Infusion ...................................................60 
5.3.6
 
Criteria for Temporarily Interrupting and Restarting Rituximab Infusion .....60 
5.3.7
 
TOXICITY MANAGEMENT PLAN FOR RITUXIMAB ....................................................61 
5.4
 
Known Toxicities to Rituximab ........................................................................61 
5.4.1
 
Prevention of Known Toxicities to Rituximab .................................................61 
5.4.2
 
Management of Known Toxicities to Rituximab ..............................................62 
5.4.3
 Infusion Reactions (Cytokine release syndrome/acute infusion reaction 
5.4.3.1
and Hypersensitivity/Allergic Reaction) ................................................62 
 Cardiovascular ............................................................................................63 
5.4.3.2
 Severe Mucocutaneous Reactions ...............................................................64 
5.4.3.3
 Hepatitis ......................................................................................................65 
5.4.3.4
 Hematologic Malignancy ............................................................................65 
5.4.3.5
 Serum Sickness ............................................................................................65 
5.4.3.6
 Tumor Lysis Syndrome ................................................................................65 
5.4.3.7
 Infection ......................................................................................................65 
5.4.3.8
 
PRIOR MEDICATIONS AND THERAPY .......................................................................66 
5.5
 
PROHIBITED MEDICATIONS AND TREATMENTS .......................................................66 
5.6
 
CONCURRENT MEDICATIONS AND THERAPY ...........................................................67 
5.7
 
Background PAH Therapy ...............................................................................67 
5.7.1
 
Immunosuppressive or Immunomodulating Drug Treatment ..........................68 
5.7.2
 Corticosteroids ............................................................................................68 
5.7.2.1
 
TOXICITY MANAGEMENT PLAN FOR CONCURRENT THERAPY: ...............................68 
5.8
 
Known Toxicities of Concurrent Therapy ........................................................68 
5.8.1
 
Prevention and Management of Known Toxicities to Concurrent Therapy ....69 
5.8.2
 
RIGHT HEART CATHETERIZATION RISKS AND MANAGEMENT GUIDELINES ............70 
5.9


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OTHER PROTOCOL STANDARD OF CARE RECOMMENDATIONS ................................70 
5.10
 
PROCEDURES FOR MONITORING SUBJECT COMPLIANCE .........................................71 
5.11
6  STUDY ASSESSMENTS ................................................................................................71 
 
ASSESSMENTS OF SAFETY .......................................................................................71 
6.1
 
ASSESSMENTS OF DISEASE ACTIVITY AND CLINICAL RESPONSE ............................71 
6.2
 
ASSESSMENTS OF MECHANISTIC ENDPOINTS ..........................................................71 
6.3
 
OTHER ASSESSMENTS .............................................................................................72 
6.4
 
EVALUATIONS BY STUDY VISIT ..............................................................................72 
6.5
 
Screening Period (Visit 1) ................................................................................72 
6.5.1
 
Randomization/Baseline Visit (Visit 2) ............................................................74 
6.5.2
 
Treatment Period .............................................................................................74 
6.5.3
 Day 0 (Visit 3) .............................................................................................74 
6.5.3.1
 Week 2/Day 14 ±4 days (Visit 4) .................................................................75 
6.5.3.2
 
Follow Up Visits ..............................................................................................76 
6.5.4
 Week 4 ±7 days (Visit 5) ..............................................................................76 
6.5.4.1
 Week 8 ± 7 days (Assessment 6) ..................................................................77 
6.5.4.2
 Week 12 ± 7 days (Visit 7)...........................................................................77 
6.5.4.3
 Week 16 ± 7 days (Assessment 8) ................................................................78 
6.5.4.4
 Week 20 ± 7 days (Assessment 9) ................................................................78 
6.5.4.5
 Week 24 ± 7 days (Visit 10).........................................................................78 
6.5.4.6
 Week 28 ± 7 days (Assessment 11) ..............................................................79 
6.5.4.7
 Week 32 ± 7 days (Assessment 12) ..............................................................79 
6.5.4.8
 Week 36 ± 7 days (Visit 13).........................................................................79 
6.5.4.9
  Week 40 ± 7 days (Assessment 14) .........................................................80 
6.5.4.10
  Week 44 ± 7 days (Assessment 15) .........................................................80 
6.5.4.11
  Week 48 ± 7 days (Visit 16) ....................................................................81 
6.5.4.12
  Week 96 (Visit 17) ..................................................................................81 
6.5.4.13
 
Quarterly Monitoring Visits .............................................................................82 
6.5.5
 
Early Endpoint Visit .........................................................................................82 
6.5.6
 
Visit Windows...................................................................................................82 
6.5.7
 
Unscheduled Visits ...........................................................................................83 
6.5.8
7  SAFETY MONITORING AND REPORTING ............................................................86 
 
OVERVIEW ..............................................................................................................86 
7.1
 
DEFINITIONS ...........................................................................................................86 
7.2
 
Adverse Event (or Adverse Experience) ..........................................................86 
7.2.1
 
Adverse Reaction and Suspected Adverse Reaction ........................................86 
7.2.2
 
Unexpected Adverse Reaction ..........................................................................86 
7.2.3
 
Serious Adverse Event ......................................................................................87 
7.2.4
 
COLLECTION AND RECORDING OF ADVERSE EVENTS .............................................87 
7.3
 
Investigational Product ....................................................................................87 
7.3.1
 
Collection Period .............................................................................................87 
7.3.2
 
Collection of Adverse Events ...........................................................................87 
7.3.3
 
Recording Adverse Events ...............................................................................88 
7.3.4
 
Recording Serious Adverse Events ..................................................................88 
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GRADING AND ATTRIBUTION OF ADVERSE EVENTS ................................................88 
7.4
 
Grading Criteria ..............................................................................................88 
7.4.1
 
Attribution Definitions .....................................................................................89 
7.4.2
 
REPORTING OF ADVERSE EVENTS ...........................................................................90 
7.5
 
Reporting of Adverse Events to DAIT/NIAID ..................................................90 
7.5.1
 Procedure for Adverse Events Requiring 24 Hour Reporting ....................90 
7.5.1.1
 Procedure for Standard Adverse Event Reporting......................................91 
7.5.1.2
 
DAIT/NIAID Reporting to the Health Authority ..............................................91 
7.5.2
 
Reporting of Adverse Events to IRBs ...............................................................91 
7.5.3
 
PREGNANCY REPORTING .........................................................................................92 
7.6
 
REPORTING OF OTHER SAFETY INFORMATION ........................................................92 
7.7
 
REVIEW OF SAFETY INFORMATION .........................................................................92 
7.8
 
Medical Monitor Review ..................................................................................92 
7.8.1
 
DSMB Review ..................................................................................................93 
7.8.2
 Temporary Halt in Enrollment for Emergency Safety Review ....................93 
7.8.2.1
8  STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN ..........................94 
 
SAMPLE SIZE ...........................................................................................................94 
8.1
 
ANALYSIS POPULATIONS ........................................................................................95 
8.2
 
Safety Population .............................................................................................95 
8.2.1
 
Intent-to-Treat Population ...............................................................................95 
8.2.2
 
Per Protocol Population ..................................................................................95 
8.2.3
 
STATISTICAL METHODS ..........................................................................................95 
8.3
 
Safety Analysis .................................................................................................95 
8.3.1
 
Efficacy Analysis ..............................................................................................96 
8.3.2
 Primary Efficacy Analysis ...........................................................................96 
8.3.2.1
 Secondary Efficacy Analysis .......................................................................97 
8.3.2.2
 
Mechanistic/Immunological Analysis ..............................................................98 
8.3.3
 
Exploratory Analysis ........................................................................................99 
8.3.4
 
INTERIM ANALYSIS .................................................................................................99 
8.4
 
Interim Analysis of Efficacy Data ....................................................................99 
8.4.1
 
Interim Analysis of Safety Data .......................................................................99 
8.4.2
 
OTHER STATISTICAL CONSIDERATIONS ................................................................100 
8.5
 
Covariates ......................................................................................................100 
8.5.1
 
Multi-center Studies .......................................................................................100 
8.5.2
 
Multiple Comparisons and Multiplicity .........................................................100 
8.5.3
 
Examination of Subgroups .............................................................................100 
8.5.4
 
Missing Data ..................................................................................................101 
8.5.5
 
Changes to the Statistical Analysis Plan .......................................................101 
8.5.6
9  ACCESS TO SOURCE DATA AND DOCUMENTS ................................................101 
10  DATA COLLECTION, QUALITY CONTROL AND QUALITY ASSURANCE .102 
11  ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD 
CLINICAL PRACTICE ..................................................................................103 


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COMPLIANCE WITH GOOD CLINICAL PRACTICES ..................................................103 
11.1
 
INSTITUTIONAL REVIEW BOARD ...........................................................................103 
11.2
 
INFORMED CONSENT .............................................................................................103 
11.3
 
DATA AND SAFETY MONITORING BOARD .............................................................104 
11.4
12  FINANCING AND INSURANCE ................................................................................104 
13  PUBLICATION POLICY .............................................................................................104 
14  REFERENCES ...............................................................................................................106 
15  APPENDICES ................................................................................................................116 
 
APPENDIX A:  MODIFIED SCLERODERMA HEALTH ASSESSMENT QUESTIONNAIRE 
15.1
(SHAQ) ................................................................................................................117 
 
APPENDIX B:  SHORT FORM – 36 HEALTH SURVEY (SF-36) .................................118 
15.2
 
APPENDIX C:  NEW YORK HEART ASSOCIATION CLASSIFICATION OF HEART 
15.3
DISEASE ................................................................................................................120 
 
APPENDIX D: SUBJECT SELF-REPORTED DEMOGRAPHICS SOURCE DOCUMENT ...121 
15.4
 
APPENDIX E:  EXPLORATORY ANALYSIS:  STANDARDIZED FICK-BASED 
15.5
PULMONARY VASCULAR RESISTANCE AND PULMONARY ARTERY CAPACITANCE122 
 
APPENDIX F: RESTORE SUB-STUDY PROTOCOL .................................................123 
15.6
 


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1 BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
 
Systemic Sclerosis – General Disease Concepts 
1.1
 
Background:  Systemic sclerosis (SSc) is a systemic connective tissue disease characterized 
by fibrosis and atrophy of the skin, joints and tendons, skeletal muscles, and internal organs 
(e.g., lungs, heart, gastrointestinal tract, and kidney) as well as by immunological 
disturbances [1]. SSc is a rare disease which is diagnosed in approximately 67 male patients 
and 265 female patients per 100,000 people each year in the United States. Internationally, 
SSc is estimated to occur in 20 people per million annually but is relatively rare in Japan and 
China. Overall, a substantial female predominance exists, with a female-to-male ratio of 3-
6:1 [2]. The limited cutaneous form of SSc has a more striking female predominance, with a 
female-to-male ratio of 10:1. SSc usually appears in women aged 30-40 years and in slightly 
older men. In roughly 85% of cases, SSc develops in individuals aged 20-60 years. Cases are 
also found among children and the elderly.  
 
The clinical manifestations of SSc include Raynaud phenomenon and skin fibrosis and, in its 
severest form, sclerosis of multiple internal organs with vascular inflammatory and fibrotic 
components. Of the numerous manifestations that affect multiple organ systems, pulmonary 
involvement produces some of the most common and serious complications of the disease. 
The major pulmonary manifestations of SSc are interstitial lung disease and systemic 
sclerosis-associated pulmonary arterial hypertension (SSc-PAH). Estimates of the prevalence 
of PAH in patients with SSc have varied widely based on the definition of PAH and the 
method of obtaining the measurements (i.e. cardiac catheterization or echocardiography) but 
is thought to be between 8 and 12%. A conservative estimate of PAH prevalence of 10% 
among patients with SSc in the United States would indicate a prevalence of 24 individuals 
per million, which may actually be higher than the number of patients affected by idiopathic 
PAH. Further, patients with SSc-PAH tend to have a worse prognosis compared to all other 
forms of PAH. One year survival rates for SSc-PAH patients range from 50-81%, which is 
considerably lower than the 88% one year survival rate for idiopathic PAH patients [3-5]. 
One study documented survival rates for individuals with SSc-PAH of 81%, 63% and 56% at 
1, 2, and 3 years from diagnosis respectively [4]. A more recent study again suggested a 3-
year survival rate of 52% in patients with SSc-PAH [6]. 
 
SSc-PAH assumes three basic forms: 1) severe SSc-PAH accompanying limited cutaneous 
SSc, 2) SSc-PAH accompanying (secondary to) interstitial lung disease, and 3) a more 
indolent form of SSc-PAH which reflects vascular pathology of SSc [7]. Although there 
appear to be different subtypes of SSc-PAH, it is not known if and how the pathogenesis 
differs with each form. In all patients with SSc, SSc-PAH significantly worsens survival and 
is the leading cause of mortality in these patients. 
 
Treatment for this disease is currently limited to the same vasodilator therapy employed in all 
forms of PAH. An evaluation of 91 patients with PAH treated with prostanoids revealed that 


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SSc-PAH patients had the worst survival of all subgroups analyzed [8]. SSc-PAH is a life-
threatening illness for which there is currently no cure. 
 
SSc-PAH Pathology: Lungs from patients with SSc-PAH exhibit a characteristic vascular 
pathology, the plexiform lesion, which is structurally similar to plexiform lesions found in 
other PAH conditions such as idiopathic PAH and HIV-associated PAH [9]. In SSc-PAH, 
both plexiform and concentric obliterative lesions stain positively for factor VIII-related 
antigen consistent with abnormal endothelial cell proliferation. Macrophages, T and B cells 
are noted clustering in and around the vascular lesions. Interestingly, the primary 
proliferative abnormalities within the pulmonary arterial walls are similar to those found in 
SSc digital arteries. Both digital and pulmonary arteries have medial and advential fibrosis 
that lead to structural luminal narrowing. 
 
Pathogenesis of SSc and SSc -PAH: Endothelial cell apoptosis may be the first event in the 
pathogenesis of the disease [10]. Anti-endothelial cell antibodies (AECA) are found in the 
circulation of SSc patients and have been posited to play a role in the development of 
vascular disease found in SSc, including PAH [11]. An instigating injury to endothelial cells 
in SSc that may trigger such autoantibody formation may be a viral infection [12]. A number 
of investigators have found evidence for viral infections, such as Epstein-Barr virus, 
parvovirus B19 and hepatitis C, E, and G in patients with SSc [13-18]. Although the role of 
cytomegalovirus (CMV) in the pathogenesis of SSc is debated [19], indirect evidence for a 
role of CMV-specific antibodies in the development of this disease has also been presented 
[20, 21]. Not only are absolute lymphocyte counts reduced in SSc [22, 23], but SSc patients 
also have relatively fewer CD4+CD25+ cells in the peripheral circulation compared to healthy 
controls [24]. In this setting of diminished regulatory T cells, a dysregulation of B cells is 
also observed [25]. Of note is that CD19 expression is increased by ≈20% on B cells in 
patients with SSc, while other B cell markers CD20, CD22 and CD40 are normally expressed 
[26]. Several different autoantibodies have been implicated in disease pathogenesis; anti-
topoisomerase I (anti-Scl-70) and anticentromere antibodies, for example, are relatively 
specific for SSc [27], whereas an antibody to Cu/Zn superoxide dismutase and anti-
topoisomerase II have been associated with localized scleroderma [28, 29]. In summary, SSc 
is an autoimmune disorder that has been associated with viral infection, endothelial damage, 
diminished regulatory T cells, dysregulated B cells, abundant mast cells, and serum 
autoantibodies.   
 
Specific Concepts Concerning SSc-PAH Pathogenesis: Very little is known about the 
pathogenesis of SSc-PAH, but it appears to be a complex and multifactorial process. Unlike 
familial PAH, SSc-PAH has not been found to be associated with a mutation of bone 
morphogenetic protein receptor type 2 (BMPR2), a member of the TGF-β superfamily of 
receptors [30]. However, it has been recognized that altered expression of TGF-β superfamily 
receptors, interacting proteins, or downstream signaling molecules occurs in SSc [31-33]. 
Defects in the balance of vasoconstrictors and vasodilators have been another research focus 
in SSc-PAH with a notable elevation of endothelin-1 (ET-1), a potent vasoconstrictor [34].
 
 


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In addition to interest in TGF-β superfamily alterations and abnormal vascular tone, there is 
significant work focusing on dysregulated immunity in the pathogenesis of SSc and SSc-
PAH. Plexiform lesions found in the arterial walls of SSc PAH patients include an 
inflammatory infiltrate [35] consisting of macrophages, T cells, B cells, and mast cells [36, 
37]. 
 
Figure 1 presents a model which integrates what is known generally about SSc pathogenesis, 
including the concept of abnormal Treg behavior, and speculates how this immune 
dysregulation could culminate in SSc-PAH. 
 
 
 
Figure 1. Immune Dysregulation in SSc (Adapted from Hasegawa et al., 2005 [38]) The 
panel on the left demonstrates that patients with SSc have a preponderance of Th2 cytokines 
which work in concert with growth factors to promote tissue fibrosis. The panel on the right 
is a model showing the putative relationship between systemic autoimmunity (possibly 
contributed to by abnormal Treg activity) and tissue fibrosis in SSc and how this could 
culminate in vascular injury and SSc-PAH. 
 
Endothelial alterations may lead to a cascade of stimulatory changes that involve many cells, 
including T cells, macrophages, mast cells, and fibroblasts. Once activated, these cells 
secrete a variety of substances, including enzymes and their inhibitors and cytokines and 
their soluble receptors. These substances lead to changes in the extracellular matrix proteins, 
including fibronectin; proteoglycans; and collagen types I, III, V, and VII which results in 
fibroproliferative changes [39]. Activation of the immune system appears to be of paramount 
importance in the pathogenesis of SSc. Antigen-activated T cells infiltrate the skin and 
produce the profibrotic cytokine IL-4. B cells may contribute to fibrosis, as deficiency of 
CD19, a B-cell transduction molecule, results in decreased fibrosis in animal models as 
described below. 
 
TH2 
B cell activation 
IL-4 
IL-6 
IL-10 
IL-13 
MCP-1 
TGF-
CTGF 
TH1 
IFN- 
TNF- 
IL-2 
IL-12 
Autoimmunity 
Tissue fibrosis 
Polymorphism or 
mutation of B cell 
response regulators 
Chronic 
B cell 
activation 
T cell 
activation 
(abnormal 
Tregs)
Cytokines 
Th2>Th1 
Cytokines 
(IL-6 & TGF- 
Breakdown of 
peripheral 
tolerance 
Vascular Injury 
SSc-PAH 
Tissue Fibrosis 


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The chemokine CXCL4 was recently found to provide a link between plasmacytoid dendritic 
cell activity and the evolution of SSc. A proteome-wide screen showed CXCL4 to be the 
only chemokine that predicted the risk and progression of SSc.  This landmark study 
demonstrated that high plasma CXCL4 levels were associated both with skin and lung 
fibrosis, as well as PAH. For this reason, we have added serial measurement of CXCL4 to 
the current study [40]. 
 
 Current Treatment for Systemic Sclerosis-Associated PAH 
1.1.1
 
Standard treatment for PAH includes therapeutics that address three major pathways 
involved in the abnormal proliferation and contraction of the smooth-muscle cells of the 
pulmonary artery including the endothelin pathway, nitric oxide pathway and prostacyclin 
pathway (Figure 2) [41]. Chronic prostacyclin infusion therapy does not provide a cure for 
PAH [42]. 
 
 
Figure 2. The Three Major Pathways Targeted by Conventional PAH Therapies [41]. 
 
Previously, the continuous infusion of the potent vasodilator prostacyclin has been 
considered maximum therapy for severe PAH [43-47]. More recently, the concept of 
combination therapy, such as an endothelin receptor blocker, or a phosphodiesterase 5 
inhibitor, with prostacyclin, is being evaluated. In addition, patients receive anticoagulation 


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and right heart failure is frequently treated with diuretics and digitalis [44]. Aside from rare 
attempts to give prednisone or cytoxan, immunotherapy has rarely been used or 
systematically studied in SSc-PAH [46-49]. Unfortunately, currently offered medical therapy 
will usually fail in time and can only serve as a bridge to lung transplantation. Another major 
limitation of current medical therapies is cost, which can be more than $100,000 per year. 
Thus, the need for a new therapeutic regimen in SSc-PAH is significant. 
 
 
Summary of Pre-Clinical and Clinical Studies 
1.2
 Pre-Clinical Studies 
1.2.1
 
CD19, a critical cell-surface signal transduction molecule of B cells, is the most positive 
response regulator of B cells and has been implicated in SSc. Transgenic mice which 
overexpress CD19 lose self tolerance and generate autoantibodies spontaneously [50]. CD19 
overexpression in the tight-skin mouse model of SSc results in increased autoantibody 
production [51] whereas CD19 deficiency significantly decreases skin fibrosis [52]. B cell 
depletion in the tight-skin mouse model was recently noted to be effective in reducing skin 
fibrosis and autoimmunity during the initiation of disease, but was not effective in 
established disease [53]. This has led Dr. Tedder and colleagues to hypothesize that chronic 
B cell activation due to augmented CD19 signaling leads to skin fibrosis and enhanced 
autoimmunity in SSc [54, 55]. 
 
The preclinical studies from our group, which were an impetus for the development of the 
current trial, broadly describe the phenomena of autoimmunity in PAH [56]. Inbred athymic 
nude rats treated with the Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitor, 
SU5416, develop significant vascular remodeling and severe PAH at Denver altitude [57]. 
Our experimental model of PAH occurs because of the absence of regulatory T cell activity. 
Unlike most published experimental models, this model does not require hypoxic conditions 
for disease initiation and progression. 
 
However, athymic animals uniquely developed severe PAH following SU5416 treatment 
when exposed to chronic hypoxia. The data shown in Figure 3 demonstrate that T cells play 
a protective role following vascular injury. Immune reconstitution of T cells with regulatory 
function prevents the development of PAH in athymic nude rats treated with SU5416 as well 
as the accumulation of B cells and anti-endothelial cell antibodies in the lungs of these 
animals. 
 


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Figure 3. Immune reconstitution of athymic rats prevents PAH development. 
Panel A: RVSP measurements in athymic and euthymic rats treated with SU5416 at d21 and 
vehicle-treated rats (athymic control, euthymic control). Immune reconstitution (IR) prior to 
SU5416 administration attenuates PAH development. Euthymic animals with normal 
immune systems regulate inflammation through the action of regulatory T cells (Tregs). 
Panel B: RVH measurements as assessed by RV/(LV+S) ratio in athymic and euthymic rats 
treated with SU5416 at d21 and vehicle-treated control rats (n=10–16 per group). Panels C 
and D: Sequential echocardiography of athymic controls, athymic SU, and athymic IR-SU at 
d21 (n=4 per group). Data are shown as means with error bars representing SEM. *P0.05. 
 
In this pre-clinical model, B cells and AECAs may be critical effectors for the development 
of experimental PAH [58]. Figure 4 demonstrates that in the absence of normal Treg 
activity, inflammation with macrophages and B cells are significant, and systemic 
inflammation is detectable in the peripheral circulation. 


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Figure 4. Evidence of anti-inflammatory effect of immune reconstitution (IR) at d7 
after SU5416 administration. A and B, Immunofluorescent images of lung sections from 
athymic SU5416-treated animals stained with CD68 (arrows) for macrophages, and with 
CD45RA (arrows) for B cells at d7 after SU5416 administration (n=8 per group). C and D, 
Morphometric analysis of macrophages (CD68) and B cells (CD45RA) in lung sections at d7 
after SU5416 administration (n=4 per group). E and F, Serum TNF- and IL-6 evaluated by 
ELISA at d7 and d21 (n=6–8 per group). Data are shown as means with error bars 
representing SEM. *P0.05. Scale bars: (A, B) 50 μm. 
 


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Figure 5. Model of immune regulation contributing to vascular health and immune 
dysregulation favoring vascular disease following endothelial injury. In this model of 
PAH, endothelial injury, causes a local immune response. With normal immune regulation, 
vascular injury does not culminate in pulmonary vascular remodeling and PAH. The absence 
of normal immune regulation results in an inappropriately exuberant inflammatory response, 
accelerated endothelial cell apoptosis, smooth muscle hypertrophy, and increased pulmonary 
vascular resistance. 
 
To summarize, in T-cell deficient animals an accumulation of B cells and macrophages was 
noted within one week of SU5416 administration. These findings were not seen in athymic 
nude rats that underwent immune reconstitution prior to SU5416 administration. These 
results are concordant with the fact that the loss of self-tolerance in animals missing normal 
Treg populations is associated with the appearance of various autoantibodies and 
autoimmune disease [59, 60]. With complete elimination of CD4+CD25hi cells, systemic 
autoimmunity occurs as manifested by multiorgan inflammation and autoantibody production 
[61]. Thus a loss of Treg-mediated self tolerance leads not only to a loss of T cell tolerance 
but also to a breakdown in B cell tolerance. This latter principle suggested that a therapy 
which addressed autoreactive B cells and possibly self-directed antibodies could be effective 
in PAH associated with immune dysregulation. As noted, this finding is relevant in SSc-PAH 
patients who likely have abnormal Treg functionality. 
 
Dysregulated B cell immunity, as a direct consequence of diminished regulatory T cell 
control in the T-cell deficient athymic rat model, is important because at this time B cells can 
be more easily targeted with a therapeutic intervention than can abnormal Treg activity. To 


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this end, the effect of B cell depletion is currently being characterized in the athymic rat 
model using SU5416. Other animal models relevant to SSc-PAH have already been treated 
with B cell depletion, and results are promising. In addition to the studies of B cell depletion 
in the tight skinned mouse discussed above, at the 2011 American Thoracic Society Meeting 
in Denver, Dr. Norbert Voelkel, an internationally-recognized PAH investigator, presented 
data showing that anti-CD20 therapy ameliorates severe PAH in a unique inflammatory 
model of PAH. In this model, PAH is induced by exposing immunocompetent rats to 
ovalbumin sensitization and vascular injury with a VEGF receptor antagonist. This regimen 
leads to significant inflammatory PAH associated with Th2 inflammation. This exciting new 
model may be directly relevant to SSc-PAH because of the noted Th2 skewing. Data using 
this model of PAH shows that B cell depletion can be significantly protective against severe 
PAH [62]. 
 Clinical Studies 
1.2.2
 
Considered generally, therapies for localized scleroderma have tended to yield more positive 
results than those for SSc. However, treatments for localized scleroderma have also been less 
vigorously investigated in randomized, controlled trials [63]. Molecular analysis 
demonstrates the involvement of CD20-positive cells in SSc [64] although histochemical 
evidence has been more inconsistent [64, 65]. Investigators comparing skin biopsy samples 
from four scleroderma patients with samples from matched controls by micro-array gene 
analysis found that B-lymphocyte-specific genes were up-regulated in skin from patients 
with SSc [64]. Immunohistochemistry of patients’ skin showed a predominance of CD20-
positive cells in a perieccrine distribution in one of the patients. In contrast, stains for CD3 
and CD138 were negative, indicating a relative absence of T cells and plasma cells, 
respectively. 
 
Lafyatis et al. treated 15 SSc patients with diffuse skin thickening with rituximab. They 
showed that, as expected, peripheral blood B cells were depleted, but also that the modest B 
cell infiltrates present in most of the skin biopsies at baseline were markedly reduced or 
completely absent on repeat skin biopsy 6 months after treatment (mean B cell score declined 
from 10.4 to 3.4). Thus, rituximab was able to deplete B cells not only in peripheral blood 
but also in affected tissues [66]. 
 
There is very little published data on the use of rituximab in SSc lung disease. A compelling 
case report of SSc-PAH was recently published [67]. The patient presented with interstitial 
lung disease, but on closer review of the case the principal and potentially life- threatening 
manifestation was SSc-PAH. This patient improved dramatically after rituximab therapy and 
would likely have qualified for the current trial.  In these and other unpublished cases, the 
effect of B cell depletion has been striking with reversal of advanced and life-threatening 
disease. While a specific antigenic target has not been identified in SSc-PAH, platelet-
derived growth factor (PDGF)-specific antibodies have recently been identified as highly 
prevalent in SSc [68]. SSc patients who received rituximab had lower titers of anti-PDGF 
antibodies, less skin fibrosis, significantly improved health assessment questionnaire scores, 
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oxygen species in vitro. Further, skin biopsies from 11 patients treated with rituximab in a 
Phase I study showed no progression of fibrosis; sequential biopsies were remarkable for a 
significant reduction of dermal myofibroblasts [69]. 
 
In PAH, even partial reversal of disease has the potential to be life-saving. In this respect, 
some patients have shown clinical improvement in their PAH with rituximab. In several 
unpublished cases (e.g. a case of Waldenstrom’s macroglobulinemia-associated PAH in 
Denver, Colorado), patients have enjoyed significant clinical improvement following 
rituximab therapy. The following is an example of one such case from Stanford University 
Medical Center: A 30 year old patient with SLE presented with Class IV heart failure 
symptoms, along with necrotizing digital ulcers. An echocardiogram revealed an RVEF of 
10% and an LVEF of 35% along with evidence of myocarditis. While the patient's left 
ventricular function improved with diuresis and pulse steroids, the patient's right heart 
systolic pressures were systemic. The patient did not respond to cyclophosphamide and was 
subsequently treated with several doses of rituximab. Within weeks, the patient's symptoms 
improved dramatically with a fall in the pulmonary artery pressures from the 80s-90s to 25 
mmHg and Class II heart failure symptoms (Dr. Roham Zamanian; personal 
communication). Another illustrative case at Stanford involved a woman with PAH 
secondary to the antiphospholipid antibody syndrome. The patient had suffered 
thromboembolic disease, and despite having undergone thromboendarterectomy twice, 
continued to have worsening symptoms of PAH. The patient was anticoagulated with 
coumadin and was treated with prostacyclin. The patient also suffered from severe 
thrombocytopenia and developed intracranial hemorrhages necessitating discontinuation of 
coumadin. The patient was treated with rituximab and enjoyed a significant improvement in 
her clinical findings with normalization of her platelet counts and downgrading to Class II 
symptoms. This improvement, attributed to rituximab, led to the conversion from intravenous 
prostacyclin to oral therapy. In summary, while there has been relatively little published to 
date on the use of rituximab in SSc, there appears to be a strong scientific and clinical basis 
for exploring this potential therapy, which is well studied in other diseases, in SSc patients 
who otherwise do not have much hope for long-term survival. 
 
Rituximab/Treatment Background 
1.3
 Current Licensing of Rituximab 
1.3.1
 
In 1997, rituximab was approved for treatment of subjects with relapsed or refractory low-
grade or follicular, B cell non-Hodgkin’s lymphoma. Rituximab is also approved for 
retreatment of lymphoma patients who have relapsed following initial rituximab therapy. 
Approved dosing is 375 mg/m2 each week for 4 or 8 doses. In March 2006, rituximab in 
combination with methotrexate was approved for use in the treatment of moderately to 
severely active rheumatoid arthritis (RA) patients who have had an inadequate response to 
one or more tumor necrosis factor (TNF) antagonist therapies. The approved dosing for RA 
is two infusions of 1000 mg given two weeks apart. In April 2011, the indications for 
rituximab (with dosing similar to non-Hodgkin’s lymphoma) were expanded to include 


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Wegener’s granulomatosis and microscopic polyangiitis in adult patients in combination with 
glucocorticoids. (See product label and prescribing information at 
http://www.Rituxan.com/rituxan/pi.) 
 
 Other Diseases in Which Rituximab Use Has Been Described 
1.3.2
 
This IgG1 chimeric monoclonal antibody was originally developed and approved for the 
treatment of non-Hodgkin’s lymphoma. In addition, rituximab has been used in the treatment 
of chronic lymphocytic leukemia [70] and Waldenström’s macroglobulinemia [71-73]. Post-
approval experience has been estimated to include more than 2,484,131 (Genentech, 2011) 
patient exposures. Although reactions frequently occur with the first infusion, most are Grade 
1 toxicity and respond to slowing or temporarily interrupting the infusion. In general, the 
drug has proven to be well tolerated, and serious reactions are very rare [74]. The period of B 
cell depletion with standard regimens of rituximab frequently averages 4 to 7 months [75, 
76]. A total of 8.4% of patients from the All Exposure RA population demonstrated a CD19 
count < lower limit of normal for 2 years or more following their last rituximab exposure. 
1.5% of these patients had CD19 counts below the lower limit of quantification for at least 2 
years. These patients are generally representative of the RA population with long-standing 
disease, previous use of multiple DMARDs, and high use of oral corticosteroids. Of note is 
the proportion of patients with low peripheral B-cell counts at baseline, prior to rituximab 
treatment, which correlates with those patients whose counts showed prolonged B cell 
depletion. The clinical relevance of prolonged peripheral B-cell depletion beyond two years 
following rituximab treatment is unknown; no safety signal has been identified in the 
prolonged B cell monitoring trials. The pattern of peripheral B cell depletion and repletion in 
granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) patients 
included in the RAVE study was similar to that previously observed in patients with RA [77].  
Effects on total immunoglobulin (Ig) levels have been much less striking, and evidence of 
immunodeficiency related to hypogammaglobulinemia has been rare. While rituximab results 
in the near-complete depletion of circulating B cells, the exact mechanism(s) of this effect 
remains poorly understood. Proposed mechanisms include induction of apoptosis, cellular 
clearance by the reticuloendothelial system through bound antibodies, complement mediated 
cell lysis, and antibody-dependent cellular cytotoxicity (ADCC) via Fc-bearing effector 
cells. One of the Fc receptors that may be involved in anti-CD20 mediated ADCC is Fc 
gamma receptor 3A (FcRIIIA), and FCGR3A polymorphisms have been shown to influence 
the response to rituximab [78, 79]. In studies of patients with lymphoma and SLE, 
homozygosity for the 158V allele predicted more complete depletion than homozygous 158F 
or heterozygous 158VF alleles.  
 
In addition to the treatment of hematologic malignancies, rituximab has been used to treat a 
variety of autoimmune disorders. Rituximab has been licensed for use in rheumatoid arthritis 
refractory to TNF antagonists in combination with methotrexate after several large clinical 
trials demonstrated a reduction in disease activity [80-82]. Rituximab has recently been 
investigated as a treatment for SLE (renal and extrarenal) in randomized, double-blind, 
placebo controlled trials. However, these studies failed to meet their primary efficacy 


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endpoints [83, 84]. Small studies and case series have examined the use of rituximab in a 
variety of the autoimmune diseases including SLE [85-88], autoimmune hemolytic anemia 
[89], immune or idiopathic thrombocytopenic purpura (ITP) [90, 91], and Sjogren’s 
syndrome [86, 92-99]. Early phase uncontrolled studies have also reported on the use of 
rituximab in other types of autoimmune disease, including, pemphigus vulgaris [100-103], 
dermatomyositis [104], polymyositis [105, 106], and others not reviewed in this document. In 
small, uncontrolled clinical studies investigating the use of rituximab in a total of 420 
patients with pemphigus vulgaris overall clinical response rates ranging from 30-90% (with 
variable definitions of response have been reported [77]. A recent report of a phase II trial in 
relapsing–remitting multiple sclerosis suggested that rituximab had beneficial effect even 
though traditionally MS is not felt to be primarily an autoantibody mediated disease [107]. 
Recently, rituximab has been demonstrated to be efficacious in inducing disease remission in 
anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis [108, 109]. 
 
 
Known and Potential Risks of Rituximab Treatment 
1.4
 
The label for rituximab includes four “black box” warnings: infusion reactions, severe 
mucocutaneous reactions, progressive multifocal leukoencephalopathy (PML), and hepatitis 
B virus reactivation.  
 
1. Infusion reactions:   Rituximab is associated with infusion-related/acute infusion 
reactions, which may be due to release of cytokines and/or other chemical mediators. 
Rituximab-induced reactions and sequelae include urticaria, hypotension, 
angioedema, hypoxia, bronchospasm, pulmonary infiltrates, acute respiratory distress 
syndrome, myocardial infarction, ventricular fibrillation, cardiogenic shock, 
anaphylactoid events, or death. Severe infusion-related reactions with fatal outcome 
have been reported during clinical trials and post-marketing use in oncology patients 
at a rate of approximately 0.04-0.07% (4 to 7 patients per 10,000). These deaths have 
primarily been related to the large B cell burden in oncology patients with resultant 
tumor lysis and cytokine storm after rituximab treatment. Approximately 80% of fatal 
reactions occur after the first infusion of rituximab, with time to onset of 30-120 
minutes. While approximately 32% of Rituxan-treated patients in RA placebo-
controlled trials experienced an adverse infusion reaction during or within 24 hours 
following their first infusion, less than 1% were serious reactions. Since rituximab 
was first approved in February 2006, there have been four cases of fatal infusion-
related reactions in RA patients treated with this agent.   
2. Severe mucocutaneous reactions:  Stevens-Johnson syndrome, lichenoid dermatitis, 
vesiculobullous dermatitis, paraneoplastic pemphigus, and toxic epidermal necrolysis 
have been reported in patients treated with rituximab. These reactions are rare, and 
the causal relationship to rituximab is difficult to determine, especially given the 
multiple medications these patients receive and the various autoimmune syndromes 
associated with non-Hodgkin’s lymphoma [110]. Reports of these events show 
variable onset time relative to rituximab exposure, with onset occurring as early as the 
day of first infusion. Some mucocutaneous reactions have been fatal. These reactions 


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have been reported very rarely in patients with autoimmune diseases treated with 
rituximab.  A fatal outcome was reported in less than 1 in 100,000 patients in the 
post-marketing setting [77]. 
3. Progressive multifocal leukoencephalopathy (PML):  PML has been reported in 
patients with autoimmune disease after receiving rituximab. PML is a demyelinating 
disease caused by the JC virus, a human polyoma virus. Approximately 80% of the 
population is seropositive for JC virus, which persists in a latent form but can 
reactivate in immunosuppressed patients. PML has been reported in a variety of 
immunosuppressed or immunodeficient patients including transplant patients, patients 
receiving chemotherapy, and HIV-infected patients independent of treatment with 
rituximab, and most recently in patients receiving natalizumab [111, 112]. Fatal cases 
of PML have also been reported in patients with hematologic malignancies and a 
variety of autoimmune diseases, including systemic lupus erythematosus (SLE, 
lupus), RA, and vasculitis following rituximab treatment. In general, the patients with 
autoimmune diseases had prior or concurrent immunosuppressive therapy. Most cases 
of PML were diagnosed within 12 months of the last infusion of rituximab. There 
have been 3 fatal reports of PML in patients with RA who had received rituximab (3 
reports in 100,000 patients with RA exposed to rituximab), with one case in a patient 
exposed to rituximab who had not received prior TNF antagonist therapy but had 
been treated with leflunomide. The role of rituximab alone in the development of 
PML is unclear. The risk of development of PML is unknown for individuals with 
SSc-PAH treated with rituximab. Recognition of PML requires a high index of 
suspicion. Patients who are considering or who have taken rituximab should be 
informed about the risks of developing PML, including that there is no effective 
treatment for PML. Patients who have been treated with rituximab should contact 
their doctor if they present with new neurological manifestations (including, but not 
limited to: confusion, dizziness, loss of balance, difficulty talking or walking or visual 
problems). Consultation with a neurologist, brain MRI, and lumbar puncture should 
be considered as clinically indicated.  
4. Hepatitis B Virus Reactivation: HBV reactivation has been reported in patients 
treated with CD20-directed cytolic antibody drugs, which include rituximab. In some 
cases this has resulted in fulminant hepatitis, hepatic failure, and death. Cases are not 
limited to hepatitis B surface antigen (HBsAg) positivity, but have also been reported 
in patients who are HBsAg negative but hepatitis B core antibody (anti-HBc) 
positive. Additionally, reactivation has occurred in patients with apparent resolved 
hepatitis B infection (HBsAg negative, anti-HBc positive and hepatitis B surface 
antibody [anti-HBs] positive). 
 
In addition to risks communicated explicitly by the “black box” warnings, the package insert 
for rituximab reports adverse reactions observed in controlled trials. Events that seem to be 
specific to use in malignancy are Tumor Lysis Syndrome (TLS), severe renal toxicity, and 
bowel obstruction/perforation. Cases of acute renal failure, hyperkalemia, hypocalcemia, 
hyperuricemia, and hyperphosphatemia, some fatal, have been reported within 24 hours after 
first infusion.  The risk of TLS correlates with number of circulating malignant cells or high 


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tumor burden. Abdominal pain, bowel obstruction, and perforation have occurred in patients 
receiving rituximab in combination with chemotherapy. Some of these cases were fatal. 
 
Adverse reactions observed in trials with RA patients have been reported separately from 
those observed in malignancy trials. Information to date reflects experience in 2578 RA 
patients treated with rituximab in controlled and long-term studies (total exposure of 5014 
patient-years). RA patients are likely to be more similar to the patients with SSc-PAH studied 
in this protocol due to the autoimmune association of the disease. Adverse events (AEs) 
occurring at a rate > 2% in RA patients who were given rituximab (or 1% greater than 
placebo), per the package insert are listed below:  
 
1. The common adverse events occurring in rituximab treated patients (greater than 10%) 
included infusion reactions, upper respiratory infection, nasopharyngitis, urinary tract 
infection, and bronchitis.     
2. Infusion reactions – In studies enrolling subjects with RA, 32% of rituximab-treated 
patients experienced an adverse reaction during or within 24 hours following the first 
infusion, compared to 23% of placebo-treated patients. This frequency decreased to 11% 
for rituximab-treated patients and 13% for placebo-treated patients for adverse events 
occurring within 24 hours following the second infusion. Serious acute infusion reactions 
were experienced by < 1% of patients in either the rituximab or placebo treatment groups. 
Acute infusion reactions requiring dose modification (stopping, interrupting, or slowing 
the infusion) occurred in 10% of rituximab treated patients and 2% of placebo treated 
patients. Acute infusion reactions (fever, chills, rigors, pruritis, urticaria/rash, 
angioedema, sneezing, throat irritation, cough, and/or bronchospasm with or without 
hypotension or hypertension) occurred with the first infusion in 27% of the rituximab 
treated patients and 19% of the placebo treated patients. Severe infusion reactions 
typically occurred during the first infusion with time to onset 30 to 120 minutes after 
starting the infusion. These reactions may include hypoxia and cardiovascular collapse, 
angioedema or bronchospasm, pulmonary infiltrates including acute respiratory distress 
syndrome, myocardial infarction, ventricular fibrillation, and cardiogenic shock. Milder 
infusion reactions include fever and chills, flushing, fatigue, pallor, headache, rhinitis, 
nausea and vomiting, tachycardia, tachypnea, dyspnea, chest pressure, and back pain. 
These reactions are similar to those described for IV immunoglobulin (IVIG). With IVIG, 
some patients are more sensitive to infusion reactions but “sensitivity” and “non-
sensitivity” are relative terms since slow infusions are tolerated by “sensitive” individuals 
[113]. Moreover, “sensitive” patients can become desensitized to the infusion reaction, 
i.e. if several infusions were given over a short period of time the infusion reaction 
disappeared or at least became less severe. There are some data indicating that 
prophylactic glucocorticoids may ameliorate these reactions [114, 115].  
3. Infections – In the pooled studies, infections occurred in 39% of rituximab treated 
patients and 34% of placebo treated patients. Serious infections occurred in 2% of 
rituximab treated patients and 1% of control patients. The most common infections were 
nasopharyngitis, upper respiratory tract infections, urinary tract infections, bronchitis, and 
sinusitis. The numbers of upper respiratory tract and rhinitis infections were slightly 
higher in the rituximab group (showed an absolute increase over placebo of at least 1%). 


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In the experience with rituximab in RA patients the most common serious infections (≥ 
0.5%) were pneumonia or lower respiratory tract infections, cellulitis, and urinary tract 
infections. Fatal serious infections included pneumonia, sepsis and colitis [116].  
4. Cardiac events – Serious cardiovascular events occurred in 1.7% of rituximab treated 
patients and 1.3% of placebo treated patients (pooled data). Cardiovascular deaths 
occurred in 0.4% of rituximab treated patients and 0% of placebo treated patients. Three 
cardiovascular deaths occurred during RA studies (all rituximab regimens) as compared 
to none in the placebo group. Myocardial infarction (MI) rates were consistent with rates 
in the general RA population. Patients with significant PAH may have baseline 
hypotension. In addition, the therapies used to treat PAH can cause hypotension. 
Exacerbation of hypotension in subjects with SSc-PAH treated with rituximab should be 
anticipated given the potential for the infusion itself to precipitate cardiac events.  
5. Hypophosphatemia and hyperuricemia – In the pooled data, newly occurring 
hypophosphatemia was observed in 12% of patients receiving rituximab compared to10% 
of patients on placebo. Newly occurring hyperuricemia was observed in 1.5% of patients 
receiving rituximab and 0.3% of patients on placebo. The RA specific data showed 
newly-occurring hypophosphatemia in 21% of patients, with newly occurring 
hyperuricemia in 2% of patients. The majority of observed hypophosphatemia occurred at 
the time of the infusions and was transient. 
6. Immunogenicity – Approximately 11% of rituximab treated RA patients tested positive 
for human anti-chimera antibody (HACA). Of 8 patients who had a serious infusion 
reaction, 2 were HACA positive. Approximately 12% of HACA positive patients who 
were re-treated with rituximab had a subsequent infusion reaction.  
 
The implications of these observed events call for further discussion. For example, 
determining the effect of rituximab on infections is problematic since patients treated with 
rituximab often have concomitant immunodeficiency and/or are on immunosuppressive 
drugs. In patients treated for B cell lymphomas, the incidence of serious infections (grade III 
or IV including sepsis) is about 2%. One reassuring finding has been the observation in 
several studies that total serum IgG and levels of protective IgG antibodies are generally 
maintained after treatment with rituximab [117, 118]. Cases of hypogammaglobulinemia 
have been reported [119-122]. Criteria supporting the use of IVIG should probably be similar 
to those used for other patients with secondary humoral immunodeficiency, for example: (i) 
low levels of protective antibodies to encapsulated organisms, viruses, or bacterial toxins 
without a response to immunization; (ii) severe hypogammaglobulinemia (serum IgG < 200 
mg/dL); and (iii) increased rate, severity, or duration of infections, especially infections of 
the respiratory system or with encapsulated organisms including, but not limited to, 
Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitides [123, 124]. 
 
Serious viral infections, either new, reactivated, or exacerbated, have been reported after use 
of rituximab including CMV, herpes simplex virus, West Nile virus, and hepatitis C. Some of 
these infections resulted in fatal outcomes up to 1 year following discontinuation of 
rituximab [116]. Most of these serious viral infections occurred in patients with hematologic 
malignancies receiving combination chemotherapy with rituximab. 
 


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An expected complication of rituximab therapy is failure to respond to vaccines. This was 
originally described in non-human primates treated with rituximab but has been confirmed in 
lymphoma patients, patients on dialysis, and patients with SLE [125-128]. Additional studies 
are needed to determine when this effect resolves. Inhibition of humoral immune responses 
poses a special problem with unusual pathogens including, but not limited to, parvovirus B19 
or infections where toxins are important. Subjects in this trial will be vaccinated against 
pneumococcal pneumonia before entry. Subjects enrolled in this trial will continue to receive 
annual influenza vaccines and will be informed that the vaccine may not be fully protective. 
Vaccination with live vaccines is not recommended, as the safety of immunization with live 
vaccines following rituximab treatment has not been studied. Non-live vaccinations should 
be given at least 4 weeks prior to administration of rituximab. 
 
Although neutropenia and thrombocytopenia have been associated with rituximab, cases are 
generally transient and do not cause clinically significant problems. Late onset neutropenia 
(LON) with rituximab has been described in patients treated with aggressive chemotherapy 
for hematological malignancies, but no such cases have been reported in patients with 
autoimmune disease. 
 
LON may also predispose one to infections. There are a number of reports of LON after 
treatment of lymphomas with rituximab [129]. In a recent report, LON was investigated in 
130 patients with B cell lymphoma receiving chemotherapy with or without rituximab [130]. 
LON was detected in 6 of 76 patients receiving rituximab and 0 of 54 patients not receiving 
rituximab. The median nadir neutrophil count for the patients who developed LON was 200; 
the lowest counts were 23 and 32. Onset occurred at an average of 175 days (77-204 days) 
post treatment and lasted an average of 14 days (11-16 days). One patient developed a buccal 
cellulitis; the other 5 patients were asymptomatic. Neutropenia responded to growth factors. 
Preliminary data suggests an association between B cell recovery and granulocyte decline. 
These changes in B cell recovery and granulocyte decline were also correlated with changes 
in the level of SDF-1, a chemokine affecting both B cell and granulocyte homeostasis. Since 
blood counts were only checked every 3 months, the authors estimated that they probably 
missed more cases of neutropenia than they found. Whether LON will be seen in non-
malignant disease treated with rituximab is unclear, but until we have more data, neutrophil 
counts should be carefully monitored.   
 
Rituximab is a humanized antibody but its variable regions are still of mouse origin. 
Therefore an immune response against rituximab (HACAs) can be seen. The clinical 
relevance of HACAs as a result of treatment with rituximab is unclear. Infusion reactions and 
rapid clearance have been linked to HACAs against other humanized monoclonal antibodies, 
including infliximab [131]. With infliximab, higher dose and/or combination with 
methotrexate reduced the occurrence of HACAs. However, 2 studies have been performed 
suggesting that subjects who develop serum antibodies to infliximab bind unique epitopes 
that do not cross-react with other antibodies, including rituximab [132, 133]. Anti-rituximab 
HACAs are rare among patients with lymphomas treated with this drug, and these HACAs 
are generally of low titer [134]. However, in a phase I/II dose escalation study of rituximab 
for SLE, high titer HACAs were detected in 6 of 17 patients [86]. Several factors may have 


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contributed to this unusual rate of HACA formation including the low dose of rituximab in 
the first two dose groups, the use of oral rather than IV steroids as prophylaxis for infusion 
reactions, and the use of low dose or no immunosuppressive drugs in many of the patients. In 
summary, while it is possible that recipients of rituximab may develop hypersensitivity, 
which may extend to other proteins of mouse origin, and thus may not be able to use other 
chimeric monoclonal antibodies or mouse-derived antibodies in the future, it has not been 
definitively shown that hypersensitivity to one chimeric molecule precludes someone from 
receiving another. For additional information on AEs observed to date with study drug and 
active controls, see the Rituxan Prescribing Information at 
http://www.rituxan.com/rituxan/pi.  
 
Rare serious adverse events (SAEs) have occurred in patients following completion of 
rituximab infusions: arthritis, disorders of blood vessels (vasculitis and lupus-like syndrome), 
lung disorders (including pleuritis, scarring of the lung and bronchiolitis obliterans), and eye 
disorders (uveitis and optic neuritis). Patients with pre-existing cardiac conditions, including 
arrhythmia and angina pectoris, have had recurrences of these cardiac events during 
rituximab infusions. Abdominal pain, bowel obstruction, and perforation have been reported 
in patients receiving rituximab in combination with chemotherapy for malignancy.  
 
Serum sickness, a syndrome consisting of one or more of the following has been reported: 
fever, joint pain, skin rash or hives, hematuria, or hypocomplementemia. The syndrome may 
occur as long as several weeks after the first infusion. This syndrome is believed to be caused 
by deposition in tissues of rituximab-anti-rituximab immune complexes [135-138].  
 
Rituximab is a Category C drug. Reproduction studies in primates at human maternal 
exposure dose levels showed no teratogenetic effects. There are no adequate and well-
controlled studies of rituximab in pregnant women. Postmarketing data indicate that B cell 
depletion can occur in infants exposed in-utero to rituximab, which generally resolves in less 
than six months. Rituximab has been detected in the serum of infants exposed in-utero. 
 
Rationale for Study 
1.5
 
The most effective treatments for SSc target the vascular complications and inflammatory 
processes that characterize this disease [139]. One of the most feared complications of SSc, 
SSc-PAH, demonstrates both vascular and inflammatory pathology [140], and yet, a 
randomized clinical trial specifically directed at this process has not been performed. Of note, 
the findings of a major study have just been published demonstrating that patients with SSc-
associated interstitial lung disease enjoy a modest but significant beneficial effect with 
cyclophosphamide treatment [141]. SSc-PAH is associated with disabling dyspnea as well as 
significant mortality. An increasing body of experimental and clinical evidence is implicating 
dysregulated B cells and pathogenic autoantibodies in the pathogenesis of SSc [25, 54, 55, 
142]. A recently described animal model of PAH has shown that autoimmune injury, 
characterized by pulmonary B cell accumulations and anti-endothelial cell antibody 
deposition on pulmonary vasculature, results in severe PAH that is similar to the human 
condition [57]. In another related preclinical model, correcting immune dysregulation 


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attenuates pulmonary B cell accumulation and antibody deposition, and greatly ameliorates 
PAH [143]. Finally, in clinical practice, rituximab has been anecdotally reported to be 
effective in several forms of PAH [144, 145] including SSc-PAH [67]. Given the strong 
scientific evidence implicating autoimmune vascular injury in disease pathogenesis, the 
expanding number of cases where rituximab has been highly effective in autoimmune-
associated PAH, the poor overall prognosis of patients with SSc-PAH, the acceptable risk-
benefit ratio of rituximab in this patient population, and the newly emerging and promising 
pre-clinical data, there is strong rationale for a trial testing the efficacy of B cell depletion in 
SSc-PAH. 
 
Change in 6MWD has been chosen as the primary efficacy endpoint for this study as it is a 
commonly used functional measure of disease severity that has been previously used by the 
FDA for licensure which will make it more comparable to past trials.  For more information 
about the change in primary endpoint from pulmonary vascular resistance (PVR) to 6MWD, 
see Section 8.1, Sample Size. A recent analysis was performed by Mathai (2012) to find the 
minimally important difference in 6MWD at 16 weeks. An analysis of 405 subjects enrolled 
in the Pulmonary Arterial Hypertension and Response to Tadalafil trial showed the estimated 
consensus minimally important difference (MID) at 16 weeks was 33m in subjects with 
PAH; therefore 33m was chosen as a minimally important distance for this trial. This study is 
also of longer duration than many previous trials in the field. Unlike vasodilating agents, B 
cell depletion may not have an immediate effect on PAH. Twenty four weeks after B cell 
depletion, vascular remodeling may have occurred resulting in disease stabilization or 
improvement or skin involvement could be improved, which could result in an increased 
6MWD. Measurement of the clinical endpoints at this time, distant from study drug 
administration, may permit detection of the largest difference in the study endpoints between 
the 2 arms. In addition, measurement of the clinical and hemodynamic endpoints at 24 weeks 
will allow an assessment of durability of response to the study intervention. A relative 
increase of 20% in 6MWD or decrease of 30% in PVR (i.e. compared to placebo-treated 
controls) would suggest disease remission and could translate into improved survival. 
 Rationale for the Entry Criteria 
1.5.1
 
The objective of this trial is to determine if rituximab has a beneficial effect on clinical 
disease progression with an acceptable level of toxicity in subjects with SSc-PAH. 
The inclusion criteria are designed to ensure that all subjects have well documented, 
relatively recent onset PAH.  
 
 Early in disease, the vascular lesion is assumed to have an inflammatory component 
potentially responsive to rituximab. It is unclear how long the inflammatory 
component persists. However, there is persistent B cell dysregulation as evidenced by 
the persistence of autoantibodies. Because lung disease in these patients may be 
heterogeneous with some areas being more actively inflamed than others, it is 
possible that B cell depletion may impact individual patients in a heterogeneous 
fashion. Patients that have more advanced and possibly irreversible disease in certain 
areas of their lungs may also have actively inflamed regions in other anatomic 


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pulmonary locations; these latter areas may be more susceptible to benefit from B cell 
depletion. 
 In order to optimize the ability to detect a treatment effect, subjects should have 
sufficient disease burden attributable to SSc-PAH to cause at least mild symptoms 
manifested by some limitation of physical exertion with maximal effort. However, the 
need to also include subjects with early SSc-PAH implies that an upper limit of the 
six minute walk distance should not be defined. The entry criteria attempt to balance 
the need to include subjects with early SSc-PAH and the need to detect a treatment 
effect of rituximab in addition to standard therapy. Therefore, subjects with Class II 
as well as Class III and IV heart failure are included in this trial. In an attempt to 
include subjects with early disease but with definitive SSc-PAH and poor prognosis, 
the study entry mPAP has been defined as a lower limit of ≥ 25 mmHg.  
 NYHA functional class IV subjects have been included in a number of previous 
clinical trials in PAH. These patients are by definition severely impaired functionally, 
and tend to have a poor prognosis. Most subjects with functional class IV disease who 
are being considered for enrollment in this study would likely have failed other 
currently available therapies. Even understanding that such patients may be at 
increased risk for complications during or immediately following the infusion of 
rituximab, the risk/benefit ratio in this setting is acceptable, especially in light of the 
additional precautions being taken to optimize the safety of these severely ill patients. 
 Subjects must be on stable doses of standard of care to ensure the changes seen 
during the study do not reflect the confounding effect of changes in concurrent PAH 
therapy, but rather use of the study drug.  
 Subjects should be competent adults able to understand the risks of participation in 
the study and capable of making medical decisions.  
 The upper age of the subjects permitted to enter this study is in line with what has 
been deemed safe in other rituximab trials in RA, but has been limited to 75 in order 
to avoid comorbidities of advanced age (for a population already at greater risk due to 
pre-existing cardiorespiratory compromise) [116]. 
 
The exclusion criteria are designed to protect the subjects’ safety by excluding those who are 
at risk for adverse events either because of the severity of their disease, their predisposition to 
infection or to the presence of co-morbid disease and to ensure a uniform study population.  
  
 Subjects with longer-standing disease (i.e. beyond 5 years) are excluded because it is 
hypothesized that the late fibroproliferative component of SSc may be less responsive 
to immunotherapy than the early inflammatory vascular lesions. In order to evaluate a 
change within 6 months of entry, subjects with steadily progressing disease are 
required. 
 Subjects must have PAH secondary to SSc only. Interstitial lung disease is a common 
pulmonary manifestation of SSc that can be associated with “secondary” pulmonary 
hypertension for reasons likely distinct from those factors causing “primary” SSc-
PAH. Therefore subjects with significant interstitial lung disease (ILD) resulting in 
secondary PAH are excluded from this trial. Subjects with mild ILD are permitted in 
the study.  Diagnostic criteria for mild ILD have been developed. Subjects with other 


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diseases resulting in PAH are excluded from this trial, as are subjects with elevated 
pulmonary capillary wedge pressures whose PAH may be the result of left heart 
failure, pulmonary venous occlusion, or mitral valve disease and not SSc.  
 Because rituximab is an immunocompromising agent, subjects who have received 
recent live vaccines within 2 months, have any evidence of severe bacterial infections 
as defined in the entry criteria or chronic infections (HIV, TB, Hepatitis B or C), will 
be excluded from this trial. 
 Rituximab has been associated with cardiovascular deaths, and cardiac disease is a 
warning specifically mentioned in the prescribing information. Therefore, a recent 
history of non-SSc-PAH-related heart disease excludes patients from entry into this 
study.  
 Patients with a prior history of anaphylaxis or IgE-mediated hypersensitivity to 
murine proteins or any component of rituximab will be excluded. In addition, in order 
to keep the population uniform in terms of disease status, the trial is targeting subjects 
who have not received prior lymphocyte depleting agents.  
 Subjects who are at increased risk for adverse events based on the presence of co-
morbid disease will be excluded from this trial including renal disease even if 
secondary to scleroderma, recent malignancy, pregnancy, and blood dyscrasias.  
 
Subjects who are unable to comply with the demands of this protocol for either physical or 
psychiatric reasons are also excluded.  
 
 Rationale for Mechanistic Studies  
 
1.5.2
 
Currently serum biomarkers are not available to reliably follow the clinical course of SSc-
PAH. The ASC01 mechanistic studies aim to evaluate several markers of disease and the 
change associated with rituximab treatment: biochemical indicators of heart failure severity 
(BNP/NT-proBNP), B cell numbers, and inflammatory markers. Because hyponatremia has 
recently been demonstrated to predict right heart failure and poor survival in PAH [146], we 
will monitor serum sodium levels throughout the trial. BNP/NT-proBNP is already an 
established marker useful in following heart failure in SSc-PAH [147] and will be used to 
follow response to therapy in this trial. Because CD19 is co-expressed on CD20 B cells, we 
expect numbers of CD19+ cells will fall following rituximab therapy. CD19+ cells will be 
measured to track the repopulation of B cells [148, 149] as well as to determine if B cell 
clones are present in SSc-PAH and what impact rituximab may have on these clones. With 
falling B cells, it is possible that serum immunoglobulins will also fall, and therefore the 
decline and eventual return of immunoglobulin subclasses will be monitored in this trial. 
With regard to following inflammatory biomarkers, both autoantibodies and serum cytokines 
will be measured on a regular basis. Anti-U1RNP, anti-U3RNP, anti-B23, anti-cardiolipin, 
anti-CENP-B have been reported to be elevated in SSc-PAH [150, 151] and will be followed 
at several time points to correlate disease activity with autoantibody levels. Because no single 
autoantibody has proven sufficient to track SSc-PAH activity, we will determine whether a 
panel of autoantibodies provides a more specific and sensitive measure. Finally, serum IL-1 


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and IL-6 are known to be elevated in severe primary pulmonary arterial hypertension [152] 
and may similarly be elevated in SSc-PAH. Other pro-inflammatory cytokines may be 
similarly elevated. Pilot studies done at Stanford University were performed using a 
multiplex analysis of serum. Preliminary evidence pointed to distinct inflammatory profiles 
in patients with PAH as compared with healthy controls, as well as sub-groups of PAH 
etiologies as compared to each other. For this reason, a similar multiplex approach for SSc-
PAH patients could identify an important inflammatory signature, as well as provide 
biomarkers of inflammatory resolution following B-cell depletion. Thus, measures of both an 
autoantibody panel and a panel of cytokines may provide a peripheral (and therefore 
accessible) assay of systemic inflammation. The mechanistic studies are designed, in a 
straightforward manner, to measure B cells, heart failure and inflammatory changes over the 
course of the clinical trial. 
2 STUDY OBJECTIVES AND PURPOSE  
The objective of this trial is to determine if rituximab has a marked beneficial effect on 
clinical disease progression relative to placebo, with minimal toxicity, in subjects with SSc-
PAH.   
 
Primary Objective 
2.1
 To compare patients with SSc-PAH treated with rituximab to those on placebo for 
change in exercise capacity, as determined by 6-minute walk distance (6MWD). 
 
Hypothesis: The treatment group will demonstrate improvement or stabilization relative to 
the placebo arm in 6MWD from baseline to 24 weeks post-treatment initiation.   
 
Secondary Objectives 
2.2
Secondary Efficacy Objectives: 
 To compare patients treated with rituximab to those on placebo for change in exercise 
capacity, as determined by 6MWD, at time points other than 24 weeks. 
 To compare patients treated with rituximab to those on placebo for change in PVR as 
assessed by right heart catheterization. 
 To compare patients treated with rituximab to those on placebo for time to clinical 
worsening, defined as first occurrence of: 
a. death,  
b. hospitalization for SSc-PAH, 
c. lung transplantation,  
d. atrial septostomy,  
e. addition of other PAH therapeutic agents, or   
f. worsening of 6MWD by > 20% and a decrease in functional class. 
 To compare patients treated with rituximab to those on placebo who changed or 
added other PAH therapeutic agents. 


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 To compare patients treated with rituximab to those on placebo for change in quality 
of life as determined by the short form 36 (SF-36) and the disability index of the 
Scleroderma Health Assessment Questionnaire (SHAQ). 
 To compare patients treated with rituximab to those on placebo for change in number 
of new digital ulcers. 
 To compare patients treated with rituximab to those on placebo for change in severity 
of Raynaud phenomenon as measured by the VAS scale of the SHAQ. 
 To compare patients treated with rituximab to those on placebo for change in DLCO 
and room air oxygen saturation at rest .  
 
A secondary objective of this study is to determine whether the effects on clinical disease 
progression are paralleled by changes in selected biomarkers.   
 
Secondary Safety Objectives: 
 To assess the safety and tolerability of rituximab for the treatment of SSc-PAH in 
patients receiving stable background medical treatment with prostanoid, endothelin 
receptor antagonist, PDE-5 inhibitor therapy, and/or guanylate cyclase stimulators. 
 
Mechanistic Study Objectives: 
 CD19 Studies 
 To determine whether the extent and/or duration of CD19+ B cell depletion 
correlates with treatment response as measured by PVR (right heart 
catheterization), exercise capacity (6MWD), and quality of life (SF-36 and 
SHAQ). 
 To monitor and correlate the reconstitution patterns of B cell subpopulations 
with treatment response. 
 To determine if the biomarkers anti-U1 RNP, anti-U3 RNP, anti-B23, anti-
cardiolipin, anti-Th/To, anti-CENP-B, RF, and other autoantibodies, and quantitative 
immunoglobulin levels, including IgG subclasses, correlate with treatment response 
as measured by PVR (right heart catheterization), exercise capacity (6MWD), and 
quality of life (SF-36 and SHAQ). 
 To determine if the biomarkers BNP/NT-proBNP, serum Na+, IL-1, IL-6, and other 
cytokines correlate with treatment response as measured by PVR (right heart 
catheterization), exercise capacity (6MWD), and quality of life (SF-36 and SHAQ).  
 To determine if B cell clones are present in SSc-PAH and what impact rituximab 
may have on these clones. 
 To determine if B cell clones present in SSc-PAH are correlated with clinical 
outcomes. 
 
 
Exploratory Objectives 
2.3
 To evaluate agreement between PVR calculated by site-based standards and a 
standardized Fick-based calculation of PVR. 


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 To assess sensitivity of the secondary efficacy analysis to standardization of the PVR 
calculation. 
 To assess the utility of a standardized derived pulmonary artery capacitance as a 
primary efficacy endpoint. 
 To evaluate the relationship between 6MWD, PVR, and other clinical measures. 
 
3 STUDY DESIGN 
 
Description of Study Design 
3.1
This is a prospective, double-blind, placebo-controlled, multi-center, randomized trial to 
evaluate the effect of rituximab on disease progression in subjects with SSc-PAH receiving 
concurrent stable-dose standard medical therapy with a prostanoid, endothelin receptor 
antagonist, phosphodiesterase 5 (PDE-5) inhibitor, and/or guanylate cyclase stimulators. The 
study will focus on assessment of clinical response and safety measures longitudinally. In 
addition, the effects of treatment with rituximab on the underlying immune mechanisms 
associated with B-cell dysregulation and pathogenic autoantibody response in this disease 
will be investigated.    
 
Subjects with SSc-PAH with elevated mean pulmonary artery pressure as measured by right 
heart catheterization will be enrolled. The diagnosis of SSc-PAH should be confirmed by a 
rheumatologist experienced in the diagnosis and treatment of systemic sclerosis in 
conjunction with a pulmonologist or cardiologist specializing in management of PAH. Both 
specialists will be part of the study team at each site.  
 
Rituximab/placebo will be administered as two IV infusions of 1000 mg each, given two 
weeks apart at Day 0 and Week 2. All subjects will receive 40mg of prednisone orally the 
night before and morning of each infusion, as well as 100 mg methylprednisolone or 
equivalent corticosteroid intravenously approximately thirty minutes prior to each infusion 
with diphenhydramine and acetaminophen orally approximately thirty to sixty minutes prior 
to each infusion of rituximab or placebo. Subjects will remain on their baseline standard 
medical regimen.  
 
Sixty eligible subjects will be accrued. Each potential study subject will provide written 
informed consent prior to screening procedures. All inclusion and exclusion criteria (Section 
4, Selection of Subjects) must be met at time of randomization prior to receipt of the first 
dose of rituximab/placebo (Day 0, Treatment Initiation).  
 
Clinical assessments and sample collection will occur at regularly scheduled visits through 
Week 48 with telephone follow-up conducted in the interim. If a subject has not recovered B 
cells by Week 48, B cell studies will be conducted quarterly until recovery is documented or 
for 2 years after initial treatment (whichever occurs first). Recovery is defined as the time 
point at which B cells recover to within 10% of baseline levels; if a baseline value is not 
available then recovery is defined as a B cell level of at least 90% of the lower limit of 


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normal. During this quarterly monitoring period of B cell recovery, AEs and SAEs will be 
assessed, providing the subject has not withdrawn consent, to capture any infectious event ≥ 
Grade 3 using the National Cancer Institute - Common Terminology Criteria for Adverse 
Events (NCI-CTCAE). No additional study-related data will be collected. Vital status on all 
subjects will be evaluated ≥ 2 years after randomization. 
 
The primary efficacy endpoint is the change in 6MWD from baseline to 24 weeks after 
treatment initiation. Hemodynamic measures will be assessed at baseline and Week 24, 
contributing to the understanding of the relationship between PVR and clinical endpoints. 
Initiation of new PAH therapy due to disease worsening prior to Week 24 will prompt an 
endpoint visit and right heart catheterization prior to initiation of the new therapy. B cell 
recovery for all will be monitored by serial assessments of B cell counts as determined by 
flow cytometry. 
 
The total duration of the study is anticipated to be approximately 8 years.  
 Stratification, Randomization, and Masking 
3.1.1
Subjects will be randomized in a 1:1 ratio to receive 2 infusions 14 days apart of either 
rituximab 1000 mg (treatment arm) or placebo (control arm). The automated randomization 
system will implement an adaptive randomization procedure to maintain balance between 
treatment groups overall, by study site, with respect to disease severity as measured by PVR 
at baseline, and with respect to time since diagnosis of SSc-PAH.  
 
To maintain study blind, the appearance of the packaging and solutions used to administer 
study drug/placebo will be identical in both study arms. 
 
All clinical and immunology laboratory staff will be masked to the treatment assignments 
until the completion of the study. In addition, immunology laboratory staff will not have 
access to any clinical results and clinical staff members, including the investigators, will not 
have access to any immunology lab results until completion of the study. B cell recovery 
status and the need for quarterly monitoring visits as defined in Section 6.5.4.12, Visit 16 
[Week 48] will be communicated to clinical staff upon completion of Visit 16 or Early 
Endpoint visit. If B cells have not recovered by two years after treatment initiation, B cell 
levels will be provided to the clinical staff. 
 
Individual treatment assignments will not be unmasked until all subjects have completed the 
study. In the event a treatment assignment needs to be unmasked for safety reasons, the site 
PI will notify the SACCC, who will consult with the DAIT medical monitor. The DAIT 
medical monitor will review the circumstances and grant approval as indicated. In the event 
of a true medical emergency necessitating immediate unmasking during off-hours, the site 
pharmacist will confer with the study PI or designee prior to release of the treatment 
assignment to emergency treating personnel. 


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Description of Endpoints 
3.2
 Primary Efficacy Endpoint 
3.2.1
The primary efficacy endpoint is change in exercise capacity, assessed by 6MWD, from 
baseline to 24 weeks after treatment initiation. 
 Secondary Efficacy Endpoints 
3.2.2
Secondary efficacy endpoints include the following clinical endpoints: 
 
1. Change in PVR measured by right heart catheterization from baseline to 24 weeks 
after treatment initiation. 
2. Exercise capacity, as determined by 6MWD, assessed longitudinally over the 48 
weeks following treatment initiation.  
3. Assessment of time to clinical worsening, censored at 48 weeks, defined as first 
occurrence of: 
a. death,  
b. hospitalization for SSc-PAH,  
c. lung transplantation,  
d. atrial septostomy,  
e. addition of other PAH therapeutic agents, or  
f. worsening of 6MWD by > 20% and a decrease in functional class. 
4. Percentage of subjects who changed or added PAH therapeutic agents at 24 and 48 
weeks. 
5. Change in quality of life as determined by the SF-36 and disability index of the 
SHAQ from baseline to 24 and 48 weeks after treatment initiation. 
6. Number of new digital ulcers assessed longitudinally over both the 24 and 48 weeks 
following treatment initiation. 
7. Severity of Raynaud phenomenon, as measured by the VAS scale of the SHAQ, 
assessed longitudinally over both the 24 and 48 weeks following treatment initiation. 
8. DLCO and oxygen saturation at rest on room air assessed longitudinally over both the 
24 and 48 weeks following treatment initiation. 
9. Change in PVR from baseline to 24 weeks after treatment initiation as a percentage of 
the baseline value. 
 
Secondary efficacy endpoints also include the following mechanistic endpoints: 
 
1. CD19 Studies 
a. CD19+ B cells (absolute number and percentage of gated lymphocytes) at 
time of B cell depletion and longitudinally over the 48 weeks following 
treatment initiation, where B cell depletion is defined as < 5 CD19+ cells/μL. 
b. Time to repopulation of CD19+ B cells, defined as the time point at which B 
cells recover to within 10% of baseline levels; if a baseline value is not 
available then recovery is defined as a B cell level of at least 90% of the lower 
limit of normal. 


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c. Reconstitution pattern of B cell subpopulations assessed longitudinally over 
the 48 weeks following treatment initiation. 
2. Level of anti-U1 RNP, anti-B23, anti-cardiolipin, anti-CENP-B, RF autoantibodies, 
and immunoglobulins, including IgG subclasses, measured by ELISA and assessed 
longitudinally over the 48 weeks following treatment initiation. 
3. Presence or absence of anti-U3 RNP, anti-Th/To, and other autoantibodies assessed 
longitudinally over the 48 weeks following treatment initiation. 
4. Level of BNP/NT-proBNP, serum Na+, IL-1, IL-6, and other cytokines assessed 
longitudinally over the 48 weeks following treatment initiation. 
5. Presence or absence of B cell clones assessed at baseline and longitudinally over the 
48 weeks following treatment initiation. 
 Secondary Safety Endpoints 
3.2.3
The safety endpoints will include the following: 
1. All NCI-CTCAE Grade 3-5 AEs, which are defined as possibly, probably, or 
definitely related to rituximab.  
2. All NCI-CTCAE Grade 3-5 adverse events. 
3. All infection related events. 
4. Treatment-related mortality, defined as death occurring at any time after 
randomization and possibly, probably, or definitely resulting from treatment given in 
the study. Study investigators will make an initial determination of the cause of death 
of any subject and if uncertainty remains, a final decision will be made by an 
independent blinded panel after reviewing all clinical and, if available, autopsy data. 
5. All-cause mortality, defined as any death occurring at any time after randomization. 
6. CD19+ B cell levels post treatment (note that analyses will be performed regularly 
per Table 6.1 through 48 weeks and quarterly thereafter as documented in Protocol 
Section 3.1, Description of Study Design). 
7. Peripheral blood cell counts, liver function, and creatinine levels after treatment. 
 Exploratory Endpoints 
3.2.4
Exploratory analysis of standardization of the PVR calculation will be based on PVR 
calculated from a standardized Fick calculation of cardiac output, which is a function of 
hemoglobin, arterial and mixed-venous saturations, and oxygen consumption according to 
the La-Farge and Miettinen formula. The cardiac output calculation is a function of assumed 
oxygen consumption according to the La-Farge calculation of values normalized for age, 
gender, heart rate. Calculation of a standardized Fick cardiac output will also allow for 
determination of the pulmonary artery capacitance (PACf). See Appendix E, Exploratory 
Analysis: Standardized Fick-based Pulmonary Vascular Resistance and Pulmonary Artery 
Capacitance for details. 
 
Additionally, an exploratory analysis of the relationship between change in PVR and change 
in 6MWD will be addressed. Relationships with other clinical endpoints, like change in NT-
proBNP, will also be explored. 


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Subject Disposition 
3.3
 Definition of Subject Completion 
3.3.1
A subject is considered to have completed the study if he/she has completed Visit 16 (Week 
48).  
 Discontinuation of Protocol-Specified Treatment Requirements 
3.3.2
Protocol-specified requirements for the treatment of subjects, including regimens for 
investigational, concomitant, or rescue medications, as well as restrictions for disallowed 
medications, will be discontinued for any individual subject under the following conditions: 
1. Any protocol-specified treatment requirement will be discontinued immediately at 
any time during the study at the request of the subject. 
2. Investigator or DAIT/NIAID will discontinue any protocol-specified treatment 
requirement if the subject’s health, safety, and/or well-being are threatened. 
3. Protocol-specified treatment requirements for rituximab will be discontinued and 
replaced with appropriate therapy for any subject who experiences any of the 
following: 
 Significant cardiopulmonary disease PROGRESSION as manifested by new or 
worsening Grade 3 or higher AEs including: 
o Incompletely controlled or life threatening arrhythmias  
o Vasovagal episode occurring either during or within 12 hours following an 
infusion 
o Sinus bradycardia unresponsive to atropine treatment as detailed in 
Section 5.4.3.2, Cardiovascular 
o Chest pain consistent with angina or evidence of myocardial infarction 
o Cardiopulmonary arrest 
o Sustained systemic hypertension requiring additional therapy above 
baseline medications 
o Sustained hypotension 
o Congestive heart failure worsening from baseline level 
o Pericardial effusion or pericarditis 
o Pneumonitis progressing above baseline level  
o Bronchospasm 
o Dyspnea progressing above baseline level 
o Stridor or other indicators of respiratory compromise  
 New symptomatic pericarditis Grade 2 or higher  
 Toxicities of rituximab, if required as per Sections 5.3.5, Criteria for 
Discontinuing Rituximab Infusion and 5.4.3, Management of Infusion 
Reactions/Known Toxicities to Rituximab 
4. Disease progression requiring change in Concurrent Medication as per Section 5.7, 
Concurrent Medications and Therapy. 


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 Follow-up Requirements for All Treated Subjects  
3.3.2.1
Independent of whether treated subjects have completed the full course of rituximab, the 
following procedures will apply:  
1. If prohibited medications as defined in Section 5.6, Prohibited Medications and 
Treatments are not needed for treatment of progressive PAH, the subject will 
complete all scheduled study visits including all exams, procedures, assessments, 
and tests according to the protocol.   
2. If prohibited medications as defined in Section 5.6, Prohibited Medications and 
Treatments (i.e. addition of new or a change in the dose of background PAH 
therapies [prostanoid, endothelin receptor antagonist, PDE-5 inhibitor, and/or 
guanylate cyclase stimulators] or surgical therapy) are needed for treatment of 
progressive PAH prior to the Week 24 visit, or if the subject withdraws consent, 
he/she will be asked to complete an Early Endpoint Visit as detailed in Section 
6.5.5, Early Endpoint Visit.  
3. In addition, if the subject has not withdrawn consent and has not achieved B cell 
recovery, follow-up clinical care must include visits at least quarterly to monitor 
recovery of B cells. Quarterly monitoring visits will continue until B cell recovery 
or for 2 years after initial treatment (whichever occurs first). Recovery is defined 
as the time point at which B cells recover to within 10% of baseline levels; if a 
baseline value is not available then recovery is defined as a B cell level of at least 
90% of the lower limit of normal. During this monitoring period, AEs and SAEs 
will be assessed, providing the subject has not withdrawn consent, to capture any 
infectious event ≥ Grade 3 using the NCI-CTCAE. No additional study-related 
data will be collected. 
 
Subjects who have discontinued their concurrent medications prior to Week 24 for toxicity 
only, and not disease progression, will continue to participate in the study. They will be 
permitted to change medication for PAH in order to maintain control of their disease and will 
complete all exams, procedures, assessments, and tests according to the protocol.  
  Subject Withdrawal from the Study 
3.3.3
When a subject is withdrawn from the study after initiating treatment but prior to B cell 
follow-up, protocol-specified treatment requirements are discontinued and study-related 
visits, exams, procedures, assessments, tests and data collection are terminated after 
completion of an Early Endpoint Visit as detailed in Section 6.5.5, Early Endpoint Visit. 
However, individual subjects will be withdrawn from the protocol under the following 
conditions: 
1. The subject withdraws consent. 
2. The investigator or DAIT/NIAID believes it is in the best interest of the subject. 
3. The study is terminated. 
 
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  Procedures for Subject Withdrawal 
3.3.3.1
Unless a subject withdraws consent and refuses, all subjects who are being withdrawn or 
elect to withdraw from the study after initiating treatment will undergo an Early Endpoint 
Visit as detailed in Section 6.5.5, Early Endpoint Visit. In addition, unless consent is 
withdrawn, B cell monitoring will be performed as outlined in Section 3.3.2.1, Follow-up 
Requirements for All Treated Subjects, #3. 
 Subject Replacement 
3.3.4
Randomized subjects who withdraw from the trial prior to starting rituximab infusion will be 
replaced. Subjects who discontinue study treatment or withdraw from the trial after initiating 
the first treatment will not be replaced. 
4 SELECTION OF SUBJECTS 
Written informed consent must be obtained prior to the subject undergoing any study-related 
procedure, including screening tests and washout periods for prohibited medications, when 
applicable. 
 
Inclusion Criteria 
4.1
Subjects who meet all of the following criteria are eligible for enrollment into the study: 
1. Subject has provided written informed consent. 
2. Subject must be between the ages of 18 and 75, inclusive at the time of 
randomization.  
3. Clinical diagnosis of systemic sclerosis (either limited or diffuse cutaneous disease). 
4. Diagnosis of SSc-PAH within the past 5 years, with a mean pulmonary arterial 
pressure (mPAP) of ≥ 25 mmHg at entry. 
5. Mean PVR of > 3 Wood units.  
6. Screening 6MWD of at least 100 meters.   
7. NYHA Functional Class II, III, or IV.  
8. Subject must be able to maintain O2 saturation ≥ 90% at rest (with or without 
oxygen). Oxygen use is permitted. 
9. Subject must be vaccinated with the pneumococcal vaccine at least 4 weeks prior to 
initiation of therapy, unless subject was vaccinated within 5 years of study entry. If 
vaccination occurred greater than 5 years prior to study entry, the subject must be 
revaccinated at least 4 weeks prior to initiation of therapy. 
10. Subjects must have been treated with background medical therapy for PAH 
(prostanoid, endothelin receptor antagonist, PDE-5 inhibitor, and/or guanylate cyclase 
stimulators) for a minimum of 8 weeks and have been on stable dose(s) of those 
medical therapy(ies) for at least 4 weeks prior to randomization with no expectation 
of change for 24 weeks after randomization.  
 
Exclusion Criteria 
4.2
Subjects who meet any of the following criteria are disqualified from enrollment in the study: 


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1. Documented PAH for greater than 5 years at the time of randomization defined as:  
 Measurement of a mPAP > 25 mmHg by right heart catheterization (RHC) at 
least 5 years previously, OR 
 Treatment with targeted background PAH therapy for > 5 years.  
2. Pulmonary Capillary Wedge Pressure (PCWP) > 15 mmHg or Left Ventricular End 
Diastolic Pressure > 15 mmHg.  
3. Persistent hypotension with systolic blood pressure (SBP) < 90 mmHg. 
4. Treatment with cyclophosphamide within 4 weeks of randomization. 
5. Treatment with immunocompromising biologic agents (including, but not limited to 
TNF inhibitors, anakinra, abatacept, and tocilizumab) within 4 weeks prior to 
treatment initiation or treatment with infliximab within 8 weeks prior to treatment 
initiation. 
6. If being treated with a non-biologic immunosuppressive or immunomodulating drug 
(including, but not limited to, methotrexate, azathioprine, mycophenolate, 
cyclosporine, hydroxychloroquine, penicillamine, and/or prednisone at ≤ 10mg daily), 
changes in dosage within 4 weeks prior to randomization. Subjects taking prednisone 
or equivalent corticosteroid > 10mg daily are excluded. 
7. Previous exposure to any lymphocyte depleting agent (Campath, ATG, etc.). 
8. Prior treatment with rituximab or other B cell depleting agents. 
9. PAH for any reason other than SSc including congenital heart disease, coronary left 
heart disease, chronic obstructive pulmonary disease, chronic thrombotic and/or 
embolic pulmonary vascular disease, or sleep apnea.  
10. History of coronary artery disease, with any of the following events within 3 years of 
randomization: significant ventricular tachy-arrhythmia, stent placement, coronary 
artery bypass surgery, and/or myocardial infarction (MI). 
11. Moderate or severe interstitial lung disease as characterized by a total lung capacity 
(TLC) of < 70% predicted, except if TLC is between 60-70% predicted and an HRCT 
within the 6 months prior to randomization shows only mild ILD.  
12. Chronic infections including, but not limited to, HIV, tuberculosis (TB), hepatitis B 
(HBV) or hepatitis C (HCV), or chest X-ray (CXR) findings consistent with TB or 
latent fungal infection. 
13. Positive serology for hepatitis B defined by positive HBV surface antigen and/or 
positive HBV core antibody, total. 
14. Positive serology for HCV antibody.  
15. A deep space infection within the past 2 years (including, but not limited to, 
meningitis, epiglottitis, endocarditis, septic arthritis, fasciitis, abdominal or pleural 
abscess, or osteomyelitis).  
16. Evidence of active infection requiring IV or PO antibiotics within 2 weeks of 
randomization.  
17. At or within 30 days of screening,  
 history of or current positive purified protein derivative tuberculin skin test 
(PPD) ( > 5mm induration, regardless of Bacille Calmette Guerin [BCG] 
vaccine administration), or positive QuantiFERON®-TB Gold In-Tube Test 
(QuantiFERON®), or historical chest x-ray unless completion of treatment 
has been documented for active TB 


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 latent TB (a positive test, a negative chest x-ray, and no symptoms or risk 
factors), unless one month of prophylaxis has been completed prior to 
inclusion 
 an indeterminate QuantiFERON® unless followed by a subsequent negative 
PPD or negative QuantiFERON® as well as a consultation with and clearance 
by local infectious disease (ID) department.  
18. Significant renal insufficiency defined as: 
 Estimated creatinine clearance < 40 mL/min (using Cockcroft-Gault formula 
based on actual body weight); OR 
 Active, untreated SSc renal crisis at the time of enrollment. 
19. Recent administration of a live vaccine (< 8 weeks) or any other immunization within 
4 weeks of treatment. 
20. History of anaphylaxis or IgE-mediated hypersensitivity to murine proteins or any 
component of rituximab.  
21. Pregnancy. 
22. Lactation. 
23. History of malignancy within the last 5 years, except for resected basal or squamous 
cell carcinoma, treated cervical dysplasia, or treated in situ cervical cancer Grade I. 
24. A woman of childbearing potential (not post-menopausal or surgically sterile) who is 
unwilling to use a medically acceptable form of birth control (including, but not 
limited to, a diaphragm, an intrauterine device (IUD), progesterone implants or 
injections, oral contraceptives, the double-barrier method, or a condom) throughout 
the duration of the study. 
25. History of non-compliance with other medical therapies. 
26. A history of alcohol or drug abuse within 1 year of randomization.  
27. Receipt of any investigational drug or device within 4 weeks before the screening 
visit, with the exception of investigational prostanoids, endothelin receptor 
antagonists, PDE-5 inhibitors, and guanylate cyclase stimulators.  
28. Recipient of lung transplant. 
29. Laboratory parameters at the screening visit showing any of the following abnormal 
results: 
 Transaminases > 2x the upper limit of normal (ULN) and/or bilirubin > 2x ULN. 
 Absolute neutrophil count (ANC) < 1,500/mm3 (or < 1.5 x 109/L). 
 Platelet count < 100,000/mm3 (or < 100 x 109/L). 
 Hemoglobin < 9 g/dL. 
30. Concurrent treatment in a clinical research study using a non-FDA approved agent, 
with the exception of an open-label study/study extension of investigational 
prostanoids, endothelin receptor antagonists, PDE-5 inhibitors, and guanylate cyclase 
stimulators, provided the open-label investigational drug will be available and dose 
will remain stable through the ASC01 primary endpoint time point of 24 weeks after 
randomization in ASC01.  
31. Any condition or treatment, which in the opinion of the investigator, places the 
subject at unacceptable risk as a participant in the trial.  


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 Coenrollment Guidelines 
4.2.1
While participating in ASC01, participating in observational registries or cohorts is 
permitted.  Subjects may not be enrolled in another clinical trial, except as noted in Exclusion 
Criterion #29.  For any co-enrollment in studies, the combined blood volumes must not 
exceed the NIH policy limit nor may the objectives or procedures confound the ASC01 study 
endpoint assessment. 
5 TREATMENT OF SUBJECTS 
 
Description of Rituximab  
5.1
 Product Description 
5.1.1
Rituxan® (rituximab) is a genetically engineered IgG1 kappa chimeric murine/human 
monoclonal antibody containing murine light- and heavy-chain variable region sequences 
and human constant region sequences. The chimeric antibody is produced by mammalian cell 
(Chinese Hamster Ovary) suspension culture in a nutrient medium containing the antibiotic 
gentamicin. Gentamicin is not detectable in the final product. Rituximab is composed of 
1,328 amino acids, and has an approximate molecular weight of 145 kilo Dalton (kD) [116]. 
The antibody reacts specifically with the CD20 antigen found on the surface of malignant 
and normal B cells, and established B cell lines. Studies have shown that rituximab binds via 
its Fc domain to human complement and lyses lymphoid B cell lines by complement-
dependent cytotoxicity through the induction of apoptosis and via antibody-dependent cell-
mediated cytotoxicity [118]. 
 
The drug is manufactured by Genentech, Inc. and by Biogen IDEC, Inc. Rituximab is 
supplied as a sterile, clear, colorless, preservative-free liquid concentrate for intravenous (IV) 
administration in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials, which must be 
diluted before administration [116].  
 Packaging and Labeling of Study Product 
5.1.2
Rituximab labeled for investigational use will be provided without charge by Genentech, Inc. 
or IDEC Pharmaceuticals. The sponsor of the trial will ensure maintenance of complete and 
accurate records of the receipt, dispensation, and disposal or return of all trial drugs in 
accordance with Title 21 CFR Parts 312.57 and 312.62 and with Genentech/IDEC 
requirements. 
 
Placebo will be supplied in packaging identical to rituximab.  
 Storage and Handling of Study Product 
5.1.3
Rituximab vials will be stored in a refrigerator at 2° to 8°C (36° to 46°F). Rituximab will not 
be used beyond the expiration date stamped on the carton and printed on the vial. Rituximab 
vials will be protected from direct sunlight. The product will not be frozen or shaken. 


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After dilution to 1-4 mg/mL in normal saline, rituximab is physically and chemically stable 
at 2° to 8°C (36° to 46°F) for 24 hours, and subsequently for 24 hours at room temperature 
provided that dilution has taken place in controlled and validated aseptic conditions.  
It is strongly recommended that higher concentrations of rituximab (4 mg/mL) be used for 
this patient population in order to minimize fluid administration. 
 
Placebo will be handled in the same fashion as described above. 
 
Dosage Regimen  
5.2
Rituximab (1000 mg) or placebo will be administered as 2 infusions given 2 weeks apart.  
 
Administration of Rituximab 
5.3
The following drugs, safety equipment, and supplies must be available during the rituximab 
infusions: 
 Oxygen 
 Oral and endotracheal airways and intubation equipment 
 Epinephrine 1:1000 solution for IV or endotracheal injection 
 Antihistamines 
 Corticosteroids 
 IV infusion solutions, tubing, catheters, and tape 
 Defibrillator with electrocardiogram (ECG) monitor 
 Dopamine 
 Atropine 
 Bronchodilators 
 
Patients will be closely monitored during the infusions. Guidance for monitoring for infusion 
reactions includes continuous heart rate monitoring during the infusions, with capability to 
detect early bradycardia. Vital signs should be monitored frequently as suggested below. 
During the first infusion, vital signs should be checked every 10 minutes during infusion for 
the first hour, and then every 15 minutes until the subject is discharged 1 hour after the 
infusion, or as clinically indicated. For the second infusion, vital signs should be checked 
every 15 minutes during infusion for the first hour, and then every 30 minutes until the 
subject is discharged 30 minutes after the infusion, or as clinically indicated. Systolic and 
diastolic pressures should be should be monitored, with mean arterial pressures (MAP) 
monitored as clinically indicated. Oxygen saturations should be measured at baseline and 
then every 30 minutes or as clinically indicated. Study centers may defer to local institutional 
standards of practice for monitoring hemodynamic and pulmonary status. 
 
Patients with significant PAH typically do not have systemic hypertension, but rather have 
low blood pressures. Because transient hypotension may occur during rituximab infusion, it 
is necessary to anticipate an exacerbation of baseline hypotension. Persistent hypotension 
with a SBP < 90 mmHg is an exclusion criterion for this study. If after randomization on the 


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day of rituximab administration prior to beginning the infusion the subject's systolic BP is < 
90 mmHg or, if measured/calculated, the  MAP is < 55 mmHg, then the guidance in Figure 
6’s algorithm below is recommended. To summarize, for a SBP < 90 mmHg or MAP < 55 
mmHg prior to starting the infusion a fluid bolus of up to 500 cc normal saline (NS) may be 
administered. If the pressures remain below these parameters and the physician is worried 
about hypotension then the infusion should not be given and the subject should be sent home. 
Another attempt to infuse the study drug may be made within 3 days. If on the second 
attempt the subject’s BP remains too low in the opinion of the investigator, then he/she will 
be withdrawn from the study. PAH specific medications should not be withheld to increase 
the blood pressure. In summary, study centers may defer to local institutional standards of 
practice for subject monitoring and clinical management during study drug infusions. 
 
Patients with Class IV or advanced Class III symptoms should receive close nursing attention 
(preferably 1:1 care) during the infusion. These patients are at high risk for hemodynamic 
compromise should they suffer a hypersensitivity reaction. Therefore, each participating 
institution will have an established algorithm for acceleration of care should a patient 
hemodynamically decompensate. It is essential that a code team can rapidly and directly 
respond to the patient’s hemodynamic deterioration and immediately transfer the patient to 
an inpatient unit for accelerated care (i.e. an off-site infusion center would not be appropriate 
for these patients). 
 
Research coordinators will contact subjects by phone within 24 hours after discharge from 
the infusion center to assess for adverse events.


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Figure 6.  Guidance algorithm for managing hypotension during rituximab infusion. 


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 Medications to Hold Prior to Study Drug Administration 
5.3.1
Since transient hypotension may occur during the rituximab infusion, all subjects taking 
antihypertensive(s) will be instructed to hold their medication(s) for 12 hours before each 
infusion in order to decrease the risk of hypotensive events. If a subject has a systolic blood 
pressure > 180 mm mercury (Hg) or diastolic blood pressure > 100 mmHg at screening, then 
the antihypertensive(s) will not be withheld prior to the infusion. However, it is uncommon 
for patients with PAH to be hypertensive. Rather, systemic hypotension is more commonly 
found in this patient group. For hypotensive patients, management will be conducted as 
outlined in Section 5.4.3.2, Cardiovascular and in Figure 6. 
 Preparation for Administration 
5.3.2
Appropriate aseptic technique will be used. The necessary amount of rituximab will be 
withdrawn and diluted to a final concentration of 1 to 4 mg/mL into an infusion bag 
containing 0.9% Sodium Chloride USP. It is strongly recommended that higher 
concentrations of rituximab (4 mg/mL) be used for this patient population in order to 
minimize fluid administration. The bag will be gently inverted to mix the solution, and any 
unused portion left in the vial will be discarded. Parenteral drug products should be inspected 
visually for particulate matter and discoloration prior to administration and should not be 
used if either is present. No incompatibilities between rituximab and polyvinyl chloride or 
polyethylene bags have been observed. 
 Pre-medication for Rituximab Infusion 
5.3.3
All subjects will receive the following pre-medication prior to each rituximab infusion to 
prevent hypersensitivity reactions and to reduce the frequency of infusion-related reactions: 
 Night before and morning of each study drug infusion: 
o Prednisone 40mg PO (or equivalent) 
 Approximately thirty minutes prior to each study drug infusion: 
o Methylprednisolone 100 mg IV (or equivalent). 
 Approximately thirty to sixty minutes prior to each study drug infusion: 
o Diphenhydramine 50 mg PO (or equivalent). Dose may be repeated every 
four hours, as needed.  
 Note: Subjects should be advised that this pre-medication given for 
the prevention and treatment of infusion-related reactions may 
cause drowsiness and impairment of driving ability prior to 
discharge. 
o Acetaminophen 650 mg PO (or equivalent). Dose may be repeated every 
four hours, as needed. 
 Administration 
5.3.4
RITUXIMAB MUST NOT BE ADMINISTERED AS AN IV PUSH OR BOLUS. 
 


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The following administration guidelines should be adhered to: 
 Attach the IV administration tubing to the IV bag of prepared rituximab by piercing 
the bag with the spike end of the tubing. 
 Attach the precision regulator extension set to the primary IV administration set. 
 Purge all tubing of air by flushing with the prepared solution, allowing only a small 
amount of solution to be discarded. 
 Identify the site for IV access. Prepare for venipuncture per institution standards of 
practice. 
 Introduce the angiocatheter into the vein, and attach to the male port of the precision 
regulator extension set. 
  Guidance for NYHA Class II or III Subjects 
5.3.4.1
 Open the roller clamp of the IV tubing, and set the precision regulator drip rate to an 
initial rate of 50 mg/hour. 
 Do not mix or dilute rituximab with other drugs. 
 If hypersensitivity or infusion-related events do not occur, escalate the infusion rate 
in 50 mg/hour increments every 30 minutes, to a maximum of 400 mg/hour. 
Subsequent infusions of rituximab can be administered at an initial rate of 100 
mg/hour, and increased by 100 mg/hour increments at 30-minute intervals, to a 
maximum rate of 400 mg/hour as tolerated. 
 
Table 5.1 Rituximab Infusion Rate for First Infusion* 
Time 
(minutes)
Drug per 
hour (mg/h) 
Infusion Rate 
(mL/h) 
0-30 
50 
12.5 
31-60 
100 
25 
61-90 
150 
37.5 
91-120 
200 
50 
121-150 
250 
62.5 
151-180 
300 
75 
181-210 
350 
87.5 
211-240 
400 
100 
241-255 
400 
100 
Note: Advance infusion rate as tolerated 
*Based on dilution of rituximab to 4 mg/mL in normal saline 
 


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Table 5.2 Rituximab Infusion Rate for Second Infusion* 
Time 
(minutes) 
Drug per 
hour (mg/h) 
Infusion Rate 
(mL/h) 
0-30 
100 
25 
31-60 
200 
50 
61-90 
300 
75 
91-120 
400 
100 
121-150 
400 
100 
151-180 
400 
100 
181-210 
400 
100 
Note: Advance infusion rate as tolerated 
* Based on dilution of rituximab to 4 mg/mL in normal saline 
 Check for extravasation at the infusion site. 
 Allow the study medication to infuse until the line of fluid is at the needle insertion 
site, ensuring that all of the medication is infused. 
  Guidance for NYHA Class IV Subjects 
5.3.4.2
Given the greater concern for potential cardiovascular events related to rituximab 
administration in this sub-group of subjects, the starting infusion rate and algorithm for 
titration of rate should be reduced to half of the standard rate outlined in Section 5.3.4.1, 
Guidance for NYHA Class II or III Subjects. Strict adherence to the 30 minute intervals of 
rate titration will be dependent upon subject tolerability of the infusion. 
 Criteria for Permanent Discontinuation of Rituximab Infusion   
5.3.5
If a subject experiences any of the events listed below, rituximab infusions will be 
discontinued, and procedures for discontinuation of protocol-specified treatments and follow-
up will be implemented (as per Section 3.3.2.1, Follow-up Requirements for all Treated 
Subjects):  
 Syndrome resembling tumor lysis syndrome 
 NCI-CTCAE Grade 3 (severe) hypersensitivity reaction 
 NCI-CTCAE Grade 3 or higher (severe/life-threatening) cytokine release 
acute infusion reaction 
 NCI-CTCAE Grade 3 or higher (severe/life-threatening) cardiac arrhythmias. 
In addition, see Protocol Section 3.3.2, Discontinuation of Protocol-Specified 
Treatment Requirements for significant cardio-pulmonary disease progression 
events warranting discontinuation of protocol-specified treatment 
requirements. 
 NCI-CTCAE Grade 3 or higher (severe/life-threatening) mucocutaneous 
reactions 


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 Significant hypotension during the infusion not responsive to standard 
medical intervention 
 Events listed in Section 3.3.2, Discontinuation of Protocol-Specified 
Treatment Requirements 
 Criteria for Withholding Rituximab Infusion 
5.3.6
Rituximab infusions are to be withheld if the subject is experiencing any of the following: 
 Fever > 38 C 
 Symptoms of an upper respiratory infection 
 Acute diarrhea 
 Signs or symptoms of intercurrent infection or serum sickness such as fever, joint 
pain, skin rash or hives, hematuria, or hypocomplementemia 
 Cytopenia as defined as ANC < 1500/mm3 (or < 1.5 x 109/L), platelets < 
100,000/mm3 (or < 100 x 109/L), or hemoglobin < 9.0 g/dL 
 Hypotension prior to initiation of infusion  
 
The infusion may be given if a subject has been afebrile for 2 days and has clear lungs. If the 
subject has been started on antibiotics, the infusion may be given after 2 days, provided 
cultures are negative. If initiation of rituximab was withheld for hypotension, the subject may 
return within 3 days for rituximab administration, as long as the return visit is within the 
windows specified below. 
 
If a subject has a rituximab infusion withheld, the following time windows for infusion will 
be applicable: 
 First Infusion: The first infusion must be given within 7 days of randomization.  
 Second Infusion: The infusion must be given 2 – 4 weeks after the first infusion; 
otherwise the subject will not receive the second infusion. 
 Criteria for Temporarily Interrupting and Restarting Rituximab Infusion 
5.3.7
The rituximab infusion should be interrupted if the patient develops worsened hypotension or 
symptomatic bradycardia. SSc-PAH patients may have baseline low blood pressure which 
the rituximab infusion may exacerbate. Please refer to Section 5.4.3.2, Cardiovascular and 
the Algorithm in Figure 6 for the suggested treatment of hypotension during rituximab 
infusions. Sinus bradycardia during rituximab infusion should be quickly responded to as 
described in Section 5.4.3.2, Cardiovascular. If additional measures seem safe and 
appropriate in order to continue the rituximab infusion, they may be implemented by the 
clinical site. In all cases, clinical judgment regarding the safety of the individual subject 
should prevail. If there is adequate response of the bradycardia and/or hypotension and the 
subject is hemodynamically stable, the infusion may be restarted The rituximab infusion 
should be interrupted for NCI-CTCAE Grade 2 or higher cytokine release reactions or Grade 
2 hypersensitivity reactions. In most cases, the infusion can be resumed at a 50% reduction in 
rate (e.g., from 100 mg/hour to 50 mg/hour) when symptoms have completely resolved. 
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have experienced NCI-CTCAE Grade 2 or less hypersensitivity infusion-related reactions 
have been able to complete the full course of rituximab therapy. Epinephrine, antihistamines, 
and corticosteroids should be available for immediate use in the event of an infusion reaction 
to rituximab (e.g., anaphylaxis). Rituximab therapy should be discontinued in the event of an 
NCI-CTCAE Grade 3 hypersensitivity or anaphylactic allergic reaction or in the event of an 
NCI-CTCAE Grade 3 cytokine release acute infusion reaction. Please refer to Section 5.4.3, 
Management of Infusion Reactions/Known Toxicities to Rituximab, for treatment guidelines. 
 
Toxicity Management Plan for Rituximab 
5.4
 Known Toxicities to Rituximab 
5.4.1
Refer to Protocol Section 1.4, Known and Potential Risks of Rituximab Treatment.  
 Prevention of Known Toxicities to Rituximab 
5.4.2
1. 
In the absence of known allergies, all subjects will be pre-medicated orally 
approximately 30 – 60 minutes prior to infusion of rituximab with acetaminophen 650 mg 
and diphenhydramine 50 mg. The pre-medication will be repeated every 4 hours, as needed. 
Subjects administered an antihistamine for the treatment or prevention of infusion-related 
reactions should be given appropriate warnings about drowsiness and impairment of driving 
ability prior to discharge. 
 
In order to reduce the frequency of infusion-related reactions, subjects will also be asked to 
take prednisone 40 mg PO (or equivalent) the night before and morning of each infusion. 
Additionally, they will be given 100 mg IV methylprednisolone (or equivalent) 
approximately thirty minutes prior to each rituximab infusion. Procedures for the 
management of infusion reactions are discussed in Section 5.4.3.1, Infusion Reactions. 
Additionally a member of the research staff will follow up with subjects within 24 hours after 
discharged from the infusion center to assess their condition. 
 
2. 
Although neutropenia and thrombocytopenia have been associated with rituximab, 
cases are generally transient and do not cause clinically significant problems. Late onset 
neutropenia with rituximab has been described in patients treated with aggressive 
chemotherapy for hematological malignancies but no such cases have been reported in 
patients with autoimmune disease. Nevertheless, to minimize the risk of potential cytopenias, 
a complete blood count will be performed and adequacy of the differential and platelet count 
will be checked prior to starting the second rituximab infusion. Counts will be re-checked as 
required in the Schedule of Events and whenever the subject develops any infection. 
Procedures for the management of cytopenias will be according to institutional practice. 
  
3. 
Most subjects at high risk for infections or viral reactivation will have been 
excluded from participation in this study. However, the following additional precautions will 
be taken to minimize the potential for significant infections and/or viral reactivation in this 
study [153-156].  


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a. Subjects will be informed about the potential effects of rituximab on 
immunocompetency including: 
 decreased response to immunization;  
 possible increased rate of infection;  
 possible neutropenia and hypogammaglobulinemia, and  
 possible reactivation of viral infections (including HBV and JC virus). 
b. Subjects and study physicians will be given the following information 
regarding immunizations: 
 Live virus vaccines are contraindicated within 8 weeks prior to beginning 
rituximab therapy. 
 All other immunizations must be completed at least 4 weeks prior to 
starting rituximab. This includes the pneumococcal vaccine, unless subject 
was vaccinated within 5 years of study entry. If vaccination occurred 
greater than 5 years prior to study entry, the subject must be revaccinated 
at least 4 weeks prior to initiation of therapy. 
 After rituximab, recommended vaccines, including yearly influenza 
vaccine, are to be administered as late as possible after the last dose of 
rituximab. If possible, influenza vaccine should be administered at least 4 
weeks prior to dosing with rituximab. 
c. Subjects will be educated about the need to recognize and report the 
symptoms of infection. Specifically, subjects will be: 
 educated about the importance of early recognition of infections and 
instructed to call the study coordinator or their health care provider if they 
have questions or if they are ill; 
 instructed to be seen by their study physician and/or local physician if they 
are having shaking chills, fever greater than 101° F, respiratory illness 
other than a mild cold, symptoms of a urinary infection, severe or 
prolonged diarrhea or abdominal pain, or if they think they may be 
developing the flu; 
 educated about symptoms of hepatitis (nausea, loss of appetite, dark urine, 
light stools), herpes zoster (new, severe, unexplained pain on one side of 
the body especially if there is a rash), and PML (confusion, lethargy, 
dizziness, difficulty talking or walking, and vision disturbances).  
d. Study physicians will administer appropriate prophylaxis for infections such 
as anti-viral chemotherapy for influenza during flu outbreaks according to 
individual institutional practice. 
 Management of Known Toxicities to Rituximab 
5.4.3
  Infusion Reactions (Cytokine release syndrome/acute infusion reaction and 
5.4.3.1
Hypersensitivity/Allergic Reaction) 
Infusion reactions (cytokine release and/or hypersensitivity reactions) may respond to 
adjustments in the infusion rate and to medical management. If a general infusion-related 


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reaction (e.g., fever, chills, myalgia, nausea, headache, rash, mild or moderate 
mucocutaneous reactions, or symptomatic hypotension) develops, the symptoms will be 
treated, and the infusion slowed or stopped as warranted. The infusion may be continued at 
one-half the previous rate once the symptoms have resolved. Hemodynamic fluctuations can 
have significance for patients with PAH and physicians trained in the care of PAH patients 
should be available in the event of an infusion reaction. For this reason, patients in this 
protocol will receive study drug with hemodynamic monitoring as described under Section 
5.3, Administration of Rituximab. For subjects who experience asymptomatic hypotension, 
the infusion rate will be temporarily slowed per the investigator’s discretion until symptoms 
resolve. 
 
The rituximab infusion should be interrupted for NCI-CTCAE Grade 2 cytokine release or 
Grade 2 hypersensitivity reactions. In most cases, the infusion can be resumed at a 50% 
reduction in rate (e.g., from 100 mg/hour to 50 mg/hour) when symptoms have completely 
resolved. Treatment of infusion-related symptoms with diphenhydramine and acetaminophen 
is recommended. Additional treatment with bronchodilators or IV saline may be indicated. 
Most subjects who have experienced non-life-threatening (NCI-CTCAE Grade 2 or less) 
hypersensitivity infusion reactions or (NCI-CTCAE Grade 2 or less) cytokine release 
reactions have been able to complete the full course of rituximab therapy.  
 
In the event of a NCI-CTCAE Grade 3 (severe) or higher hypersensitivity reaction, or a NCI-
CTCAE Grade 3 (severe) or higher cytokine release acute infusion reaction, the infusion 
should be permanently discontinued and the subject should receive medical treatment. 
Epinephrine, antihistamines, corticosteroids, albuterol, oxygen, and emergency resuscitation 
equipment should be kept at the bedside during the infusion of rituximab for immediate use 
in the event of a hypersensitivity reaction to rituximab (e.g., anaphylaxis). Subjects who 
experience an NCI-CTCAE Grade 3 (severe) or higher hypersensitivity reaction, or an NCI-
CTCAE Grade 3 (severe) or higher cytokine release acute infusion reaction will receive no 
additional rituximab, and procedures for discontinuation of protocol-specified treatments and 
follow-up will be implemented (as per Section 3.3.2.1, Follow-up Requirements for All 
Treated Subjects). If it is medically indicated, a subject will be hospitalized for infusion-
related reactions. 
 Cardiovascular 
5.4.3.2
Infusions should be discontinued in the event of an NCI-CTCAE Grade 3 (serious) or Grade 
4 (life-threatening) cardiac arrhythmia. For subjects who develop an NCI-CTCAE Grade 3 or 
higher arrhythmias, rituximab will be permanently discontinued, and procedures for 
discontinuation of protocol-specified treatments and follow-up will be implemented (as per 
Section 3.3.2.1, Follow-up Requirements for All Treated Subjects). In addition, see Protocol 
Section 3.3.2, Discontinuation of Protocol-Specified Treatment Requirements for significant 
cardio-pulmonary disease progression events warranting discontinuation of protocol-
specified treatment requirements.  
 
Sinus bradycardia can be dangerous in PAH patients whose cardiac output can be heart rate-
dependent. A vasovagal response to rituximab infusion can result in sinus bradycardia and 


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should be immediately be responded to. If a patient develops symptomatic bradycardia 
evidenced by lightheadedness, change in consciousness, worsening hypotension or other 
symptoms, then the subject should receive 0.5 mg of atropine IV with repeat dosing not to 
exceed 3 mg. If patient develops symptomatic bradycardia, that is unresponsive to atropine, 
then the infusion should not be resumed. During a period of bradycardia the infusion should 
be suspended. If the bradycardia resolves the infusion may be re-started at 50% the original 
infusion rate. 
 
If during the rituximab infusion the SBP drops below 90 mmHg or MAP drops below 55 
mmHg, then the guidance in algorithm in Figure 6 may be followed per the investigator’s 
clinical judgment. Different interventions may be utilized depending on the individual 
investigator’s clinical judgment. To summarize the algorithm, the infusion may be 
interrupted and a fluid bolus of up to 500 cc normal saline administered. If the SBP rises to > 
90 mmHg and MAP rises to ≥ 55 mmHg the infusion may be resumed at half the infusion 
rate. If the SBP does not rise to > 90 mmHg and MAP to ≥ 55 mmHg, another fluid bolus of 
up to 500 cc, and/or low dose dopamine may be considered. If the BP does not stabilize, 
clinical judgment should prevail as to when to discontinue study drug infusion. At any time if 
clinically indicated, the subject should be stabilized and the infusion withheld, slowed, or 
discontinued as appropriate per the guidance provided. Consideration must be given to 
administration of multiple fluid boluses (≥ 1 Liter) dependent on subject’s cardiopulmonary 
status. Procedures for follow-up per Section 3.3.2.1, Follow-up Requirements for All Treated 
Subjects should be followed. 
 
In addition to hypotension, subjects with SSc-PAH may be sensitive to fluid overload. 
Investigators need to be cognizant of the total fluid load when administering the rituximab 
infusion. It is strongly recommended that higher concentrations of rituximab (4 mg/mL) be 
used to minimize fluid administration. 
 
As stated in Section 5.1.3, Storage and Handling of Study Product, rituximab is physically 
and chemically stable at 2° to 8°C (36° to 46°F) for 24 hours after dilution and subsequently 
for 24 hours at room temperature after dilution provided that dilution has taken place in 
controlled and validated aseptic conditions. The interventions listed above and in the 
Algorithm in Figure 6 to manage cardiovascular complications once the infusion has begun 
may take place over a 24 hour period. Subjects should be continuously monitored until they 
have completed the infusion or until it is determined that the infusion must be discontinued 
for toxicity. 
 Severe Mucocutaneous Reactions 
5.4.3.3
Subjects experiencing an NCI-CTCAE Grade 3 (severe) or higher mucocutaneous reaction 
(to be reported under such categories as Dermatology/Skin, Gastrointestinal, or 
Renal/Genitourinary in the NCI-CTCAE dependent upon manifestations) should not receive 
additional rituximab and should seek prompt medical evaluation. Skin biopsy may help to 
distinguish among different mucocutaneous reactions and to guide subsequent treatment. 
Severe mucocutaneous reactions may require topical or systemic corticosteroid therapy. The 
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been determined. If a subject develops an NCI-CTCAE Grade 3 (severe) or higher severe 
mucocutaneous reaction, rituximab will be discontinued, and procedures for discontinuation 
of protocol-specified treatments and follow-up will be implemented (as per Section 3.3.2.1, 
Follow-up Requirements for All Treated Subjects). 
 Hepatitis 
5.4.3.4
If an enrolled subject develops hepatitis B, rituximab will be discontinued, and procedures 
for discontinuation of protocol-specified treatments and follow-up will be implemented (as 
per Section 3.3.2.1, Follow-up Requirements for All Treated Subjects). Patients may have 
elevated transaminases secondary to endothelin antagonists. Hepatitis attributed to this class 
of agents should be responded to according to manufacturers’ suggestions. 
  Hematologic Malignancy 
5.4.3.5
If an enrolled subject develops a hematologic malignancy, rituximab will be discontinued, 
and procedures for discontinuation of protocol-specified treatments and follow-up will be 
implemented (as per Section 3.3.2.1, Follow-up Requirements for All Treated Subjects).  
  Serum Sickness 
5.4.3.6
Should serum sickness occur, the infusion will be stopped permanently, and procedures for 
discontinuation of protocol-specified treatments and follow-up will be implemented (as per 
Section 3.3.2.1, Follow-up Requirements for All Treated Subjects). The subject will be given, 
if judged clinically appropriate, antihistamines for symptomatic relief of urticaria and 
nonsteroidal anti-inflammatory drugs for relief of pain. In some cases, a short course of 
moderate dose corticosteroids may be required for relief of joint pain or fever. If a serum 
sickness-like reaction occurs, then additional studies will be performed at the time of clinical 
presentation to evaluate its possible mechanisms. These studies may include measurement of 
serum cytokines, C3 and C4 levels, a urinalysis, and a skin biopsy with indirect 
immunofluorescence (if appropriate). 
  Tumor Lysis Syndrome 
5.4.3.7
In the event a syndrome resembling tumor lysis syndrome, the rituximab infusion should be 
discontinued. The subject should not be retreated with rituximab, and procedures for 
discontinuation of protocol-specified treatments and follow-up should be implemented (as 
per Section 3.3.2.1, Follow-up Requirements for All Treated Subjects). 
  Infection  
5.4.3.8
Subjects will be instructed to contact study personnel and be seen by their study physician 
and/or local physician if they have a fever ≥101 F or other signs and symptoms of an 
infection. For subjects who develop severe or frequent infections, the need for IVIG will be 
evaluated by each investigator (see Section 5.6, Prohibited Medications and Treatments). 
Local institutional policies for the use of IVIG will apply. Physicians will use their discretion 
to treat infections according to standard of care.  


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Prior Medications and Therapy 
5.5
Entry criteria with medications and therapy are described in Section 3.1, Description of Study 
Design, Section 4.1, Inclusion Criteria, and Section 4.2, Exclusion Criteria.  
 
Prohibited Medications and Treatments 
5.6
All subjects are to have access to any care deemed medically necessary, but administration of 
the following medications, for the purposes of this study, are prohibited and will be 
considered either protocol deviations or reasons for early endpoint unless otherwise noted 
below or in Section 5.7, Concurrent Medications and Therapy: 
 
 Addition of new medical therapies for PAH or a change in the dose of background 
PAH therapies (prostanoid, endothelin receptor antagonist, PDE-5 inhibitor, 
and/or guanylate cyclase stimulators) unless the change in therapy is due to 
toxicity of prior medications prior to week 24 except for an increase of up to 10% 
in prostacyclin therapy as described in Section 5.7, Concurrent Medications and 
Therapy 
 Surgical therapy for PAH        
 Any systemic corticosteroids, except for 
o prednisone (or equivalent) detailed in Section 5.7.2.1, Corticosteroids, or  
o corticosteroid premedications as specified in Section 5.3.3, Pre-medication for 
Rituximab Infusion, or 
o the methylprednisone (or equivalent) administered for the treatment of severe 
mucocutaneous reactions as per Section 5.4.3.3, Severe Mucocutaneous 
Reactions and of serum sickness manifestations as per Section 5.4.3.6, Serum 
Sickness.   
*Corticosteroid doses may be adjusted within the 0-10 mg daily range, as 
clinically indicated, as long as they are stable for 4 weeks prior to Weeks 24 and 
48 endpoint assessments.  
 Any biologic immunosuppressive agent, including rituximab or other CD20 
depleting agents. 
 IVIG between 4 weeks prior to Day 0 and 4 weeks after Week 2 
 New non-biologic immunosuppressive or immunomodulating drug therapy prior 
to week 24 or changes in concurrent non-biologic immunosuppressive or 
immunomodulating drug therapy prior to week 24 except for the management of 
toxicity 
 Any investigational agent other than investigational prostanoids, endothelin 
receptor antagonists, PDE-5 inhibitors, and guanylate cyclase stimulators. The 
latter investigational agents are those drugs in one of these four classes that are 
already being used for the treatment of PAH, but are not currently licensed for 
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Concurrent Medications and Therapy 
5.7
 Background PAH Therapy 
5.7.1
From the time of study entry, subjects are to remain on background PAH medical therapy 
with either a single agent or a combination of prostanoid, endothelin receptor antagonist, 
PDE-5 inhibitor, and/or guanylate cyclase stimulators as per the entry criteria. Doses must 
remain stable through the week 24 primary endpoint visit.  
 
Investigational prostanoids, endothelin receptor antagonists and PDE-5 inhibitors are 
permitted if the drugs are already in use for the treatment of PAH but are not currently 
licensed for this use by the FDA. 
 
Permitted Dose modifications: 
 Toxicity management for any of the four classes of drugs 
 Increase in prostacyclin dose of up to 10% for worsening disease independent of 
route of administration. 
 
Other PAH medications, such as diuretics and anti-coagulation, should be managed by the 
treating physician according to institutional practice. Changes in diuretics and anti-
coagulants are permitted throughout the duration of the study. 
 
Stabilization/clinical improvement in PAH should result in no change to background therapy 
for the duration of the subject’s participation in the study. 
 
If a change in medical (beyond the 10% increase in prostacyclin dose) or surgical therapy is 
necessary for the treatment of progressive PAH before the week 24 primary endpoint visit 
then the subject will complete an Early Endpoint visit (see Section 6.5.6) and procedures in 
Section 3.3.2.1, Follow-up Requirements for All Treated Subjects numbers 2 and 3 will be 
followed. 
 
After the week 24 endpoint visit, the doses of background therapies for PAH may be 
increased and other agents may be added as needed to treat worsening PAH if the subject 
experiences any one of the following: 
 a deterioration in functional class and a decrease in 6MWD by > 20%, 
or 
 the development of signs and symptoms of clinical right heart failure, such as 
worsening edema, ascites, and/or weight gain,  
or 
 other objective data suggesting clinical deterioration, such as worsening 
cardiopulmonary hemodynamics as evidenced by a ≥ 20% decrease in cardiac 
output, a ≥ 20% increase in mPAP, or a ≥ 20% increase in PVR. 
 


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 Immunosuppressive or Immunomodulating Drug Treatment 
5.7.2
Concurrent therapy with non-biologic immunosuppressive or immunomodulating drugs 
(including, but not limited to, methotrexate, hydroxychloroquine, penicillamine, 
mycophenolate, azathioprine or cyclosporine) is permitted. Doses must remain stable for at 
least the four weeks prior to randomization through at least the 24 week primary endpoint 
visit unless adjustment is needed for the management of toxicity. 
 
Doses of concurrent non-biologic immunosuppressive or immunomodulating drugs may be 
increased or new non-biologic immunosuppressive or immunomodulating drugs may be 
initiated for worsening SSc disease manifestations after the week 24 endpoint visit. 
 Corticosteroids 
5.7.2.1
Concurrent therapy prednisone (or equivalent corticosteroid) at ≤ 10mg daily is permitted. 
 Corticosteroid doses may be adjusted with within the 0-10 mg daily range, as 
clinically indicated, as long as they are stable for 4 weeks prior to the weeks 24 and 
48 endpoint assessments. 
 Intra-articular corticosteroid injections may be administered. 
 To accommodate the occasional use of extra corticosteroids by patients for reasons 
not associated with SSc, increases of up to 60 mg/day that are decreased back to the 
baseline dose within 2 weeks are permitted. Only 2 such increases are permitted 
during the study. Subjects must be back to baseline doses of corticosteroid by 4 
weeks prior to the week 24 and week 48 visits. 
 
Toxicity Management Plan for Concurrent Therapy: 
5.8
If discontinuation of an agent is required due to toxicity, the study physician will exercise 
clinical judgment in terms of whether to prescribe another agent within the same therapeutic 
class, add an agent from another therapeutic class, or continue with other background 
therapies already in place. 
 Known Toxicities of Concurrent Therapy 
5.8.1
Endothelin receptor antagonists have the potential to induce severe liver injury and damage 
to the fetus. Elevations in serum aminotransferases and bilirubin are markers for potential 
serious liver injury. It is suggested that these laboratory tests be measured prior to the 
initiation of any of the endothelin receptor antagonists. ERAs are classified by the FDA as 
Category X drugs; pregnancy must be excluded prior to treatment initiation and prevented 
once treatment has begun. Endothelin receptor antagonists cause a dose dependent decrease 
in hemoglobin. In individuals with severe CHF, endothelin receptor antagonists have been 
associated with increased fluid retention and hospitalization for exacerbation of CHF.  
 
Phosphodiesterase inhibitors potentiate the hypotensive effects of nitrates and therefore are 
contraindicated in patients taking organic nitrates either regularly or intermittently. This class 
of medication has vasodilator properties and may result in transient decreases in blood 
pressure and nose bleeds. Caution should be exercised in prescribing PDE-5 inhibitors in 


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individuals with a history of coronary artery disease, myocardial infarction, or retinitis 
pigmentosa. 
 
Epoprostenol is a potent vasodilator and additional reductions in blood pressure may be seen 
when an individual receives concurrent antihypertensives, diuretics or other vasodilators. 
There is the potential for epoprostenol to increase bleeding in individuals on anti-platelet 
agents or anticoagulants. Most of the adverse events experienced by subjects using 
epoprostenol are related to its vasodilatory effect and include nausea, vomiting, headache, 
hypotension, foot/bone pain, and flushing. 
 
Riociguat is a guanylate cyclase stimulator indicated for the treatment of adults with PAH 
(WHO Group 1) to improve exercise capacity, clinical worsening and WHO functional class.  
Riociguat is a pregnancy Category X drug and as such is contraindicated for during 
pregnancy as it may cause fetal harm. Females of reproductive capacity must be enrolled in 
the Adempas REMS program. Pregnancy testing should be performed monthly during 
treatment and female patients must agree to acceptable methods of contraception while 
taking this medication. Co-administration of riociguat with nitrates or nitric oxide donors is 
contraindicated as is concomitant administration of riociguat with PDE inhibitors, either 
specific PDE-5 inhibitors or nonspecific PDE inhibiters. Riociguat may cause hypotension. 
Serious bleeding was observed in clinical trials in individuals receiving riociguat compared 
to placebo including serious hemoptysis. Smoking, strong CYP and P-gp/BCRP inhibitors 
have the potential to change the exposure to riociguat. 
 Prevention and Management of Known Toxicities to Concurrent Therapy 
5.8.2
It is suggested that subjects receiving endothelin receptor antagonists have liver enzymes and 
a bilirubin tested monthly and a CBC checked every 3 months for anemia. If a subject is 
found to have elevated liver enzymes or falling hemoglobin, the subject will be managed in 
accordance with common clinical practice. Guidelines may be found in the package inserts of 
the relevant medications. The study physician will exercise clinical judgment in determining 
whether it is necessary to discontinue a medication for toxicity. Study physicians should be 
aware and subjects should be educated on the drug interactions with endothelin receptor 
antagonists including hormonal contraceptives, ketoconazole, warfarin, rifampicin, and 
statins. If subjects become pregnant during the course of the trial they will be withdrawn. 
Procedures for withdrawal as detailed in Sections 3.3.3, Subject Withdrawal from the Study 
and 6.5.5, Early Endpoint Visit will be followed. The pregnancy will be reported as detailed 
in Section 7.6, Pregnancy Reporting. 
 
Female subjects receiving riociguat therapy should have monthly pregnancy testing and 
agree to acceptable methods of contraception. If a subject becomes pregnant during the 
course of the trial, they will be withdrawn. Procedures for withdrawal as detailed in Sections 
3.3.3, Subject Withdrawal from the Study and 6.5.5, Early Endpoint Visit will be followed. 
The pregnancy will be reported as detailed in Section 7.6, Pregnancy Reporting.  Study 
physicians should be aware and subjects should be educated on the drug interactions with 
riociguat. Co-administration of nitrates, nitric oxide donors and phophodiesterase inhibitors 
are contraindicated. Blood pressure should be monitored and doses adjusted as needed if 


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subjects are taking concurrent strong cytochrome CYP inhibitors and P-gp/BCRP inhibitors 
such as azole anti-mycotics or HIV protease inhibitors. Subjects should be educated on the 
possibility of serious bleeding.  
 
Right Heart Catheterization Risks and Management Guidelines 
5.9
Most published reports of adverse events related to RHC have been based on single-center 
experiences with relatively small sample sizes. In the 1980s, Fuster and colleagues [157] 
conducted a long-term, retrospective, follow-up study on 120 patients, and reported a 4.2% 
fatality rate associated with RHC in patients with pulmonary arterial hypertension (PAH). In 
the same decade, a national registry of 187 patients with PAH from 32 centers reported a 
complication rate of 5.3%, with no deaths or sustained morbid events related to RHC 
diagnostic procedures [158]. 
 
Two decades later, a study was conducted to investigate the complication rates from RHC in 
patients with PAH from 15 experienced PAH centers, in both the United States and Europe 
[159]. This 5-year retrospective and a 6-month prospective evaluation of serious adverse 
events related to RHC procedures in patients with PAH, defined as mean pulmonary artery 
pressure > 25 mmHg at rest, revealed that the safety of the procedure has improved 
significantly. A total of 7218 RHC procedures were performed (21% prospective). The 
overall number of serious adverse events was 76 (1.1%). There were 4 fatalities reported 
associated with the procedure (0.055%). The remaining 74 nonfatal incidents were classified 
as mild or moderate in severity and resolved either spontaneously or with minor 
interventions. The most frequent complications were hematoma at venipuncture sites, 
pneumothoraces, arrhythmias, vasovagal episodes, and hypotensive episodes. The most 
frequent complications were related to venous access (n = 29, 38%). Internal jugular (IJ) was 
reported to be the preferred access site (73%) that was associated with a low rate of 
complications (0.3%). 
 
Measures to minimize the risks associated with RHC include the use of the IJ approach 
whenever feasible, with ultrasound guidance for direct visualization of the vein. The use of 
fluoroscopy should also be implemented whenever feasible. Direct visualization under 
fluoroscopy can minimize arrhythmic events, which are more prevalent in patients with an 
enlarged right atrium or ventricle, including those with PAH. Measurement of the pulmonary 
capillary wedge pressure is safer if performed using fluoroscopy for direct visualization. 
Fluoroscopy does involve exposure to radiation. Although the radiation exposure can vary 
from person to person, the whole-body radiation will be about 200 mrem during each RHC. 
 
Other Protocol Standard of Care Recommendations 
5.10
Supplemental oxygen should be provided to subjects in order to keep their oxygen saturation 
at or above 90% at all times.  
 
Subjects should receive influenza vaccines annually per institutional practice preferably not 
within 4 weeks of study drug infusion.  


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Procedures for Monitoring Subject Compliance 
5.11
Since the medication is administered as an IV infusion, study personnel will assure 
compliance with treatment. Personnel who administer the drug will document its 
administration. Study personnel will also monitor patients for compliance with baseline PAH 
stable dose medical therapy.  
6 STUDY ASSESSMENTS 
 
Assessments of Safety 
6.1
To assess safety in this population, peripheral blood cell counts, liver function, and serum 
creatinine will be monitored before enrollment and at frequent intervals thereafter. Physical 
examinations will be conducted before enrollment and at frequent intervals thereafter. The 
information from these assessments and subject interviews will be used to characterize the 
frequency of all adverse events Grade 2 or above, all serious adverse events, 
granulocytopenias, and treatment-related adverse events of NCI-CTCAE Grade 3 or higher 
(see Section 7, Safety Monitoring and Reporting). 
 
Recovery of B cells will be monitored by assessing B cell levels at baseline and serially post-
treatment until reconstitution is documented. In addition, quantitative immunoglobulin levels, 
including IgG subclasses, will be serially monitored. Urine or serum pregnancy tests per 
institutional policy will be performed for all women of child-bearing potential at screening 
and prior to each rituximab infusion.  
 
Assessments of Disease Activity and Clinical Response 
6.2
Change in 6MWD as a measure of exercise capacity is the primary efficacy assessment in 
this trial. Disease activity will be monitored by checking autoantibodies and evaluating 
clinical outcomes including change in PVR measured by right heart catheterization and 
measuring room air oxygen saturation by pulse oximetry at screening and serially throughout 
the trial. Changes in severity of Raynaud phenomenon as measured by the VAS scale of the 
SHAQ, and change in digital ulcer count will be evaluated. Change in DLCO, a measure of 
disease activity as well as safety, will be followed. BNP/NT-proBNP and serum Na+ will be 
followed as non-invasive means of assessing heart failure. Change in quality of life will be 
determined by serial administration of the SF-36 and the SHAQ.  
 
Assessments of Mechanistic Endpoints  
6.3
The CD19+ B cell assays will be performed by the Human Immune Monitoring Center 
(HIMC) at Stanford University. B cell panels may include, but are not limited to IgD FITC, 
CD24 PE, CD19 PerCP-Cy5.5, CD28 PE-Cy7, CD27 APC, CD20 APC-H7, CD3 V450, 
CD45 Pacific Orange.  
 


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With a fraction of the PBMCs analyzed by flow cytometry for the above B cell panels, a sub-
study will measure and monitor B cell clonality utilizing massively parallel V-D-J 
pyrosequencing.  
 
IL-1 and IL-6 concentrations, as well as other cytokines, will be measured using multiplex 
assays [40].  
 
Quantitative immunoglobulin levels, including IgG subclasses, will be measured using a 
Luminex kit on the same instrument. Viable cells will be assayed by flow cytometry to assess 
the number of CD19+ cells in the phenotyping panel using a LSRII platform.  
 
The autoantibody biomarkers and RF will be analyzed by the Immunoassay Core Laboratory 
at the University of Pittsburgh.  
 
Subjects will be consented for permission to store any leftover blood samples to be used for 
future IRB-approved research to study the disease SSc-PAH, the immune system, and the 
effect of treatment on this disease.  
 
Other Assessments  
6.4
Rituximab serum levels (PK) and HACA levels will be collected at baseline, Week 24, and 
Week 36, and analyzed by Genentech. 
 
Evaluations by Study Visit 
6.5
The visit schedule and evaluation procedures for the screening, baseline, treatment, and 
follow-up period are found in the Schedule of Events, Table 6.1.  
 
Note: all subjective assessments completed by the subject (SHAQ, SF-36) must be done at 
the beginning of a visit, prior to any other study related procedures. 
 Screening Period (Visit 1) 
6.5.1
Unless otherwise specified below, the screening evaluations must be performed within 28 
days of the first treatment visit (Day 0), with the exception of the 6MWT which must be 
completed within 14 days of the first treatment visit (Day 0). This study will be explained in 
lay language to each potential participant. Each participant will sign an informed consent 
before committing to study screening procedures. 
 
Note: if the Screening physical exam, oxygen saturation, chemistry panel, and pregnancy test 
are performed within 7 days of Day 0, they do not need to be repeated during the 
Randomization/Baseline or Day 0 visits unless clinically indicated. 
 
Assessments:  
 Obtain written informed consent from subject, and provide the subject a copy of 
the signed consent form prior to any screening procedures being performed 


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 Medical history, including Family History of Autoimmune Disease 
 Demographics 
 Record all concomitant medications 
 Full physical exam including vital signs 
 Weight and height  
 NYHA Functional Class Assessment 
 Right heart catheterization 
 12-lead ECG (unless done concomitantly with RHC)  
 Pulmonary Function Tests: Spirometry, DLCO, TLC 
 Chest X-Ray: posteroanterior (PA) and lateral 
 6-minute walk test (6MWT) 
 Hematology: CBC with differential count and platelet count (hematocrit, 
hemoglobin, platelets, WBC, bands, eosinophils, lymphocytes, monocytes, 
neutrophils) 
 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, 
creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein 
 Infectious Disease Screen: HIV antibody; hepatitis B core antibody total, hepatitis 
B surface antibody, hepatitis B surface antigen; hepatitis C antibody with a 
hepatitis C virus (HCV) ribonucleic acid (RNA) (polymerase chain reaction 
[PCR]) if antibody positive 
 TB testing (PPD or QuantiFERON®-TB Gold In-Tube Test [QFT-G_IT]), unless 
performed within 30 days prior to screening and documented as negative in the 
subject’s records, or unless subject is known to have a positive or indeterminate 
test and has documentation of appropriate therapy. 
 Urinalysis 
 Pregnancy test (urine or serum per institutional policy): only if a subject is a 
female of childbearing potential 
 Vaccinations: All immunizations must be completed at least 4 weeks prior to 
starting rituximab/placebo. The subject must be vaccinated with the 
pneumococcal vaccine at least 4 weeks prior to initiation of therapy, unless the 
subject was vaccinated within 5 years of study entry. If vaccination occurred 
greater than 5 years prior to study entry, the subject must be revaccinated at least 
4 weeks prior to initiation of therapy. Depending on the season, influenza vaccine 
should be administered at least 4 weeks prior to dosing with rituximab/placebo. 
Otherwise, it should be administered as late as possible in the flu season to still be 
effective after the last dose of rituximab/placebo. 
 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe). Measure first at room air, then add/titrate oxygen if needed to 
maintain O2 saturation ≥ 90%. The room air oxygen saturation measurement will 
be conducted at the discretion of the study physician. The measurement should 
not be performed if subject safety will be compromised. 


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 Randomization/Baseline Visit (Visit 2) 
6.5.2
The randomization/baseline visit evaluations may be combined with the Day 0 visit, or done 
within the 7 days prior to the first rituximab infusion. They are not required for eligibility 
verification, and therefore, they may be done after randomization. 
 
Note: if the Screening chemistry panel was drawn within 7 days of Day 0, it does not need to 
be repeated during this Randomization/Baseline visit unless clinically indicated. 
 
Assessments:  
 SF-36 
 SHAQ 
 Digital Ulcer Count 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
 Hematology: CBC with differential count and platelet count (hematocrit, 
hemoglobin, platelets, WBC, bands, eosinophils, lymphocytes, monocytes, 
neutrophils), CD19+ B cells 
 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, 
creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, 
BNP/NT-proBNP 
 CD19+ B cell studies: blood tested locally and sent to Stanford Human Immune 
Processing Center for the Stanford Human Immune Monitoring Center   
 Autoantibodies: anti-U1 RNP, anti-U3 RNP, anti-B23, anti-cardiolipin, anti-
Th/To, anti-CENP-B, RF, and any other autoantibodies: frozen serum sent to 
University of Pittsburgh Immunoassay Core Laboratory. 
 Serum Immunoglobulins: quantitative immunoglobulin levels including IgG 
subclasses: frozen serum sent to Stanford Human Immune Processing Center for 
the Stanford Human Immune Monitoring Center 
 IL-1, IL-6, and other cytokine assays: frozen serum sent to Stanford Human 
Immune Processing Center for the Stanford Human Immune Monitoring Center 
 HACA and rituximab serum level (PK): batched and sent to Covance 
Laboratories. 
 Treatment Period 
6.5.3
  Day 0 (Visit 3) 
6.5.3.1
This visit is to occur within 7 days of the randomization/baseline visit, and 
assessments/procedures listed under the randomization/baseline visit may be done on Day 0 
or within the 7 day allowed window as scheduling allows. All assessments listed under the 
Randomization/Baseline and Day 0 Visits must be completed prior to infusion of the 
first dose of rituximab or placebo. 
 


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Note: if the Screening physical exam, oxygen saturation, and pregnancy test were performed 
within 7 days of Day 0, they do not need to be repeated during this Day 0 visit unless 
clinically indicated. 
 
Assessments: 
 Abbreviated Physical Examination with vital signs 
 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe). Measure first at room air, then add/titrate oxygen if needed to 
maintain O2 saturation ≥ 90%. The room air oxygen saturation measurement will 
be conducted at the discretion of the study physician. The measurement should 
not be performed if subject safety will be compromised. 
 Pregnancy test (urine or serum per institutional policy): only if a subject is a 
female of childbearing potential 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
 
Study Drug Infusion Procedures: 
The pharmacy is to store, handle, and prepare the study drug (rituximab or placebo) 
according to the recommendations listed in Sections 5.1.3, Storage and Handling of Study 
Product, 5.2, Dosage Regimen, and 5.3.2, Preparation for Administration, and per the 
prescribing information. 
 
In addition, sites will verify that they have the appropriate safety equipment and supplies in 
the infusion area available per Section 5.3, Administration of Rituximab. 
 
The first rituximab or placebo infusion will be administered at the Day 0 visit. The infusion 
should be performed after all other clinical assessments for this visit. Please refer to 
Section 5.3.1 for medications to hold prior to infusion, Section 5.3.3 for pre-medications and 
Section 5.3.4 for study drug administration guidelines.  
 
After the completion of the first infusion of study drug, the subject will be reassessed and 
vital signs repeated every 15 minutes until 1 hour after the infusion. If the subject is stable, 
the subject will be discharged home. 
 
 Telephone Assessment: A member of the research staff will contact the subject 
within 24 hours after discharge from the infusion unit to assess their condition. 
  Week 2/Day 14 ±4 days (Visit 4) 
6.5.3.2
This is the second rituximab/placebo infusion visit, to occur within 14 ± 4 days of Day 0. 
Please refer to Section 5.3.1 for medications to hold prior to infusion, Section 5.3.3 for pre-
medications and Section 5.3.4 for study drug administration guidelines.  
All visit assessments must be performed prior to study drug infusion. 
 
 


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Assessments: 
 Abbreviated Physical Examination with Vital Signs 
 Hematology: CBC with differential count and platelet count (hematocrit, 
hemoglobin, platelets, WBC, bands, eosinophils, lymphocytes, monocytes, 
neutrophils) 
o Note: hemoglobin, platelets, lymphocytes, neutrophils, and bands results 
from a hematology specimen drawn within 24 hours of the infusion must 
be reviewed prior to initiating the study drug infusion (see Section 5.3.6, 
Criteria for Withholding Rituximab Infusion).   
 Pregnancy test (urine or serum per institutional policy): only if a subject is a 
female of childbearing potential. 
 CD19+ B cell studies 
 Serum Immunoglobulins 
 IL-1, IL-6, and other cytokine assays 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
 
Refer to Study Drug Infusion Procedures in Protocol Section 5.3, Administration of 
Rituximab. After the completion of the second infusion of study drug, the subject will have 
vital signs repeated. The subject will be re-assessed at 30 minutes post-infusion. If the subject 
is stable, the subject will be discharged home.  
 
 Telephone Assessment: A member of the research staff will contact the subject 
within 24 hours after discharge from the infusion unit to assess their condition.  
 Follow Up Visits 
6.5.4
 Week 4 ±7 days (Visit 5) 
6.5.4.1
Assessments:  
 Abbreviated Physical Examination with Vital Signs 
 NYHA Functional Class Assessment 
 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe). Measure first at room air, then add/titrate oxygen if needed to 
maintain O2 saturation ≥ 90%. The room air oxygen saturation measurement will 
be conducted at the discretion of the study physician. The measurement should 
not be performed if subject safety will be compromised. 
 SHAQ 
 6MWT: The subject should complete the assessment with the same amount of 
supplemental oxygen used to complete the Screening 6MWT unless safety will be 
impacted (if the 6MWD decreased from the Screening 6MWD by more than 
20%, repeat the 6MWT within 7 days, but not on the same day) 
 Digital Ulcer Count 


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 Hematology: CBC with differential count and platelet count (hematocrit, hemoglobin, 
platelets, WBC, bands, eosinophils, lymphocytes, monocytes, neutrophils) 
 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, 
creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, 
BNP/NT-proBNP 
 CD19+ B cell studies 
 Serum Immunoglobulins 
 IL-1, IL-6, and other cytokine assays 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 8 ± 7 days (Assessment 6) 
6.5.4.2
The Week 8 assessment will be conducted via telephone. If safety concerns arise, the subject 
should be asked to come to the site for an unscheduled visit. 
 
Assessments:  
 Digital Ulcer Count Assessment 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 12 ± 7 days (Visit 7) 
6.5.4.3
Assessments:  
 Abbreviated Physical Examination with Vital Signs 
 NYHA Functional Class Assessment 
 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe).  Measure first at room air, then add/titrate oxygen if needed to 
maintain O2 saturation ≥ 90%. The room air oxygen saturation measurement will 
be conducted at the discretion of the study physician. The measurement should 
not be performed if subject safety will be compromised. 
 SHAQ 
 6MWT: The subject should complete the assessment with the same amount of 
supplemental oxygen used to complete the Screening 6MWT unless safety will be 
impacted (if the 6MWD decreased from the Screening 6MWD by more than 
20%, repeat the 6MWT within 7 days, but not on the same day) 
 Digital Ulcer Count 
 Hematology: CBC with differential count and platelet count (hematocrit, 
hemoglobin, platelets, WBC, bands, eosinophils, lymphocytes, monocytes, 
neutrophils) 
 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, 
creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, 
BNP/NT-proBNP 
 CD19+ B cell studies 
 Autoantibodies 


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 Serum Immunoglobulins 
 IL-1, IL-6, and other cytokine assays 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 16 ± 7 days (Assessment 8) 
6.5.4.4
The Week 16 assessment will be conducted via telephone. If safety concerns arise, the 
subject should be asked to come to the site for an unscheduled visit. 
 
Assessments:  
 Digital Ulcer Count Assessment 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 20 ± 7 days (Assessment 9) 
6.5.4.5
The Week 20 assessment will be conducted via telephone. If safety concerns arise, the 
subject should be asked to come to the site for an unscheduled visit. 
 
Assessments:  
 Digital Ulcer Count Assessment 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 24 ± 7 days (Visit 10) 
6.5.4.6
Assessments:  
 Full Physical Examination with Vital Signs 
 NYHA Functional Class Assessment 
 Right heart catheterization: The subject should be on the same amount of 
supplemental oxygen used during the Screening RHC unless safety will be 
impacted. The methods of calculating pulmonary capillary wedge mean pressure 
and cardiac output should be the same as that used for the Screening RHC and, if 
feasible, the procedure should be scheduled at approximately the same time of 
day as the Screening RHC. 
 Pulmonary Function Tests: DLCO only 
 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe). Measure first at room air, then add/titrate oxygen if needed to 
maintain O2 saturation ≥ 90%. The room air oxygen saturation measurement will 
be conducted at the discretion of the study physician. The measurement should 
not be performed if subject safety will be compromised. 
 SF-36 
 SHAQ 


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 6MWT: The subject should complete the assessment with the same amount of 
supplemental oxygen used to complete the Screening 6MWT unless safety will be 
impacted (if the 6MWD decreased from the Screening 6MWD by more than 
20%, repeat the 6MWT within 7 days, but not on the same day) 
 Digital Ulcer Count 
 Hematology: CBC with differential count and platelet count (hematocrit, 
hemoglobin, platelets, WBC, bands, eosinophils, lymphocytes, monocytes, 
neutrophils) 
 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, 
creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, 
BNP/NT-proBNP 
 CD19+ B cell studies 
 Autoantibodies 
 Serum Immunoglobulins 
 IL-1, IL-6, and other cytokine assays 
 HACA and rituximab serum level (PK). Batched and sent to Covance 
Laboratories. 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 28 ± 7 days (Assessment 11) 
6.5.4.7
The Week 28 assessment will be conducted via telephone. If safety concerns arise, the 
subject should be asked to come to the site for an unscheduled visit. 
 
Assessments:  
 Digital Ulcer Count Assessment 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 32 ± 7 days (Assessment 12) 
6.5.4.8
The Week 32 assessment will be conducted via telephone. If safety concerns arise, the 
subject should be asked to come to the site for an unscheduled visit. 
 
Assessments:  
 Digital Ulcer Count Assessment 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 36 ± 7 days (Visit 13)  
6.5.4.9
Assessments:  
 Abbreviated Physical Examination with Vital Signs 
 NYHA Functional Class Assessment 


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 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe). Measure first at room air, then add/titrate oxygen if needed to 
maintain O2 saturation ≥ 90%. The room air oxygen saturation measurement will 
be conducted at the discretion of the study physician. The measurement should 
not be performed if subject safety will be compromised. 
 SHAQ 
 6MWT: The subject should complete the assessment with the same amount of 
supplemental oxygen used to complete Screening the 6MWT unless safety will be 
impacted (if the 6MWD decreased from the Screening 6MWD by more than 
20%, repeat the 6MWT within 7 days, but not on the same day) 
 Digital Ulcer Count 
 Hematology: CBC with differential count and platelet count (hematocrit, 
hemoglobin, platelets, WBC, bands, eosinophils, lymphocytes, monocytes, 
neutrophils) 
 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, 
creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, 
BNP/NT-proBNP 
 CD19+ B cell studies 
 Serum Immunoglobulins 
 IL-1, IL-6, and other cytokine assays 
 HACA and rituximab serum level (PK). Batched and sent to Covance 
Laboratories. 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 40 ± 7 days (Assessment 14) 
6.5.4.10
 
The Week 40 assessment will be conducted via telephone. If safety concerns arise, the 
subject should be asked to come to the site for an unscheduled visit. 
 
Assessments:  
 Digital Ulcer Count Assessment 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
  Week 44 ± 7 days (Assessment 15) 
6.5.4.11
The Week 44 assessment will be conducted via telephone. If safety concerns arise, the 
subject should be asked to come to the site for an unscheduled visit. 
 
Assessments:  
 Digital Ulcer Count Assessment 
 Concomitant Medication Assessment 
 Adverse Event Assessment 


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  Week 48 ± 7 days (Visit 16) 
6.5.4.12
Assessments:  
 Full Physical Examination with Vital Signs 
 NYHA Functional Class Assessment 
 Pulmonary Function Tests: DLCO only 
 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe). Measure first at room air, then add/titrate oxygen if needed to 
maintain O2 saturation ≥ 90%. The room air oxygen saturation measurement will 
be conducted at the discretion of the study physician. The measurement should 
not be performed if subject safety will be compromised. 
 6MWT: The subject should complete the assessment with the same amount of 
supplemental oxygen used to complete the Screening 6MWT unless safety will be 
impacted (if the 6MWD decreased from the Screening 6MWD by more than 
20%, repeat the 6MWT within 7 days, but not on the same day) 
 SF-36 
 SHAQ 
 Digital Ulcer Count 
 Hematology: CBC with differential count and platelet count (hematocrit, 
hemoglobin, platelets, WBC, bands, eosinophils, lymphocytes, monocytes, 
neutrophils), CD19+ B cells 
 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, 
creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, 
BNP/NT-proBNP 
 CD19+ B cell studies 
 Autoantibodies 
 Serum Immunoglobulins 
 IL-1, IL-6, and other cytokine assays 
 Concomitant Medication Assessment 
 Adverse Event Assessment 
 Week 104 (Visit 17) 
6.5.4.13
An assessment of vital status approximately two years post randomization will be conducted 
via telephone or review of public records or health registries (to which the subject has 
consented to share this data).  
 
For subjects who completed their participation prior to the approval of protocol version 8.0, 
every possible effort to contact these subjects to consent them for this assessment should be 
made. 
 
Assessments:  
 Vital status 
 Treatment Satisfaction assessment 


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 Quarterly Monitoring Visits 
6.5.5
Subjects who complete the Week 48 visit or who reach endpoint early after receiving any 
study drug, but whose B cell numbers have not recovered, will be followed at least quarterly 
to monitor recovery. Quarterly monitoring visits (12 weeks ± 14 days from the last visit) will 
consist of obtaining a B cell count (at both the central lab and at Stanford Human Immune 
Processing Center for the Stanford Human Immune Monitoring Center) and CBC (at the 
central lab), and will continue until recovery or for 2 years after initial treatment (whichever 
occurs first). Recovery is defined as the time point at which B cells recover to within 10% of 
baseline levels; if a baseline value is not available then recovery is defined as a B cell level of 
at least 90% of the lower limit of normal. During this monitoring period, AEs and SAEs will 
be assessed, providing the subject has not withdrawn consent, only to capture any infectious 
event > Grade 3 using the NCI-CTCAE. No additional study-related data will be collected. 
 Early Endpoint Visit 
6.5.6
Subjects who withdraw early from the Baseline to Week 48 portion of the study will be asked 
to complete an Early Endpoint Visit. All scheduled exams, procedures, and laboratory tests 
scheduled for Week 24 will be performed at this visit, unless the early withdrawal is after 
that time, in which case all scheduled exams, procedures, and laboratory tests scheduled for 
Week 48 will be performed. If prohibited medications or treatments as defined in Section 5.6, 
Prohibited Medications and Treatments are required for treatment of progressive PAH, the 
Early Endpoint Visit, including the repeat right heart catheterization if withdrawal occurs 
prior to 24 weeks, should occur prior to the change in medication or therapy.   
 
If the subject received any study drug prior to early endpoint and his/her B cell numbers have 
not recovered, that subject requires quarterly monitoring as described in Section 6.5.5, 
Quarterly Monitoring Visits.  
 
Data from subjects who initiate rituximab therapy, but do not complete all study visits, will 
still be included in the Intent-to-Treat (ITT) and safety analyses. 
 Visit Windows 
6.5.7
The screening visit has a window of 28 days between signing of the informed consent 
document and initiation of study treatment. This visit may be divided into several visits as 
scheduling dictates to complete all assessments and procedures. The baseline/randomization 
visit must be performed within 7 days of start of study treatment (Day 0). These assessments 
are not required for eligibility evaluation and so may be completed after randomization. 
Assessments and procedures listed under the baseline/randomization visit may be done on 
Day 0 as scheduling allows, so long as all assessments are completed prior to the first dose of 
rituximab treatment. The Week 2 visit (second infusion of rituximab) must be completed 
within 14 ± 4 days of Day 0. All monthly follow-up visits and phone assessments have a visit 
window of ± 7 days.  
 


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Whenever possible, a rescheduled visit should remain within the designated visit window. 
The coordinating center should be notified if the study procedures for any scheduled visit 
cannot be performed within the designated window. 
 Unscheduled Visits 
6.5.8
If disease activity increases or other concerns arise between regularly scheduled visits, 
subjects should be instructed to contact study personnel to determine whether an 
“unscheduled” visit should be completed. The following evaluations will be performed at 
each unscheduled visit: 
 
 Adverse Event Assessment 
 Concomitant medication assessment 
 Physical Examination 
 Oxygen Saturation (forehead or ear probe must be used for saturations < 95% by 
finger probe). Measure first at room air, then add/titrate oxygen if needed to maintain 
O2 saturation ≥ 90%. The room air oxygen saturation measurement will be conducted 
at the discretion of the study physician. The measurement should not be performed if 
subject safety will be compromised. 
 Other evaluations may be performed at the investigator’s discretion 
 
If the unscheduled visit is due to an increase in SSc-PAH disease activity, these additional 
evaluations should also be performed: 
 
 NYHA Functional Class Assessment 
 6MWT: The subject should complete the assessment with the same amount of 
supplemental oxygen used to complete the Screening 6MWT unless safety will be 
impacted (if the 6MWD decreased from the Screening 6MWD by more than 20%, 
repeat the 6MWT within 7 days, but not on the same day) 
 SF-36 
 SHAQ 
 Digital Ulcer Count 
 Hematology: CBC with differential count and platelet count (hematocrit, hemoglobin, 
platelets, WBC, bands, eosinophils, lymphocytes, monocytes, neutrophils) 
 CD19+ B cell studies  
 Autoantibodies 
 Serum Immunoglobulins 
 IL-1, IL-6, and other cytokine assays 
 


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Table 6.1, Schedule of Evaluations 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
 
 
Description 
Screen Base-
line1 
Day 
01 
Week 
2 
Week 
4 
Week 
8 
Week 
12 
Week 
16 
Week 
20 
Week 
24 
Week 
28 
Week 
32 
Week 
36 
Week 
40 
Week 
44 
Week 
48 
Week 
104 
Quar-
terly 
Un-
sched
-uled
Visit window 
-28 
days 
-7  
days 
 
±4 
days 
±7 
days 
±7 
days 
±7 
days 
±7 
days 
±7 
days 
±7 
days 
±7  
days 
±7 
days 
±7 
days 
±7 
days 
±7 
days 
±7 
days 
 
±14 
days 
Type of Visit 
Clinic Clinic Clinic
Clinic
Clinic Phone Clinic
Phone
Phone
Clinic
Phone Phone
Clinic
Phone
Phone
Clinic
Phone Clinic Clinic
Clinical Draw (mL) 
21.5 
11 
2 
8 
11 
0 
11 
0 
0 
11 
0 
0 
11 
0 
0 
13 
0 
3 
3 
Research Draw (mL) 
0 
41 
0 
29  
29  
0 
33 
0 
0 
38  
0 
0 
34  
0 
0 
33  
0 
24 
33 
Visit Draw Total (mL) 
21.5 
52  
2 
37 
40  
0 
44 
0 
0 
49  
0  
0 
45  
0 
0 
46  
0 
27 
36 
General Assessments 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination& Vitals2 
X 
 
X22 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Vaccinations17 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NYHA Functional Class Assessment 
X 
 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24
Right Heart Catheterization20 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Pulmonary Function Tests3 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
Oxygen Saturation4 
X 
 
X22 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
ECG5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chest X-Ray 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TB testing21 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6MWT 6,18 
X18 
 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24
SF-36  
 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
X24
SHAQ 
 
X 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24
Digital Ulcer Count 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X24
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
Adverse Event Assessment 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X23 
X 
Post-infusion Phone Assessment7 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment of Vital Status26 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Treatment Satisfaction26 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Laboratory Assessments 
Hematology8 
X 
X 
 
X25 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
X 
X24
CD19+ B cell count (local) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chemistry9 
X9 
X22 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
Infectious Disease Screen10 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy Test (if applicable)11 
X 
 
X22 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD19+ B Cell Studies12 (24mL) 
 
X 
 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
X
 
X
X24
Autoantibodies13  (4mL) 
 
X 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
X24
Serum Igs14 & IL-1, IL-6,& Other 
Cytokine Assays15 (5mL) 
 
X 
 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24
HACA and PK Assays19 (3-5mL) 
 
X 
 
 
 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
Study Medication  
Rituximab/Placebo16 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1 Baseline and Day 0 assessments may be combined into one visit as scheduling dictates so long as they are completed within 7 days of the first rituximab infusion.  
2 Physical Examination: Full PE (including height, weight, vital signs, general appearance, skin, head/eyes/ears/neck/throat, respiratory/chest, cardiovascular, abdominal, 
neurological, lymph nodes, musculoskeletal/extremities) at Screen, Week 24, and Week 48. Abbreviated PE (including weight, vital signs, skin, respiratory/chest, 
cardiovascular, abdominal, lower extremity edema) at all other clinic visits.  
3 Pulmonary Function Tests: Height must be measured at time of PFTs. Full PFTs with spirometry, DLCO, and TLC at Screening. DLCO only at Weeks 24 and 48. Sites to 
send absolute values to Rho to convert for Hgb and altitude adjustments.  
4 Oxygen Saturation: forehead or ear probe must be used for saturations < 95% by finger probe at rest. Measure first at room air, then add/titrate oxygen if needed to maintain 
O2 saturation ≥ 90%. The room air oxygen saturation measurement will be conducted at the discretion of the study physician. The measurement should not be performed if 
subject safety will be compromised.   
5 ECG: If not done with RHC (center-specific).  
6 6MWT: Subject should complete assessment with same amount of supplemental oxygen used to complete the Screening 6MWT unless safety will be impacted (if the 
6MWD decreased from the Screening 6MWD by more than 20%, repeat the 6MWT within 7 days, but not on the same day). 
7 A member of the research staff will contact the subject within 24 hours after discharge from the infusion to assess for adverse events 
8 Hematology: CBC with differential count and platelet count (hematocrit, hemoglobin, platelet count, WBC, bands, eosinophils, lymphocytes, monocytes, and neutrophils). 
At Baseline, Week 48, and during Quarterly monitoring, CD19+ cells will also be enumerated. 
9 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, 
BNP/NT-proBNP. Note: BNP/NT-proBNP is not collected at screening.  
10 Infectious Disease Screen: HIV antibody, hepatitis B surface antibody and surface antigen, HCV antibody with HCV RNA ( PCR) if antibody positive.  
11 Pregnancy Test: For women of child-bearing potential a pregnancy test (urine or serum per institutional policy) must be obtained at screening and within 72 hours of each 
rituximab infusion.  
12 CD19+ B Cell Studies: Blood sent to Stanford Human Immune Processing Center for the Stanford Human Immune Monitoring Center.  
13 Autoantibodies: will include but are not limited to Anti-U1 RNP, anti-U3 RNP, anti-B23, anti-cardiolipin, anti-Th/To, anti-CENP-B, and RF. Frozen serum sent to 
University of Pittsburgh Immunoassay Core Laboratory.  
14 Serum Immunoglobulins: Quantitative Immunoglobulin Levels including IgG subclasses (frozen serum sent to Stanford Human Immune Processing Center for the 
Stanford Human Immune Monitoring Center).  
15 IL-1, IL-6, and Other Cytokine Assays: Frozen serum sent to Stanford Human Immune Processing Center for the Stanford Human Immune Monitoring Center.  
16 Rituximab: Per Protocol Section 5, Treatment of Subjects.  
17 Vaccinations: All immunizations must be completed at least 4 weeks prior to starting rituximab/placebo. Subject must be vaccinated with the pneumococcal vaccine at 
least 4 weeks prior to initiation of therapy, unless subject was vaccinated within 5 years of study entry. If vaccination occurred greater than 5 years prior to study entry, the 
subject must be revaccinated at least 4 weeks prior to initiation of therapy. Depending on the season, influenza vaccine should be administered at least 4 weeks prior to 
dosing with rituximab/placebo. Otherwise, it should be administered as late as possible in the flu season to still be effective after the last dose of rituximab/placebo. 
18 The 6MWT must be completed within 14 days of the first treatment visit (Day 0).  
19 HACA and PK Assays: HACA and rituximab serum level (PK). Batched and sent to Covance Laboratories. 
20 When completing the repeat RHC, the subject should be on the same amount of supplemental oxygen used during the Screening RHC unless safety will be impacted. The 
method of calculating cardiac output should be consistent with that used at the Screening RHC. 
21TB testing (PPD or QuantiFERON®-TB Gold In-Tube Test [QFT-G_IT]): unless performed within 30 days prior to screening and documented as negative in the subject’s 
records or unless subject is known to have a positive test and has documentation of appropriate therapy. 
22 If the Screening physical exam, oxygen saturation, and chemistry panel are performed within 7 days of Day 0, they do not need to be repeated during the 
Randomization/Baseline or Day 0 visits unless clinically indicated. 
23 Only infectious events ≥ NCI-CTCAE grade 3 will be reported. 
24 Required only if the unscheduled visit is due to an increase in SSc-PAH disease activity; see Section 6.5.8, Unscheduled Visit for further details. 
25 Hemoglobin, platelets, lymphocytes, neutrophils, and bands results from a hematology specimen drawn within 24 hours of the infusion must be reviewed prior to initiating 
the study drug infusion (see Section 5.3.6, Criteria for Withholding Rituximab Infusion).  
26 A member of the research staff will obtain verbal consent to conduct an assessment of vital status and treatment satisfaction approximately two years post randomization 
for each subject. If the subject cannot be reached, a review of public records or health registries should be completed.  


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7 SAFETY MONITORING AND REPORTING 
 
Overview 
7.1
This section defines the types of safety data that will be collected under this protocol and 
outlines the procedures for appropriately collecting, grading, recording, and reporting that 
data. Adverse events that are classified as serious according to the definition of health 
authorities must be reported promptly (per Section 7.5, Reporting of Adverse Events) and 
appropriately to the investigational new drug application (IND) sponsor (DAIT/NIAID), 
principal investigators in the trial, Institutional Review Boards (IRBs), and health authorities. 
Information in this section complies with ICH Guideline E2A: Clinical Safety Data 
Management: Definitions and Standards for Expedited Reporting, ICH Guideline E-6: 
Guideline for Good Clinical Practice, and applies the standards set forth in the National 
Cancer Institute (NCI), Common Terminology Criteria for Adverse Events (CTCAE), Version 
4.0: http://ctep.cancer.gov/reporting/ctc.html. 
 
Definitions 
7.2
 Adverse Event (or Adverse Experience) 
7.2.1
Any untoward or unfavorable medical occurrence in a human subject, including any 
abnormal sign, symptom, or disease, temporally associated with the subject’s participation in 
the research, whether or not considered related to the subject’s participation in the research 
(modified from the definition of adverse events in the 1996 International Conference on 
Harmonization E-6 Guidelines for Good Clinical Practice)." [From OHRP "Guidance on 
Reviewing and Reporting Unanticipated Problems Involving Risks to Subjects or Others and 
Adverse Events (1/15/07)" < http://www.hhs.gov/ohrp/policy/advevntguid.pdf> 
 Adverse Reaction and Suspected Adverse Reaction 
7.2.2
An adverse reaction means any adverse event caused by a drug. Adverse reactions are a 
subset of all suspected adverse reactions for which there is reason to conclude that the drug 
caused the event. 
Suspected adverse reaction (SAR) means any adverse event for which there is a reasonable 
possibility that the drug caused the adverse event. For the purposes of safety reporting, 
‘reasonable possibility’ means there is evidence to suggest a causal relationship between the 
drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty 
about causality than adverse reaction, which means any adverse event caused by a drug (21 
CFR 312.32(a)). 
 Unexpected Adverse Reaction 
7.2.3
A SAR is considered “unexpected” if it is not listed in the investigator brochure or is not 
listed at the specificity or severity that has been observed. 


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 Serious Adverse Event 
7.2.4
An AE or SAR is considered “serious” if, in the view of either the investigator or 
DAIT/NIAID, it results in any of the following outcomes (21 CFR 312.32(a)): 
1. Death 
2. A life-threatening event: An AE or SAR is considered “life-threatening” if, in the 
view of either the investigator or DAIT/NIAID, its occurrence places the subject at 
immediate risk of death. It does not include an AE or SAR that, had it occurred in a 
more severe form, might have caused death.  
3. Inpatient hospitalization or prolongation of existing hospitalization 
4. Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
5. Congenital anomaly or birth defect 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the subject and may require medical or surgical intervention to prevent 
one of the outcomes listed above. 
 
 Collection and Recording of Adverse Events 
7.3
 Investigational Product 
7.3.1
The primary investigational product in this protocol is rituximab/placebo. In addition, 
subjects in this protocol are required to receive background medical therapy for PAH 
(prostanoid, endothelin receptor antagonist, PDE-5 inhibitor, and/or guanylate cyclase 
stimulators [riociguat (Adempas)]). For purposes of reporting safety information on the 
MedWatch form, these drugs will be considered concurrent study mandated therapy.  
 Collection Period 
7.3.2
Adverse events NCI-CTCAE grade 2 and above will be collected from the time the subject 
signs the informed consent until he/she initiates study intervention or until he/she is 
determined to be ineligible to receive study intervention, if the investigator determines that 
the adverse event is related to a study-mandated procedure, treatment, or change in treatment. 
 
For all participants, adverse events NCI-CTCAE grade 2 and above will be collected from 
the time of initiation of study intervention (i.e., the administration of the first dose of study 
drug/study drug as defined in Section 6.5.3.1, Visit 3 [Day 0]) until he/she completes study 
participation or until 30 days after he/she prematurely withdraws (without withdrawing 
consent) or is withdrawn from the study. 
 Collection of Adverse Events 
7.3.3
Adverse events (including SAEs) may be discovered through any of these methods: 
 Observing the participant. 


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 Questioning the participant in an objective manner. 
 Receiving an unsolicited complaint from the participant. 
 In addition, an abnormal value or result from a clinical or laboratory evaluation 
(including, but not limited to, a radiograph, an ultrasound, or an 
electrocardiogram) can also indicate an adverse event, as defined in Section 7.4, 
Grading and Attribution of Adverse Events. 
 Recording Adverse Events 
7.3.4
Throughout the study, the investigator will record and grade adverse events NCI-CTCAE 
grade 2 and above on the appropriate AE electronic case report form (AE eCRF) regardless 
of their severity or relation to study medication or study procedure.  
 
Once recorded, an AE will be followed until it resolves with or without sequelae, or until the 
end of study participation, or until 30 days after the subject prematurely withdraws (without 
withdrawing consent)/or is withdrawn from the study, whichever occurs first. 
 Recording Serious Adverse Events 
7.3.5
Serious AEs will be recorded on the appropriate AE eCRF and on the SAE eCRF. All 
requested information on the AE eCRF and SAE eCRF should be provided, if available, for 
submission to the Statistical and Clinical Coordinating Center (SACCC) and DAIT/NIAID. 
New SAEs will be recorded until end of study participation OR for 30 days after premature 
withdrawal OR as in Section 3.3.2.1, Follow-up Requirements for All Treated Subjects for 
subjects for whom B cells have not recovered.  
Once recorded, an SAE will be followed until it resolves with or without sequelae OR until 
30 days after the end of study participation UNLESS it is a Grade 3 or higher infection in an 
individual for whom B cells have not recovered (as per Section 3.3.2.1, Follow-up 
Requirements for All Treated Subjects), in which case it will be followed until resolution. 
 
Grading and Attribution of Adverse Events 
7.4
 Grading Criteria 
7.4.1
The study site will grade the severity of adverse events experienced by the study subjects 
according to the criteria set forth in the National Cancer Institute’s Common Terminology 
Criteria for Adverse Events Version (CTCAE) 4.0. This document (referred to herein as the 
NCI-CTCAE manual) provides a common language to describe levels of severity, to analyze 
and interpret data, and to articulate the clinical significance of all adverse events. The NCI- 
CTCAE has been reviewed by the Protocol Chair(s) and has been deemed appropriate for the 
subject population to be studied in this protocol. 
Adverse events will be graded on a scale from 1 to 5 according to the following standards in 
the NCI-CTCAE manual: 


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Grade 1 = mild adverse event. 
Grade 2 = moderate adverse event. 
Grade 3 = severe and undesirable adverse event. 
Grade 4 = life-threatening or disabling adverse event. 
Grade 5 = death. 
If NCI-CTCAE criteria are defined for grading an abnormal value or result from a clinical or 
laboratory evaluation (including, but not limited to, a radiograph, an ultrasound, or an 
electrocardiogram), then a treatment-emergent adverse event is defined as an increase in 
grade from Baseline (Day 0) or from the last post-baseline value that doesn’t meet grading 
criteria. Changes in grade from screening to Baseline (Day 0) will also be recorded as 
adverse events but are not treatment-emergent. If a specific event or result from a given 
clinical or laboratory evaluation is not included in the NCI-CTCAE manual, then an 
abnormal result would be considered an adverse event if changes in therapy or monitoring 
are implemented. 
 Attribution Definitions 
7.4.2
The relation, or attribution, of an adverse event to an investigational product will initially be 
determined by the site investigator. The site investigator will also record the initial 
determination of attribution on the appropriate AE eCRF. The relation of an adverse event to 
the study intervention will be determined using the descriptors and definitions provided in 
Table 7.4.2. Final determination of attribution for safety reporting will be decided by 
DAIT/NIAID. 
 
For additional information and a printable version of the NCI-CTCAE manual, consult the 
NCI-CTCAE web site: http://ctep.cancer.gov/reporting/ctc.html. 
 
Table 7.4.2. NCI-CTCAE attribution of adverse events 
Code 
Descriptor 
Relationship (to primary investigational product and/or other 
concurrent mandated study therapy) 
Unrelated Categories  
1 
Unrelated 
The adverse event is clearly not related. 
2 
Unlikely 
The adverse event is unlikely related. 
Related Categories 
3 
Possible 
The adverse event has a reasonable possibility to be related; 
there is evidence to suggest a causal relationship. 
4 
Probable 
The adverse event is likely related. 
5 
Definite 
The adverse event is clearly related. 
 


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Reporting of Adverse Events 
7.5
 Reporting of Adverse Events to DAIT/NIAID 
7.5.1
This section describes the responsibilities of the site investigator to report adverse events to 
the SACCC. Timely reporting of adverse events is required by 21 CFR and ICH E6 
guidelines. For this study, adverse events of NCI-CTCAE Grade 2 and higher will be 
reported. 
 
Unless otherwise noted below in Section 7.5.1.1 as requiring 24 hour reporting, AEs must be 
recorder on the appropriate AE eCRF within five (5) days of the site learning of the event(s). 
 Procedure for Adverse Events Requiring 24 Hour Reporting 
7.5.1.1
The adverse events that are bulleted below must be reported by site investigators to the 
SACCC regardless of relationship or expectedness to study intervention within a 24 hour 
period of discovering the adverse event: 
 All SAEs per 21 CFR 312.32 definitions (see Section 7.2.4, Serious Adverse 
Event). 
 All clinical events with a NCI-CTCAE Grade 3 or greater deemed possibly, 
probably, or definitely related to rituximab; 
 Note: clinical events include clinical signs/symptoms and diagnoses 
and laboratory abnormalities with clinical consequence (defined as the 
requirement for intervention, correction, increased monitoring, or 
further evaluation). 
 Administration of atropine, dopamine, or other inotropic agents within 24 hours of 
rituximab/placebo infusion; 
 Any event that the site considers Serious but is not easily categorized. 
Elective hospitalizations or hospital admissions for the purpose of conduct of protocol-
mandated procedures are not to be reported as an SAE unless hospitalization is prolonged 
due to complications. 
The following process for reporting of the adverse events bulleted above ensures compliance 
with the ICH guidelines and the Food and Drug Administration (FDA) CFR regulations. 
When an investigator identifies such an adverse event, he or she must notify the SACCC 
within 24 hours of discovering the adverse event, and complete and submit the AE/SAE 
eCRF within 1 business day following initial notification. The SACCC is responsible for 
notifying DAIT/NIAID upon receipt of the site’s notification of the adverse event and 
sending a SAE report form to DAIT/NIAID within 2 business days after receipt of the 
AE/SAE eCRF from the site. 


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 Procedure for Standard Adverse Event Reporting 
7.5.1.2
All other adverse events (Section 7.3.3, Collecting Adverse Events) must be recorded by the 
site on the appropriate AE eCRF within 5 business days of the site learning of the adverse 
event(s). 
 DAIT/NIAID Reporting to the Health Authority 
7.5.2
After an adverse event requiring 24 hour reporting (per Section 7.5.1.1, Procedure for 
Adverse Events Requiring 24 Hour Reporting) is submitted by the site investigator and 
assessed by DAIT/NIAID, there are two options for DAIT/NIAID to report the adverse event 
to the appropriate health authorities: 
 Annual IND Report. This option applies if the adverse event is classified as one of 
the following: 
o Serious, expected, suspected adverse reactions (see Section 7.2.2, Adverse 
Reaction and Suspected Adverse Reaction, and Section 7.2.3, Unexpected 
Adverse Reaction). 
o Serious and not a suspected adverse reaction (see Section 7.2.2, Adverse 
Reaction and Suspected Adverse Reaction). 
Note that all adverse events (not just those requiring 24 hour reporting) will be 
reported in the Annual IND Report. 
 Expedited Safety Report. This option applies if the adverse event is classified as one 
of the following: 
o Serious and unexpected suspected adverse reaction (see Section 7.2.2, Adverse 
Reaction and Suspected Adverse Reaction and Section 7.2.3, Unexpected 
Adverse Reaction). 
o Aggregate analysis of adverse events that suggests a causal relationship to 
rituximab. 
o Any findings from clinical or epidemiological studies, analysis of data pooled 
across multiple studies, published or unpublished scientific papers, or from 
animal or in vitro testing that would result in a safety-related change in the 
protocol, informed consent, investigator brochure or other aspects of the 
overall conduct of the trial will be reported. 
Safety Reports must be reported by DAIT/NIAID to the appropriate health 
authorities within 15 calendar days; fatal or immediately life-threatening serious, 
unexpected, suspected adverse reactions must be reported within 7 calendar days. 
 Safety reports will be provided to Genentech, Inc. per the terms negotiated in the 
Clinical Trial Agreement (CTA). 
 Reporting of Adverse Events to IRBs 
7.5.3
All investigators must report adverse events, including expedited reports, in a timely fashion 
to their respective IRBs in accordance with applicable regulations and guidelines. 
All IND Safety Reports to the FDA will be distributed by the DAIT/NIAID or designee to all 
participating institutions for site IRB submission. 


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Pregnancy Reporting 
7.6
This study includes pregnancy information as safety data. Information about any pregnancy 
should be reported promptly to the SACCC on the same timeline as an SAE (Section 7.5.1.1, 
Procedure for Adverse Events Requiring 24 Hour Reporting). 
All pregnancies identified during the study must be followed to conclusion and the outcome 
of each must be reported. The investigator should be informed immediately of any pregnancy 
in a study subject or a partner of a study subject. A pregnant subject should be instructed to 
stop taking study medication. The investigator should report to the SACCC all pregnancies 
within 1 business day (as described in Section 7.5.1.1, Procedure for Adverse Events 
Requiring 24 Hour Reporting) using the Pregnancy eCRF. The investigator should counsel 
the subject and discuss the risks of continuing with the pregnancy and the possible effects on 
the fetus. Monitoring of the pregnant subject should continue until the conclusion of the 
pregnancy, and an updated Pregnancy eCRF detailing the outcome of the pregnancy should 
be submitted to the SACCC. When possible, similar information should be obtained for a 
pregnancy occurring in a partner of a study subject. 
Information requested about the delivery will include: 
 Subject’s enrollment ID 
 Gestational age at delivery 
 Birth weight, length, and head circumference 
 Gender 
 Appearance, pulse, grimace, activity, and respiration (APGAR) score at 1 minute, 
5 minutes, and 24 hours after birth, if available 
 Any abnormalities. 
Should the pregnancy result in a congenital abnormality or birth defect, an SAE must be 
submitted to the SACCC using the SAE reporting procedures described above. 
 
Reporting of Other Safety Information 
7.7
An investigator should promptly notify the SACCC when an “unanticipated problem 
involving risks to subjects or others” is identified, which is not otherwise reportable as an 
adverse event. 
 
Review of Safety Information 
7.8
 Medical Monitor Review 
7.8.1
The DAIT Medical Monitor will receive monthly reports from the SACCC compiling new 
and accumulating information on AEs, SAEs, and pregnancies recorded by the sites on 
appropriate eCRFs. 
In addition, the Medical Monitor will receive SAE and pregnancy reports for review and 
triage after the SACCC is made aware of these events (See Sections 7.5.1, Reporting of 
Adverse Events to DAIT/NIAID, and 7.6, Pregnancy Reporting). 


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 DSMB Review 
7.8.2
The Data and Safety Monitoring Board (DSMB) will review accumulating safety data at least 
yearly during planned DSMB Data Review Meetings. Data for the planned safety reviews 
will include, at a minimum, a listing of all reported AEs and SAEs. To ensure patient safety 
between Data Review Meetings, the DSMB will be informed of all Expedited Safety Reports 
in a timely manner. 
In addition to the pre-scheduled data reviews and planned safety monitoring, the DSMB may 
be called upon for ad hoc reviews or emergency meetings. The DSMB will review any event 
that potentially impacts safety at the request of the protocol chair or DAIT/NIAID. In 
addition, the following events will trigger both a comprehensive DSMB Emergency Safety 
Review and a temporary halt in enrollment: 
a. Any immediately life threatening event or death that occurs in the study which is 
unexpected and possibly, probably, or definitely related to study intervention. 
b. Any event, including those listed in Section 3.3.2, Discontinuation of Protocol-
Specified Treatment Requirements, that results in permanent discontinuation of 
infusion of study intervention occurring in 2 of the first 5 subjects, 4 of the first 15 
subjects, 6 of the first 30 subjects, or a total of 8 events after 30 subjects have been 
enrolled in the trial. 
c. Any occurrence of PML. 
d. Clinical Grade 3 or higher events or SAEs which are possibly, probably, or definitely 
related to study treatment but do not result in discontinuation of study treatment will 
be reviewed whenever the ratio of events to enrolled subjects is 20% (e.g. 9 events for 
47 subjects, 10 events for 48-52 subjects, 11 events for 53-57 subjects, etc.).  
o Note: clinical events include clinical signs/symptoms and diagnoses and 
laboratory abnormalities with clinical consequence (defined as the 
requirement for intervention, correction, increased monitoring, or further 
evaluation). 
The DSMB will have discretion to recommend actions regarding study conduct and 
continuation as a consequence of any planned or unplanned monitoring activity. 
 Temporary Halt in Enrollment for Emergency Safety Review 
7.8.2.1
A temporary halt in enrollment will occur for the events listed in Section 7.8.2, DSMB 
Review with the exception of 7.8.2.d. For events falling under 7.8.2.d, a halt in enrollment 
will occur if the DSMB Emergency Safety Review is not completed within 2 weeks. In the 
event that the study temporarily halts enrollment, no new subjects will be consented or start 
on therapy with rituximab or placebo; and subjects already on rituximab or placebo will 
continue on therapy unless they are the focus of the DSMB review. Subjects in the screening 
phase of the study may continue to undergo minimal risk procedures (e.g. blood tests), but 
more than minimal risk procedures should be deferred. Randomization will not occur until 
the DSMB review is complete. The FDA will be notified of any halt in enrollment. After 


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careful review of the data, the DSMB will make recommendations regarding study conduct 
and/or continuation. 
8 STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN 
 
Sample Size 
8.1
As of May 2014, 30 subjects had enrolled in the study.  The slow pace of enrollment 
prompted a redesign of the trial with the objective of reducing the sample size to enable 
completion of the study within 2 years while still retaining the potential to address a 
clinically relevant question. Sixty was the largest sample size felt to be attainable (down from 
80).   
 
Through version 5.0 of the protocol, the primary endpoint was the change in PVR from 
baseline to Week 24, defining a 20-30% improvement from baseline as clinically 
significant. Assuming a baseline mean of 810 dyne-s/cm5, a drop at Week 24 equal to 251 
dyne-s/cm5  would be considered clinically significant.  Power was 85% for a design with 80 
subjects, assuming the baseline mean was the same for both arms (i.e. 810 dyne-s/cm5), a 
standard deviation in the change from baseline of 350 dyne-s/cm5, and the percent change 
from baseline equals 0% and 30% for placebo and rituximab arms, respectively. 
 
Contrary to the original assumption that the mean baseline PVR was 810 dyne-s/cm5, review 
of the data revealed that the mean baseline PVR for the enrolled subjects was approximately 
553 dyne-s/cm5.  Hence, relative to an assumed population baseline mean of 553 dyne-s/cm5, 
the difference of 251 dyne-s/cm5 used in the original power/sample size computation would 
equate to an improvement of 46%.  The magnitude of the drop is much larger than the 
clinically meaningful effect and is overly optimistic (or completely unrealistic). This degree 
of change is not needed to demonstrate clinical efficacy. Further, if the baseline mean is 
smaller than originally anticipated, a larger (not smaller) study would be needed to detect a 
clinically meaningful effect.   
 
The 6MWD has been the most utilized endpoint in PAH trials, and a 33m improvement from 
baseline is considered clinically significant [160].  For sample size calculations, we assume a 
change from baseline of 0m for the placebo arm and 33m for the rituximab arm.   An effect 
size of this magnitude is attainable in a 24 week period, and has been seen in as little as 12 
weeks in studies using vasodilators.  Assuming a standard deviation of 58m (a best guess 
based on prior studies) and a 20% drop out rate, ASC01 would be powered at 50% to detect a 
treatment group difference of 33m for this phase II study.  If a significant result is found for 
this primary efficacy endpoint and no safety signals emerge, this study could provide a 
definitive conclusion about this treatment.  In order to achieve 80% power, the true 
difference between treatment groups would have to be at least 50m.  While other studies 
evaluating other drugs have seen this magnitude of difference in similar timeframes, we have 
no data on rituximab to suggest the true difference is at least 50m. Furthermore, rituximab 
would be interesting as a therapy option if the effect size is as small as 33m.  


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Analysis Populations 
8.2
 Safety Population 
8.2.1
The Safety Population (SP), which will be used for all safety analyses, will include all 
subjects for whom study treatment is initiated. 
 Intent-to-Treat Population  
8.2.2
The Intent-to-Treat (ITT) population will include all randomized subjects who initiate 
rituximab therapy and meet entry criteria. Randomized subjects who withdraw from the trial 
prior to starting rituximab infusion will be excluded from the ITT analysis set and replaced. 
The primary efficacy analyses will be based on the ITT population, as will key secondary 
efficacy and mechanistic analyses. Subjects who, for whatever reason, do not complete their 
assigned therapy will be included in the ITT population in the groups to which they were 
randomized. 
 Per Protocol Population 
8.2.3
The Per Protocol (PP) population will be defined as those subjects who comply with the 
assigned treatment protocol by completing two IV infusions of 1000mg of 
Rituximab/Placebo at Visit 3 and Visit 4 as well as completing primary endpoint evaluation 
(6MWD ) at Visit 10 with no major protocol deviations that would affect their efficacy 
outcome. Subjects who are withdrawn from follow-up prior to Visit 10 will not be included 
in the PP population. A masked data review panel will evaluate deviations from the protocol 
including, for example, violations of entry criteria, departures from assigned treatment 
regimen, or administration of study procedures outside the specified visit windows. The 
panel may exclude subjects with serious protocol deviations from the PP population. Both the 
primary analyses and all secondary analyses will be replicated on the PP population. 
 
Statistical Methods 
8.3
In presenting data from this trial, continuous data will be summarized in tables listing the 
mean, standard deviation or standard error, median, and number of subjects in a group. 
Categorical data will be summarized in tables listing the frequency and the percentage of 
subjects in a group. These summaries will be presented separately for subjects on the two 
treatment arms.  
 Safety Analysis 
8.3.1
All safety analyses will be performed using the Safety Population.  
AEs including changes in laboratory values will be graded according to the National Cancer 
Institute’s Common Terminology Criteria for Adverse Events Version (CTCAE) 4.0 
(http://ctep.cancer.gov/reporting/ctc.html). The frequency of AEs will be summarized by 
system organ class, preferred term, severity (grade), and relationship to study treatment. For 
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preferred term at the greatest severity. For these summaries, relationship to study treatment 
will be categorized as either treatment related (possibly, probably, or definitely related to 
study medication) or unrelated. Similar analyses will be performed for SAEs.  
For all reported adverse events and SAEs, the risk of the event in each arm will be described 
using event rates and an analysis based on a Poisson regression model will be conducted to 
compare the rate of each type of event in the treatment groups. The proportion of subjects 
experiencing at least one event will be reported and the treatment groups compared based on 
Fisher’s Exact Test.  
Treatment-related mortality, defined as death occurring at any time after randomization and 
possibly, probably, or definitely resulting from study treatment, and all-cause mortality, 
defined as any death occurring after randomization, will be summarized. The proportion of 
subjects dying in each treatment group will be reported and the treatment groups compared 
based on Fisher’s Exact Test. 
Laboratory parameters will be summarized both overall and by treatment group using 
appropriate descriptive statistics. For quantitative safety parameters, change from the last 
pre-randomization assessment to the final visit assessment will be summarized overall and by 
treatment group. For qualitative parameters, descriptive information on shifts in pre- to post-
randomization findings will be provided. In addition, abnormal and clinically significant 
abnormalities will be summarized and listed separately. 
All safety comparisons and associated p-values are considered exploratory, not as formal 
tests of hypothesis. As such no adjustments will be made for multiple comparisons and all p-
values must be interpreted cautiously. 
Detailed listings of AEs, deaths, and safety-related laboratory values will be generated as 
specified in the Statistical Analysis Plan (SAP). 
 Efficacy Analysis 
8.3.2
  Primary Efficacy Analysis 
8.3.2.1
The primary efficacy analysis for this study is designed to test the scientific hypothesis that 
rituximab has a beneficial clinical impact on SSc-PAH as measured by improvement or 
stabilization in 6MWD between baseline to 24 weeks in patients treated with standard of care 
plus rituximab than in patients treated with standard of care plus placebo. Operationally for 
this study, this scientific hypothesis will be examined by formally testing the following 
statistical hypothesis: 
 
H0: 
The mean change in 6MWD between baseline and 24 weeks does not differ 
between individuals treated with rituximab and those treated with placebo 
 
(δrituximab = δplacebo where δx = mean 6MWD at 24 weeks – mean 6MWD at 
baseline) 
 
versus 


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HA:  
The mean change in 6MWD between baseline and 24 weeks is more 
beneficial in those individuals treated with rituximab than in those treated 
with placebo 
 
(δrituximab < δplacebo where δx = mean 6MWD at 24 weeks – mean 6MWD at 
baseline) 
 
While this hypothesis is inherently one-sided, two-sided tests will be used in the analysis. 
This primary hypothesis will be tested using a repeated measures random effects model to 
model change from baseline in 6MWD as the primary outcome and treatment as the primary 
predictor, controlling for visit day, limitations by musculoskeletal disease, baseline measures 
of 6MWD, and a treatment by visit day interaction  A random slope and intercept will be fit 
for each subject.  An unstructured covariance matrix will be assumed for the random effects. 
More details on this analysis and sensitivity analyses will be defined in the Statistical 
Analysis Plan (SAP). The primary efficacy analysis will be based on the ITT population. 
  Secondary Efficacy Analysis 
8.3.2.2
All secondary analyses will be conducted in an exploratory fashion with p-values and 
confidence intervals presented as descriptive statistics with no adjustments for multiple 
comparisons. Tests will be two-sided and interval estimates will be generated at the 95% 
confidence level. As part of secondary analyses, appropriate contrasts will be constructed 
using model-based approaches. Larger models will be constructed for these comparisons with 
consideration of additional covariates that are found to differ between the treatment groups. 
Covariates may include, but are not limited to: study center, method of cardiac output, 
gender, disease duration and severity, and concomitant therapy use. All analyses will be 
repeated using the Per Protocol Population.  
 
Change from baseline in PVR at Week 24 and percent change from baseline in PVR at Week 
24 will be analyzed using an analysis of covariance, adjusting for treatment, method of 
cardiac output, and baseline PVR. 
 
For the outcome time to clinical worsening, the two groups will be compared using survival 
approaches. Graphical summaries of time to clinical worsening will be generated using 
Kaplan-Meier survival curves with a log-rank test used to test for differences in the two arms. 
The two arms will also be compared after control of appropriate covariates using a Cox 
proportional hazards model. 
 
For the proportion of subjects who changed or added PAH therapeutic agents, the treatment 
two arms will be compared using a logistic regression model, controlling for appropriate 
covariates. This will be done at the following time points: prior to week 24 and after week 
24. 
 
Longitudinal changes in continuous outcome measures including the subscales of the SF-36, 
the SHAQ, and DLCO will be compared across the two arms using linear mixed models.  
Initially the models will be used to describe changes between baseline and 24 weeks and 


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baseline and 48 weeks including only treatment, baseline levels of the outcome of interest, 
and an indicator if the measurement was affected by changed or new PAH therapeutic agents, 
with added models developed to assess the effect of other covariates. 
 
Longitudinal changes in the six-minute walk distance at week 48 will be assessed using a 
similar model as the primary endpoint, but including all data through 48 weeks. This model 
will address any subject who had their 6MWD affected by new or changed PAH therapeutic 
agents after the 24 week time point.  Additionally, the analysis will be conducted on the ITT 
population will additionally add limited by any musculoskeletal disease (yes/no) as a 
covariate. Additional models may be developed to assess the effect of other covariates. 
 
Longitudinal change in the oxygen saturation will be compared across the two arms using 
generalized estimating equations. An ordinal response variable will be used to account for the 
subjects that cannot perform the room air assessment. Initially the model will be used to 
describe changes between baseline and 24 weeks and baseline and 48 weeks including only 
treatment, baseline levels of oxygen saturation, and an indicator if the measurement was 
affected by changed or new PAH therapeutic agents, with added models developed to assess 
the effect of other covariates. However, if all subjects can perform the room air assessment, 
the data will be analyzed as a continuous model, following the method described above for 
the six-minute walk distance. 
 
Longitudinal change in number of new digital ulcers will be compared across the two arms 
using Poisson marginal models for longitudinal data. Initially the models will be used to 
describe changes between baseline and 24 weeks and baseline and 48 weeks including only 
treatment, baseline levels of the outcome of interest, and an indicator if the measurement was 
affected by changed or new PAH therapeutic agents , with added models developed to assess 
the effect of other covariates. 
 Mechanistic/Immunological Analysis 
8.3.3
Because of the exploratory nature of the analyses, descriptive statistics and plots (including, 
but not limited to, those described subsequently) will be used to gain an understanding of the 
data prior to developing any statistical models. Means, medians, standard deviations, 
minimums and maximums will be computed for each continuous biomarker at each time 
point for placebo and rituximab groups and separately for treatment responders / non-
responders. For dichotomous biomarkers, frequencies and percents will be computed at each 
time point for placebo and rituximab groups and separately for treatment responders / non-
responders. Treatment response, to be defined in the statistical analysis plan (SAP), will be 
based on PVR, exercise capacity as measured by 6MWD, or quality of life as measured by 
SF-36 and SHAQ. To gain a better understanding of trends over time, summary statistics 
(e.g. means, medians, or percents) will be plotted versus time at the relevant time points. 
Plots for individual subjects may also be useful. 


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 Exploratory Analysis 
8.3.4
Agreement between a secondary endpoint (PVR calculated by site-based standards) and the 
standardized Fick-based calculation of PVR (PVRf) will be assessed using the method of 
Bland and Altman, separately for baseline and Week 24 right heart catheterization. Analyses 
will also be subset on method of calculation of cardiac output (thermodilution vs. Fick) if 
found to be a significant predictor in the ANCOVA model. 
 
This secondary efficacy analysis will be repeated for PVRf. Change from baseline in the 
standardized Fick-based calculation of pulmonary artery capacitance (PACf) will also be 
analyzed in the same manner as the PVR efficacy analysis. 
 
Additionally, the relationship between change in PVR and change in 6MWD at Week 24 will 
be analyzed using a Spearman’s rank correlation coefficient. Relationships between 
additional clinical outcomes and 6MWD or PVR can also be provided.  These clinical 
outcomes will be addressed in the SAP. 
 
The analyses of Treatment Satisfaction responses will also be considered exploratory.  
Questions will include: 
1. Do you think you benefited by being in the study? 
2. Have you noticed any long term benefits from study treatment?  
3. Do you think you received rituximab or placebo? 
 
All exploratory analyses will be conducted on subjects in the ITT population with available 
data. 
 
Interim Analysis 
8.4
Results of interim analyses will be reported to the DSMB for planned Data Review Meetings. 
Reports prepared for these meetings will focus on study conduct and subject safety and may 
include information on enrollment, randomization, site activation status, protocol deviations, 
subject status and demographics, and safety analyses.  
 Interim Analysis of Efficacy Data 
8.4.1
There will not be any interim analysis on efficacy data for this trial. 
 Interim Analysis of Safety Data 
8.4.2
The DSMB will periodically review safety data as described in Section 7.8.2, DSMB Review. 
The safety analyses for this trial will be descriptive rather than inferential. Detailed listings 
and summary tabulations of adverse events and safety-related laboratory values will be 
generated as specified in the Statistical Analysis Plan. The safety analyses will be completed 
using the safety population without stratification. 
No formal stopping rules for safety endpoints are specified. 


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Other Statistical Considerations 
8.5
 Covariates 
8.5.1
The primary analysis will be adjusted for as stated above. 
If the analyses described in Section 8.5.4, Examination of Subgroups yield covariates that 
differ significantly between treatment groups, then, as a secondary analysis, these covariates 
may also be considered for adjustment in the models specified for the primary analysis. 
Covariates that may be considered include, but are not limited to: age, study center, gender, 
race, duration of SSc-PAH disease, baseline and concomitant medication use, undergoing 
cardiopulmonary rehabilitation (yes/no), non-biologic immunosuppressive or 
immunomodulating drug use (yes/no), and non-biologic immunosuppressive or 
immunomodulating drug change after 6 months (yes/no). 
 Multi-center Studies 
8.5.2
Due to the small sample size and large number of sites needed for this study, study center 
will not be included as a covariate in the analysis.  However, if evidence of a study center 
effect exists and there are appropriate sample sizes at each site, a sensitivity analysis will be 
performed including study center in the efficacy analyses. All safety analyses will be based 
on data pooled across all centers with no adjustment or stratification by center. 
 Multiple Comparisons and Multiplicity 
8.5.3
This study has a single primary analysis designed to assess the efficacy of rituximab in 
clinical response as measured by change in PVR. Consequently, no adjustments for 
multiplicity are needed for Type I error protection. The secondary efficacy analyses are 
considered to be descriptive with p-values and confidence intervals presented as descriptive 
measures of strength of evidence rather than formal statistical inference. Therefore, no 
multiplicity adjustments are needed for this study. 
 Examination of Subgroups 
8.5.4
Secondary analyses of the primary and secondary objectives may be conducted for the 
following subgroups: 
 Race 
 Ethnicity 
 Gender 
 Baseline PVR 
 Baseline 6MWD 
 NYHA Functional Class 
 Duration of SSc-PAH disease 
 
Additionally, exploratory analyses of responder subgroups and subgroups defined through 
the mechanistic and immunological studies will be conducted. 


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 Missing Data 
8.5.5
Standard procedures will be used to ensure that data are as complete and accurate as possible. 
In analyses, a full accounting will be made for all data items. Because this is a Phase II trial 
that is underpowered, there will not be any additional handling of missing data for the 
primary analysis.  However, sensitivity analyses will be performed to assess the robustness of 
the conclusions under different assumptions. For example, missing data due to death of a 
subject prior to Week 24 would be handled differently than missing data due to a missed 
visit. 
As described in Section 3.3.3.1, Follow-up Requirements for All Treated Subjects, every 
effort will be made to obtain 6MWD for patients withdrawing prior to the 24 week visit. For 
the primary analysis, this observation will be included in the repeated measures model and 
will contribute to the estimated mean change from baseline at Week 24.  
Secondary analyses will be performed on all available data; no imputation will be performed 
on missing data, recognizing that these exploratory analyses could be biased in an unknown 
direction by non-ignorably missing data.  
If data are missing for the primary endpoint, sensitivity analyses will be conducted to assess 
whether conclusions are robust to alternative analytic approaches for handling these data. If 
data are missing for other key endpoints, sensitivity analyses could also be conducted, if 
deemed appropriate. Details of the sensitivity analyses will be provided in the Statistical 
Analysis Plan. 
 Changes to the Statistical Analysis Plan 
8.5.6
A detailed description of the planned analyses will be provided in a SAP to be completed and 
signed off prior to the completion of the trial. Major changes from this protocol will be noted 
in the SAP. If there is sufficient reason to do so, revised plans may be issued during the 
course of the study. Changes to the SAP that are made subsequent to database lock will be 
documented in the clinical study report. 
9 ACCESS TO SOURCE DATA AND DOCUMENTS 
Each participating site will maintain the highest degree of confidentiality permitted for the 
clinical and research information obtained from subjects participating in this clinical trial. 
Medical and research records should be maintained at each site in the strictest confidence. 
However, as a part of the quality assurance and legal responsibilities of an investigation, each 
site must permit authorized representatives of the IND sponsor, the SACCC, Genentech, Inc., 
and health authorities to examine (and when required by applicable law, to copy) clinical 
records for the purposes of quality assurance reviews, audits, and evaluation of the study 
safety and progress. Unless required by the laws permitting copying of records, only the 
coded identity associated with documents or other subject data may be copied (obscuring any 
personally identifying information). Authorized representatives as noted above are bound to 
maintain the strict confidentiality of medical and research information that may be linked to 
identified individuals. Participating sites will normally be notified in advance of auditing 
visits. 


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All subject records and study documentation will be kept after the protocol is completed. 
This will include all documentation of AEs, records of study drug receipt and dispensation, 
and all IRB correspondence. All study records will be kept for at least 2 years after the 
investigation is completed. 
10 DATA COLLECTION, QUALITY CONTROL AND QUALITY 
ASSURANCE 
The investigator is required to keep accurate records to ensure the conduct of the study is 
fully documented. The period of record retention should be consistent with the record 
retention policies of the sponsoring agency or applicable regulatory agencies. However, in 
certain instances, documents should be retained for a longer period if required by the 
applicable regulatory agency or by the National Institutes of Health. 
 
The investigator will report all protocol deviations to DAIT and the SACCC per the 
instructions in the ACE Manual of Operations. The SACCC will forward reports of protocol 
deviations to the responsible DAIT/NIAID medical officer for review as specified in the 
ACE Manual of Operations. 
 
The SACCC is responsible for regular inspection of the conduct of the trial, for verifying 
adherence to the protocol, and for confirming the completeness, consistency and accuracy of 
all documented data. 
 
Data will be obtained from a variety of sources including, but not limited to laboratory 
notebooks, automated instrument output files, and clinical subject charts. Data from these 
source materials will be transmitted to the SACCC via one of two mechanisms. Data 
collected electronically at central laboratories will be transferred electronically directly from 
the laboratory to the SACCC using standard secure data transfer procedures. Data collected 
at the clinical sites will be transmitted to the SACCC using an internet-based remote data 
entry system. Clinical site personnel use an internet browser to key data into electronic CRFs 
(eCRFs); each eCRF page is submitted to the SACCC data server electronically as the page 
is completed. Univariate data validation tests are performed as the data are keyed. The central 
database, which resides on the SACCC server, is backed up nightly; backup tapes are saved 
in a secure, off-site location. At any time, authorized site personnel may log in to the remote 
data entry system, review and correct previously entered data, or key additional data. The 
data will be further validated per the study data validation plan via a series of computerized 
and manual edit checks, and all relevant data queries will be raised and resolved on an 
ongoing basis. Complete, clean data will be frozen to prevent further inadvertent 
modifications. All discrepancies will be reviewed and any resulting queries will be resolved 
with the investigators and amended in the database. All elements of data entry (i.e., time, 
date, verbatim text, and the person performing the data entry) will be recorded in an 
electronic audit trail to allow all data changes in the database to be monitored and maintained 
in accordance with federal regulations. 
 


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The SACCC will periodically visit the participating clinical sites and audit the source 
documents in order to validate the data in the SACCC central database. Data will be provided 
using the subject’s screening or enrollment number; the SACCC will not collect personally 
identifying information such as the subject’s name or social security number. Subjects will 
provide demographic information such as race, ethnicity, and birth date. 
Data collected by the SACCC will be held in the strictest confidence, and are protected from 
access that could reveal personally identifying information about any subject in the trial. 
11 ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD 
CLINICAL PRACTICE 
The study will be conducted according to Good Clinical Practice (GCP) guidelines, U.S. 21 
CFR Part 50 – Protection of Human Subjects, and Part 56 – Institutional Review Boards. 
 
Compliance with Good Clinical Practices 
11.1
This trial will be conducted in compliance with the protocol, current GCPs recommended by 
the International Conference on Harmonization (ICH) and the applicable regulatory 
requirements for participating institutions. These include the tenets of the Declaration of 
Helsinki and review and approval by the appropriate ethics review committee or IRBs of 
participating organizations. The SACCC will assure compliance through a program of quality 
assurance audits performed both at participating sites and within the SACCC for data quality 
and adherence to protocol requirements. The SACCC is operated by Rho Federal Systems 
Division, Inc. (RhoFED), Chapel Hill, North Carolina under a contract from DAIT/NIAID. 
 
Institutional Review Board 
11.2
Each participating institution must provide for the review and approval of this protocol and 
associated informed consent documents by an appropriate ethics review committee or IRB. 
Any amendments to the protocol or consent materials must be approved by the IRB and 
submitted to the FDA before they are placed into use. In both the United States and in other 
countries, only institutions holding a current Federal Wide Assurance issued by the Office of 
Human Research Protection (OHRP) at the Department of Health and Human Services 
(DHHS) may participate. 
 
The investigator will inform the IRB of serious or unexpected AEs that might occur during 
the study and are likely to affect the safety of the subjects, or the conduct of the study. The 
investigators will comply fully with all IRB requirements for both the reporting of AEs, 
protocol or consent form changes, as well as any new information pertaining to the use of the 
study medication that might affect the conduct of the study.  
 
Informed Consent 
11.3
The principles of informed consent in the current edition of the Declaration of Helsinki, as 
well as compliance with all IRB requirements, will be implemented in the study, before any 
protocol-specified procedures are carried out. A standard consent form for subject 
participation will be provided with the protocol to each institution. Any modifications to the 


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standard information in the template will require review and approval by the SACCC and 
DAIT/NIAID. Informed consent will be obtained in accordance with 21 CFR 50.52. 
Information may be given to subjects in oral, written or video form by the investigator. All 
prospective subjects will be given ample time to read the consent form, and ask questions, 
before signing.  
 
If subjects are to be enrolled who do not speak and read English, the consent materials must 
be translated into the language appropriate for the enrolling subject. Translated documents 
must be certified to contain the complete descriptions provided in the English version of the 
document. If an interpreter is used to provide or assist in describing the consent materials to 
an enrolling subject, the interpreter must also sign the consent materials certifying their 
involvement with the consent process. 
 
After completion, a copy of the signed consent form will be given to the subject. The original 
signed consent form will be kept on file in the subject’s study chart, available for inspection 
by regulatory authorities, both federal and institutional. 
 
Data and Safety Monitoring Board 
11.4
The responsibility for reviewing the ethical conduct of the study and for monitoring reports 
of evidence of adverse or beneficial effect is assigned to the DAIT Autoimmunity Data and 
Safety Monitoring Board (DSMB). The DSMB is an independent group composed of 
biomedical ethic experts, physicians, and other scientists who are responsible for continuing 
review of study information. The DSMB makes recommendations to DAIT/NIAID on issues 
affecting the course and conduct of this clinical study. 
12 FINANCING AND INSURANCE 
Participating institutions must comply with their institution’s policies on compensation, 
insurance, and indemnity. Institutions must have adequate liability insurance coverage to 
satisfy their local and national requirements for study participation. 
13 PUBLICATION POLICY 
The Autoimmunity Centers of Excellence (ACE) policy on publication of study results will 
apply to this study. Authorized participants may find details regarding the policy statement 
on the ACE internet website at http://www.rhoworld.com. Study investigators are 
encouraged to communicate and publish study results with prior notification of DAIT/ 
NIAID and Genentech, Inc. The following procedure is suggested: 
 
1. Manuscripts, abstracts, posters and other material for public distribution will be 
submitted to DAIT/NIAID, and Genentech, Inc. at least 30 days prior to submission 
for publication or public presentation. 
2. DAIT/NIAID, and Genentech, Inc. will review and comment on the proposed 
material within 30 days. 
3. DAIT/NIAID, and/or Genentech, Inc. may ask that confidential information be 
deleted or redacted in this case where a patent may be filed or where confidential 


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information is involved. Publication or presentation may be delayed up to 60 
additional days in order to file a patent application. 


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14  REFERENCES 
 
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Sakkas, L.I., New developments in the pathogenesis of systemic sclerosis. 
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Allcock, R.J., et al., A study of the prevalence of systemic sclerosis in northeast 
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Koh, E.T., et al., Pulmonary hypertension in systemic sclerosis: an analysis of 17 
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Mukerjee, D., et al., Prevalence and outcome in systemic sclerosis associated 
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Stupi, A.M., et al., Pulmonary hypertension in the CREST syndrome variant of 
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Lefevre, G., et al., Survival and prognostic factors in systemic sclerosis-associated 
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Denton, C.P. and C.M. Black, Pulmonary hypertension in systemic sclerosis. Rheum 
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Kuhn, K.P., et al., Outcome in 91 consecutive patients with pulmonary arterial 
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Cool, C.D., et al., Pathogenesis and evolution of plexiform lesions in pulmonary 
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Sgonc, R., et al., Endothelial cell apoptosis is a primary pathogenetic event 
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Wusirika, R., et al., The assessment of anti-endothelial cell antibodies in 
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and western blot analysis in 49 patients with scleroderma. Am J Clin Pathol, 2003. 
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Vercellotti, G.M., Effects of viral activation of the vessel wall on inflammation and 
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pulmonary arterial hypertension. Am J Respir Crit Care Med, 2008. 177(12): p. 
1364-9. 
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Yap, L.B., et al., The natriuretic peptides and their role in disorders of right heart 
dysfunction and pulmonary hypertension. Clin Biochem, 2004. 37(10): p. 847-56. 
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Roll, P., et al., Regeneration of B cell subsets after transient B cell depletion using 
anti-CD20 antibodies in rheumatoid arthritis. Arthritis Rheum, 2006. 54(8): p. 2377-
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Sidner, R.A., et al., In vivo human B-cell subset recovery after in vivo depletion with 
rituximab, anti-human CD20 monoclonal antibody. Hum Antibodies, 2004. 13(3): p. 
55-62. 
150. 
Steen, V. and T.A. Medsger, Jr., Predictors of isolated pulmonary hypertension in 
patients with systemic sclerosis and limited cutaneous involvement. Arthritis Rheum, 
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151. 
Ulanet, D.B., et al., Autoantibodies against B23, a nucleolar phosphoprotein, occur 
in scleroderma and are associated with pulmonary hypertension. Arthritis Rheum, 
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Humbert, M., et al., Increased interleukin-1 and interleukin-6 serum concentrations 
in severe primary pulmonary hypertension. Am J Respir Crit Care Med, 1995. 
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Calabrese, L.H., et al., Progressive multifocal leukoencephalopathy in rheumatic 
diseases: Evolving clinical and pathologic patterns of disease. Arthritis Rheum, 
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154. 
Calabrese, L.H. and N. Zein, Biologic agents and liver toxicity: an added concern or 
therapeutic opportunity? Nat Clin Pract Rheumatol, 2007. 
155. 
Calabrese, L.H., N.N. Zein, and D. Vassilopoulos, Hepatitis B virus (HBV) 
reactivation with immunosuppressive therapy in rheumatic diseases: assessment and 
preventive strategies. Ann Rheum Dis, 2006. 65(8): p. 983-9. 
156. 
Smolen, J.S., et al., Consensus statement on the use of rituximab in patients with 
rheumatoid arthritis. Ann Rheum Dis, 2007. 66(2): p. 143-50. 
157. 
Fuster, V., et al., Primary pulmonary hypertension: natural history and the 
importance of thrombosis. Circulation, 1984. 70(4): p. 580-7. 
158. 
Rich, S., et al., Primary pulmonary hypertension. A national prospective study. Ann 
Intern Med, 1987. 107(2): p. 216-23. 


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159. 
Hoeper, M.M., et al., Complications of right heart catheterization procedures in 
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160. 
Mathai, S.C., et al., The minimal important difference in the 6-minute walk test for 
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161. 
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Hemnes, A.R. and H.C. Champion, Right heart function and haemodynamics in 
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p. 181-90; viii. 
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Chung, L., Liu, J., Parsons, L., Hassoun, P., McGoon, M., Badesch, D., Nicolls, M., 
Zamanian, R.T., Characterization of connective tissue disease-associated pulmonary 
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Wilkins, M.R., et al., Sildenafil versus Endothelin Receptor Antagonist for Pulmonary 
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Lammers, A.E., A.A. Hislop, and S.G. Haworth, Prognostic value of B-type 
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Reesink, H.J., et al., Brain natriuretic peptide as noninvasive marker of the severity of 
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Ventetuolo, C.E., et al., Surrogate and combined end points in pulmonary arterial 
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Catalano, O., et al., Intra-observer and interobserver reproducibility of right ventricle 
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Linde, C., et al., Randomized trial of cardiac resynchronization in mildly symptomatic 
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Li, A., et al., Acute adverse reactions to magnetic resonance contrast media--
gadolinium chelates. Br J Radiol, 2006. 79(941): p. 368-71. 
 
 


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15 APPENDICES 
 
Appendix A:  Modified Scleroderma Health Assessment Questionnaire (SHAQ)  
 
Appendix B:  Short Form – 36 Health Survey (SF-36) 
 
Appendix C:  New York Heart Association Classification of Heart Disease 
 
Appendix D:  Subject Self-Reported Demographics Source Document 
 
Appendix E:  Exploratory Analysis:  Standardized Fick-based Pulmonary Vascular 
Resistance and Pulmonary Artery Capacitance 
 
Appendix F: RESTORE Sub-study Protocol 
 


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Appendix A:  Modified Scleroderma Health Assessment Questionnaire (SHAQ)  
15.1
 
 


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Appendix B:  Short Form – 36 Health Survey (SF-36) 
15.2
 
 


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Appendix C:  New York Heart Association Classification of Heart Disease 
15.3
 
Functional Capacity  
Objective Assessment  
Class I. Patients with cardiac disease but without resulting limitation of 
physical activity. Ordinary physical activity does not cause undue fatigue, 
palpitation, dyspnea, or anginal pain. 
A. No objective evidence of 
cardiovascular disease. 
Class II. Patients with cardiac disease resulting in slight limitation of 
physical activity. They are comfortable at rest. Ordinary physical activity 
results in fatigue, palpitation, dyspnea, or anginal pain. 
B. Objective evidence of 
minimal cardiovascular 
disease. 
Class III. Patients with cardiac disease resulting in marked limitation of 
physical activity. They are comfortable at rest. Less than ordinary activity 
causes fatigue, palpitation, dyspnea, or anginal pain. 
C. Objective evidence of 
moderately severe 
cardiovascular disease. 
Class IV. Patients with cardiac disease resulting in inability to carry on 
any physical activity without discomfort. Symptoms of heart failure or the 
anginal syndrome may be present even at rest. If any physical activity is 
undertaken, discomfort is increased. 
D. Objective evidence of 
severe cardiovascular 
disease 
 
The Criteria Committee of the New York Heart Association. Nomenclature and Criteria for 
Diagnosis of Diseases of the Heart and Great Vessels. 9th ed. Boston, Mass: Little, Brown & 
Co; 1994:253-256. 
 
 


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Appendix D: Subject Self-Reported Demographics Source Document 
15.4
 
 


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 Appendix E:  Exploratory Analysis:  Standardized Fick-based Pulmonary 
15.5
Vascular Resistance and Pulmonary Artery Capacitance 
Standardized Fick-based pulmonary vascular resistance (PVRf, in Woods Units) and 
pulmonary artery capacitance (PACf, in mL/mmHg) are calculated as follows: 
 
PVR = TPG / CO 
 
where TPG = Transpulmonary Gradient (mmHg) 
 
 
CO = Cardiac Output (L/min) 
 
TPG = mPAP – PCWP 
 
where mPAP = mean pulmonary artery pressure (mmHg) 
 
 
PCWP = mean pulmonary capillary wedge pressure (mmHg) 
 
Thus, the Fick-based calculation of PVR is:  
PVRf = (mPAP – PCWP) / COf 
 
where COf = Fick cardiac output (L/min) 
 
COf = VO2 / ((Ca – Cv) x cHb x 13.6) 
 
where VO2 = oxygen consumption according to LaFarge and Meittinen 
(mL/min)[161] 
 
 
Ca = arterial oxygen saturation (%) (systemic) 
 
 
Cv = venous oxygen saturation (%) (pulmonary artery) 
 
 
cHb = hemoglobin concentration (g/L) 
 
VO2(males) = (138.1 – 11.49 lnAge + 0.378 HR) x BSA 
VO2(females) = (138.1 – 17.04 lnAge + 0.378 HR) x BSA 
 
where lnAge = natural log of age in years 
 
 
HR = heart rate (beats/min) 
 
 
BSA = body surface area (m2) 
 
The DuBois and DuBois formula is used to calculate body surface area. 
BSA (m²) = 0.20247 x Height(m)0.725 x Weight(kg)0.425 
 
Pulmonary artery capacitance is calculated as follows. 
PACf = SVf / PP 
 
where SV = stroke volume (mL) 
 
 
PP = pulse pressure (mmHg) 
 
 
SVf = COf x 1000 / HR  
PP = systolic PAP – diastolic PAP 
 
where PAP is the pulmonary artery pressure 
 


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Appendix F: RESTORE Sub-study Protocol 
15.6
 
 
 
Sub-study Protocol Title:  Right Ventricular Response to Rituximab in 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension – A 
Magnetic Resonance Imaging Sub-study 
 
Short Title: RESTORE Sub-Study 
 
 
 
RESTORE Sub-study Sponsor: 
VERA MOULTON WALL CENTER FOR PULMONARY VASCULAR DISEASE, 
STANFORD UNIVERSITY 
 
 
 
 
 
 
RESTORE Protocol Chair: 
David N. Rosenthal, MD 
Stanford University School of Medicine 
Division of Pediatric Cardiology 
750 Welch Road #305  
Palo Alto, CA 94304-5731  
Phone: 650-723-7913  
Fax: 650-725-8343  
Email: davidnr@stanford.edu 
 
RESTORE Protocol Co-Chairs: 
Roham T. Zamanian, MD, FCCP 
Stanford University School of Medicine 
Vera Moulton Wall Center for Pulmonary 
Vascular Disease 
 
Division of Pulmonary & Critical Care Medicine 
300 Pasteur Dr. Room H3143 
Stanford, Ca 94305 
Phone: 650-725-5495 
Fax: 650-725-5489 
E-mail: zamanian@stanford.edu 
Jeffery A. Feinstein, MD, MPH 
Lucile Packard Children’s Hospital 
Vera Moulton Wall Center for Pulmonary 
Vascular Disease 
Division of Pediatric Cardiology 
750 Welch Rd. #305  
Palo Alto, CA 94304-5731  
Phone:  650-723-7913  
Fax: 650-725-8343  
E-mail: jaf@stanford.edu 


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Sub-study Protocol Synopsis 
Title of Sub-study Protocol:   Right Ventricular Response to Rituximab in Systemic Sclerosis Associated Pulmonary Arterial 
Hypertension – A Magnetic Resonance Imaging Sub-study (RESTORE Sub-Study) 
Investigators:   David N Rosenthal, MD; Roham T Zamanian, MD, FCCP; Jeffrey A Feinstein, MD, MPH 
Objectives:  
The primary objective of the RESTORE sub-study is to evaluate the therapeutic effect of rituximab on the right ventricle (RV) 
in patients with SSc-PAH. Changes in right ventricular end diastolic volume index (RVEDVI) and stroke volume (SV) 
determined by cardiac MRI will be used as surrogates of RV function and prognosis. We hypothesize that RVEDVI and SV 
will reduce 24 weeks following rituximab therapy as compared to the placebo control. 
Criteria for Evaluation: 
All cardiac MRI (CMRI) endpoints will be assessed at baseline and at the Week 24 follow-up (or at time of discontinuation 
from the study) in 20 patients from each arm (80 studies total). The change in RVEDVI, RV mass, and SV will be analyzed 
and compared between placebo and rituximab cohorts. These sub-study endpoints will be used to augment the hemodynamic 
findings of the main study (ASC01). It is likely that comparison between hemodynamic response and surrogates of RV 
function by cardiac MRI will be valuable. Correlation of hemodynamic and CMRI parameters may allow for further 
determination of mechanisms of therapeutic effect of rituximab in this study. 
Study Design: 
Enrollment for the RESTORE sub-study will parallel that of the ASC01 protocol. The sub-study will enroll patients who have 
consented for the primary clinical study across all centers that participate in the RESTORE sub-study. The sub-study is 
designed to compare 20 rituximab with 20 placebo treated patients at baseline and at the Week 24 follow up. All ASC01 study 
inclusion and exclusion criteria apply. Furthermore, there are additional exclusion criteria that pertain only to the RESTORE 
sub-study: 1) known hypersensitivity to Gadolinium, 2) inability to tolerate or cooperate with MRI, 3) morbid obesity, and 4) 
presence of metallic objects or pacemakers.  
 
Efficacy Endpoints: 
The primary efficacy endpoint will be change in RVEDVI measured by cardiac MRI from baseline to 24 weeks after 
treatment initiation. 
The secondary efficacy endpoints will be as follows: 
1. Change in RV mass measured by cardiac MRI from baseline to 24 weeks after treatment initiation. 
2. Change in SV measured by cardiac MRI from baseline to 24 weeks after treatment initiation. 
Images and Analyses: 
Cardiac MR imaging will be performed on any 1.5 T scanner (standard clinical equipment will meet these specifications) with 
simultaneous ECG recording. Image acquisition is based on routine MR protocols for traditional volumetric parameters 
supplemented with a delayed enhancement perfusion protocol (gadolinium). The detailed MRI protocol is specified in the 
Manual of Procedures (MOP), but will include short axis and four chamber views of the heart, and MRI angiography in the 
pulmonary artery and aorta. This will be followed by contrast enhanced imaging using gadolinium to determine myocardial 
perfusion in a four chamber view, and to evaluate for late enhancement using four chamber and short axis views.  
Image analysis will be carried out off-line by investigators at Stanford University who are blinded to treatment arm, using 
validated MRI software tools such as QMass/QFlow (Medis Medical Imaging Systems, Raleigh, NC).  
Sample Size and Statistical Analyses:  
This sub-study will include 20 subjects treated with rituximab and 20 subjects treated with placebo. 
The primary efficacy analysis of change in RVEDVI from baseline to 24 weeks will be performed on the intention-to-treat 
(ITT) population. Analysis of covariance (ANCOVA) will be used to model baseline to Week 24 change in RVEDVI as the 
primary outcome and treatment as the primary predictor, controlling for center and baseline value of RVEDVI. 
Secondary analyses will include repeating the primary analysis for the following outcomes: change in RV mass from baseline 


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to 24 weeks and change in SV from baseline to 24 weeks. 


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1     Hypothesis 
Primary Hypothesis: Right ventricular end-diastolic volume index (RVEDVI), as 
determined by cardiac MRI, will reduce 24 weeks following rituximab therapy as compared 
to placebo control, in subjects with SSc-PAH participating in ACE Protocol ASC01. 
 
Secondary Hypotheses:   
 Right ventricular mass, as determined by cardiac MRI, will reduce 24 weeks 
following rituximab therapy as compared to placebo control, in subjects with SSc-
PAH participating in ACE Protocol ASC01. 
 Stroke volume, as determined by cardiac MRI, will reduce 24 weeks following 
rituximab therapy as compared to placebo control, in subjects with SSc-PAH 
participating in ACE Protocol ASC01. 
 
Exploratory Hypotheses: 
 Cardiac output, as determined by cardiac MRI, will agree with the main study 
assessment of cardiac output as well as the standardized Fick-based calculation of 
cardiac output 
 Right ventricular end diastolic volume, as determined by cardiac MRI, will correlate 
with the standardized Fick-based calculation of pulmonary artery capacitance 
2     Background and Rationale 
2.1     Background 
Pulmonary arterial hypertension (PAH) is a debilitating, progressive, and fatal complication 
of systemic sclerosis (SSc). Epidemiologic data show that patients with SSc-associated PAH 
(SSc-PAH) have a poor prognosis. While hemodynamic parameters such as right atrial 
pressure and cardiac index are predictors of survival in WHO group I PAH, they are 
relatively crude and indirect markers of RV performance in PAH [162, 163]. Interestingly, 
we have recently demonstrated that in comparison to other diseases with associated PAH, 
SSc-PAH is characterized by less severe hemodynamic perturbation, but also a more robust 
neurohormonal activation and worse 1-year survival [164]. These findings suggest that the 
right ventricular response to PAH in systemic sclerosis may differ from that seen in other 
etiologies of PAH and potentially plays an important role in shaping the natural history of 
this form of PAH.  
 
Recent studies have demonstrated the utility of MRI based protocols for evaluation of right 
ventricular performance in PAH [165, 166]. Protocols which evaluate RV structure and 
function supplement the routine hemodynamic measures obtained during cardiac 
catheterization and add additional dimensions to the evaluation of disease and response to 
therapies. On the one hand, the measures from MRI are more directly related to the 
pathophysiology of right ventricular injury than are the less direct (but widely employed) 
measures of right atrial pressure and cardiac index, or the even further removed index of 6-
minute walk distance (6MWD). Thus, these MRI measures, while perhaps less directly 
clinically applicable than 6MWD, do offer the potential for a more precise delineation of the 
cardiac impact of a given therapy such as rituximab. These data will be complementary to 


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pulmonary vascular resistance (PVR), which is a more pure vascular measure. Additionally, 
the reproducibility of the MRI measures of RV function is very high, so relatively small 
changes can be detected and thus MRI offers the hope of detecting early cardiac remodeling 
changes even if a full clinical impact is not yet demonstrable.  
2.2     Rationale 
PAH is a cardiopulmonary disease manifested by increased PVR due to obliterative 
arteriopathy. The increase in PVR creates increased pressure work for the right ventricle. 
Clinical evidence suggests that the capacity of the right ventricle to respond to this increase 
in afterload by remodeling may be a critical determinant of disease progression and overall 
prognosis [167-169]. Although right heart catheterization (RHC) is the gold standard for 
diagnosis of PAH, the hemodynamic measures which are obtained during RHC do not appear 
to provide comprehensive evaluation of RV function nor do they offer accurate prognosis for 
all forms of PAH [170]. For example, patients with PAH resulting from congenital heart 
diseases are typically characterized by very poor hemodynamics, yet these patients have 
among the best survival of any PAH cohort. One potential explanation for this is the long-
standing and early onset of adverse hemodynamics in such patients, which may allow for 
more effective right ventricular remodeling. 
 
Specifically relevant to the ASC01 study is the role of RV performance in SSc-PAH. Using a 
large multicenter PAH registry, we have recently demonstrated that SSc-PAH (Chung, 
Zamanian unpublished data) patients had the most favorable hemodynamics as demonstrated 
by lower mean pulmonary artery pressure, higher cardiac index and lower PVR when 
compared to idiopathic PAH and other autoimmune-associated PAH. Despite this apparently 
favorable hemodynamic profile, the 1 year survival for SSc-PAH was worse when compared 
with the other cohorts. This again supports the idea that it is not hemodynamic profile alone 
that determines prognosis, but rather the cardiac response to these hemodynamics in the form 
of RV remodeling. 
 
Cardiac MRI allows for a comprehensive non-invasive evaluation of RV chamber size, 
myocardial mass and systolic function, providing information which is complementary to 
that obtained by cardiac catheterization. Evaluation of baseline RV status using cardiac MRI 
in SSc-PAH and the response to rituximab will help determine the role which RV remodeling 
plays in shaping the overall patient outcome in PAH, and may also demonstrate a mechanism 
by which rituximab affects disease outcome, if such effect is found. 
 
The RESTORE sub-study will further help delineate the utility of cardiac MRI in studying 
treatment response in PAH. While there have been some data evaluating this, there is not yet 
adequate data to specify which markers of RV remodeling will be most useful in evaluating 
the evolution of patients with PAH. The RESTORE sub-study, by providing MRI data in a 
well-characterized set of patients, will help better define this question as well. 
2.3     Significance 
 
This sub-study is likely to enhance the capacity of the ASC01 study in evaluation of response 
of SSc-PAH to rituximab therapy by evaluating a set of outcomes which are complementary 


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to secondary study aims, but which are more focused on right ventricular performance. This 
will assist in evaluating the factors contributing to the clinical outcomes observed in the main 
study by providing data which help clarify the cardiac responses during therapy. These data 
will help clarify the contribution to outcome that arises from the vascular outcome of PVR. 
3     Study Overview 
3.1     Overall Study Design 
The RESTORE sub-study will include all sites participating in the primary ASC01 study that 
have the capability to perform the required MRI studies. Enrollment within the RESTORE 
sub-study will consist of consecutive ASC01 study subjects at each site, provided that the 
subject provides consent for this separate sub-study. This should diminish any unintentional 
bias in patient selection at each site. Thus, both placebo and rituximab arms will be included 
in the RESTORE sub-study. Twenty subjects from each arm will be recruited into this sub-
study. Each subject will be studied at baseline (i.e. prior to initiation of study drug) and at 
Week 24, or at time of discontinuation. Subjects in RESTORE will undergo comprehensive 
cardiac MRI evaluation. Given that the primary comparison is a pre/post match, and that the 
MRI interpretation is performed by individuals blinded to the treatment assignment, it is not 
likely to introduce bias if one or more sites cannot participate in the RESTORE sub-study. 
However, should this be the case, the overall study population will be compared to the sub-
population captured at these sites with respect to baseline characteristics. Since the 
RESTORE sub-study will use the same entry criteria as the parent protocol, there should not 
be a problem with differential patient selection, with the exception of the additional exclusion 
criteria specified below that relate to the contra-indications to MRI. While it does not appear 
likely that these criteria will bias the sub-study population, the data analysis will again 
compare the baseline characteristics of study subjects for the parent protocol to that of the 
RESTORE sub-study. 
3.2     Study Population 
3.2.1     Inclusion Criteria  
Each participant included in this sub-study must be a participant in and meet the inclusion 
criteria for the ASC01 clinical protocol.  In addition, written informed consent for the 
RESTORE sub-study must be provided. 
3.2.2     Exclusion Criteria   
Each participant included in this sub-study must not meet any of the exclusion criteria for the 
ASC01 clinical protocol. In addition, subjects who meet any of the following are disqualified 
from enrollment in the sub-study:  
 Known hypersensitivity to Gadolinium 
 Inability to tolerate or cooperate with MRI (small spaces/claustrophobia) 
 Morbid obesity 
 Presence of metallic objects or pacemaker 


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3.3     Recruitment 
Recruitment for this sub-study will be limited to subjects who have consented and elected to 
participate in the ASC01 clinical protocol. Replacement of subjects in this sub-study will 
occur in the same manner as the parent protocol. Randomized subjects who withdraw from 
the trial prior to starting rituximab infusion will be replaced. Subjects who discontinue study 
treatment or withdraw from the trial after initiating the first treatment may not be replaced.     
4     Study Design  
4.1     Description of Study Design 
Twenty subjects from each treatment arm, distributed among all participating sites, will be 
recruited for this sub-study. Each subject will be studied at baseline (i.e. prior to initiation of 
study drug) and after 24 weeks, or at time of discontinuation. In addition to the data 
collection and testing specified in the ASC01 protocol, subjects in RESTORE will undergo 
comprehensive cardiac MRI evaluation as described in the RESTORE Manual of Procedures 
(MOP). In brief, this will include short axis, body axial, four-chamber and three-chamber 
cardiac-gated MRI sequences; pulmonary and aortic angiography in the proximal main 
pulmonary artery and each of the proximal branch pulmonary arteries; and the ascending 
aorta; measurement of myocardial perfusion using gadolinium enhanced cardiac MRI in the 
same views specified above, and measurement of delayed gadolinium enhancement in those 
views. 
4.2     Endpoints 
The primary efficacy endpoint will be change in RVEDVI measured by cardiac MRI from 
baseline to 24 weeks after treatment initiation. 
 
The secondary efficacy endpoints will be as follows: 
 Change in RV mass measured by cardiac MRI from baseline to 24 weeks after 
treatment initiation. 
 Change in SV measured by cardiac MRI from baseline to 24 weeks after treatment 
initiation. 
 
An exploratory endpoint is also defined: 
 Cardiac output measured by cardiac MRI 
5     Study Objectives 
The primary objective of the RESTORE sub-study is to evaluate the therapeutic effect of 
rituximab on the right ventricle (RV) in patients with SSc-PAH. To achieve this objective, 
we have developed the following hypotheses: 
 
Primary Hypothesis: Right ventricular end-diastolic volume index, as determined by 
cardiac MRI, will reduce 24 weeks following rituximab therapy as compared to placebo 
control, in subjects with SSc-PAH participating in ACE Protocol ASC01. 
 


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Secondary Hypotheses:   
 Right ventricular mass, as determined by cardiac MRI, will reduce 24 weeks 
following rituximab therapy as compared to placebo control, in subjects with SSc-
PAH participating in ACE Protocol ASC01. 
 Stroke volume, as determined by cardiac MRI, will reduce 24 weeks following 
rituximab therapy as compared to placebo control, in subjects with SSc-PAH 
participating in ACE Protocol ASC01. 
 
Exploratory Hypotheses: 
 Cardiac output, as determined by cardiac MRI, will agree with the main study 
assessment of cardiac output as well as the standardized Fick-based calculation of 
cardiac output 
 Right ventricular end diastolic volume, as determined by cardiac MRI, will correlate 
with the standardized Fick-based calculation of pulmonary artery capacitance 
6     Treatment of Subjects and Study Measurements 
6.1     Subject Clinical Considerations 
Consideration must be given to the need for rate adjustment of intravenous Flolan® during 
the MRI procedures for those subjects requiring this concomitant background SSc-PAH 
therapy. Given the requirement for IV extension tubing from the subject to infusion pump 
while in the MRI scanner, Flolan® flow rate adjustment (increase) will be required to 
compensate for the longer tubing length to allow for consistent dosing.  
 
Further guidance on this issue may be found in the RESTORE MOP. 
6.2     Imaging Process 
Cardiac MR imaging will be performed using any 1.5 T scanner with simultaneous 
ECG recording, according to a protocol for correction of phase offset errors. All images will 
be obtained using breath-hold in end-expiration. Acquisition will be performed including the 
following sequences: 
 Short-axis cine images of the heart from apex to base, covering the whole LV 
and RV.  
 Body axial cine images of the heart, extending from the surface of the diaphragm to 
the superior margin of the pulmonary valve. 
 Long axis cine images of the heart, extending from the surface of the diaphragm to 
the superior margin of the pulmonary valve. 
 Angiography (flow imaging) will be obtained in the aorta from an imaging plane 
located perpendicular to the long axis of the ascending aorta, with the plane located 
just distal to the sino-tubular ridge, and distal also to the origin of the coronary 
arteries. 
 Angiography (flow imaging) will be obtained in the main pulmonary artery from an 
imaging plane located perpendicular to the long axis of the main pulmonary artery, 
with the plane located midway between the pulmonary valve and the bifurcation of 
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 Angiography (flow imaging) will be obtained in each of the branch pulmonary 
arteries from an imaging plane perpendicular to the long axis of the applicable artery, 
with the plane located midway between the origin of the artery and the origin of the 
first branch from that artery. 
 Myocardial perfusion imaging during gadolinium infusion will be obtained using 
short axis views of the heart, as per the baseline cine imaging. 
 Delayed imaging after injection of gadolinium will be performed using both short 
axis and axial views of the heart. 
 
Full details of the procedures can be found in the RESTORE MOP. 
6.3     Imaging Analyses 
All cardiac MRI images obtained during the RESTORE sub-study will be provided to the 
Stanford laboratory for interpretation as described in additional detail in the following 
section. Images will be shipped on digital media (CD/DVD).  
 
All cardiac MRI studies will be analyzed in the same fashion, using commercial software. 
MRI studies will be analyzed at one time after the conclusion of the study (after last patient 
last visit). 
 
Studies will be analyzed by one interpreter, either one of the sub-study investigators or 
personnel trained directly by these investigators. A test set of images will be completed in 
duplicate and intra-observer variability will be calculated. If this variability is not acceptable 
(i.e. coefficient of variation < 15%) [171], the interpreter will review interpretation criteria 
and then repeat the intra-observer variability using the test set (these images will be obtained 
using the sub-study protocol, but may not include sub-study subjects). The interpreter of 
images will not have access to treatment assignment nor to the sequence of the studies, but 
the 2 studies for each subject will be reviewed as a pair. 
 
For interpretation of the MRI, diastole and systole will be assigned manually using the 
largest and smallest volume of both the RV and LV (this may result in separate assignment of 
diastole and systole for the 2 ventricles). The diastolic endocardial and epicardial contours of 
both the RV and LV will be traced manually by one of the study interpreters, and will be 
processed using software to obtain RV and LV mass, and end-diastolic volume. The chamber 
volumes will also be manually traced at end-systole, yielding end-systolic volumes. In the 
LV, papillary muscles will be traced separately, and thus incorporated into the mass rather 
than volume calculation. Due to difficulties in obtaining consistent tracings with this method 
in the RV, the RV tracings will exclude papillary muscles. LV and RV volumes/mass will be 
determined using short axis images. Ejection fraction will be calculated by determining the 
difference between diastolic and systolic volume for each chamber, and dividing by the 
diastolic volume of the corresponding chamber. For measurement of flow, the angiography 
(phase contrast) images will be used, with flow measured in the ascending aorta and main 
pulmonary artery. For all parameters, the values will be normalized by body surface area (i.e. 
expressed as indexed values). 
 


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For each study, a data collection form will be completed and signed by the study interpreter 
at the time of the interpretation. This form will be retained at Stanford, with the information 
entered in an Excel spreadsheet and transferred to the SACCC electronically. 
7     Data Management 
7.1     Tracking Images and Quality Control Procedures  
Clinical sites are responsible for collecting, labeling, tracking, and shipping the images to 
Stanford University as specified in the RESTORE MOP. The exception is the first cardiac 
MRI study conducted at each site for this sub-study, which will be sent to the SACCC to 
forward to Stanford without awaiting protocol completion, to allow a blinded quality control 
review. After an image is collected for a subject, the clinical site is responsible for entering 
the subject ID and accession numbers into the web-based tracking system (RhoLAB) 
operated by the SACCC. The RhoLAB tracking system will notify the Stanford laboratory 
that a CD/DVD is being shipped. The information supplied to Stanford by the SACCC will 
contain accession numbers, but not subject ID numbers. The only links between subject ID 
numbers and accession numbers will be at the clinical site and at the SACCC. These links 
will allow cardiac MRI results to be linked to clinical outcomes through the SACCC. As 
CDs/DVDs are received at Stanford, the receiving facility must log into the RhoLAB 
tracking system and verify that the CDs/DVDs were received and document the condition of 
the CDs/DVDs at the time of receipt. After analyses are completed at Stanford, the analysis 
results will be supplied by Stanford to the SACCC electronically using accession numbers as 
unique identifiers.  
 
Upon arrival at Stanford the images will also be logged and stored in a dedicated, locked file. 
The actual CD/DVD will be retained at Stanford in the event that re-interpretation is 
required. In addition, this image may be required for quality control purposes. 
 
The intra-observer variability will be determined at the onset of the sub-study interpretation. 
This will be accomplished by having the reader interpret a set of 5 studies in duplicate, 
allowing two weeks between the initial and repeat interpretation. For this purpose, the 2 
interpretations will be sent to the SACCC for measurement of the coefficient of variation 
between the duplicate readings. If this does not meet the standard specified in Section 6.3, 
Imaging Analysis, then the individual will review interpretation criteria and repeat the quality 
control process.  
7.2     Database Management  
All data will be maintained in hard copy form at Stanford and transmitted to the SACCC 
electronically. Upon receipt of such data, the SACCC will store it in a central database on the 
SACCC network that provides protection of subject confidentiality and allows the cardiac 
MRI study data to be merged with the clinical data from the ASC01 study.  
7.3     Retention of Documents 
The Stanford database ensures integrity of the data through multiple levels of password 
protection and routine backups of all data. All data and subject material will be password 


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protected on the local database, which will only be accessible to the Protocol Chair and Co-
Chairs. All data will be subsequently stored on the SACCC network, which will allow the 
RESTORE sub-study data to be merged with the clinical data from the ASC01 study. 
 
All study-related documents will be maintained in the core facility for 6 years, or longer if 
required by the FDA. 
8     Statistical Considerations and Analytic Plan 
8.1     Subject Selection or Allocation 
The RESTORE sub-study will include all sites participating in the primary ASC01 study that 
have the capability to perform the required MRI studies. Enrollment within the RESTORE 
sub-study will consist of consecutive patients at each site, provided that the patient provides 
consent for this separate sub-study. With regard to patient treatment assignment, all data will 
be analyzed on the basis of intention-to-treat, in the identical manner as the parent protocol. 
Any patient excluded from analysis in the parent protocol will also be excluded from analysis 
in the RESTORE sub-study. 
8.2     Sample Size 
The sample size for this sub-study is determined in large part by the size of the parent 
protocol and budget considerations. With these factors in mind, a sample size of 20 subjects 
in each arm (40 total subjects) is feasible.  
 
Data from van Wolferen indicate that in subjects with pulmonary hypertension, RVEDVI 
determined by cardiac MRI was 85 +/- 25 ml/m2. Assuming that the standard deviation does 
not change during the sub-study, and that there is a 10% (8.5 ml/m2) mean reduction in right 
ventricular volume index over the placebo group, one obtains the following relationship 
between the percent reduction and the expected power:  
 


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 A mean reduction of 10% would power this sub-study at 27.8%. A recent study of 
resynchronization therapy for left ventricular failure demonstrated a reduction in left 
ventricular systolic volume index of approximately 15% of the baseline value; if this level is 
found, the power would increase to 47.6% [172]. 
 
Since the main purpose of the RESTORE sub-study is to get a better understanding of how 
rituximab affects RVEDVI for subsequent research studies, this level of power will suffice. 
8.3     Statistical Analysis of Study Objectives 
The primary aim of this sub-study is a comparison of the change in RVEDVI between the 
rituximab and the placebo arm of the parent trial, in subjects who have cardiac MRI at 
baseline and at Week 24, or at time of discontinuation. This comparison will be made using 
an ANCOVA model with change in RVEDVI as the response and treatment, center, and 
baseline value as covariates. It is not anticipated that non-parametric tests will be required, 
but the data distribution will be evaluated and suitable data transformation will be performed 
as needed. However, based upon experience in other studies using similar outcomes, such is 
not likely to be required [165, 166]. A p-value of 0.05 will be taken as the threshold for 
significance. For the secondary outcomes of RV mass and stroke volume, a similar analysis 
will be performed. 
9     Safety Monitoring and Reporting 
Power for Various Percent Changes from Baseline in RVEDVI when Sample Size = 40
Assuming Mean of one Group = 85 and 1-sided test at alpha=0.05
Power
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
Percent Difference in Change from Baseline
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24


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Monitoring and reporting of any adverse events associated with MRI procedures for this sub-
study will be the responsibility of the clinical sites and will be reported to the IND holder 
using the standard mechanisms implemented as described in Section 7, Safety Monitoring 
and Reporting, of the ASC01 clinical protocol which is incorporated by reference for this 
sub-study. 
9.1     Risk Analysis 
This is an observational protocol that presents the incremental risk associated with 
performing 2 cardiac MRI studies in the study population. This is substantially the same as 
the risk of the administration of gadolinium, as the other MRI risks are minor (although 
claustrophobia is not uncommon, and may require termination of the protocol). For subjects 
with normal renal function, adverse effects from gadolinium are reported in 0.48% of 
subjects [173]. Most reactions were minor; there was one case of anaphylactic shock in 9528 
doses of gadolinium. In patients with pre-existing renal failure, there is a risk of provoking 
renal toxicity. However, this risk is low at the doses of gadolinium proposed in this protocol 
and is extremely low in the patient population specified in the parent protocol, which 
excludes subjects with significant renal pathology. Of concern regarding use of gadolinium 
are reports of the development of nephrogenic systemic fibrosis in subjects with severe renal 
failure who receive gadolinium. However, this risk is again low or absent in the subjects 
targeted by the parent protocol, as the risk of nephrogenic systemic fibrosis emerges only 
when the creatinine clearance is in the 15-29 mL/min range, or in patients who are dependent 
upon dialysis. 
10     Access to Source Data and Documents 
The requirements outlined in Section 9, Access to Source Data and Documents, of the 
ASC01 clinical protocol are incorporated by reference for this sub-study.   
11     Quality Assurance 
The requirements outlined in Section 10, Data Collection, Quality Control and Quality 
Assurance, of the ASC01 clinical protocol are incorporated by reference for this sub-study. 
12     Other Administrative Procedures 
The administrative procedures outlined in Section 11, Ethical Considerations and 
Compliance with Good Clinical Practice, of the ASC01 clinical protocol are incorporated by 
reference for this sub-study.   
 

SAP_000.pdf: 
DAIT/Rho STATISTICAL ANALYSIS PLAN 
 08 March 2019 FINAL 
 
 
Protocol # ASC01 
A Randomized, Double-blind, Placebo-Controlled, Phase II Multicenter 
Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment 
of Systemic Sclerosis-Associated Pulmonary Arterial Hypertension 
(SSc-PAH) 
Short Title:  Rituximab for Treatment of SSc-PAH 
 
 
VERSION: 
1.0 
DATE: 
08 March 2019 
SPONSOR: 
Division of Allergy, Immunology, and Transplantation 
National Institute of Allergy and Infectious Diseases – NIH 
5601 Fishers Lane 
Rockville, MD 20852 
Telephone:  (301) 496-5717 
Fax:  (301) 402-3573 
 
 
PREPARED BY: 
Rho 
Rho Building 
6330 Quadrangle Drive 
Chapel Hill, NC 27517 
Telephone:  (919) 408-8000 
Fax:  (919) 408-0999 
 
 
 


Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
DAIT/Rho STATISTICAL ANALYSIS PLAN 
ACKNOWLEDGMENT AND SIGNATURE SHEET 
 
 A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for 
the Treatment of Systemic Sclerosis-Associated Pulmonary Arterial 
Hypertension (SSc-PAH) 
 
Approved: 
__________________________________ Date: 
_______________ 
 
Mark Nicolls, MD 
 
 
 
Protocol Chair 
 
 
 
Approved: 
__________________________________ Date: 
_______________ 
 
David Badesch, MD 
 
 
 
Protocol Chair 
 
 
 
Approved: 
__________________________________ Date: 
_______________ 
 
Ellen Goldmuntz, MD, PhD 
 
 
 
NIAID Medical Monitor 
 
 
 
Approved: 
__________________________________ Date: 
_______________ 
 
Lynette Keyes-Elstein, DrPH 
 
 
 
Rho Scientist 
 
 
 
Approved: 
__________________________________ Date: 
_______________ 
 
Ashley Pinckney, MS 
 
 
 
Rho Statistician 
 
 
 
 
 
 
Version: 1.0 
Page  2 of 36 
 


Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
Document History 
 
Version 
 
Date 
 
Change(s) 
 
Author 
0.1 
16Jan2019 
Initial Draft 
Ashley Pinckney 
0.2 
01Mar2019 
Second Draft 
Ashley Pinckney 
1.0 
08Mar2019 
Final Draft 
Ashley Pinckney 
 
Version: 1.0 
Page  3 of 36 
 


Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
Table of Contents 
 
1. 
PROTOCOL SYNOPSIS ............................................................................................... 6 
2. 
INTRODUCTION ......................................................................................................... 12 
3. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS ..................................... 13 
4. 
ANALYSIS SAMPLES ................................................................................................ 14 
4.1. 
Safety Population ............................................................................................. 14 
4.2. 
Modified Intent-to-Treat Population ................................................................. 14 
4.3. 
Per Protocol Populations ................................................................................. 14 
5. 
STUDY SUBJECTS .................................................................................................... 15 
5.1. 
Disposition of Subjects .................................................................................... 15 
5.2. 
Demographic and Other Baseline Characteristics ........................................... 15 
6. 
STUDY OPERATIONS ................................................................................................ 16 
6.1. 
Protocol Deviations .......................................................................................... 16 
6.2. 
Treatment Adherence ...................................................................................... 16 
7. 
ENDPOINT EVALUATION .......................................................................................... 17 
7.1. 
Overview of Efficacy Analysis Methods ........................................................... 17 
7.1.1. Multicenter Studies ...................................................................................... 17 
7.1.2. Assessment Time Windows ........................................................................ 17 
7.2. 
Primary Endpoint ............................................................................................. 17 
7.2.1. Computation of the Primary Endpoint ......................................................... 17 
7.2.2. Primary Analysis of the Primary Endpoint ................................................... 18 
7.2.3. Sensitivity Analyses of the Primary Endpoint .............................................. 18 
7.3. 
Secondary Endpoints ....................................................................................... 19 
7.3.1. Pulmonary Vascular Resistance ................................................................. 19 
7.3.2. Six Minute Walk Distance ........................................................................... 20 
7.3.3. Clinical Worsening ...................................................................................... 20 
7.3.4. Addition of PAH medications ....................................................................... 21 
7.3.5. Quality of Life: SF-36 .................................................................................. 22 
7.3.6. Quality of Life: HAQ-DI ................................................................................ 23 
7.3.7. Digital Ulcers ............................................................................................... 23 
7.3.8. Severity of Raynaud’s Phenomenon ........................................................... 24 
7.3.9. DLCO .......................................................................................................... 24 
7.3.10. Oxygen Saturation ...................................................................................... 24 
7.4. 
Examination of Subgroups ............................................................................... 25 
8. 
SAFETY EVALUATION .............................................................................................. 26 
8.1. 
Overview of Safety Analysis Methods ............................................................. 26 
8.2. 
Adverse Events ................................................................................................ 26 
8.3. 
Deaths and Serious Adverse Events ............................................................... 27 
8.3.1. Serious Adverse Events .............................................................................. 27 
8.3.2. Deaths ......................................................................................................... 27 
8.4. 
Clinical Laboratory Evaluation ......................................................................... 28 
8.5. 
Vital Signs, Physical Findings, and Other Observations Related to Safety ..... 29 
8.5.1. Vital Signs ................................................................................................... 29 
8.5.2. Physical Examinations ................................................................................ 29 
 
Version: 1.0 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
9. 
OTHER ANALYSES ................................................................................................... 30 
9.1. 
Use of Medications .......................................................................................... 30 
9.1.1. Prior and Concomitant Medications ............................................................ 30 
9.1.2. Background PAH Medications .................................................................... 30 
10. 
INTERIM ANALYSES AND DATA MONITORING ..................................................... 30 
11. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL ........................... 31 
12. 
REFERENCES ............................................................................................................ 31 
13. 
APPENDICES ............................................................................................................. 32 
13.1. 
Study Flow Chart ............................................................................................. 32 
13.2. 
Schedule of Events .......................................................................................... 33 
14. 
ATTACHMENTS ......................................................................................................... 36 
14.1. 
Scoring of the SF-36 ........................................................................................ 36 
 
1. 
PROTOCOL SYNOPSIS ............................................................................................... 6 
2. 
INTRODUCTION ......................................................................................................... 12 
3. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS ..................................... 13 
4. 
ANALYSIS SAMPLES ................................................................................................ 14 
5. 
STUDY SUBJECTS .................................................................................................... 15 
6. 
STUDY OPERATIONS ................................................................................................ 16 
7. 
ENDPOINT EVALUATION .......................................................................................... 17 
8. 
SAFETY EVALUATION .............................................................................................. 26 
9. 
OTHER ANALYSES ................................................................................................... 30 
10. 
INTERIM ANALYSES AND DATA MONITORING ..................................................... 30 
11. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL ........................... 31 
12. 
REFERENCES ............................................................................................................ 31 
13. 
APPENDICES ............................................................................................................. 32 
14. 
ATTACHMENTS ......................................................................................................... 36 
 
 
 
Version: 1.0 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
1. 
PROTOCOL SYNOPSIS 
Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment of 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension (SSc-PAH) 
ACE Protocol Number:  ASC01  
Protocol Chair(s):  Mark Nicolls, MD; David Badesch, MD 
IND Holder: DAIT/NIAID/NIH 
Primary Objective: The primary objective of this study is to compare patients with SSc-
PAH treated with rituximab to those on placebo for change in exercise capacity, as 
determined by six minute walk distance (6MWD). 
Secondary Efficacy Objectives:  
• 
To compare patients treated with rituximab to those on placebo for change in 
exercise capacity, as determined by 6MWD, at time points other than 24 weeks. 
• 
To compare patients treated with rituximab to those on placebo for change in PVR 
as assessed by right heart catheterization. 
• 
To compare patients treated with rituximab to those on placebo for time to clinical 
worsening, defined as first occurrence of: 
a. death,  
b. hospitalization for SSc-PAH, 
c. lung transplantation,  
d. atrial septostomy,  
e. addition of other PAH therapeutic agents, or   
f. 
worsening of 6MWD by > 20% and a decrease in functional class. 
• 
To compare patients treated with rituximab to those on placebo who changed or 
added other PAH therapeutic agents. 
• 
To compare patients treated with rituximab to those on placebo for change in quality 
of life as determined by the short form 36 (SF-36) and the disability index of the 
Scleroderma Health Assessment Questionnaire (SHAQ). 
• 
To compare patients treated with rituximab to those on placebo for change in 
number of new digital ulcers. 
• 
To compare patients treated with rituximab to those on placebo for change in 
severity of Raynaud phenomenon as measured by the VAS scale of the SHAQ. 
• 
To compare patients treated with rituximab to those on placebo for change in DLCO 
and room air oxygen saturation at rest.  
 
A secondary objective of this study is to determine whether the effects on clinical disease 
progression are paralleled by changes in selected biomarkers.   
Secondary Safety Objectives: 
• To assess the safety and tolerability of rituximab for the treatment of SSc-PAH in 
patients receiving stable background medical treatment with prostanoid, endothelin 
receptor antagonist, PDE-5 inhibitor therapy, and/or guanylate cyclase stimulators. 
 
 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment of 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension (SSc-PAH) 
Mechanistic Study Objectives: 
• 
CD19 Studies 
 
To determine whether the extent and/or duration of CD19+ B cell depletion 
correlates with treatment response as measured by exercise capacity (6MWD),  
PVR (right heart catheterization), and quality of life (SF-36 and SHAQ). 
 
To monitor and correlate the reconstitution patterns of B cell subpopulations with 
treatment response 
• 
To determine if the biomarkers anti-U1 RNP, anti-U3 RNP, anti-B23, anti-
cardiolipin, anti-Th/To, anti-CENP-B, RF, and other autoantibodies, and quantitative 
immunoglobulin levels, including IgG subclasses, correlate with treatment response 
as measured by PVR (right heart catheterization), exercise capacity (6MWD), and 
quality of life (SF-36 and SHAQ). 
• 
To determine if the biomarkers BNP/NT-proBNP, serum Na+, IL-1, IL-6, and other 
cytokines correlate with treatment response as measured by PVR (right heart 
catheterization), exercise capacity (6MWD), and quality of life (SF-36 and SHAQ).   
• 
To determine if B cell clones are present in SSc-PAH and what impact rituximab 
may have on these clones. 
• 
To determine if B cell clones present in SSc-PAH are correlated with clinical 
outcomes. 
 
Exploratory Objectives: 
• 
To evaluate agreement between PVR calculated by site-based standards and a 
standardized Fick-based calculation of PVR. 
• 
To assess sensitivity of the secondary efficacy analysis to standardization of the 
PVR calculation. 
• 
To assess the utility of a standardized derived pulmonary artery capacitance as a 
primary efficacy endpoint. 
• 
To evaluate the relationship between 6MWD, PVR, and other clinical measures. 
Study Arms:   
Subjects will be randomized in a 1:1 ratio to receive either: 
 
2 infusions of rituximab, 1000 mg each, given 2 weeks apart, or 
 
2 infusions of placebo given 2 weeks apart 
Study Design:  This is a prospective, double-blind, placebo-controlled, multi-center, 
randomized trial to evaluate the effect of rituximab on disease progression in subjects with 
SSc-PAH receiving concurrent stable-dose standard medical therapy with a prostanoid, 
endothelin receptor antagonist, phosphodiesterase 5 (PDE-5) inhibitor, and/or guanylate 
cyclase stimulators. The study will focus on assessment of clinical response and safety 
measures longitudinally. In addition, the effects of treatment with rituximab on the 
underlying immune mechanisms associated with B-cell dysregulation and pathogenic 
autoantibody response in this disease will be investigated.   
 
Subjects with SSc-PAH with elevated mean pulmonary artery pressure as measured by 
right heart catheterization will be enrolled. The diagnosis of SSc-PAH should be confirmed  
 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment of 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension (SSc-PAH) 
by a rheumatologist experienced in the diagnosis and treatment of systemic sclerosis in 
conjunction with a pulmonologist or cardiologist specializing in management of PAH. Both 
specialists will be part of the study team at each site.  
 
Rituximab/placebo will be administered as two IV infusions of 1000 mg each, given two 
weeks apart at Day 0 and Week 2. All subjects will receive 40mg of prednisone orally the 
night before and morning of each infusion, as well as 100 mg methylprednisolone or 
equivalent corticosteroid intravenously thirty minutes prior to each infusion, with 
diphenhydramine and acetaminophen orally thirty to sixty minutes prior to each infusion of 
rituximab or placebo. Subjects will remain on their baseline standard medical regimen.  
 
Sixty eligible subjects will be accrued. Each potential study subject will provide written 
informed consent prior to screening procedures. All inclusion and exclusion criteria 
(Section 4, Selection of Subjects) must be met at time of randomization prior to receipt of 
the first dose of rituximab/placebo (Day 0, Treatment Initiation).  
 
Clinical assessments and sample collection will occur regularly through Week 48 with 
telephone follow-up conducted intermittently. If a subject has not recovered B cells by 
Week 48, B cell studies will be conducted quarterly until recovery is documented or for 2 
years after initial treatment (whichever occurs first). Recovery is defined as the time point 
at which B cells recover to within 10% of baseline levels; if a baseline value is not 
available then recovery is defined as a B cell level of at least 90% of the lower limit of 
normal. During this quarterly monitoring period of B cell recovery, AEs and SAEs will be 
assessed, providing the subject has not withdrawn consent, to capture any infectious 
event ≥ Grade 3 using the National Cancer Institute—Common Terminology Criteria for 
Adverse Events (NCI-CTCAE). No additional study-related data will be collected. 
 
The primary efficacy endpoint is the change in 6MWD from baseline to 24 weeks after 
treatment initiation. Hemodynamic measures will be assessed at baseline and Week 24, 
contributing to the understanding of the relationship between PVR and clinical endpoints. 
Time to clinical worsening will be assessed as a secondary outcome measure through 
Week 48, contributing to the understanding of the relationship between PVR and clinical 
endpoints. Initiation of new therapy due to disease worsening prior to Week 24 will prompt 
an endpoint visit and right heart catheterization prior to initiation of the new therapy. B cell 
recovery for all will be monitored by serial assessments of B cell counts as determined by 
flow cytometry. 
 
The total duration of the study is anticipated to be approximately 8 years.  
Study Population:  Any individual 18 to 75 years of age, who has been diagnosed with 
SSc-PAH within the past 5 years (as defined by a clinical diagnosis of SSc with a mPAP ≥ 
25 mmHg), a NYHA Functional Class of II, III, or IV, a mean PVR of > 3 Wood units, and 
who meets all entry criteria is eligible for randomization.  
Primary Efficacy Endpoint: Change in exercise capacity, assessed by 6MWD, from 
baseline to 24 weeks after treatment initiation. 
 
 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment of 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension (SSc-PAH) 
Secondary Efficacy Endpoints: 
Secondary efficacy endpoints include the following clinical endpoints: 
1. Change in PVR measured by right heart catheterization, from baseline to 24 weeks 
after treatment initiation. 
2. Exercise capacity, as determined by 6MWD, assessed longitudinally over the 48 
weeks following treatment initiation.   
3. Assessment of time to clinical worsening, censored at 48 weeks, defined as first 
occurrence of: 
a. death,  
b. hospitalization for SSc-PAH,  
c. lung transplantation,  
d. atrial septostomy,  
e. addition of other PAH therapeutic agents, or  
f. 
worsening of 6MWD by > 20% and a decrease in functional class. 
4. Percentage of subjects who changed or added PAH therapeutic agents at 24 and 
48 weeks. 
5. Change in quality of life as determined by the SF-36 and disability index of the 
SHAQ from baseline to 24 and 48 weeks after treatment initiation. 
6. Number of new digital ulcers assessed longitudinally over both the 24 and 48 weeks 
following treatment initiation. 
7. Severity of Raynaud phenomenon, as measured by the VAS scale of the SHAQ, 
assessed longitudinally over both the 24 and 48 weeks following treatment initiation. 
8. DLCO and oxygen saturation at rest on room air assessed longitudinally over both 
the 24 and 48 weeks following treatment initiation. 
9. Change in PVR from baseline to 24 weeks after treatment initiation as a percentage 
of the baseline value. 
 
Secondary efficacy endpoints also include the following mechanistic endpoints: 
1. CD19 Studies 
a. CD19+ B cells (absolute number and percentage of gated lymphocytes) at 
time of B cell depletion and longitudinally over the 48 weeks following 
treatment initiation, where B cell depletion is defined as <5 CD19+ cells/μL. 
b. Time to repopulation of CD19+ B cells, defined as the time point at which B 
cells recover to within 10% of baseline levels; if a baseline value is not 
available, then recovery is defined as a B cell level of at least 90% of the 
lower limit of normal. 
c. Reconstitution pattern of B cell subpopulations assessed longitudinally over 
the 48 weeks following treatment initiation. 
2. Level of anti-U1 RNP, anti-B23, anti-cardiolipin, anti-CENP-B, RF autoantibodies, 
and immunoglobulins, including IgG subclasses, measured by ELISA and assessed 
longitudinally over the 48 weeks following treatment initiation. 
3. Presence or absence of anti-U3 RNP, anti-Th/To, and other autoantibodies 
assessed longitudinally over the 48 weeks following treatment initiation. 
 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment of 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension (SSc-PAH) 
4. Level of BNP/NT-proBNP, serum Na+, IL-1, IL-6, and other cytokines assessed 
longitudinally over the 48 weeks following treatment initiation. 
5. Presence or absence of B cell clones assessed at baseline and longitudinally over 
the 48 weeks following treatment initiation. 
 
Secondary Safety Endpoints: 
1. All NCI-CTCAE Grade 3-5 AEs, which are defined as possibly, probably, or 
definitely related to rituximab infusion.  
2. All NCI-CTCAE Grade 3-5 adverse events. 
3. All infection related events. 
4. Treatment-related mortality, defined as death occurring at any time after 
randomization and possibly, probably, or definitely resulting from treatment given in 
the study. Study investigators will make an initial determination of the cause of 
death of any subject and if uncertainty remains, a final decision will be made by an 
independent blinded panel after reviewing all clinical and, if available, autopsy data. 
5. All-cause mortality, defined as any death occurring at any time after randomization. 
6. CD19+ B cell levels post treatment (note that analyses will be performed regularly 
per Table 6.1 through 48 weeks and quarterly thereafter as documented in Section 
3.1, Description of Study Design). 
7. Peripheral blood cell counts, liver function, and creatinine levels after treatment. 
 
Exploratory Endpoints: 
Exploratory analysis of standardization of the PVR calculation will be based on PVR 
calculated from a standardized Fick calculation of cardiac output, which is a function of 
hemoglobin, arterial and mixed-venous saturations, and oxygen consumption according to 
the La-Farge and Miettinen formula. The cardiac output calculation is a function of 
assumed oxygen consumption according to the La-Farge calculation of values normalized 
for age, gender, heart rate. Calculation of a standardized Fick cardiac output will also allow 
for determination of the pulmonary artery capacitance (PACf). See Appendix E, 
Exploratory Analysis: Standardized Fick-based Pulmonary Vascular Resistance and 
Pulmonary Artery Capacitance for details. 
Additionally, an exploratory analysis of the relationship between change in PVR and 
change in 6MWD will be addressed. Relationships with other clinical endpoints, like 
change in NTproBNP, will also be explored. 
Sample Size:  The study will include 30 subjects treated with rituximab and 30 subjects 
treated with placebo.   
Data Analyses:  The primary efficacy analysis of change in 6MWD from baseline to 24 
weeks will be performed on the intention-to-treat (ITT) population. and will be tested using 
a repeated measures random effects model to model change from baseline in 6MWD as 
the primary outcome and treatment as the primary predictor, controlling for visit day, 
limitations by musculoskeletal disease, baseline levels of 6MWD, and a treatment by visit 
day interaction.  A random slope and intercept will be fit for each subject.  An unstructured 
covariance matrix will be assumed for the random effects. Additional covariates will be 
considered for this model as part of secondary analysis. 
 
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Title of the Protocol:  A Randomized, Double-blind, Placebo-Controlled, Phase II 
Multicenter Trial of a Monoclonal Antibody to CD20 (Rituximab) for the Treatment of 
Systemic Sclerosis-Associated Pulmonary Arterial Hypertension (SSc-PAH) 
 
Secondary analyses will include repeating the primary analysis in the per protocol 
population, subgroup analyses of the primary outcome, safety analyses, exploratory 
analyses of the secondary endpoints, and exploratory analyses of the mechanistic 
endpoints. 
Safety Stopping Rules: In addition to the pre-scheduled data reviews and planned safety 
monitoring, the DSMB may be called upon for ad hoc reviews or emergency meetings. 
The DSMB will review any event that potentially impacts safety at the request of the 
protocol chair or DAIT/NIAID. In addition, the following events will trigger both a 
comprehensive DSMB Emergency Safety Review and a temporary halt in enrollment: 
a. Any immediately life threatening event or death that occurs in the study which is 
unexpected and possibly, probably, or definitely related to study intervention. 
b. Any event, including those listed in Section 3.3.2, Discontinuation of Protocol-
Specified Treatment Requirements, that results in permanent discontinuation of 
infusion of study intervention occurring in 2 of the first 5 subjects, 4 of the first 15 
subjects, 6 of the first 30 subjects, or a total of 8 events after 30 subjects have been 
enrolled in the trial. 
c. Any occurrence of PML. 
d. Clinical Grade 3 or higher events or SAEs which are possibly, probably, or definitely 
related to study treatment but do not result in discontinuation of study treatment will 
be reviewed whenever the ratio of events to enrolled subjects is 20% (e.g. 9 events 
for 47 subjects, 10 events for 48-52 subjects, 11 events for 53-57 subjects, etc.).  
o Note: clinical events include clinical signs/symptoms and diagnoses and 
laboratory abnormalities with clinical consequence (defined as the 
requirement for intervention, correction, increased monitoring, or further 
evaluation). 
The DSMB will have discretion to recommend actions regarding study conduct and 
continuation as a consequence of any planned or unplanned monitoring activity. 
A temporary halt in enrollment will occur for the events listed above with the exception of 
event d. For events falling under d, a halt in enrollment will occur if the DSMB Emergency 
Safety Review is not completed within 2 weeks. In the event that the study temporarily 
halts enrollment, no new subjects will be consented or start on therapy with rituximab or 
placebo; and subjects already on rituximab or placebo will continue on therapy unless they 
are the focus of the DSMB review. Subjects in the screening phase of the study may 
continue to undergo minimal risk procedures (e.g. blood tests), but more than minimal risk 
procedures should be deferred. Randomization will not occur until the DSMB review is 
complete. The FDA will be notified of any halt in enrollment. After careful review of the 
data, the DSMB will make recommendations regarding study conduct and/or continuation. 
 
 
 
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2. 
INTRODUCTION 
This statistical analysis plan includes pre-planned analyses related to the study objectives 
outlined in the protocol. 
 
 
 
 
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3. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS 
The following analyses and reporting conventions will be used: 
• 
Categorical variables will be summarized using counts (n) and percentages (%) and will 
be presented in the form “n (%).”  Percentages will be rounded to one decimal place. 
• 
Numeric variables will be summarized using n, mean, standard deviation (SD), median, 
minimum (min), maximum (max).  The min/max will be reported at the same level of 
significance as original data.  The mean and median will be reported at one more 
significant digit than the precision of the data, and SD will be reported at two more 
significant digits than the precision of the data. 
• 
The median will be reported as the average of the two middle numbers if the dataset 
contains an even number of observations. 
• 
Test statistics including t and z test statistics will be reported to two decimal places.  
• 
P-values will be reported to three decimal places if greater than or equal to 0.001.  If less 
than 0.001, the value will be reported as “<0.001.”  A p-value can be reported as “1.000” 
only if it is exactly 1.000 without rounding.  A p-value can be reported as “0.000” only if it 
is exactly 0.000 without rounding. 
If departures from these general conventions are present in the specific evaluations section of 
this SAP, then those conventions will take precedence over these general conventions.  
 
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4. 
ANALYSIS SAMPLES 
4.1. 
Safety Population 
The Safety Population (SP), which will be used for all safety analyses, will include all subjects 
for whom study treatment is initiated. 
4.2. 
Modified Intent-to-Treat Population  
The Modified Intent-to-Treat (ITT) population will include all randomized subjects who initiate 
rituximab therapy and meet entry criteria. Randomized subjects who withdraw from the trial 
prior to starting rituximab infusion will be excluded from the ITT analysis set and replaced. The 
primary efficacy analyses will be based on the ITT population, as will key secondary efficacy 
and mechanistic analyses. Subjects who, for whatever reason, do not complete their assigned 
therapy will be included in the ITT population in the groups to which they were randomized. 
4.3. 
Per Protocol Populations 
The Per Protocol (PP) population will be defined as those subjects who comply with the 
assigned treatment protocol by completing two IV infusions of 1000mg of Rituximab/Placebo at 
Visit 3 and Visit 4 as well as completing primary endpoint evaluation (6MWD) at Visit 10 with no 
major protocol deviations that would affect their efficacy outcome. Subjects who are withdrawn 
from follow-up prior to Visit 10 will not be included in the PP population. A masked data review 
panel will evaluate deviations from the protocol including, for example, violations of entry 
criteria, departures from assigned treatment regimen, or administration of study procedures 
outside the specified visit windows. The panel may exclude subjects with serious protocol 
deviations from the PP population. Both the primary analyses and all secondary analyses will 
be replicated on the PP population. 
A second Per Protocol (PP2) population will be also be defined.  This population will be defined 
as eligible subjects who comply with the assigned treatment protocol by completing two IV 
infusions of 1000mg of Rituximab/Placebo at Visit 3 and Visit 4. 
 
 
 
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5. 
STUDY SUBJECTS 
5.1. 
Disposition of Subjects  
The disposition of all enrolled subjects will be summarized in tables and listed. 
The numbers and percentages of subjects randomized, in each analysis sample, and 
completing selected study weeks (Day 0; Weeks 2, 24 and 48), as well as reasons for early 
termination from the study will be presented.  For subjects discontinuing study treatment early, 
the reasons for discontinuing study treatment early will also be presented.   
Subject status will also be shown graphically by subject. Each subject will be represented by a 
line with events such as treatment, early study withdrawal, first addition/change in PAH 
medications, death, etc. shown over time in the study.   
5.2. 
Demographic and Other Baseline Characteristics  
Summary descriptive statistics for baseline and demographic characteristics will be reported for 
all analysis samples.  Characteristics to be summarized include: age, race, ethnicity, sex, body 
weight and height at screening, duration of SSc-PAH disease, PVR (by type), right atrial mean 
pressure, mean pulmonary artery pressure, cardiac output, pulmonary capillary wedge mean 
pressure, 6MWD, NYHA Functional Class, digital ulcer count, DLCO, FVC, TLC, HAQ-DI 
score, oxygen saturation, autoantibody status, number of PAH medications, oxygen use, 
limited or diffuse SSc, duration of SSc, smoking history, and BNP/pro-NTBNP. 
Demographic and baseline characteristic data will also be presented in data listings by 
treatment group and subject. 
 
 
 
 
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6. 
STUDY OPERATIONS 
6.1. 
Protocol Deviations 
Major protocol deviations will be listed by site with information such as type of deviation, 
whether notification to the IRB was required, date of occurrence, and the reason for the 
deviation.  Protocol deviations will be summarized in tabular format by type of deviation.   
6.2. 
Treatment Adherence 
Subjects will be randomized into either the rituximab or placebo arm. Rituximab or placebo will 
be administered as two IV infusions of 1000 mg each, given two weeks apart at Day 0 and 
Week 2. Study treatment information is collected on the Rituximab/Placebo Infusion CRF page. 
Percent compliance for study drug (rituximab or placebo) will be computed for both study visits 
and overall as: 
% Compliance = Total dose received  (mg)
Total dose planned (mg)  × 100 
Where: 
Total dose received (mg) =  concentration of infusion (mg/mL) ×  total volume infused (mL) 
 
For each study arm and overall, percent compliance for study drug will be summarized 
descriptively by mean, standard deviation, median, minimum and maximum. 
Compliance data for study drug will be listed by treatment group, subject, and infusion visit and 
will include use of pre-infusion medications, duration of infusion, information on incomplete or 
interrupted infusions, actual and planned dose, as well as concomitant therapy during study 
drug administration. 
 
 
 
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7. 
ENDPOINT EVALUATION 
7.1. 
Overview of Efficacy Analysis Methods 
7.1.1. 
Multicenter Studies 
Due to the small sample size and large number of sites needed for this study, study center will 
not be included as a covariate in the analysis.  However, if evidence of a study center effect 
exists and there are appropriate sample sizes at each site, a sensitivity analysis will be 
performed including study center in the efficacy analyses. All safety analyses will be based on 
data pooled across all centers with no adjustment or stratification by center.  
7.1.2. 
Assessment Time Windows 
Allowable visit windows for all scheduled visits are provided in Table 7-1. 
Table 7-1 
Visit Windows 
Visit 
Window 
Screening 
-28 Days 
Baseline 
-7 Days 
Day 0 
 
Week 2 
± 4 Days 
Weeks 4, 8, 12, 16, 20, 
24, 28, 32, 36, 40, 44, 48 
± 7 Days 
Quarterly Monitoring 
± 14 Days 
 
Unscheduled visits may also occur throughout the study. All data will be included in analyses, 
regardless of time of assessment. 
7.2. 
Primary Endpoint 
7.2.1. 
Computation of the Primary Endpoint 
The primary efficacy endpoint is change in exercise capacity, assessed by the Six Minute Walk 
Distance (6MWD), from baseline to 24 weeks after treatment initiation. The 6MWD will be 
performed at Screening and Weeks 4, 12, 24, 36, and 48; additionally, if the 6MWD decreased 
from the Screening 6MWD by more than 20%, a subject should repeat the 6MWD within 7 days, 
but not on the same day. A 6MWD may also be performed at an unscheduled visit.     
The 6MWD measures the distance a subject is able to walk over a total of six minutes on a 
hard, flat surface. The goal is for the subject to walk as far as possible in six minutes. The 
subject is allowed to self-pace and rest as needed as they traverse back and forth along a 
marked walkway. The total distance walked, in meters, will be recorded for each subject on the 
6-Minute Walk Test CRF page. Information on whether a subject has a musculoskeletal disorder 
that limits ambulation (yes/no) will also be collected on this CRF page.  
If a subject’s 6MWD decreased from Screening by more than 20% and a repeat 6MWD was 
performed, then the better of the two values will be used in the primary analysis. Missing data 
will not be imputed. 
 
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7.2.2. 
Primary Analysis of the Primary Endpoint 
The primary efficacy analysis for this study is designed to test the scientific hypothesis that 
rituximab has a beneficial clinical impact on SSc-PAH as measured by improvement or 
stabilization in 6MWD between baseline to 24 weeks in patients treated with standard of care 
plus rituximab than in patients treated with standard of care plus placebo. Operationally for this 
study, this scientific hypothesis will be examined by formally testing the following statistical 
hypothesis: 
H0: 
The mean change in 6MWD between baseline and 24 weeks does not differ 
between individuals treated with rituximab and those treated with placebo 
 
(δrituximab = δplacebo where δx = mean 6MWD at 24 weeks – mean 6MWD at 
baseline) 
versus 
HA:  
The mean change in 6MWD between baseline and 24 weeks is more beneficial 
in those individuals treated with rituximab than in those treated with placebo 
 
(δrituximab < δplacebo where δx = mean 6MWD at 24 weeks – mean 6MWD at 
baseline) 
While this hypothesis is inherently one-sided, a two-sided test at α=0.05 will be used to 
evaluate the treatment effect at 24 weeks. This primary hypothesis will be tested using a 
repeated measures random effect model to model the distance walked as a function of 
treatment, visit week and a treatment by visit week interaction. Additionally, a quadratic term for 
visit week will be included in the model.  A random slope and intercept will be fit for each 
subject using a separate unstructured covariance matrix for each treatment group.  The model 
will include only 6MWD data up to Week 24. All available data up to Week 24 will be used for 
subjects who terminate early from the study. All unscheduled visits that occur prior to Week 24 
will also be included in the analysis. 
Model estimates and standard errors of the distance walked and change from baseline in 
distance walked will be presented at baseline and Week 24, along with the differences 
between arms. An appropriate contrast will be used to test the difference (baseline to Week 24) 
between treatment arms. 
Distance walked and change from baseline in distance walked with be shown graphically by 
treatment arm. Spaghetti plots and summary statistics including 25th percentile, mean, median, 
and 75th percentile will be plotted for each visit by treatment group. Additionally, mean 
predicted values from the model will be plotted over time, alongside the actual distance walked 
values for each study visit. 
The primary efficacy analysis will be based on the ITT population. 
 
7.2.3. 
Sensitivity Analyses of the Primary Endpoint 
The following sensitivity analyses will be performed: 
1. Primary analyses performed on both PP populations. 
2. If a subject’s 6MWD decreased from Screening by more than 20% and a repeat 6MWD 
was performed, then models will be develop using both SMWD values and also using 
just the second, confirmatory, value in the model. 
3. Changes in background PAH medications prior to Week 24 (yes/no) will be added as a 
covariate to the model. 
4. Missing data will not be imputed for the primary endpoint.  However, sensitivity 
analyses may be performed to assess the robustness of the conclusions under different 
 
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assumptions. For example, missing data due to death of a subject prior to Week 24 
would be handled differently than missing data due to a missed visit. A joint 
longitudinal/survival model to evaluate trends in clinical assessments under different 
assumptions about the underlying missing data mechanism may be considered, if 
appropriate.    
5. Demographic and baseline characteristics that differ meaningfully between the two 
treatment groups (with or without a p-value <0.05) may be included in a covariate-
adjusted model, as appropriate. 
6. Time-varying covariates, like oxygen use during SMWD, may be added as appropriate.  
7.3. 
Secondary Endpoints 
All secondary analyses will be conducted in an exploratory fashion with p-values and 
confidence intervals presented as descriptive statistics with no adjustments for multiple 
comparisons. Interval estimates will be generated at the 95% confidence level. Analyses will be 
conducted on the ITT population and the PP populations. Missing data will not be imputed.  
7.3.1. 
Pulmonary Vascular Resistance  
Endpoint:  A right heart catheterization (RHC) will be performed at Screening and Week 24. 
Data for the RHC will be collected on the Right Heart Catheterization CRF page. 
During a RHC a catheter is guided to the right side of the heart and then into the pulmonary 
artery; blood flow through the heart is observed and is used to measure pressures in a 
subject’s heart and lungs. Pulmonary vascular resistance (PVR) will be collected in Wood Units 
and method of cardiac output (indirect or estimated Fick, direct Fick, or thermodilution) will be 
collected as well. Multiple types of PVR (indirect or estimated Fick, direct Fick, or 
thermodilution) may be collected for a subject. Higher PVR values indicate worse disease 
status. 
PVR will be derived in two ways: 
1. First, as noted previously, multiple types of PVR (indirect Fick, direct Fick, or 
thermodilution) may be collected for a subject. The PVR value used for analysis will be 
chosen in the following order, as available from the Right Heart Catheterization CRF 
page: 1. thermodilution, 2. direct Fick, 3. indirect Fick. Both the screening and Week 24 
RHC must use the same method of cardiac output. If the method used for screening and 
Week 24 do not match, only the baseline measure will be included in model-based 
analyses. 
2. Second, PVR will be derived using the formula:  
𝑃𝑃𝑃𝑃𝑃𝑃 = (𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 –  𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃)/𝐶𝐶𝐶𝐶,   where 
mPAP = mean pulmonary artery pressure (mmHg) 
PCWP = mean pulmonary capillary wedge pressure (mmHg) 
CO = Cardiac Output (L/min) 
Similar to the first method, method of cardiac output will be chosen in the following order, 
as available: 1. thermodilution, 2. direct Fick, 3. indirect Fick. PCWP can be recorded at 
end expiration or by a computer-generated mean.  If both are recorded, at end expiration 
will be used.  
For each derived PVR variable, change from Screening to Week 24 in the PVR and percent 
change from Screening to Week 24 in the PVR will be computed for each subject. Missing 
values will not be imputed.  
Analysis: For each derived PVR variable, the following analyses are planned.  
 
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1. Descriptive statistics will be presented for PVR, percent change from Screening, and 
change from Screening in PVR for each treatment group and overall. Spaghetti plots 
and summary statistics including 25th percentile, mean, median, and 75th percentile will 
be plotted for each visit by treatment group. Separate summaries will be prepared for all 
subjects with available data at either time point and for the subset of subjects with 
measurements at both time points.  
2. Additionally, a repeated measures mixed model will be fit with PVR as the outcome and 
treatment, visit week (actual observed week), and their interaction as predictors. 
Random intercepts and slopes will be fit for each subject using an unstructured 
covariance structure for each treatment group. Model estimates and standard errors for 
PVR and change from baseline in PVR will be presented at baseline and Week 24, 
along with the differences between arms. An appropriate contrast will be used to test 
the difference (baseline to Week 24) between treatment arms. 
 
A separate analysis is planned to simultaneously evaluate the all reported PVR (indirect Fick, 
direct Fick, or thermodilution) measures for a subject without the prioritization rules described 
above. A multivariable repeated measures mixed model will be fit with PVR as the outcome 
and treatment, type of PVR (indirect or estimated Fick, direct Fick, or thermodilution), visit week 
(actual observed week) and interaction terms for type*treatment and type*treatment*week. 
Random intercepts and slopes will be fit for each subject using an unstructured covariance 
structure for each PVR type and treatment group. In addition, separate residual variance 
components will be estimated for each type of PVR.  
Note: PVR is measured only at screening and Week 24. Subjects lost prior to Week 24 will 
contribute only screening data. If subjects are not lost completely at random, findings from 
these analyses may be biased as they will reflect changes in PVR in the subset of individuals 
who made it to Week 24. 
Analogous analyses will be repeated on the percent change from Screening in PVR at Week 
24. 
7.3.2. 
Six Minute Walk Distance 
Endpoint: Exercise capacity, as determined by 6MWD, will be assessed as defined in Section 
7.2.1, Computation of the Primary Endpoint.  
Changes in PAH medication use will be recorded on the Initiation of New PAH Medications and 
Therapy CRF page, and will be used to determine whether subjects had changes in their 
background PAH medications before and/or after the Week 24 visit (yes/no).  
Analysis: Analyses analogous to those specified for the primary endpoint will be performed for 
longitudinal changes in the 6MWD at Week 48.  See Section 7.2.2, Primary Analysis of the 
Primary Endpoint, for more details on the analyses.  Data for this model will include all 
available data to Week 48.  Additionally, the model will include a covariate to indicate changes 
in PAH therapeutic agents before and/or after the 24 week time point (yes/no). The interaction 
between this covariate and treatment will be included if significant (p-value<0.05). Spaghetti 
plots of the data will also be assessed to define any time trends that may need to be reflected 
in the model. 
Additional models may be developed to assess the effect of other covariates. 
7.3.3. 
Clinical Worsening 
Endpoint: Assessment of time to clinical worsening, censored at 48 weeks, will be defined as 
first occurrence of the following: death, hospitalization for SSc-PAH, lung transplantation, atrial 
septostomy, addition of other PAH therapeutic agents, or worsening of 6MWD by > 20% and 
 
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an increase in functional class. The components of clinical worsening will be assessed as 
follows. 
a. Death: Date of death will be collected on the Post-Study Follow-Up/Week 104 
Vital Status CRF page. 
b. Hospitalization for SSc-PAH: Date of hospitalization for SSc-PAH will be 
collected on the Serious Adverse Event CRF page and determined from SAEs 
that had seriousness criteria of “Required hospitalization or prolongation of 
existing hospitalization” and that were marked as being possibly, probably, or 
definitely related to worsening disease on the Adverse Events CRF page.  Time 
to the event will be derived from the date that the AE became serious. If a subject 
has multiple hospitalizations for SSc-PAH, then the first date will be used. 
i. A second clinical worsening endpoint will be similarly defined except that 
“hospitalizations for SSc-PAH” will be evaluated by a blinded review panel 
to exclude those events relating only to a subject’s SSc and not PAH. 
c. Lung transplantation: Date of lung transplant will be collected on the Initiation of 
New PAH Medications and Therapy CRF page.    
d. Atrial septostomy: Date of atrial septostomy will be collected on the Initiation of 
New PAH Medications and Therapy CRF page. 
e. Addition of other PAH therapeutic agents:  Date of new medications or doses of 
PAH medications changed for the treatment of PAH will be collected on the 
Initiation of New PAH Medications and Therapy CRF page. 
f. 
Worsening of 6MWD by > 20% and an increase in functional class:  The 6MWD 
and the NYHA Functional Class Assessment will be performed at Screening and 
Weeks 4, 12, 24, 36, and 48; they may also be performed at an unscheduled 
visit.  Data for the 6MWD will be collected on the 6-Minute Walk Test CRF page 
and data on functional class will be collected on the NYHA Functional Class 
Assessment CRF.  The first date at which the 6MWD has decreased by >20% 
from the Screening walk distance and the NYHA Functional Class Assessment 
has increased a class from Screening will be recorded. If a subject’s 6MWD 
decreased from Screening by more than 20% and a repeat 6MWD was 
performed, then the better of the two values will be used in the analysis.  If a 
subject is NYHA Class IV at baseline, then only a decrease of 20% in the 6MWD 
will be required. 
 
Time to clinical worsening will be defined as the first date that meets any of the above criteria 
and will be calculated in study days as: date of first event – date of randomization. If a subject 
does not experience a relevant event by Week 48 or if the date of death is after the 48 week 
follow-up period, then time to clinical worsening will equal the duration of follow-up in the main 
study for that subject. Appropriate censor variables will be defined to indicate whether the time-
to variable represents an event time or a censor time. 
Analysis: Time to clinical worsening and type of clinical worsening reached will be summarized 
by treatment group and overall. Kaplan-Meier curves, including 95% confidence intervals, will 
be created for each treatment arm and will be compared using the log-rank test. A Cox 
proportional hazards model may be fit to assess the effect of treatment on time to clinical 
worsening, if appropriate. Additional covariates may be added to the model as appropriate. 
Analyses will be run using the definition of hospitalization of SSc-PAH as defined in both point 
B and B.i above. 
7.3.4. 
Addition of PAH medications 
Endpoint: New medications or doses of PAH medications changed for the treatment of PAH 
will be collected on the Initiation of New PAH Medications and Therapy CRF page. Date of the 
 
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new or modified medication along with reason for the change will be collected for the first time 
a PAH medication is modified or added. 
Indicator variables for new or modified PAH therapeutic agents will be derived for two time 
points: prior to Week 24 and after Week 24. Time to the addition or modification of PAH 
medications will be defined in study days as: date of the first time a PAH medication is modified 
or added – date of randomization.  
Analysis: The number and proportion of subjects who changed or added PAH therapeutic 
agents prior to Week 24 and after Week 24 will be summarized by treatment group and overall, 
along with the reason for changes to the background PAH medications. Time (in days) to the 
addition or modification of PAH medications will also be summarized by treatment group and 
overall. 
Kaplan-Meier curves, including 95% confidence intervals, will be created for each treatment 
arm and will be compared using the log-rank test.  
7.3.5. 
Quality of Life: SF-36  
Endpoint: Change in quality of life as determined by the Short Form Health Survey (SF-36) will 
be assessed at Baseline and Weeks 24 and 48.  Data for the SF-36 will be captured on the 
Short Form – 36 Health Survey CRF page. The SF-36 is a 36-item, patient-reported survey of 
patient health.  Higher scores indicate better outcomes while lower scores indicate more 
disability.  
The following SF-36 scale scores will be calculated: Physical Functioning, Role-Physical, 
Bodily Pain, General Health, Vitality, Social Functioning, Role-Emotional, and Mental Health.  
From these scale scores, two summary scores that aggregate measures will be created.  The 
Physical Component Score is comprised of the Physical Functioning Scale, the Role-Physical 
Scale, the Bodily Pain Scale, and the General Health Scale.  The Mental Component Score is 
comprised of the Vitality Scale, the Social Functioning Scale, the Role-Emotional Scale, and 
the Mental Health Scale. 
Norm-based scores will also be generated for the eight SF-36 scales and the two summary 
measures.  All eight SF-36 scales and the two summary scores will be normalized to have a 
mean of 50 and a standard deviation of 10 for the 1998 general US population.  The advantage 
of the standardization is that results for one scale can be meaningfully compared with the other 
scales, and the scores have a direct interpretation in relation to the distribution of scores in the 
1998 general US population.  Specifically, scores above or below 50 are above or below the 
average, respectively, for the 1998 general US population.  Because the standard deviation is 
10 for all 8 scales, each one point difference in scores is equivalent to one-tenth of a standard 
deviation unit or an effect size of 0.10.  See Appendix 14.1, Scoring the SF-36 Version 2, of 
this SAP for details regarding calculation of these scores. 
Change from baseline in the SF-36 Physical Component Summary (PCS) and Mental 
Component Score (MCS) domains at Weeks 24 and 48 will be computed for each subject. 
Missing data will not be imputed. 
Analysis: Longitudinal changes in the SF-36 at Weeks 24 and 48 will be compared across the 
two arms using linear mixed models. A repeated measures random effect model with MCS or 
PCS as the outcome, and treatment, visit week (actual observed week), and their interaction as 
predictors. Additionally, a quadratic term for visit week may be included in the model, if 
significant.  Random intercepts and slopes will be fit for each subject using an unstructured 
covariance structure for each treatment group. 
 
Descriptive statistics will be presented for the MCS and PCS and change from baseline in the 
MCS and PCS for each treatment group and overall by study visit. MCS and PCS and change 
from baseline in MCS and PCS will be shown graphically by treatment arm. Spaghetti plots and 
 
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08 March 2019 
 
summary statistics including 25th percentile, mean, median, and 75th percentile will be plotted 
for each visit by treatment group.  
Model estimates and standard errors of the MCS and PCS and change from baseline in the 
MCS and PCS will be presented at baseline and Weeks 24 and 48, along with the differences 
between arms. Appropriate contrasts will be used to test the difference (baseline to Week 24 or 
Week 48) between treatment arms. Mean predicted values from the model will be plotted over 
time, alongside the actual values for each study visit. 
Additional models may be developed to assess the effect of other covariates, as necessary.  
7.3.6. 
Quality of Life: HAQ-DI  
Endpoint: Change in quality of life as determined by the Disability Index of the Scleroderma 
Health Assessment Questionnaire (HAQ-DI) will be completed at Baseline and Weeks 4, 12, 
24, 36, and 48.  Data for the HAQ-DI will be collected on the SHAQ (Scleroderma Health 
Assessment Questionnaire) CRF page. 
Eight domain scores are computed as the maximum value over all variables in the domain. 
Domains are defined as follows: Dressing/Grooming (Questions 1A, 1B), Arising (Questions 
2A, 2B), Eating (Questions 3A, 3B, 3C), Walking (Questions 4A, 4B), Hygiene (Questions 5A, 
5B, 5C), Reach (Questions 6A, 6B), Grip (Questions 7A, 7B, 7C), and Activities (Questions 8A, 
8B, 8C). Each of the questions are answered on a 4-point scale, where 0 = without any 
difficulty, 1 = with some difficulty, 2 = with much difficulty, and 3 = unable to do. The total score, 
HAQ-DI, is then computed as the mean over all the non-missing domain scores. If less than 6 
domain scores are available, HAQ-DI is missing. Higher scores indicate more disability. 
Change from baseline in the HAQ-DI score will also be calculated for each visit. Missing values 
will not be imputed. Use of devices will not be included in the scoring. 
Analysis: Analyses analogous to those specified in Section 7.3.5, Quality of Life: SF-36, will be 
performed for the HAQ-DI. 
7.3.7. 
Digital Ulcers 
Endpoint: The total number of digital ulcers will be captured at the Baseline visit, and 
subsequently the number of new digital ulcers since the subject's last study visit will be 
captured at the Week 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 visits and recorded on the 
Digital Ulcer Count CRF page. 
The total number of digital ulcers present on the dorsal and palmar surfaces for both the left 
and right fingers will be captured at the baseline study visit. The number of new digital ulcers 
since the last study visit (including any ulcers that have appeared and healed since the last 
study visit) on the dorsal and palmar surfaces for both the left and right fingers will be captured 
at the post-baseline study visits.  A variable for the total number of digital ulcers on both hands 
will be summed from the number present on the dorsal and palmar surfaces for both the left 
and right fingers.   
Rates (per week) of new digital ulcers for each treatment for each of two periods will be 
calculated for each subject, the two observations will be defined as: 
1) the period including assessments at weeks 4, 8, 12, 16, 20, and 24 (Period=1) 
2) the period including assessments at weeks 28, 32, 36, 40, 44, and 48 (Period=2). 
For each period, we will define: 
1) Total number of new digital ulcers on both hands = sum over all assessments for the 
period 
2) Period Weeks = Sum of weeks between assessments over all intervals in the period 
(based on actual dates of the visits).  
• 
the natural log(period weeks) will also be derived 
 
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3) PAH Meds = indicator if PAH medications were changed or modified in the treatment 
period 
Analysis:  The number of digital ulcers at screening and then the number of new digital ulcers 
at the follow-up visits will be summarized by treatment group and overall.  
Longitudinal rates of the number of new digital ulcers at Weeks 24 and 48 will be compared 
across the two arms using Poisson marginal models for longitudinal data.  Initially, the Poisson 
model will be used to describe the rate of new digital ulcers (per week) as the outcome, and  
only treatment, number of digital ulcers at Baseline, and an indicator if the measurement was 
affected by changed or new PAH therapeutic agents as covariates. The SAS procedure GEE 
will be used to fit the Poisson model; an unstructured working correlation matrix will be used. 
Period weeks will be used as the offset variable to represent the different time intervals. 
Estimated rates of new digital ulcers (per week) for each treatment group and period will be 
derived. The rate of new digital ulcers will be compared across treatment groups for each time 
period. Additionally, within each treatment group, the differences in rates between period 1 and 
period 2 will be compared, and the differences in the rates between period 1 and period 2 will 
be compared across treatment arms.  
Additional models may be developed to assess the effect of other covariates as appropriate.  
7.3.8. 
Severity of Raynaud’s Phenomenon 
Endpoint: Change in quality of life as determined by the Scleroderma Health Assessment 
Questionnaire (SHAQ) will be completed at Baseline and Weeks 4, 12, 24, 36, and 48.  Data 
for the SHAQ will be collected on the SHAQ (Scleroderma Health Assessment Questionnaire) 
CRF page. 
Severity of Raynaud’s phenomenon will be measured by a VAS scale of the SHAQ. The SHAQ 
VAS scales include a question asking, “In the past week, how much has your Raynaud’s 
Phenomenon interfered with your activities?” Subjects are asked to place a mark on a 15 cm 
line, scaled from 0 (does not interfere) to 100 (very severe limitation), to describe the severity 
of their Raynaud’s phenomenon in the past week.  The distance from the left edge of the line to 
the vertical line placed by the subject will be measured in centimeters; VAS scores will be 
converted to a 0 to 100 scale.  Change from baseline in the VAS score will be calculated. 
Analysis:  Analyses analogous to those specified in Section 7.3.5, Quality of Life: SF-36, will 
be performed for the severity of Raynaud’s phenomenon. 
7.3.9. 
DLCO 
Endpoint: Pulmonary function tests will be performed at Screening, Week 24, and Week 48; 
only DLCO will be collected at the Week 24 and 48 visits.  Data for DLCO will be collected on 
the Pulmonary Function Tests CRF pages. Lower DLCO values indicate worse disease activity. 
DLCO will be calculated according to the Global Lung Function Initiative (GLI) all-age reference 
values and corrected for hemoglobin. The hemoglobin used for adjustment should be done 
within two weeks of the PFT test and will be recorded on the CRF page. If a hemoglobin 
measure is not available, the unadjusted value will not be used. The % predicted DLCO is 
computed as the measured DLCO divided by the computed predicted value and multiplied 
by 100%. Change from baseline in the % predicted DLCO will be calculated.  
Analysis: Analyses analogous to those specified in Section 7.3.5, Quality of Life: SF-36, will be 
performed for DLCO. 
7.3.10. Oxygen Saturation 
Endpoint: Oxygen saturation will be recorded at Screening, Day 0, Weeks 4, 12, 24, 36, and 
48, and will be collected on the Oxygen Saturation CRF page.  If possible, oxygen saturation 
 
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will be measured on room air; if oxygen saturation is <90% on room air, then the flow rate 
required to maintain saturation ≥90% will be recorded.  If a subject is unable to have oxygen 
saturation measured on room air and supplemental oxygen is required, then the flow rate 
required to maintain saturation ≥90% will be recorded.  
Additionally, oxygen saturation is recorded at Screening and Weeks 4, 12, 24, 36, and 48 on 
the 6-Minute Walk Test CRF page, using the “pre walk” values. Oxygen saturation values and 
whether the value was recorded on room air or supplemental oxygen will be recorded.  If 
oxygen saturation is measured on supplemental oxygen, then the flow rate used will be 
recorded as well.  
Analysis:  Oxygen saturation on room air and flow rate required to maintain saturation ≥90% 
the will be summarized by treatment group and overall by study visit.  
7.4. 
Examination of Subgroups 
Secondary analyses of the primary and secondary objectives may be conducted for the 
following subgroups, including, but not limited to: 
• 
Race 
• 
Ethnicity 
• 
Gender 
• 
Baseline PVR 
• 
Baseline 6MWD 
• 
NYHA Functional Class 
• 
Duration of SSc-PAH disease 
Additionally, exploratory analyses of responder subgroups and subgroups defined through the 
mechanistic and immunological studies may also be conducted. 
 
 
 
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8. 
SAFETY EVALUATION 
8.1. 
Overview of Safety Analysis Methods 
All safety analyses will be carried out using the safety sample defined in Section 4 unless 
otherwise noted.  Missing safety information will not be imputed.  These analyses will not be 
stratified by site.   
Listings will be prepared for applicable safety measurements.  All listings will be sorted in order 
of treatment, subject identifier (ID), and time of assessment (e.g., visit, time, and/or event).} 
8.2. 
Adverse Events 
All AEs will be classified by system organ class (SOC) and preferred term, according to a 
standardized thesaurus (Medical Dictionary for Regulatory Activities [MedDRA] version 13.1).  
The severity of AEs will be classified using the National Cancer Institute’s (NCI’s) Common 
Toxicity Criteria for Adverse Events (CTCAE) version.  Each AE is entered on the electronic 
case report form (eCRF) once at the highest severity. As such, no additional data manipulation 
is needed to identify events. 
Adverse events NCI-CTCAE grade 2 and above will be collected from the time the subject 
signs the informed consent until he/she initiates study intervention or until he/she is determined 
to be ineligible to receive study intervention, if the investigator determines that the adverse 
event is related to a study-mandated procedure, treatment, or change in treatment.  
For all participants, adverse events NCI-CTCAE grade 2 and above will be collected from the 
time of initiation of study intervention (i.e., the administration of the first dose of study 
drug/study drug) until he/she completes study participation or until 30 days after he/she 
prematurely withdraws (without withdrawing consent) or is withdrawn from the study. During 
the quarterly monitoring period of the study, only NCI-CTCAE grade 3 and higher infections are 
collected on the adverse events CRF page. Grade 3 and higher infections that occur during the 
quarterly monitoring period will be listed separately and will not be included in data tabulations, 
unless otherwise specified. 
Treatment-emergent AEs will be identified as those with an onset date on or after the first dose 
of study medication.  If the start of the AE in relation to the start of study medication cannot be 
established (e.g., the start date for the AE is missing), then the AE will be considered 
treatment-emergent.  All data tabulations will be of only treatment-emergent events unless 
otherwise specified; non-treatment-emergent AEs will be listed. 
An overall summary table will be developed to report the number of events and the number and 
percentage of subjects having at least one event in the following categories:  
• 
AEs 
• 
AEs requiring 24-hour reporting to the sponsor 
• 
AEs indicated as serious 
• 
AEs that lead to study drug discontinuation 
• 
AEs with an outcome of death 
• 
AEs by relationship to a study drug 
• 
AEs reported by maximum severity 
• 
All NCI-CTCAE Grade 3-5 AEs, which are defined as possibly, probably, or definitely related 
to rituximab 
• 
All NCI-CTCAE Grade 3-5 adverse events 
 
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• 
All infection related AEs 
In addition, AEs classified by MedDRA SOC and preferred term will be summarized for each 
treatment group and overall for each of the following: 
• 
All AEs 
• 
AEs by maximum severity  
• 
AEs by relationship to study drug: relationship to study treatment will be categorized as 
either treatment related (possibly, probably, or definitely related to study medication) or 
unrelated 
• 
All infection related AEs 
Summary tables will present the total number of events as well as the number and percentage 
of subjects experiencing the events.  If a subject experiences the same AE on multiple 
occasions, the event will be counted once for each occurrence when reporting the number of 
AEs.  When reporting the number of subjects experiencing the events, a subject will only be 
counted once if they experience an event within the particular SOC or preferred term.  
Percentages will be based on the number of subjects in the safety population. 
AEs classified by MedDRA system organ class and preferred term will be summarized for each 
treatment group and overall, and incidence rates for AEs classified by MedDRA SOC and 
preferred term will be provided for each treatment group and overall. The incidence rate for a 
SOC or preferred term will be defined as the number of AEs divided by the person-time of 
exposure aggregated in each treatment group, and an analysis based on a Poisson regression 
model will be conducted to compare the rate of each type of event in the treatment groups. The 
proportion of subjects in each study arm experiencing each type of AE will be compared using 
a Fisher’s Exact test. 
Separate data listings will be provided for the following subsets: 
• 
Treatment-related AEs  
• 
AEs leading to study drug discontinuation 
• 
Grade 3-5 AEs which are defined as possibly, probably, or definitely related to rituximab 
• 
All Grade 3-5 AEs 
• 
Infections 
• 
Grade 3 and higher infections that occurred during the quarterly monitoring period 
• 
AEs occurring from informed consent and prior to treatment initiation 
8.3. 
Deaths and Serious Adverse Events 
8.3.1. 
Serious Adverse Events 
Serious adverse events (SAEs) and events requiring 24-hour reporting to the sponsor will be 
summarized by MedDRA SOC and preferred term in a manner analogous to what is described 
in Section 8.3. SAE summary tables by MedDRA SOC and preferred term will be provided by 
severity and relationship to study therapy as well. 
8.3.2. 
Deaths 
Any death that occurs while a subject is active in the main study (up to Week 48) will be 
collected and reported as a SAE, and any death that occurs after main study completion will 
only be reported as a SAE if the subject is active in quarterly monitoring for B cells and if the 
cause of death is determined to be a Grade 3 or higher infection. Additionally, an assessment 
of vital status approximately two years post randomization will be conducted via telephone or 
 
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review of public records or health registries (to which the subject has consented to share this 
data). Any deaths discovered from this review will be recorded on the Post-Study Follow-Up/ 
Week 104 Vital Status CRF page. 
Treatment-related mortality will be defined as death occurring  after randomization and before 
Week 48, and possibly, probably, or definitely resulting from study treatment.  Treatment-
related mortality can only be assessed if a death was reported as a SAE; relationship to study 
treatment was not collected for deaths recorded on the Post-Study Follow-Up/ Week 104 Vital 
Status CRF page.  As such, treatment-related mortality will be summarized at Week 48.  
All-cause mortality, defined as any death occurring after randomization, will be summarized at 
Week 48 and Week 104.    
In addition, time to all-cause mortality will be defined as the number of months post-
randomization until death from any cause. If death does not occur during the post- 
randomization study period (up to Week 104), then time to all-cause mortality will be set equal 
to Week 104 or the day of the Week 104 vital status telephone call, whichever occurred first. 
Appropriate censor variables will be defined to indicate whether the time-to variables represent 
event times or a censor times. Kaplan-Meier survival curves from each treatment arm with be 
compared using the log-rank test. 
Separate displays listing and summarizing death, including time to death and cause of death, 
will also be created 
8.4. 
Clinical Laboratory Evaluation 
Clinical laboratory measurements include serum chemistry and hematology. Subjects 
randomized prior to March 19, 2013 had serum chemistry and hematology results analyzed by 
their sites local lab. Data for these labs is collected on the Hematology and Chemistry CRF 
pages. Subjects randomized after March 19, 2013 have serum chemistry and hematology labs 
run by the central lab, ICON. Results will be converted to standardized units where possible. 
For numeric laboratory results, descriptive statistics of laboratory values and the change from 
baseline of laboratory values will be presented for each treatment group and overall.  For 
categorical laboratory results, the number and percentage of subjects reporting each result will 
be presented for each treatment group and overall.   
Laboratory data will be plotted to show patterns over time.  Summary statistics including 25th 
percentile, median, and 75th percentile will be plotted for each visit by treatment group.  Lines 
connecting individual subject results from subjects with grade 2 or higher values will be overlaid 
on each figure. For lab results that are not gradable, results from subjects with values outside 
of 2 *upper limit of normal or 0.5*lower limit of normal will be overlaid. Tests with qualitative 
results (such as “present” or “positive”) will not be plotted. Reference ranges from the central 
lab will be used. 
In addition, abnormal and clinically significant abnormalities will be summarized and listed 
separately. 
 
 
 
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8.5. 
Vital Signs, Physical Findings, and Other Observations Related to Safety 
8.5.1. 
Vital Signs 
Descriptive statistics of vital signs results and change from baseline of vital signs will be 
summarized for each treatment group and overall.  Data listings sorted by treatment group, 
subject, vital sign parameter, and time of assessment will be provided for vital signs 
measurements. Data will be plotted to show patterns over time.  Summary statistics including 
25th percentile, median, and 75th percentile will be plotted for each visit by treatment group. 
8.5.2. 
Physical Examinations 
Data listings will be provided for physical examination results and sorted by treatment group, 
subject, body system, and time of assessment. 
 
 
 
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9. 
OTHER ANALYSES 
9.1. 
Use of Medications 
9.1.1. 
Prior and Concomitant Medications 
Medications will be coded according to the World Health Organization (WHO) Drug Dictionary 
(version 2010.03).  Medications reported on the Prior/Concomitant Medications CRF will be 
categorized for analysis as prior or after study treatment by comparing the medication start and 
stop dates with the first and last dose of study medication dates.  Prior medications will have 
both the medication start and stop dates prior to the first dose of study medication date.  
Concomitant medications will have a medication start date on or after the first dose of study 
medication. Medications taken for the treatment of PAH will be summarizes separately, see 
Section 9.1.2 below. 
The number and percentage of subjects receiving prior and concomitant medications will be 
presented overall and by medication class.  When reporting the number of subjects receiving 
the medication, a subject will only be counted once if they ever received the medication within 
the medication class.  Percentages will be based on the number of subjects in the analysis 
population.   
9.1.2. 
Background PAH Medications 
Background PAH medications are recorded on the Background PAH Medications CRF page at 
Screening and when the medication is modified or a new medication is added for the treatment 
of PAH during the course of the study. Medications will be coded according to the World Health 
Organization (WHO) Drug Dictionary (version 2010.03). 
From the time of study entry, subjects are to remain on background PAH medical therapy with 
either a single agent or a combination of prostanoid, endothelin receptor antagonist, PDE-5 
inhibitor, and/or guanylate cyclase stimulators as per the entry criteria. Doses must remain 
stable through the week 24 primary endpoint visit. 
The type and total number of PAH medications that subjects are on at screening will be 
summarized by treatment group and overall.  
See Section 7.3.4, Addition of PAH medications, for more information on analyses related to 
background PAH medications. 
10. 
INTERIM ANALYSES AND DATA MONITORING 
The progress of the study will be monitored by the Data and Safety Monitoring Board (DSMB).  
The Autoimmune DSMB will be chartered to review safety data and to make recommendations 
regarding continuation, termination, or modification of the study.  The DSMB will formally 
review the safety data at least yearly.  The discontinuation of study treatment will also be 
periodically reported to the DSMB.  
In addition, safety data will be reviewed by the DSMB when an event occurs that is of sufficient 
concern to the National Institute of Allergy and Infectious Diseases (NIAID) medical monitor or 
protocol chair to warrant review, or when an event occurs that could contribute to a predefined 
stopping rule specified in the protocol.  
Findings will be reported to Institutional Review Boards (IRBs) and health authorities. 
 
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11. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL 
1. Section 7.2.2: Primary endpoint:   
a. Actual values rather than change from baseline in 6MWD will be used in the 
model. 
b. Remove baseline measures of 6MWD as covariate 
c. Use visit week rather than visit day 
d. Addition of quadratic term for visit week 
e. Remove limitations from musculoskeletal disease as a covariate. The number of 
“yes” occurrences is too sparse to inform the analysis.   
2. Section 7.3.3: Clinical worsening: Protocol says “worsening of 6MWD by > 20% and a 
decrease in functional class” vs. corrected “worsening of 6MWD by > 20% and an 
increase in functional class” 
3. Section 7.3.4: Analyses changed from using a logistic regression model to analyzing 
time to first change or addition of PAH medications. 
 
12. 
REFERENCES 
 
 
 
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13. 
APPENDICES  
13.1. Study Flow Chart 
FLOW DIAGRAM OF PROTOCOL 
 
Visit 1 
 
 
Screening  
Day -28 to -1 
 
Visit 2 
Baseline 
Day -7 to 0 
 
 
  
 
 
 
 
 
Visit 3 
Day 0 
 
 
 
  
 
 
 
Visit 4 
Week 2 
 
 
 
 
 
 
Assessments 5-10  
Weeks 4-24 
 
 
 
 
 
 
Assessments 11-16  
Weeks 28-48 
 
 
 
 
 
B Cell Recovery 
 
 
 
 
 
Assessment 17 
Week 104 
Screening:  Eligibility determined per assessments listed in Table 6.1. 
Baseline Evaluations: Baseline and Day 0 assessments (Table 6.1) may 
be combined into 1 visit as scheduling dictates so long as they are 
completed within 7 days of the first infusion 
1st Rituximab Infusion 
Subjects receive 1000 mg IV 
rituximab. 
Clinical and disease assessments (including exercise capacity [6MWD]), 
questionnaires, and laboratory assessments will be completed per Table 
6.1. 
 
For subjects who have not withdrawn consent and who have received 
any rituximab and B cells have not recovered, follow-up clinical care 
must include visits at least quarterly to monitor recovery of B cells.   
 
Randomize 60 subjects 
1st Placebo Infusion 
Subjects receive IV placebo.  
Pretreated with corticosteroids, 
2nd Rituximab Infusion 
Subjects receive 1000 mg IV 
rituximab.  
P
t
t d
ith
ti
t
id
2nd Placebo Infusion 
Subjects receive IV placebo.  
Pretreated with corticosteroids,  
di h
h d
i
d
Clinical and disease assessments, questionnaires, and laboratory 
assessments will be completed per Table 6.1. 
 
Two year post randomization assessment to be completed on vital status 
and treatment satisfaction for all subjects.  
 
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13.2. Schedule of Events 
Table 6.1, Schedule of Evaluations 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
 
 
Description 
Screen Base-
line1 
Day  
01 
Week  
2 
Week  
4 
Week 
8 
Week 
12 
Week 
16 
Week 
20 
Week 
24 
Week 
28 
Week 
32 
Week 
36 
Week 
40 
Week 
44 
Week 
48 
Week 
104 
Quar-
terly 
Un-
sched
-uled 
Visit window 
-28 
days 
-7  
days 
 
±4  
days 
±7  
days 
±7 
days 
±7  
days 
±7  
days 
±7  
days 
±7  
days 
±7  
days 
±7  
days 
±7  
days 
±7  
days 
±7  
days 
±7  
days 
 
±14 
days 
Type of Visit 
Clinic Clinic Clinic Clinic Clinic Phone Clinic Phone Phone Clinic Phone Phone Clinic Phone Phone Clinic Phone Clinic Clinic 
Clinical Draw (mL) 
21.5 
11 
2 
8 
11 
0 
11 
0 
0 
11 
0 
0 
11 
0 
0 
13 
0 
3 
3 
Research Draw (mL) 
0 
41 
0 
29  
29  
0 
33 
0 
0 
38  
0 
0 
34  
0 
0 
33  
0 
24 
33 
Visit Draw Total (mL) 
21.5 
52  
2 
37 
40  
0 
44 
0 
0 
49  
0  
0 
45  
0 
0 
46  
0 
27 
36 
General Assessments 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination& Vitals2 
X 
 
X22 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Vaccinations17 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NYHA Functional Class Assessment 
X 
 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24 
Right Heart Catheterization20 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Pulmonary Function Tests3 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
Oxygen Saturation4 
X 
 
X22 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
ECG5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chest X-Ray 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TB testing21 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6MWT 6,18 
X18 
 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24 
SF-36  
 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
X24 
SHAQ 
 
X 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24 
Digital Ulcer Count 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X24 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
Adverse Event Assessment 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X23 
X 
Post-infusion Phone Assessment7 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment of Vital Status26 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Treatment Satisfaction26 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Laboratory Assessments 
Hematology8 
X 
X 
 
X25 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
X 
X24 
CD19+ B cell count (local) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chemistry9 
X9 
X22 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
Infectious Disease Screen10 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy Test (if applicable)11 
X 
 
X22 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD19+ B Cell Studies12 (24mL) 
 
X 
 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
X 
X24 
Autoantibodies13  (4mL) 
 
X 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
X24 
 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
 
1 
Baseli
ne 
and 
Day 0 
assessments may be combined into one visit as scheduling dictates so long as they are completed within 7 days of the first rituximab infusion.  
2 Physical Examination: Full PE (including height, weight, vital signs, general appearance, skin, head/eyes/ears/neck/throat, respiratory/chest, cardiovascular, abdominal, neurological, 
lymph nodes, musculoskeletal/extremities) at Screen, Week 24, and Week 48. Abbreviated PE (including weight, vital signs, skin, respiratory/chest, cardiovascular, abdominal, lower 
extremity edema) at all other clinic visits.  
3 Pulmonary Function Tests: Height must be measured at time of PFTs. Full PFTs with spirometry, DLCO, and TLC at Screening. DLCO only at Weeks 24 and 48. Sites to send absolute 
values to Rho to convert for Hgb and altitude adjustments.  
4 Oxygen Saturation: forehead or ear probe must be used for saturations < 95% by finger probe at rest. Measure first at room air, then add/titrate oxygen if needed to maintain O2 saturation 
≥ 90%. The room air oxygen saturation measurement will be conducted at the discretion of the study physician. The measurement should not be performed if subject safety will be 
compromised.   
5 ECG: If not done with RHC (center-specific).  
6 6MWT: Subject should complete assessment with same amount of supplemental oxygen used to complete the Screening 6MWT unless safety will be impacted (if the 6MWD decreased 
from the Screening 6MWD by more than 20%, repeat the 6MWT within 7 days, but not on the same day). 
7 A member of the research staff will contact the subject within 24 hours after discharge from the infusion to assess for adverse events 
8 Hematology: CBC with differential count and platelet count (hematocrit, hemoglobin, platelet count, WBC, bands, eosinophils, lymphocytes, monocytes, and neutrophils). At Baseline, 
Week 48, and during Quarterly monitoring, CD19+ cells will also be enumerated. 
9 Chemistry: albumin, alkaline phosphatase (ALP), ALT/SGPT, AST/SGOT, BUN, creatinine, potassium, sodium, total and indirect bilirubin, total CO2, total protein, BNP/NT-proBNP. 
Note: BNP/NT-proBNP is not collected at screening.  
10 Infectious Disease Screen: HIV antibody, hepatitis B surface antibody and surface antigen, HCV antibody with HCV RNA ( PCR) if antibody positive.  
11 Pregnancy Test: For women of child-bearing potential a pregnancy test (urine or serum per institutional policy) must be obtained at screening and within 72 hours of each rituximab 
infusion.  
12 CD19+ B Cell Studies: Blood sent to Stanford Human Immune Processing Center for the Stanford Human Immune Monitoring Center.  
13 Autoantibodies: will include but are not limited to Anti-U1 RNP, anti-U3 RNP, anti-B23, anti-cardiolipin, anti-Th/To, anti-CENP-B, and RF. Frozen serum sent to University of 
Pittsburgh Immunoassay Core Laboratory.  
14 Serum Immunoglobulins: Quantitative Immunoglobulin Levels including IgG subclasses (frozen serum sent to Stanford Human Immune Processing Center for the Stanford Human 
Immune Monitoring Center).  
15 IL-1, IL-6, and Other Cytokine Assays: Frozen serum sent to Stanford Human Immune Processing Center for the Stanford Human Immune Monitoring Center.  
16 Rituximab: Per Protocol Section 5, Treatment of Subjects.  
17 Vaccinations: All immunizations must be completed at least 4 weeks prior to starting rituximab/placebo. Subject must be vaccinated with the pneumococcal vaccine at least 4 weeks 
prior to initiation of therapy, unless subject was vaccinated within 5 years of study entry. If vaccination occurred greater than 5 years prior to study entry, the subject must be 
revaccinated at least 4 weeks prior to initiation of therapy. Depending on the season, influenza vaccine should be administered at least 4 weeks prior to dosing with rituximab/placebo. 
Otherwise, it should be administered as late as possible in the flu season to still be effective after the last dose of rituximab/placebo. 
18 The 6MWT must be completed within 14 days of the first treatment visit (Day 0).  
19 HACA and PK Assays: HACA and rituximab serum level (PK). Batched and sent to Covance Laboratories. 
20 When completing the repeat RHC, the subject should be on the same amount of supplemental oxygen used during the Screening RHC unless safety will be impacted. The method of 
calculating cardiac output should be consistent with that used at the Screening RHC. 
21TB testing (PPD or QuantiFERON®-TB Gold In-Tube Test [QFT-G_IT]): unless performed within 30 days prior to screening and documented as negative in the subject’s records or 
unless subject is known to have a positive test and has documentation of appropriate therapy. 
22 If the Screening physical exam, oxygen saturation, and chemistry panel are performed within 7 days of Day 0, they do not need to be repeated during the Randomization/Baseline or Day 
0 visits unless clinically indicated. 
Serum Igs14 & IL-1, IL-6,& Other 
Cytokine Assays15 (5mL) 
 
X 
 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X24 
HACA and PK Assays19 (3-5mL) 
 
X 
 
 
 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
Study Medication  
Rituximab/Placebo16 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
23 Only infectious events ≥ NCI-CTCAE grade 3 will be reported. 
24 Required only if the unscheduled visit is due to an increase in SSc-PAH disease activity; see Section 6.5.8, Unscheduled Visit for further details. 
25 Hemoglobin, platelets, lymphocytes, neutrophils, and bands results from a hematology specimen drawn within 24 hours of the infusion must be reviewed prior to initiating the study 
drug infusion (see Section 5.3.6, Criteria for Withholding Rituximab Infusion).  
26 A member of the research staff will obtain verbal consent to conduct an assessment of vital status and treatment satisfaction approximately two years post randomization for each subject. 
If the subject cannot be reached, a review of public records or health registries should be completed.  
 
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Autoimmunity Centers of Excellence 
Statistical Analysis Plan 
Protocol No. ASC01 
08 March 2019 
 
14. 
ATTACHMENTS 
 
14.1. Scoring of the SF-36 
 
 
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