A Pilot Study to Evaluate the Safety and Efficacy of Oral Treprostinil in the Treatment of Calcinosis in Patients With Systemic Sclerosis
Phase: PHASE2
Conditions: Systemic Sclerosis, Calcinosis
Sponsor: Stanford University

1 
PROTOCOL 
STUDY TITLE: A Pilot Study to Evaluate the Safety and Efficacy 
of Oral Treprostinil in the Treatment of Calcinosis in Patients with 
Systemic Sclerosis 
STUDY DRUG: Oral Treprostinil 
SUPPORT PROVIDED BY: United Therapeutics 
SPONSOR INVESTIGATOR: Lorinda Chung, MD, MS 
SUB-INVESTIGATOR: Antonia Valenzuela, MD, MS 
INSTITUTION: Stanford University, Departments of Medicine, Division of 
Immunology and Rheumatology 
NCT02663895 
VERSION: 2.0 (December 1, 2016) 

2 
 
Research personnel and contact information 
 
Sponsor Investigator: 
Lorinda Chung, MD, MS 
Stanford University School of Medicine 
3801 Miranda Ave. 
Palo Alto VA Health Care System 
Palo Alto, CA 94304 
Phone: (650) 493-5000 ext. 62042 
Fax: (650) 849-1213 
E-mail: shauwei@stanford.edu 
 
Co-investigator: 
David Fiorentino, MD, PhD 
Stanford University, Department of Dermatology 
900 Blake Wilbur Dr. 
Stanford, CA  94305-5334 
Phone: (650) 498-6120 
Fax: (650) 723-7996 
Email: fiorentino@stanford.edu 
 
Kathryn J. Stevens, MD 
Associate Professor 
Radiology and Orthopedics Musculoskeletal Imaging 
Stanford University Medical Center 
300 Pasteur Dr., Room S-062A 
Stanford, CA  94305 
Phone (650) 725-8638 
Fax (650) 725-7296 
kate.stevens@stanford.edu 
 
 
Sub-investigators: 
Antonia Valenzuela, MD, MS 
Stanford University School of Medicine 
1000 Welch Road 
Suite 203 MC 5755 
Palo Alto, CA 94304 
Phone: (650) 7990560 
Fax: (650) 849-1213 
E-mail: antoniav@stanford.edu 
 
Kathleen Kolstad, MD 
Stanford University School of Medicine 
1000 Welch Road 
Suite 203 MC 5755 

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Palo Alto, CA 94304 
Fax: (650) 849-1213 
E-mail: kkolstad@stanford.edu 
 
Additional sub-sites: 
None 
 
Study Coordinator: 
, MD, MS 
Stanford University School of Medicine 
 
 
Palo Alto, CA 
 
Phone: 
 
Fax: 
 
E-mail: 
 
 
Pharmacy Contact: 
, PRM 
Stanford University Medical Center,
 
Stanford, CA 
 
Phone: 
 
Fax: 
 
Email: 
 
 
Research Agreement Contact: 
, Research Process Manager 
Stanford University School of Medicine 
 
Menlo Park, CA 
 
Phone: 
 
Fax: 
 
Email: 
 

4 
 
 
1. Background: 
Calcinosis cutis is the deposition of calcium in the skin and subcutaneous tissues. It is a common 
and potentially debilitating manifestation of systemic sclerosis (SSc), affecting almost one 
quarter of these patients. Several studies have found an association between calcinosis and 
vascular manifestations of SSc including digital ulcers (DU) [1] [2] and acro-osteolysis.[3] We 
confirmed this in a retrospective multi-center international cohort study of 5280 patients with 
SSc where DUs were the strongest predictor of calcinosis in multivariate analysis (OR 3.7, 
95%CI 2.6-5.3, p<0.0001). Interestingly, we also found a novel association between calcinosis 
and osteoporosis (OR 3.9, 95%CI 2.1-7.4, p<0.0001). 
 
Treprostinil delivered by continuous subcutaneous infusion was effective in both the healing and 
prevention of DU in patients with SSc in an open-label, single-center clinical trial of 5 (of 12) 
patients who were able to tolerate the medication.[4] A study of 148 subjects with DU showed 
that the administration of oral treprostinil up to 16 mg twice daily for 20 weeks was associated 
with a small but statistically insignificant reduction in net ulcer burden in comparison to 
placebo.[5] Additionally, preliminary observations in the Pulmonary Hypertension Assessment 
and Recognition of Outcomes in Scleroderma (PHAROS) registry have found that two patients 
with SSc-PAH and calcinosis treated with subcutaneous treprostinil for PAH experienced 
approximately 50% radiographic improvement in their calcinosis lesions after 6 months of 
therapy (Shapiro et al., unpublished data). 
 
We developed and validated a novel radiographic scoring system to assess the severity of 
calcinosis affecting the hands of patients with SSc that accounts for area coverage, density, and 
anatomic location. This scoring system is feasible and was found to have excellent intra- and 
inter-rater reliability with intra-class correlation coefficients (ICC) of .93 (.89-.97) and .89 (.86- 
.92), respectively.[6] 
 
Given that calcinosis is a frequent, debilitating complication of SSc associated with digital 
vascular ischemia with no effective therapies, a clinical trial, using novel outcome measures, 
testing the safety and efficacy of a powerful vasodilator for the treatment of calcinosis is 
warranted. 
 
2. Primary/Secondary objectives: 
Our main objective is to conduct an open-label proof-of-concept study with the following 
primary and secondary endpoints. 
Primary endpoints: 
• To assess the safety and tolerability of oral treprostinil using TID dosing in SSc patients 
with calcinosis affecting the hands. 
• To assess the efficacy of oral treprostinil in stabilizing the calcinosis burden or reducing 
the radiographic progression of calcinosis over one year. 
Secondary endpoints: 
• To assess the effect of oral treprostinil on the change in the Scleroderma Health 
Assessment Questionnaire, the Cochin Hand Functional Scale, SF-36, Mawdsley 
Calcinosis Questionnaire, Raynaud Condition Score, and patient and physician 
assessment of calcinosis severity over one year. 

5 
 
• To assess changes in blood flow using SPY perfusion machine following treatment with 
treprostinil at 1 year compared to baseline. 
• To assess changes in vascular and SSc-PAH associated biomarkers following treatment 
with treprostinil at 1 year compared to baseline. 
• To assess bone markers P1NP and C-telopeptide levels at baseline and at 1 year 
• To assess baseline cortical area and cortical porosity measured by HR-pQCT in SSc 
patients with calcinosis. 
• To assess osteoclastogenesis using PBMCs cultures at baseline and at 1-year 
 
3. Hypothesis: 
We hypothesize that calcinosis is a result of microvascular injury and ischemic damage, and that 
therefore treprostinil may be beneficial in the treatment of calcinosis in patients with SSc. 
 
4. Study design: 
This prospective open-label trial will enroll 12 patients with SSc and at least one calcinotic 
lesion of the hands that is palpable on physical examination and also measureable on hand 
radiographs. Each subject will undergo a screening evaluation 4 weeks before treatment with the 
study drug is initiated. Each subject will receive treprostinil 0.125 mg TID orally, which will be 
increased by 0.125 mg TID every 3 to 4 days as tolerated. Follow-up evaluations will be 
performed every 3 months over a 12-month period of time. 
 
5. Study population: 
The population for this study will consist of adult SSc patients with evidence of at least one 
calcinotic lesion of the hands that is palpable on physical examination and also measureable on 
hand radiographs. 
 
Key inclusion criteria: 
• Signed written informed consent 
• Age > 18 years of age 
• Diagnosis of limited or diffuse cutaneous systemic sclerosis (SSc) according to the 
revised 2013 ACR/EULAR classification criteria for SSc[7] 
• Radiological and physical examination evidence of at least one subcutaneous calcium 
deposition in the hands that is clinically apparent as part of routine clinical care. 
• If female of childbearing potential, the patient must have a negative pregnancy test at 
screening and baseline visits 
• Oral corticosteroids (≤ 10 mg/day of prednisone or equivalent) and NSAIDs are 
permitted if the patient is on a stable dose regimen for ≥ 2 weeks prior to screening and 
throughout the study 
• Calcium channel blockers, alpha-1-antagonists, ACE-inhibitors, angiotensin receptor 
blockers, and protein-pump inhibitors are permitted as long as the doses are stable for 4 
weeks prior to screening and throughout the study 
• Women of childbearing potential must agree to use adequate contraception when sexually 
active with any combination of at least 2 effective methods of birth control (except for 
women who have a partner who is sterile, i.e. due to vasectomy) 

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Key Exclusion Criteria: 
• Rheumatic disease other than SSc 
• Patients with pulmonary arterial hypertension (PAH), NYHA Class III or IV, as 
determined by right heart catheterization or on PAH approved medications for PAH 
• Patients with moderate or severe hepatic impairment (Child Pugh Class C), or 
transaminase elevation (ALT or AST) > 3 x the upper limit of normal at screening visit 
• Patients with diverticulosis 
• Hemoglobin < 75% of the lower limit of the normal range 
• Systolic blood pressure < 95 mmHg or diastolic blood pressure < 50 mmHg 
• 
Patients who are hemodynamically unstable, or have acute renal, cardiac or pulmonary 
failure, or any life-threatening condition. 
• 
Concurrent malignancy except non-melanoma skin cancers 
• Patients receiving specific (sildenafil, tadalafil) or unspecific phosphodiesterase-5 
inhibitors (dipyridamole, theophylline), endothelin receptor antagonists, prostanoids, 
riociguat, or NO donors (nitrates) within 4 weeks of screening 
• Patients receiving bisphosphonates, warfarin, colchicine, minocycline, intravenous 
immunoglobulins, or biological agents including abatacept or rituximab within 4 weeks 
of screening 
• Patients receiving local treatments for calcinosis of the hands including surgical removal 
or intralesional steroid injections within 12 weeks of screening or throughout the study. 
• Patients who have participated in another clinical trial of an investigative agent within 30 
days of screening (or 5 half-lives of the investigational drug, whichever is longer) 
• Pregnant or nursing women 
• Patients with a history of drug or alcohol abuse within 6 months of screening 
• Any medical condition that, in the opinion of the investigator, might interfere with the 
subject’s participation in the study or poses an added risk for the subject 
• Inability to comply with study and follow-up procedures 
 
6. Description of the treatment: 
United Therapeutics will provide treprostinil labeled for investigational use. The 
Sponsor/Investigator of the trial will ensure maintenance of complete and accurate records of the 
receipt, dispensation, and disposal or return of all trial drug in accordance with Title 21 Code of 
Federal Regulations (C.F.R.), Part 312.57 and 312.62 and United Therapeutics requirements. 
 
Treprostinil 
Treprostinil (Orenitram®) is a prostacyclin vasodilator indicated for the treatment of pulmonary 
arterial hypertension (WHO Group 1) to improve exercise capacity. The chemical name of 
Orenitram® is Acetic acid, 2-[[(1R,2R,3aS,9aS)-2,3,3a,4,9,9a-hexahydro-2-hydroxy-1-[(3S)-3- 
hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]-, complexed with 2,2’-iminobis[ethanol] (1:1). The 
molecular formula is C23H34O5C4H11NO2, and its molecular weight is 495.65. Orenitram® is an 
extended release osmotic tablet for oral administration, and it is formulated as the diolamine salt 
of treprostinil, a tricyclic benzindene analogue of prostacyclin. It is available in the following 
four strengths: 0.125 mg (white tablet imprinted with UT 0.125), 0.25 mg (green tablet imprinted 
with UT 0.25), 1 mg (yellow tablet imprinted with UT 1), and 2.5 mg (pink tablet imprinted with 
UT 2.5). The formulations also contain xylitol, maltodextrin, sodium lauryl sulfate, magnesium 

7 
 
stearate, cellulose acetate, triethyl citrate, polyvinyl alcohol, titanium dioxide, polyethylene 
glycol, and talc. In addition tablets may contain colorants FD&C Blue #2, iron oxide yellow, and 
iron oxide red. The imprinting ink contains shellac glaze, ethanol, isopropyl alcohol USP, iron 
oxide black, n-butyl alcohol, propylene glycol, and ammonium hydroxide. 
 
Mechanism of Action 
The major pharmacologic actions of treprostinil are direct vasodilation of pulmonary and 
systemic arterial vascular beds, inhibition of platelet aggregation, and inhibition of smooth 
muscle cell proliferation. 
 
Storage 
Treprostinil must be stored at 25°C (77°F); excursions 15°C to 30°C (59°F to 86°F). For further 
details, see the Investigator Brochure and the Orenitram Package Insert.[8] 
 
Dosage and Administration 
Initiate treprostinil at 0.125 mg TID (every 8 ± 2 h), with dose escalation of an additional 0.125 
mg TID every 3 to 4 days as tolerated. No dose changes will be allowed for 5 days before the 
month 12 visit. [9] Give with food, swallow tablets whole, and use only intact tablets; do not 
crush, split, or chew. The maximum dose, dose escalation or titration will be done at the 
Investigator’s discretion based upon individual subject tolerability. Patients with mild hepatic 
impairment (Child Pugh Class A) should initiate treprostinil at 0.125 mg TID, and increment at 
0.125 mg TID every 6 to 8 days.[8] 
 
Criteria for Treatment Discontinuation 
Treatment will be discontinued in the following circumstances: 
• Patients who wish to withdraw from further participation 
• Patients with a serious or life-threatening adverse event that in the opinion of the 
investigator is directly attributable to the study drug 
• The patient deviated from the protocol 
• The patient’s behavior is likely to undermine the validity of his / her results 
• Patients who have a positive pregnancy test during the study 
• Patients who develop any condition that in the opinion of the investigator could be 
worsened by further treatment with the study drug 
 
Criteria for Stopping Rules 
The study may be stopped at any time if, in the opinion of the Investigator and / or Sponsor, 
continuation of the study represents a serious medical risk to the subjects. This may include, but is 
not limited to, the presence of serious, life-threatening, or fatal adverse events or adverse events that 
are unacceptable in nature, severity, or frequency. The Sponsor reserves the right to discontinue the 
study for any reason at any time. 
 
Down titration of study medication 
Following the 12-month study assessments, patients will be titrated off study drug. Subjects 
discontinuing treprostinil, upon exiting the study at the End of Study visit or at early termination, 
who are receiving doses greater than 2 mg TID, should be tapered in 25% increments every 24 hours 
based on their End of Study dose prior to discontinuation. Once a dose of 2 mg TID or less is 

8 
 
reached, the subject can discontinue treprostinil. For example, a subject who reached an End of Study 
dose between 6 and 12 mg TID would decrease by 2 mg TID every 24 hours, and a subject who 
reached an End of Study dose between 2 and 6 mg TID would decrease by 1 mg TID every 24 hours. 
 
7. Estimated duration: 14 months 
 
8. Time and events schedule and description of assessments: 
Assessments: 
Screening Visit (1 month prior to treatment): 
• Informed consent 
• Review of inclusion and exclusion criteria 
• Complete medical history including scleroderma diagnosis 
• Review of concomitant medications 
• Complete physical examination including examination of calcinosis, digital ulcers, vital 
signs, height/weight, and Modified Rodnan skin score (MRSS) 
• Electrocardiogram (EKG) 
• Laboratory tests: CBC with differential, comprehensive metabolic panel, and urinalysis 
• Urine pregnancy test (females of child bearing potential only) 
• Review of adverse events 
Baseline Visit (0 month): 
• Review of concomitant medications 
• Complete physical examination with vital signs, and MRSS 
• Examination of calcinosis and digital ulcers 
• Laboratory tests: CBC with differential, comprehensive metabolic panel, and urinalysis 
• Urine pregnancy test (females of child bearing potential only) 
• Questionnaires: SHAQ, Cochin hand functional scale, SF-36 assessment of health status, 
Mawdsley Calcinosis Questionnaire, Physician/Patient Global Assessment of calcinosis 
severity, and Raynaud’s Condition Score. 
• Review of adverse events 
• Study drug dispense/return 
• Radiological assessment of calcinosis 
• Skin biopsy 
• Blood for biomarker analyses 
• SPY assessment of superficial blood flow 
• Anthropometric measurements 
• High resolution peripheral quantitative computed tomography (HRpQCT) 
• Handgrip strength measurement 
At 3 months visit: 
• Review of concomitant medications 
• Examination of calcinosis and digital ulcers 
• Urine pregnancy test (females of child bearing potential only) 
• Questionnaires: SHAQ, Cochin hand functional scale, SF-36 assessment of health status, 
Mawdsley Calcinosis Questionnaire, Physician/Patient Global Assessment of calcinosis 
severity, and Raynaud’s Condition Score. 

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• Blood for biomarker analyses 
• Review of adverse events 
• Study drug dispense/return 
At 6 months visit: 
• Review of concomitant medications 
• Examination of calcinosis and digital ulcers 
• Complete physical examination with vital signs, and MRSS 
• Laboratory tests: CBC with differential, comprehensive metabolic panel, and urinalysis 
• Urine pregnancy test (females of child bearing potential only) 
• Questionnaires: SHAQ, Cochin hand functional scale, SF-36 assessment of health status, 
Mawdsley Calcinosis Questionnaire, Physician/Patient Global Assessment of calcinosis 
severity, and Raynaud’s Condition Score. 
• Review of adverse events 
• Study drug dispense/return 
At 9 months visit: 
• Review of concomitant medications 
• Examination of calcinosis and digital ulcers 
• Urine pregnancy test (females of child bearing potential only) 
• Questionnaires: SHAQ, Cochin hand functional scale, SF-36 assessment of health status, 
Mawdsley Calcinosis Questionnaire, Physician/Patient Global Assessment of calcinosis 
severity, and Raynaud’s Condition Score. 
• Review of adverse events 
• Study drug dispense/return 
At 12 months visit: 
• Review of concomitant medications 
• Examination of calcinosis and digital ulcers 
• Urine pregnancy test (females of child bearing potential only) 
• Questionnaires: SHAQ, Cochin hand functional scale, SF-36 assessment of health status, 
Mawdsley Calcinosis Questionnaire, Physician/Patient Global Assessment of calcinosis 
severity, and Raynaud’s Condition Score. 
• Review of adverse events 
• Study drug dispense/return 
• Complete physical examination with vital signs, and MRSS 
• Laboratory tests: CBC with differential, comprehensive metabolic panel, and urinalysis 
• Radiological assessment of calcinosis 
• Skin biopsy 
• Blood for biomarker analyses 
• SPY assessment of superficial blood flow 
• EKG 
• High resolution peripheral quantitative computed tomography (HRpQCT) 
At 13-months visit: 
• Review of concomitant medications 
• Examination of calcinosis and digital ulcers 

10 
 
• Review of adverse events 
• Study drug return 
• Complete physical examination with vital signs 

11 
 
Study Flowchart: 
Study phase 
Pre- 
treatment 
phase 
Treatment phase 
Follow-up 
phase 
Visit 
Screening 
Baseline 
1 
2 
3 
End 
Follow-up 
Month 
-1 
0 
3 
6 
9 
12 
13 
Informed consent 
X 
Inclusion/exclusion criteria 
X 
Medical history including SSc 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
X 
X 
X 
X 
X 
Examination of calcinosis and 
digital ulcers 
X 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
X 
Height/Weight 
X 
EKG 
X 
X 
Laboratory tests* 
X 
X 
X 
X 
Urine pregnancy test 
X 
X 
X 
X 
X 
X 
Radiological assessment of 
calcinosis 
X 
X 
Scleroderma Health 
Assessment Questionnaire 
(SHAQ) 
X 
X 
X 
X 
X 
Cochin Hand Functional Scale 
X 
X 
X 
X 
X 
Mawdsley Calcinosis 
Questionnaire 
X 
X 
X 
X 
X 
SF-36 assessment of health 
status 
X 
X 
X 
X 
X 
Raynaud’s Condition Score 
X 
X 
X 
X 
X 
Physician/patient global 
assessment of calcinosis 
severity 
X 
X 
X 
X 
X 
MRSS 
X 
X 
X 
X 
Skin biopsies 
X 
X 
Blood for biomarker analyses 
X 
X 
X 
PBMC 
X 
X 
P1NP and C-telopeptide 
X 
X 
SPY 
X 
X 
XtremeCT II scans 
X 
X 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
Study drug dispense/return 
X 
X 
X 
X 
X 
X 
*CBC, CMP, UA 

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Description of assessments: 
 
Medical history including SSc: Significant past or present illnesses, current prescription or 
nonprescription medications (including vitamins and herbal products), and history of allergies or 
idiosyncratic responses to drugs should be noted. 
 
Physical examination: A complete physical examination will be conducted by a physician at 
Screening, 6 months, and at end of study. Any significant changes to the subject’s medical 
condition, physical examination, and concomitant medications should be documented throughout 
the course of the study. Any untoward medical experience should be recorded as an adverse 
event. 
 
Examination of calcinosis and digital ulcers: Investigator will perform a thorough clinical 
examination looking for calcinotic deposits at each visit. We will use case report forms with 
picture images of the hands to record calcinosis and digital ulcers at each evaluation (Appendix 
A). We will define digital ulcer as an area with visually discernable depth and a loss of 
continuity of epithelial coverage in the volar aspect of the finger and distal from the proximal 
interphalangeal joints, which could be denuded (active) or covered by a scab or necrotic tissue 
(indeterminate). At each visit, the status of each digital ulcer will be rated as “A,” a current 
active digital ulcer, “H”, a completely healed ulcer, or “I,” an ulcer with indeterminate status, as 
previously defined.[10] 
 
Vital Signs: Systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature 
(°C) will be measured prior to assessments and after sitting for 5 minutes at screening, baseline, 
6 months, and 12 months. Vital signs should also be assessed in the case of abnormal clinical 
signs and symptoms. 
 
Electrocardiogram (ECG): Twelve-lead ECGs will be recorded after at least 5 minutes rest in the 
semi-recumbent position at screening and at the end of the study at 12 months. Recordings 
should include lead II as a rhythm strip and contain at least 5 QRS complexes. ECG parameters 
collected (after at least 5 minutes rest) include heart rate, and PR interval, QT interval, QRS 
duration and any clinically significant abnormalities. 
 
Laboratory tests: Blood and urine samples for the measurement and evaluation of CBC, CMP, 
and urinalysis (UA) will be collected at screening, 6 months and at the end of the study, and 
analyzed at Stanford laboratory. 
 
Urine pregnancy test: urine beta-HCG will be measured in all female in reproductive age at all 
visits. 
 
Radiological examination of calcinosis: Patients will have plain radiographs of the hands at 
baseline and at 1 year. Dr. Kathryn Stevens and Dr. Lorinda Chung will serve as the blinded X- 
ray assessors. 

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Scleroderma Health Assessment Questionnaire (SHAQ): The SHAQ is a patient self- 
administered instrument, which has been previously validated in SSc and demonstrates 
meaningful clinical changes in the course of the disease over time. It is comprised of the HAQ- 
DI, a self-administered 20-question instrument that assesses a patient’s level of functional ability 
and five scleroderma visual analogue scale (VAS) measurements to evaluate symptoms specific 
to SSc (Appendix B). [11-13] 
 
Cochin Hand Function Scale (CHFS): CHFS is a questionnaire derived from 18 validated 
questions to assess functional disability and handicap due to hand involvement in rheumatoid 
arthritis. Each answer is scored on a scale of 0 (no difficulty) to 5 (impossible to do), with a 
maximum score of 90. A higher score indicates worse disability or handicap. The CHFS has 
been demonstrated as a reliable and valid assessment of hand function at the activity level in 
persons with SSc (Appendix C).[14, 15] 
 
Mawdsley Calcinosis Questionnaire: The content of this questionnaire is patient-generated and 
includes 17 questions related to the impact of calcinosis rated from 0 (no limitation) to 10 (worst 
limitation possible) (Appendix D). 
 
The Medical Outcomes Study 36-Item Short Form Health Survey (SF-36): The SF-36 is one of 
the most widely used instruments to assess quality of life in patients with systemic illnesses. It is 
a self-administered questionnaire covering eight areas: physical function, physical role, bodily 
pain, general health, vitality, social function, emotional role, and mental health. For each area, 
the score ranges from 0 (poorer health status) to 100 (better health status) (Appendix E). [16-24] 
 
Patient and Physician global assessments: Patients and physicians will each independently rate 
the severity of calcinosis on a 10 cm VAS. The term “severity” will be used to measure the 
extent of disease activity and associated disability or discomfort the patient experiences during 
the indicated time period (Appendix F).[25] 
 
Raynaud’s Condition Score (RCS): We will measure changes in Raynaud’s phenomenon (RP) by 
using the RCS, a self-assessment of RP activity using a 0–10 ordinal scale. The RCS 
incorporates the cumulative daily frequency, duration, severity, and impact of RP attacks 
(Appendix G).[25] 
 
Modified Rodnan Skin Score (mRSS): The mRSS measures skin tightness and is the sum 
of scores from 17 surface anatomic areas (fingers, hands, forearms, arms, feet, legs, and thighs 
bilaterally, and face, chest, and abdomen singly) rated on a 0–3 scale (0=normal skin; 1=mild 
thickness; 2=moderate thickness; 3=severe thickness with inability to pinch the skin into a fold). 
The skin score will be the sum of the individual skin assessment scores and ranges from 0 (best 
possible outcome) to 51 (worst possible outcome) (Appendix H). [26-28] 
 
Skin biopsies: We will collect two side-by-side skin samples to assess for vascular changes on 
histopathology and gene expression changes following treatment with treprostinil: one at 
baseline and one at the end of the study. This part of the study will be optional for participating 
patients. Skin biopsies will be obtained from a standard site on the forearm (extensor surface of 
forearm approximately 10 cm proximal to the olecranon, which is chosen for its uniform 

14 
 
involvement in patients with SSc). Two adjacent 5 mm punch biopsies (taken full thickness to 
subcutaneous fat) will be harvested from the site. This involves selecting an area to biopsy and 
wiping the skin with alcohol. Next, 1-2 cc of lidocaine with 1:100,000 epinephrine is introduced 
into the skin using a 30G needle. Two punch biopsies will then be taken and processed as 
described in appendix I. Next, the wound will be sutured with 1-2 4.0 nylon sutures. Wounds 
will be dressed with polysporin and a bandage. Verbal wound care instructions will be given. 
The first biopsy will be bisected: one half will be flash frozen in liquid nitrogen and half will be 
embedded in paraffin. The second biopsy will also be bisected: one half will be placed in RNA 
later, and the other half will be placed in a cryo-tube. (See appendix I for details) 
 
Research blood: We will collect serum and peripheral blood samples to assess changes in 
vascular and SSc-associated biomarkers following treatment with treprostinil. Blood samples for 
the assessment of biomarkers will be drawn at baseline, 3 months, and end of study visit. Please 
see appendix for a complete list of biomarkers (Appendix J). 
 
P1NP and C-telopeptide: Procollagen Type I Intact N Terminal Propeptide (P1NP), a marker of 
bone formation, and C-telopeptide, a marker of bone resorption, will be measured at baseline and 
12 months to provide information about the rate of bone turnover, and to determine whether 
patients with calcinosis have high turnover rates. 
 
PBMCs: PBMCs will be obtained for each participant at baseline to assess for abnormal 
osteoclastogenesis. A recent publication investigated whether acro-osteolysis (bone resorption of 
distal phalanges), a clinical feature often associated with digital ulcers and calcinosis[3], was 
associated with abnormal osteoclastogenesis in SSc patients. The authors found that hypoxia 
may enhance the formation of osteoclasts particularly in SSc patients with acro-osteolysis. We 
hypothesize that SSc patients with calcinosis will similarly demonstrate increased 
osteoclastogenesis, resulting in enhanced propensity to osteoporosis/bone loss. [29] We will 
obtain PBMCs at baseline and 12 months. 
 
SPY: The SPY Near-Infrared Perfusion Assessment System (distributed by LifeCell Corp., 
Branchburg, N.J.; manufactured by Novadaq Technologies Inc., Richmond, BC, Canada) is an 
imaging technology that utilizes indocyanine green (ICG), and allows real-time visual 
assessment of superficial blood flow.[30] 
 
XtremeCT II scan: The latest-generation high-resolution peripheral quantitative computed 
tomography (HR-pQCT) XtremeCT II scan is a novel technology with dramatically improved 
spatial resolution. It is able to define the cortical and trabecular surfaces of the bones in a three- 
dimensional fashion, and therefore provides information on bone microarchitecture as well as 
bone density. The XtremeCT II machine at Stanford University is one of 10 in the United States. 
We will perform HR-pQCT at baseline and at 12 months to assess for changes in cortical area 
and cortical porosity. 
 
Safety: During the study, the primary assessment of safety will be changes in vital signs, clinical 
laboratory parameters, EKG, and the development of adverse events. 
 
9. Safety monitoring and reporting 

15 
 
Definitions: 
Adverse Event (AE): An AE is any untoward medical experience occurring to a subject during a 
clinical trial whether or not it is related to the study drug. An AE may include an intercurrent 
illness, injury, or any other concomitant impairment of the subject’s health, as well as abnormal 
laboratory findings if deemed to have clinical significance. AEs may also include worsening of 
an existing symptom or condition or post-treatment events that occur as a result of protocol- 
mandated procedures. 
 
Severe Adverse Event (SAE): A SAE is an AE occurring at any dose that results in any of the 
following outcomes: 
• Death 
• A life-threatening AE 
• Inpatient hospitalization or prolongation of existing hospitalization 
• A persistent or significant disability / incapacity 
• A congenital anomaly / birth defect 
 
In addition, 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 require medical / surgical intervention to prevent one of the outcomes 
listed above. Examples of such medical events include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in 
hospitalization, or the development of drug dependency or drug abuse. Life threatening means that 
the subject was, in the view of the Investigator, at immediate risk of death from the event as it 
occurred. It does not mean that the event, had it occurred in a more severe form, might have caused 
death. 
 
Reporting Responsibilities: 
In the event of an adverse event, the first concern will be for the safety of the patients. 
Investigators are required to collect and document all adverse events (AEs) and non-related 
serious adverse events (SAEs). All SAEs, regardless of expectedness or causality, must be 
reported to the Sponsor by fax (+ 1 919-313-1297 or other appropriate number) within 24 hours 
of awareness. A completed SAE report form along with any relevant hospital records and 
autopsy reports should be faxed to the Drug Safety Department at United Therapeutics 
Corporation. A follow-up SAE report form must be forwarded to the Drug Safety Department at 
United Therapeutics Corporation within 48 hours of the receipt of any new / updated 
information. The Investigator must also promptly notify their Investigational Review Board 
(IRB) or Ethics Committee (EC) of the SAE, including any follow-up information, in accordance 
with applicable national regulations and guidelines set forth by the IRB or EC. All documents 
related to AEs (serious, non serious, related, or not) will be readily available for review, should 
the need arise. 
 
An AE or SAE occurring during the study must be documented in the subject’s source 
documents and on the appropriate CRF page. Information relating to the AE such as onset and 
cessation date and times, intensity, seriousness, relationship to study drug, and outcome is also to 
be documented in the CRF. Where possible, AEs should be recorded using standard medical 
terminology. If several signs or symptoms are clearly related to a medically defined diagnosis or 

16 
 
syndrome, the diagnosis or syndrome should be recorded on the CRF page, not the individual 
signs and symptoms. 
 
All AEs should be followed until either resolution (or return to normal or baseline values), until 
they are judged by the Investigator to no longer be clinically significant, or for at least 4 weeks if 
the AE extends beyond the final visit. All SAEs that occur during the study will be followed until 
resolution, death, or the subject is lost to follow-up even if they are ongoing more than 4 weeks 
after completion of the final visit. Supplemental measurements and / or evaluations may be 
necessary to investigate fully the nature and / or causality of an AE or SAE. This may include 
additional laboratory tests, diagnostic procedures, or consultation with other healthcare 
professionals. CRF pages should be updated. 
 
10. Statistical Considerations 
Statistical analysis: 
Descriptive statistics and frequency distributions of all variables of interest will be reported as 
proportions (%) for categorical variables and as mean ± standard deviation or median (range) for 
continuous variables. Baseline to 12-month differences and 95% confidence intervals will be 
calculated for scored outcomes (x-ray score, MRSS, physician’s global assessment by VAS, and 
quality of life measurements). For the primary efficacy endpoint, mean change and standard 
deviation in calcinosis burden assessed by radiograph from baseline to 12-month visit, we will 
use Student’s t-test. The mean rate of change of calcinosis in radiograph will be calculated with 
the following formula: (Year 1 XR score – Baseline XR score)/time. XR score is defined as: sum 
of scores for 22 weighted areas affecting each hand: %area coverage (0-100) X density (1-3) X 
weight for each area.[6] 
 
Sample size calculation: 
We will need 9 paired radiographs (9 patients) at 1-year to provide >80% power to detect a mean 
change in x-ray score of 12.0 (equivalent to a minimally significant change in score of 25%) with 
a SD of 8.2 with moderate correlation r=0.5 using a two-sided test at alpha 0.05 level. We 
estimate a 15% dropout rate based on prior clinical trials. Hence, the total number of subjects 
needed to obtain 9/0.85 subjects who complete the study is 11 subjects. Also, due to the small 
sample size, we plan to use Wilcoxon signed rank test to analyze the effects. Thus, we anticipate 
losing about 10% of power due to using a non-parametric test. As a result, the sample size is 
further adjusted to 11/0.9 = 12. Statistical significance will be defined as p ≤ 0.05. 

17 
 
Appendix A. CRF for assessment of digital ulcers and calcinosis 
 
 
 
 
 

18 
 
Appendix B. Scleroderma Health Assessment Questionnaire (SHAQ) 
 
In this section we are interested in learning how your illness affects your ability to function in 
daily life.  Please feel free to add comments. 
 
Please check the one response that best describes your usual abilities 
 
IN THE PAST SEVEN DAYS: 
Without 
ANY 
difficulty 
With 
SOME 
difficulty 
With 
MUCH 
difficulty 
UNABLE 
to do 
DRESSING & GROOMING 
Are you able to: 
- Dress yourself, including tying 
  
 
  
 
  
 
  
 
shoelaces and doing buttons 
- Shampoo your hair? 
  
 
  
 
  
 
  
 
ARISING 
Are you able to: 
- Stand up from an armless 
  
 
  
 
  
 
  
 
straight chair? 
- Get in and out of bed? 
  
 
  
 
  
 
  
 
EATING 
Are you able to: 
- Cut your meat? 
  
 
  
 
  
 
  
 
- Lift a full glass to your mouth? 
  
 
  
 
  
 
  
 
- Open a new milk carton? 
  
 
  
 
  
 
  
 
WALKING 
Are you able to: 
- Walk outdoors on flat ground? 
  
 
  
 
  
 
  
 
- Climb up five stairs? 
  
 
  
 
  
 
  
 

19 
 
Please check any AIDS or DEVICES that you usually use for any of these activities: 
  
 
Cane 
  
 
  
 
Walker 
  
 
  
 
Crutches 
  
 
  
 
Wheelchair 
  
 
Devices for dressing (button hook, zipper pull, etc.) 
Built up or special utensils 
Special or built-up chair 
Other (specify: 
) 
 
 
Please check any categories for which you usually need ASSISTANCE FROM ANOTHER 
PERSON: 
  
 
Dressing and grooming 
  
 
  
 
Arising 
  
 
Eating 
Walking 
 
 
 
 
 
 
 
 
Patient Signature 
Date 

20 
 
 
 
 
Are you able to: 
Without 
ANY 
difficulty 
With 
SOME 
difficulty 
With 
MUCH 
difficulty 
UNABLE 
to do 
 
- Wash and dry your entire body? 
- Take a tub bath? 
  
 
  
 
  
 
  
 
  
 
  
 
  
 
  
 
- Get on and off the toilet? 
  
 
  
 
  
 
  
 
Are you able to : 
- Reach and get down a 5 pound 
object (such as a bag of sugar) 
from just over your head? 
  
 
  
 
  
 
  
 
- Bend down and pick up clothing 
off the floor? 
  
 
  
 
  
 
  
 
Are you able to: 
- Open car doors? 
  
 
  
 
  
 
  
 
- Open jars that have been 
previously opened? 
  
 
  
 
  
 
  
 
- Turn faucets on and off? 
  
 
  
 
  
 
  
 
Are you able to: 
- Run errands and shop? 
  
 
  
 
  
 
  
 
- Get in and out of a car? 
  
 
  
 
  
 
  
 
- Do chores such as vacuuming or 
  
 
  
 
  
 
  
 
yardwork? 
 
 
Please check any AIDS or DEVICES that you usually use for any of these activities: 
  
 Raised Toilet Seats 
  
 Bathtub Bar 
  
 Bathtub Seat 
  
 Long-Handled Appliances for Reach 
  
 Jar Opener (for jars previously opened) 
  
   Long-Handled Appliances in Bathroom 
  
   Other (specify: 
) 

21 
 
Please check any categories for which you usually need HELP FROM ANOTHER PERSON: 
  
 
Hygiene 
  
 
Gripping and Opening Things 
  
 
Reach 
  
 
Errands and Chores 
 
 
 
 
Patient Signature 
Date 
 
 
 
We are also interested in learning whether or not you are affected by pain because of your 
illness. You do not have to answer the questions that do not apply to you. 
 
How much pain have you had because of your illness IN THE PAST WEEK ? 
PLACE A MARK ON THE LINE TO INDICATE THE SEVERITY OF THE PAIN. 
 
NO PAIN 
VERY SEVERE 
PAIN 
├────────────────────────────────────────────────┤ 
0 
100 
 
IN THE PAST WEEK, how much have your intestinal problems interfered with 
your daily activities? 
PLACE A MARK ON THE LINE TO INDICATE THE LIMITATION OF ACTIVITY. 
 
INTESTINAL PROBLEMS 
VERY SEVERE 
DO NOT LIMIT ACTIVITIES 
LIMITATION 
├────────────────────────────────────────────────┤ 
0 
100 
 
IN THE PAST WEEK, how much have your breathing problems interfered with 
your daily activities? 
PLACE A MARK ON THE LINE TO INDICATE THE LIMITATION OF ACTIVITY. 
 
BREATHING PROBLEMS 
VERY SEVERE 
DO NOT LIMIT ACTIVITIES 
LIMITATION 
├────────────────────────────────────────────────┤ 
0 
100 
 
 
IN THE PAST WEEK, how much has Raynaud’s interfered with your daily activities? 
PLACE A MARK ON THE LINE TO INDICATE THE LIMITATION OF ACTIVITY. 
 
RAYNAUD’S DOES 
VERY SEVERE 

22 
 
NOT LIMIT ACTIVITIES 
LIMITATION 
├────────────────────────────────────────────────┤ 
0 
100 
 
IN THE PAST WEEK, how much have your finger ulcers interfered with your 
daily activities? 
PLACE A MARK ON THE LINE TO INDICATE THE LIMITATION OF ACTIVITY. 
 
FINGER ULCERS 
VERY SEVERE 
DO NOT LIMIT ACTIVITIES 
LIMITATION 
├────────────────────────────────────────────────┤ 
0 
100 
Overall, considering how much pain, discomfort, limitations in your daily life and 
other changes in your body and life, how severe would you rate your disease today? 
PLACE A MARK ON THE LINE TO INDICATE THE LIMITATION OF ACTIVITY. 
 
NO DISEASE 
VERY SEVERE 
LIMITATION 
├────────────────────────────────────────────────┤ 
0 
100 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patient Signature 
Date 

23 
 
Appendix C. Cochin Hand Functional Scale 
 
Answers to the questions 
0 = yes, without difficulty 
1 = yes, with a little difficulty 
2 = yes, with some difficulty 
3 = yes, with much difficulty 
4 = nearly impossible to do 
5 = impossible to do 
 
In the kitchen 
1. Can you hold a bowl? 
2. Can you grasp a full bottle and raise it? 
3. Can you hold a plate full of food? 
4. Can you pour liquid from a bottle into a glass? 
5. Can you unscrew the lid from a jar that has been opened before? 
6. Can you cut meat with a knife? 
7. Can you prick things well with a fork? 
8. Can you peel fruit? 
 
Dressing 
9. Can you button your shirt? 
10. Can you open and close a zip? 
 
Hygiene 
11. Can you squeeze a new tube of toothpaste? 
12. Can you hold a toothbrush efficiently? 
 
At the office 
13. Can you write a short sentence with an ordinary pen? 
14. Can you write a letter with an ordinary pen? 
 
Other 
15. Can you turn a round door knob? 
16. Can you cut a piece of paper with scissors? 
17. Can you pick up coins from a table top? 
18. Can you turn a key in a lock? 
 
 
 
 
 
Subject signature 
Date 

24 
 
Appendix D. Mawdsley Calcinosis Questionnaire 
 
Patient Name or Reference  
   
Current geographical location   
   
 
Hemisphere/season 
   
 
Month/Day   
 
 
Part A. 
1. a. How many calcinosis lesions (open or closed) do you ACTUALLY have today?   
 
1. b. How many calcinosis do you FEEL that you have today?   
 
2. a. How many digital ulcers do you have today?   
 
2. b. How many of these digital ulcers do you think are related to calcinosis?   
 
 
Part B. 
In the past TWO WEEKS, what is the worst degree that… 
 
1. Your Raynaud’s has interfered with daily activities? 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 
 
2. Your DIGITAL ULCERS interfered with daily activities? 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 
 
3. You experienced PAIN from your calcinosis? 
No Pain 
Worst Possible Pain 
├────────────────────────────────────────────────┤ 
0 
10 
 
4. You felt any areas of your calcinosis getting TIGHTER or having more pressure? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 
 
5. You felt any areas of your calcinosis GROWING under your skin? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 

25 
 
6. You felt any areas of your calcinosis THROBBING? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 
 
7. Your calcinosis has been TENDER to TOUCH? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 
 
8. You felt the need to PROTECT areas of your calcinosis? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 
 
9. You have been FEARFUL or WORRIED that any of your calcinosis areas are INFECTED? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 
 
10. You have been worried that a calcinoisis wound MIGHT NOT HEAL? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 
 
11. Your calcinosis interfered with ability to CARE FOR SELF? 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 
 
12. Your calcinosis interfered with your ability to USE YOUR HANDS? 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 
 
13. Your calcinosis interfered with WALKING? 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 
 
Test question: Your calcinosis interfered with your ability to USE YOUR HANDS or to WALK? 
No Limitation 
Maximum limitation/ 
Worst possible 

26 
 
├────────────────────────────────────────────────┤ 
0 
10 
 
14. Your calcinosis interfered with your ABILITY TO WORK (paid or unpaid) 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 
 
15. Your calcinosis made you FEEL DOWN, DEPRESSED or HOPELESS? 
Not at all 
Maximum 
├────────────────────────────────────────────────┤ 
0 
10 
 
16. Your calcinosis interfered with your ability to enjoy and ‘be there’ for your FRIENDS and 
FAMILY? 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 
 
17. Your calcinosis interfered with your ability to enjoy RECREATIONAL ACTIVITIES 
(hobbies, sports, etc)? 
No Limitation 
Maximum limitation/ 
Worst possible 
├────────────────────────────────────────────────┤ 
0 
10 

27 
 
Appendix E. Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) 
 
Instructions for completing the questionnaire: Please answer every question. Some questions 
may look like others, but each one is different. Please take the time to read and answer each 
question carefully by filling in the bubble that best represents your response. 
 
1. In general, would you say your health is: 
 
❑ Excellent 
❑ Very good 
❑ Good 
❑ Fair 
❑ Poor 
 
2. Compared to one year ago, how would you rate your health in general now? 
 
❑ Much better now than a year ago 
❑ Somewhat better now than a year ago 
❑ About the same as one year ago 
❑ Somewhat worse now than one year ago 
❑ Much worse now than one year ago 
 
3. The following items are about activities you might do during a typical day. Does your health 
now limit you in these activities? If so, how much? 
 
a. Vigorous activities, such as running, lifting heavy objects, participating in strenuous sports. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
b. Moderate activities, such as moving a table, pushing a vacuum cleaner, bowling, or playing 
golf? 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
c. Lifting or carrying groceries. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
d. Climbing several flights of stairs. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
e. Climbing one flight of stairs. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 

28 
 
 
f. Bending, kneeling or stooping. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
g. Walking more than one mile. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
h. Walking several blocks. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
i. Walking one block. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
j. Bathing or dressing yourself. 
❑ Yes, limited a lot. 
❑ Yes, limited a little. 
❑ No, not limited at all. 
 
4. During the past 4 weeks, have you had any of the following problems with your work or other 
regular daily activities as a result of your physical health? 
 
a. Cut down the amount of time you spent on work or other activities? 
 Yes  No 
 
b. Accomplished less than you would like? 
 Yes  No 
 
c. Were limited in the kind of work or other activities 
 Yes  No 
 
d. Had difficulty performing the work or other activities (for example, it took extra time) 
 Yes  No 
 
5. During the past 4 weeks, have you had any of the following problems with your work or other 
regular daily activities as a result of any emotional problems (such as feeling depressed or 
anxious)? 
 
a. Cut down the amount of time you spent on work or other activities? 
 Yes  No 
 
b. Accomplished less than you would like 
 Yes  No 

29 
 
c. Didn't do work or other activities as carefully as usual 
 Yes  No 
6. During the past 4 weeks, to what extent has your physical health or emotional problems 
interfered with your normal social activities with family, friends, neighbors, or groups? 
 
❑ Not at all 
❑ Slightly 
❑ Moderately 
❑ Quite a bit 
❑ Extremely 
 
7. How much bodily pain have you had during the past 4 weeks? 
 
❑ Not at all 
❑ Slightly 
❑ Moderately 
❑ Quite a bit 
❑ Extremely 
 
8. During the past 4 weeks, how much did pain interfere with your normal work (including both 
work outside the home and housework)? 
 
❑ Not at all 
❑ Slightly 
❑ Moderately 
❑ Quite a bit 
❑ Extremely 
 
9. These questions are about how you feel and how things have been with you during the past 4 
weeks. For each question, please give the one answer that comes closest to the way you have 
been feeling. How much of the time during the past 4 weeks. 
 
a. did you feel full of pep? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
b. have you been a very nervous person? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
c. have you felt so down in the dumps nothing could cheer you up? 
❑ All of the time 
❑ Most of the time 

30 
 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
d. have you felt calm and peaceful? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
e. did you have a lot of energy? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
f. have you felt downhearted and blue? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
g. did you feel worn out? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
h. have you been a happy person? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
i. did you feel tired? 
❑ All of the time 
❑ Most of the time 
❑ A good bit of the time 
❑ Some of the time 
❑ A little of the time 

31 
 
❑ None of the time 
10. During the past 4 weeks, how much of the time has your physical health or emotional 
problems interfered with your social activities (like visiting friends, relatives, etc.)? 
❑ All of the time 
❑ Most of the time 
❑ Some of the time 
❑ A little of the time 
❑ None of the time 
 
11. How TRUE or FALSE is each of the following statements for you? 
 
a. I seem to get sick a little easier than other people 
❑ Definitely true 
❑ Mostly true 
❑ Don't know 
❑ Mostly false 
❑ Definitely false 
 
b. I am as healthy as anybody I know 
❑ Definitely true 
❑ Mostly true 
❑ Don't know 
❑ Mostly false 
❑ Definitely false 
 
c. I expect my health to get worse 
❑ Definitely true 
❑ Mostly true 
❑ Don't know 
❑ Mostly false 
❑ Definitely false 
 
d. My health is excellent 
❑ Definitely true 
❑ Mostly true 
❑ Don't know 
❑ Mostly false 
❑ Definitely false 

32 
 
Appendix F. Patient and physician calcinosis global assessment 
Patient Calcinosis Global Assessment 
 
Please rate the overall activity of your calcinosis today by placing a mark on the line below. 
 
 
 
 
cm 
0 
5 
10 
No evidence of 
Extremely active or severe 
calcinosis 
 
calcinosis 
 
 
 
 
 
 
 
Patient Signature 
Date 
 
 
 
 
Physician Calcinosis Global Assessment 
 
 
Overall, considering how much pain, discomfort, limitations in your patient’s daily life, and 
other changes in his/her body and life, how severe would you rate your patient’s calcinosis 
today? Please place a mark on the line below. 
 
 
 
 
cm 
0 
5 
10 
No evidence of 
Extremely active or severe 
calcinosis 
 
calcinosis 
 
 
 
 
 
 
 
Physician Signature 
Date 

33 
 
Appendix G. Raynaud condition score 
 
RAYNAUD’S CONDITION SCORE (RCS): The Day’s RCS or Raynaud’s Condition Score is 
the patient’s rating of how much difficulty you had with your Raynaud’s TODAY. Consider 
how many attacks you had and how long they lasted. Consider how much pain, numbness, or 
other symptoms the Raynaud’s caused in your fingers (including painful sores) and how much 
the Raynaud’s alone affected the use of your hands today. 
 
 
No Difficulty 
Extreme Difficulty 
⏐⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⏐ 
0 
10 
 
Input number from 1.0 to 10.0, in increments of 0.10 

34 
 
Appendix H. Modified Rodnan Skin Score (MRSS) 
 
 
SCLERODERMA 
Score from 0 (normal thickness) to 3 (severe thickening) 
Face 
Anterior chest 
Abdomen 
LEFT 
RIGHT 
Fingers 
Dorsum of hands 
Forearms 
Upper arms 
Thighs 
Lower legs 
Dorsum of feet 
TOTAL: 
 
 
 
 
Evaluator’s Signature 
Date 

35 
 
Appendix I. Skin tissue sample collection instructions 
 
Snap freezing, or flash freezing, is the process by which samples are lowered to temperatures 
below -80°C very rapidly liquid nitrogen. Snap freezing achieves the same endpoint as slow rate- 
controlled freezing, but at a much faster rate. This procedure is intended to ensure that tissue 
samples collected will be frozen in a safe and efficient manner while eliminating the risks of 
contamination and variation in molecular integrity. The following protocol describes a general 
procedure for snap-freezing. For nonstandard sample types, always refer to the tissue – specific 
protocols. 
 
This procedure describes the sample collection, storage and transport of tissue collected for RNA 
testing. 
 
Materials 
 
A. 
CryoStor™CS10 freezing medium (Stem Cell Technologies Cat#07930) 
B. 
2ml CryoVials or 2ml sample tube 
C. 
4mm Acu-Punch biopsy tool 
D. 
Disposable gloves 
E. 
Isopropanol filled freezing vessel 
F. 
-80°C Freezer 
G. 
Liquid nitrogen 
H. 
Liquid nitrogen canister 
I. 
Cutting board/surface 
J. 
Long metal forceps 
K. 
Scalpel/razor blades 
L. 
Shipping Log 
M. 
Sample Storage Box 
 
Safety implications 
 
Adhere to proper use of PPE. General precautions for biohazardous materials are to be observed. 
All disposable supplies should be disposed per the institution’s waste protocol. 
 
Procedure 
 
Collecting Biopsy 
 
1. Properly label (1) 2ml sample tube AND (1) Cryo-tube with 1ml CryoStor™ CS10 
freezing medium. 
2. Using a 4mm Acu-Punch biopsy tool , collect tissue from the non-dominant arm or upper 
buttock area. 
 
For ATAC-seq 
 
3. Dissect a small sample from the biopsy (approximately 1 cubic mm). 

36 
 
4. Place in prelabeled Cryo-tube with 1ml CryoStor™ 
5. Freeze by placing in an isopropanol filled freezing vessel 
6. Place vessel in -80°C freezer*. 
*Goal is 1 degree decrement per hour 
7. Store at -80ºC for short term storage or liquid nitrogen for long term storage. 
 
For RNA extraction 
 
8. Place remaining biopsy in prelabeled 2ml sample tube. 
9. Snap freeze the biopsy by holding sample tube with long metal forceps and immersing 
approximately halfway down the height of the liquid nitrogen in the canister for at least 
30 seconds. 
10. Transfer sample tube to -80°C freezer for storage. 
11. It is preferable to ship the samples on dry ice as soon as possible after collection. 

37 
 
Appendix J. Biomarkers list 
 
Ang-1 
Ang-2 
MMP-2, MMP-9 
NT-proBNP, PIGF 
VEGFR1, sRAGE 
GLUT-1 
Ficolin-1 
MBL 
H62 plex (includes VEGF, PDGFBB, bFGF,IL-13, HGF, IL-6, IL-1β, IL-2, IL-4, IL-5, IL-8, 
IL-10, IL-12, IL-13,[TNF]-α,[IFN]-γ) 

38 
 
References: 
1. 
Avouac J, Mogavero G, Guerini H, Drape JL, Mathieu A, Kahan A, et al. Predictive 
factors of hand radiographic lesions in systemic sclerosis: a prospective study. Annals of the 
rheumatic diseases. 2011 Apr;70(4):630-3. PubMed PMID: 21131648. Epub 2010/12/07. eng. 
2. 
Koutaissoff S, Vanthuyne M, Smith V, De Langhe E, Depresseux G, Westhovens R, et 
al. Hand radiological damage in systemic sclerosis: comparison with a control group and clinical 
and functional correlations. Seminars in arthritis and rheumatism. 2011 Apr;40(5):455-60. 
PubMed PMID: 20864145. Epub 2010/09/25. eng. 
3. 
Johnstone EM, Hutchinson CE, Vail A, Chevance A, Herrick AL. Acro-osteolysis in 
systemic sclerosis is associated with digital ischaemia and severe calcinosis. Rheumatology 
(Oxford, England). 2012 Dec;51(12):2234-8. PubMed PMID: 22923763. Epub 2012/08/28. eng. 
4. 
Chung L, Fiorentino D. A pilot trial of treprostinil for the treatment and prevention of 
digital ulcers in patients with systemic sclerosis. Journal of the American Academy of 
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