Conditions: Lymphangioleiomyomatosis (LAM)
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 12
Healthy volunteers: no

Brief summary:
The investigators will perform a two-center phase I trial of celecoxib (COX-2 inhibitor) administered at 200mg by mouth daily for 6 months. Up to 12 adult women with LAM will be recruited (between 4-8 at each site). The Specific Aims are:

Aim 1: To investigate whether, in LAM patients, celecoxib is safe and well tolerated, and has evidence of clinical benefit.

Aim 2: To investigate the potential value of a novel biomarker of LAM, quantitative measurement of the number of TSC2 mutant LAM cells per ml of blood, to assess disease severity.

Detailed description:
Background: Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction, kidney angiomyolipomas (AMLs), and LAM cell growth within the axial lymphatics and multiple other organs and surfaces. LAM occurs both sporadically and in association with tuberous sclerosis complex (TSC). Sirolimus (rapamycin), an mTORC1 inhibitor, has been shown to stabilize lung function decline and decrease angiomyolipoma tumor size in both TSC and sporadic LAM patients. However, cessation of rapamycin therapy results in recurrent decline in lung function, and regrowth of angiomyolipoma, suggesting that continuous use may be required to maintain its beneficial effects. Recently the investigators have discovered that cyclo-oxygenase (COX) function is altered in cells lacking TSC2, including in a LAM patient-derived angiomyolipoma cell line. COX-2 levels are increased, prostaglandin metabolite levels are increased, and treatment with COX-2 inhibitors are effective in reducing tumor size in two different Tsc mouse models, one a native tumor, and the other a xenograft model. Furthermore, rapamycin does not affect these differences in COX-2 expression or prostaglandin metabolites.

Objectives/Hypothesis: Our preclinical studies indicate that celecoxib (a COX-2 specific inhibitor) decreases the size of TSC2-deficient tumors in Tsc models. Hence the investigators propose this Pilot Clinical Trial to test the safety and tolerability of celecoxib in patients with LAM, with preliminary assessment of potential benefit using multiple approaches.

Specific aims: The primary endpoint of this pilot trial is to test the safety and tolerability of treatment with celecoxib in patients with mild-to-moderate LAM, who are not currently on sirolimus; and to assess the potential benefit of this treatment using the following: 1. Spirometry, 2. MRI measurement of angiomyolipoma size, 3. St. George's Respiratory Questionnaire, 4. VEGF-D serum levels. The investigators will assess Exhaled breath condensate prostaglandin metabolites to confirm effects of celecoxib. The investigators will also develop a novel biomarker of LAM to assess response, quantitative measurement of the number of TSC2 mutant circulating LAM cells, by next generation sequencing.

Study design: The investigators will perform a pilot clinical trial to investigate the safety and tolerability of celecoxib therapy as a single agent for patients with LAM. LAM subjects who are not taking everolimus or rapamycin will be treated with celecoxib at 200mg PO QD for 6 months. They will be monitored for respiratory function and angiomyolipoma size. At the end of the 6 month period, celecoxib will be discontinued, and subjects will be monitored for another 6 months.

Clinical Impact: Sirolimus is the only medical therapy shown to reduce tumor size and stabilize lung function in patients with LAM and TSC-LAM. Although sirolimus has clear benefits, results from the MILES trial suggest that continuous therapy in some form is required, as the rate of decline in lung function resumed when sirolimus was discontinued. The investigators hope that celecoxib will show benefit with minimal toxicity in this trial, and provide an alternative approach for the long term prophylactic/preventive treatment of patients with mild-to-moderate LAM. Our study will include patients with TSC LAM, which often appears to be more slowly progressive than sporadic LAM, and hence long term therapy with celecoxib may have particular benefit in the TSC LAM population. In addition, the investigators will develop a quantitative measure of circulating LAM cell levels as part of this trial.

Arms:
arm 1: Celecoxib 200mg PO QD for 6 months

Interventions: intervention 1: Celecoxib
Intervention descriptions: intervention 1: We will perform a pilot clinical trial to investigate the safety and tolerability of celecoxib therapy as a single agent for patients with LAM. LAM subjects who are not taking everolimus or rapamycin will be treated with celecoxib at 200mg PO QD for 6 months. They will be monitored for respiratory function and angiomyolipoma size. At the end of the 6 month period, celecoxib will be discontinued, and subjects will be monitored for another 6 months.

Protocol document:
Prot_SAP_000.pdf: 
April 3, 2017 
1 
Protocol Title:  A Pilot Clinical Trial of COX-2 inhibition in LAM and TSC 
Version Date: 3 Apr 2017 
NCT #:  02484664 
PI:  David Kwiatkowski, MD, PhD 


April 3, 2017 
2 
 
 
 
 
 
 
 
 
 
A Pilot Clinical Trial of COX-2 inhibition in LAM and TSC 
 
 
Abbreviated Protocol Title: COLA Trial 
    
Identifying words: LAM, COX-2, Celecoxib, Circulating LAM cells 
 
Principal Investigator:  
David Kwiatkowski, MD, PhD 
 
 
 
 
1 Blackfan Circle, Rm. 6-013 
Brigham and Women’s Hospital 
Boston, MA  02115 
617 355 9005 
 
Associate Investigators:  
Elizabeth Henske, MD 
Joel Moss, MD, PhD 
 
 
 
    
Souheil El-Chemaly, MD 
 
 
 
    
Hilary Goldberg, MD 
 
 
 
 
Ivan Rosas, MD 
 
 
 
 
Tracey Doyle, MD 
 
 
 
 
Gary M. Hunninghake, MD 
 
 
 
 
Research Contact:   
Souheil El-Chemaly, MD 
 
 
Duration of study:   
2 years 
 
Number and type of subjects: 12 adult female subjects diagnosed with lymphangioleiomyomatosis 
 
Ionizing radiation:   
No  
 
Project uses IND/IDE:  
IND/IDE Exempt- Exemption letter attached 
 
Multi-Institutional Project: Yes 
 
Participating Institutions:  Brigham and Women’s Hospital, Boston, MA; Division of Intramural Research 
National Heart, Lung, and Blood Institute, Bethesda, MD.  
 
 
 
 
 
 
 


April 3, 2017 
3 
 
 
 
 
 
 
 
 
 
I- 
Background and significance 
 
A- 
Overview 
Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease which affects almost exclusively 
women. LAM is characterized pathologically by proliferation of abnormal smooth muscle cells in nodules in the 
lung parenchyma, which typically have mutations in TSC2, and by cystic changes within the lung parenchyma 
(McCormack and Henske 2010, Taveira-DaSilva et al. 2010, Henske and McCormack 2012). Cystic lung 
changes are seen by chest CT scans in up to 80% of women with TSC of age > 40 years (Cudzilo et al. 2013). 
LAM is also commonly diagnosed in women who do not have clinical features of TSC or germ line mutations 
in TSC1 or TSC2, and is called sporadic LAM. Inactivating mutations of both alleles of either TSC1 or TSC2 
have been found in LAM cells from both TSC-LAM and sporadic LAM patients (Carsillo et al. 2000, Badri et 
al. 2013). Approximately 60% of women with the sporadic form of LAM also have renal angiomyolipoma 
(McCormack and Henske 2010).  The presence of the same TSC2 mutation in LAM cells and renal 
angiomyolipoma cells from women with sporadic LAM, but not in normal tissues, indicates that they are part of 
the same clonal neoplasm (Carsillo et al. 2000).  This has led to the model that LAM is a systemic disease with 
neoplastic cells that spread to the lungs via a metastatic mechanism (Crino et al. 2006, Henske and McCormack 
2012). 63% of TSC LAM patients defined radiographically developed pulmonary symptoms, and 12.5% died 
from LAM in a recent large series (Cudzilo et al. 2013). Thus, LAM is a major contributor to morbidity and 
mortality in the TSC population. 
The protein products of TSC1 and TSC2, hamartin and tuberin, respectively, along with the TBC1D7 protein 
form a protein complex called the TSC complex that has GTPase activity toward the small GTPase Rheb (Ras 
homologue enriched in brain) (Dibble and Manning 2013). Loss of tuberin or hamartin inactivates the TSC 
protein complex, and leads to constitutive high levels of Rheb-GTP, which leads to hyperactivation of the 
mammalian target of Rapamycin complex 1 (mTORC1) (Kwiatkowski and Manning 2005, Dibble and Manning 
2013). mTORC1 is recognized as a master regulator of cell growth, with many anabolic effects that expand cell 
mass and all the critical building blocks required for cell growth, including NADPH, glycolytic intermediates, 
lipids, nucleotides, and ribosomes (Duvel et al. 2010, Dibble and Manning 2013). In the randomized controlled 
MILES (Multicenter International LAM Efficacy of Sirolimus) trial, the mTORC1 inhibitor rapamycin 
stabilized lung function and improved symptoms in LAM patients (McCormack et al. 2011).  However, lung 
function in treated subjects declined when rapamycin was discontinued.  Furthermore, renal angiomyolipomas 
that had responded to sirolimus also re-grew when the drug was discontinued (Bissler et al. 2008).  Thus, other 
approaches to LAM treatment and/or control are urgently needed. 
Although regulation of mTORC1 activity is recognized as the major canonical function of the TSC protein 
complex, multiple other effects occur in cells lacking the TSC complex, which appear to be independent of 
mTORC1 (Henske and McCormack 2012).  We have recently demonstrated that loss of TSC2 increases COX-2 
and PTGIS levels, and prostaglandin biosynthesis in a rapamycin-insensitive manner, but dependent on 
mTORC2 and EGFR (Li et al, 2014). In addition, celecoxib, a selective COX-2 inhibitor, reduced renal tumor 
incidence in Tsc2+/- mice, and aspirin suppressed tumor progression in a xenograft tumor model utilizing LAM 
patient-derived angiomyolipoma cells (Li et al. 2014). This recently published study motivates the current Pilot 
Clinical Trial proposal of COX-2 inhibition as a novel therapeutic strategy for LAM. 
 
b- In vitro and preclinical evidence 


April 3, 2017 
4 
 
Preliminary studies 
Our recent publication provides the rationale for this clinical trial (Li et al. 2014).  
 
Loss of TSC2 upregulates both COX-2 and prostacyclin synthase expression and prostaglandin production, 
independent of mTORC1 (Li et al. 2014) 
Prostaglandins are products of prostaglandin-endoperoxide synthases (PTGSs) 1 and 2, more commonly 
known as COX-1 and COX-2. COX-1 and COX-2 convert arachidonic acid released from membrane 
phospholipids into PGH2. Prostaglandin I2 (prostacyclin) synthase (PTGIS) then produces prostacyclin (PGI2) 
from PGH2. We had previously noted that both COX-2 (PTGS2) and PTGIS expression were significantly 
increased, by 2- and 40-fold, respectively, in TSC2-deficient angiomyolipoma (101) cells relative to TSC2-
addback cells, through microarray analysis (Lee et al. 2010). To validate these findings, we performed real-time 
RT-PCR analysis. TSC2-deficient cells exhibited a 101-fold increase of PTGS2, and a 15-fold increase of 
PTGIS in comparison to addback TSC2-expressing cells (P < 0.001). 
 
Effects of COX inhibitors on COX expression, PG levels, and proliferation (Li et al. 2014) 
To assess the effects of COX inhibition on TSC2 deficient cells, we treated the angiomyolipoma (101) cells 
with Sulindac (COX-1 inhibitor), NS398 (COX-2 inhibitor), or aspirin (an irreversible COX-1 and COX-2 
inhibitor that acetylates COX-2, resulting in production of 15-epi-LXA4) for 24 h. NS398 and aspirin reduced 
COX-2 and COX-1 levels without affecting phosphorylation of p44/42–MAPK or S6. Aspirin significantly 
decreased PGE2 levels and furthermore, reduced proliferation of the 101 cells in vitro  (Li et al. 2014) 
 
Effects of COX inhibitors in Tsc mouse models (Li et al. 2014) 
We then assessed the effects of COX inhibition on TSC2 null tumor development in vivo.  First, we 
used the COX-2–specific inhibitor Celecoxib to treat Tsc2+/- mice, which spontaneously develop renal 
cystadenomas (Onda et al. 1999). Celecoxib treatment (0.1% wt/wt in chow) for 4 months, beginning at 1 m of 
age, reduced the volume of renal cystadenomas in Tsc2+/- mice by 50% at age 5m relative to vehicle control (p 
= 0.0002).  Then, we assessed the effect of aspirin in a xenograft tumor model using Tsc2-deficient ELT3 cells, 
modified to express luciferase. Aspirin treatment for 3 weeks decreased bioluminescent signal as well as tumor 
size. Aspirin-treated tumors also had reduced expression of COX-2 and c-Myc, and had increased levels of 
cleaved-caspase-3 and cleaved-PARP. Aspirin-treated mice bearing ELT3 xenograft tumors had markedly 
reduced urinary levels of PGE2, consistent with strong inhibition of each of COX-1 and COX-2, as expected. 
Together, these data indicate that COX inhibition reduces tumor growth in two different in vivo Tsc models (Li 
et al. 2014).  
 
Evidence for COX-2 activation in vivo in LAM (Li et al. 2014) 
We then examined COX-2 expression directly in LAM lung samples. LAM lungs were found to express 
higher levels of COX-2 in comparison with control lungs by immunohistochemistry. COX-2 expression was 
seen in cells also expressing smooth muscle actin and phospho-S6 in LAM nodules, commonly called LAM 
cells (Li et al. 2014).  
To gain some insight into the functional effects of COX-2 expression in LAM, 15-epi-lipoxin A4 
(LXA4), a product of aspirin-acetylated COX-2, was measured in exhaled breath condensate (EBC) from three 
LAM subjects. LXA4 was detected in the LAM EBCs and levels were increased following transient aspirin 
treatment. We also hypothesized that LAM cell activity might increase serum PG metabolites in LAM patients.  
The mean serum PGE2 level of LAM patients (27.8 pg/ml) was higher than that of healthy women (19.6 pg/ml; 
P = 0.0021). In addition, the mean serum 6-keto-PGF1 level of LAM patients (192 pg/ml) was also higher 
than the mean of healthy women (82.6 pg/ml; P = 0.0006).  These results suggest that COX-2 activity is high in 
LAM cells in vivo (Li et al. 2014). 
 
Use of next-generation sequencing (NGS) for TSC2 mutation detection 


April 3, 2017 
5 
 
We have employed NGS for sensitive detection of TSC1/TSC2 mutations in multiple settings.  This 
includes on microdissected LAM tissue, in collaboration with Lucia Schuger (Badri et al. 2013), analysis of 
cultured skin tumor cells, in collaboration with Tom Darling (Tyburczy et al. 2014), and on a large number of 
TSC patients with no mutation identified (data not shown).  In brief we either prepare large amplicons from 
TSC1/TSC2, or perform hybrid capture of those genomic regions using Agilent probe sets, to create NGS 
libraries highly enriched for the TSC1/TSC2 regions.  Following sequencing on the Illumina 2500 machine, we 
use a set of standard pipeline tools to align sequences to the human genome.  We use our own specialized 
scripts written in python and Matlab to detect all sequence variants present at allele frequency > 1%, and then 
review all calls using IGV.  By this methodology we have identified several sequence variants that are present 
at 1-2% frequency, and validated them by secondary methods (Badri et al. 2013, Tyburczy et al. 2014). 
 
Current clinical use and toxicities of celecoxib: implications for the COLA trial 
Celecoxib is a COX-2 selective non-steroidal anti-inflammatory drug (NSAID).  It is used to treat 
osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and several other inflammatory conditions.  It has 
been in clinical use in the USA for 16 years.  Celecoxib is generally well tolerated, with mild to moderate upper 
GI complaints being the most common adverse event (McCormack 2011).  Although celecoxib has been 
associated with an increased risk of cardiovascular events, including myocardial infarction (MI) and stroke, this 
has been inconsistent in meta-analyses (McCormack 2011).  Nonetheless, due to this potential risk, LAM 
patients with a history of MI or stroke will not be entered on this trial. Celecoxib and other NSAIDs may 
increase risk of serious gastrointestinal (GI) ulceration, bleeding, and perforation.  To minimize this risk, LAM 
subjects on this protocol will not take any other NSAID, and omeprazole will be permitted for use concurrently 
for any LAM subjects on this trial.  In addition, LAM patients with a prior history of ulcer disease or GI 
bleeding will be excluded from this protocol.  Celecoxib and other non-steroidal anti-inflammatory drugs 
(NSAIDs) are known to reversibly affect renal function.  For this reason, subjects with impairment of renal 
function will be excluded from the trial, and renal function will be monitored among subjects on this trial. 
 
c- Rationale and hypothesis 
 
 
As shown above, we have demonstrated that COX-2 and prostacyclin synthase expression is markedly 
increased in TSC2 deficient cells, and have also shown that prostaglandin metabolites are altered in 
angiomyolipoma cells.  We have shown that treatment with a COX-2 inhibitor changes the PG metabolite 
profile of the angiomyolipoma cells, and inhibits their growth in vitro.  Furthermore we have shown that COX-2 
inhibition reduces growth of TSC2 deficient cells in vivo in two Tsc mouse models.  Thus, we hypothesize that 
COX-2 inhibition may be effective for TSC and LAM patients in reducing LAM and angiomyolipoma 
cell growth, and in stabilizing lung function.   
We further hypothesize that COX-2 inhibition will decrease the number of circulating TSC2-
mutant LAM cells.  Previous studies from the Moss laboratory at NIH have shown that circulating LAM cells 
can be isolated by a combination of density gradient separation and FACS separation using antibodies to CD45 
and CD235a (Crooks et al. 2004, Cai et al. 2014).  We will use this same methodology followed by NGS to 
identify mutations in TSC2 in patients enrolled on this study.  We will then use similar methods to quantify the 
number of circulating LAM cells in the subjects enrolled on this trial, developing a novel biomarker of LAM.   
Since this method works on a blood sample, it is non-invasive. 
 
II- 
Specific Aims 
The main objective of COLA: A Pilot Clinical Trial of COX-2 inhibition LAM and TSC is to test the safety 
and tolerability of celecoxib in subjects with LAM, and to test in a pilot manner its potential therapeutic 
effectiveness.  If successful, this pilot study will enable future phase II/III studies formally evaluating efficacy 
of this therapy for LAM.  A second objective of this study is to develop a novel biomarker of LAM, the number 
of circulating LAM cells per ml of blood. 
Specific Aims 


April 3, 2017 
6 
 
Aim 1: To investigate whether, in LAM patients, celecoxib is safe and well tolerated, and has evidence of 
clinical benefit.  LAM patients diagnosed by consensus criteria (Johnson et al. 2010) and with mild to moderate 
disease will be treated with celecoxib at 200mg PO QD. Assessments will include history and physical 
examination, chemistries, complete blood count, renal function assessment, and health status questionnaire 
during 6 months of treatment. To assess benefit, we will measure pulmonary function, angiomyolipoma size, 
VEGF-D levels, and quality of life using the St. George’s Respiratory Questionnaire, before and after therapy 
with celecoxib.  Twelve LAM patients at the two sites (BWH and NIH) will be enrolled on this study.  
Aim 2:  To investigate the potential value of novel biomarkers of LAM, quantitative measurement of the 
number of TSC2 mutant LAM cells per ml of blood and/or identification of TSC2 mutations in plasma 
cell-free DNA, to assess disease severity.  We will develop a next gen sequencing (NGS) assay on FACS 
sorted circulating LAM cells and/or plasma cell-free DNA to quantitatively determine the number of TSC2 
mutant LAM cells per ml of blood and/or the amount of TSC2 mutant DNA molecules per ml of plasma.  
 
 
III- 
Subject selection 
a- Inclusion and exclusion criteria 
We will perform a two-center phase I trial of celecoxib (COX-2 inhibitor) administered at 200mg by mouth 
daily for 6 months.  The two centers are BWH and the National Institutes of Health Clinical Center.  Up to 12 
adult women with LAM will be recruited at both sites, and up to 8 subjects at BWH (between 4-8 at each site). 
The study will use the following eligibility criteria. 
 
Inclusion criteria 
a. Female of age 18 to 69 
b. Ability to give informed consent 
c. Definite diagnosis of LAM 
Typical cystic change on CT scan of the chest plus one of the following i) biopsy or cytology of any 
tissue demonstrating LAM, ii) angiomyolipoma, chylothorax, clinical or genetic diagnosis of 
tuberous sclerosis, iii) serum VEGF-D > 800pg/ml 
d. post-bronchodilator forced expiratory volume in one second ≥ 70% of  predicted and DLCO ≥ 
70% predicted during baseline visit.  
e. Women of childbearing potential must agree to use two forms of barrier contraception after 
screening visit, for the duration of study participation and for 30 days after last dose. 
 
Exclusion criteria 
a. History of intolerance to non-steroidal anti-inflammatory drugs (NSAIDs) 
b. History of current regular use (daily most days of the week) of NSAIDs 
c. History of use of rapamycin or everolimus 
d. Uncontrolled intercurrent illness 
e. Pregnant, breast feeding or planning to become pregnant in the next 2 years 
f. Significant hematological (platelet count <100.000/µl or hepatic abnormalities (Liver function 
tests >2 times normal).  
g. Use of an investigational drug within 30 days of study start 
h. Inability to attend scheduled clinic visits 
i. Inability to give informed consent 
j. Inability to perform spirometry 
k. Creatinine > 1.0 mg/dl or eGFR < 60 ml/min  
l. Pneumothorax within past 8 weeks 
m. History of malignancy in the last 2 years other than basal cell skin cancer 
n. Use of estrogen containing medication within 30 days of enrolment 
o. Currently taking doxycycline, metformin, lupron or simvastatin 


April 3, 2017 
7 
 
p. Unable to undergo MRI 
q. History of seizure within the last year 
r. History of hepatitis or known active hepatitis B or C, or HIV positive serology 
s. Angiomyolipoma of diameter > 4 cm 
t. History of vascular disease, including myocardial infarction or stroke 
u. History of ulcers or GI bleeding 
v. Allergy to sulfonamides, unless subject has previously used Celocoxib without any adverse 
reactions.  
w. Age older than 70 
 
b- Source of subjects and recruitment methods 
 
Participants will be referred by physicians who care for LAM patients at participating institutions. Care will be 
taken to allow potential subjects to feel free from tension and subtle coercion. The investigators will advise 
potential subjects that a study is being done, and briefly describe the study to ascertain interest prior to 
identifying themselves as being involved. Investigators will emphasize that participation is voluntary and that 
not participating will not jeopardize future care.  
The study investigators will also contact providers outside of these institutions who may be caring for patients 
with LAM to notify them of the clinical trial design, inclusion and exclusion criteria. This contact will be in the 
form of a letter to referring physicians. Such physicians will be provided with contact information of key 
personnel in the trial in order to refer potential subjects for participation.  
Research staff will seek to enroll any potentially qualified adults regardless of age, ethnic, religious or racial 
background, and economic status.  
Subjects will be recruited from other past studies. We will also post an advertisement on the Brigham and 
Women’s website and on clinicaltrials.gov as well as use flyers to advertise the study in the clinics and events 
for LAM patients. 
 
IV- 
Subject enrollment 
 
The primary specialist or heath care provider, usually a physician, who is known to the potential subject and has 
first- hand knowledge of the patient’s medical history must (1) give approval for his/her patient to be contacted 
for research purposes (2) initially introduce the study to the patient and (3) obtain the patient’s permission to be 
contacted by study staff. A nurse or study coordinator will review the informed consent with the subject to 
avoid the possibility of coercion or bias since subjects will be recruited from Investigator’s own practice. 
Subjects will be reminded that refusal to participate in this trial will not change the quality of care that she will 
receive at BWH. Study participation is voluntary and the subject will be advised that she can withdraw at any 
time.  
 
The subject will be given the opportunity to take the consent home to consider participation. The subject will be 
asked to read the informed consent form prior to the physician or study coordinator asking the subject any 
questions concerning their health. Written, informed consent will be obtained after one of the investigators has 
reviewed the protocol verbally in detail with the subject. The physician will inform the subject that she has the 
alternative not to participate in the study. Subjects will receive a signed copy of the consent form for their 
records. After all questions are answered, and only after the consent form is signed by the patient and one of the 
MDs listed on the application will study screening commence. Subjects unable to give consent will not be 
approached.  
 
 
Participation in other trials 


April 3, 2017 
8 
 
Subjects will be allowed to participate in other research studies, as long as they do not involve the use of an 
investigational therapy.  
 
Inclusion of Women and Minorities in this study 
Adult women (>18 years of age) without any restrictions on age, race or ethnic group are eligible to participate 
in this trial. We will make every attempt to reach women of all ethnic groups, by advertising broadly through 
patient groups, LAM foundation and the internet.  
 
 
V- 
Study procedures 
Prior to enrolling in the study subjects must undergo the following tests to determine eligibility and baseline 
characteristics.  
 
Screening and baseline testing 
Timing prior to start of therapy 
Informed consent 
To be obtained before entry 
 
Medical history and physical 
examination 
Including weight, height, SaO2, 
vital signs (heart rate, blood 
pressure, temperature) 
Within 28 days 
Hematology 
Complete blood count and 
differential (CBCD) 
Within 28 days 
Serum chemistries 
Must include alkaline 
phosphatase, AST, ALT, 
bilirubin, serum creatinine, 
calicium and magnesium 
Within 28 days 
Urine test 
Including pregnancy test 
Within 28 days 
Electrocardiogram (ECG) 
 
Within 28 days 
Pulmonary function test 
Spirometry, Post 
Bronchodilator spirometry, 
TLC, DLCO 
Within 28 days 
Abdominal MRI 
 
Within 3 months 
 
Study Visit Summary 
 
Study Procedures 
All study procedures will be performed in accordance with accepted standards of practice. Relevant changes in 
clinical status and medication use, symptom history and adverse events will be reviewed at each study visit and 
in between study visit by a phone call placed from the research team to subjects enrolled in the study.  
 
Blood samples (chemistry, CBC) and biomarkers (VEGF-D, circulating TSC2 mutant cells) will be obtained via 
blood draws at indicated study visits. In addition, urine pregnancy testing will be performed at each study visit 
during the course of drug administration. Pulmonary function testing will be performed in accordance with ATS 
guidelines (Anonymous 1991) and according the schedule outlined in Table 1. Abdominal MRI will be 
performed at enrollment –if not done in the past 3 months-, and if angiomyolipoma or any other kidney 
abnormalities are detected, follow-up MRI will be performed at the discretion of the study team as indicated in 
Table 1. The St. Georges Respiratory Questionnaire (SGRQ) will also be administered and scored in accordance 
with published guidelines (Barr et al. 2000).  Information on hospitalization and side effects will be obtained via 
direct subject interviews and medical record review.  
Table 1: study visits and testing 
Week 


April 3, 2017 
9 
 
 
Baseline 
8 
16 
24 
Visit number 
1 
2 
3 
4/early 
termination 
Informed consent 
X 
 
 
 
History and physical 
X 
X 
X 
X 
Concurrent Medication 
Reconciliation 
X 
X 
X 
X 
Adverse Event Review 
X 
X 
X 
X 
Drug Administration Record  
X 
X 
X 
X 
Liver, renal, glucose, 
cholesterol  
X 
X 
X 
X 
EKG 
X 
 
 
 
Urine pregnancy  
X 
X 
X 
X 
CBC diff  
X 
X 
X 
X 
MRI abdomen  
X 
 
 
X 
Full PFT  
X 
 
 
X 
St George’s questionnaire  
X 
 
 
X 
Stored serum and plasma 
X 
X 
X 
X 
Exhaled breath condensate 
X 
 
 
X 
measurement of TSC2 mutant 
cells  
X 
 
 
X 
Study Windows 
The screening to first dose period will be 28 days. All other study visit windows will be +/- one week.  
 
Study visit procedures 
Screening Visit (Visit 1) 
The Screening Visit will take about 5 hours.   
The following testing will be performed at the screening visit: 
• Medical history 
• Full physical exam 
• Medication reconciliation 
• Blood collection as outlined in table 1 
• Urine pregnancy testing 
• EKG 
• MRI of the abdomen if not done in the last 3 months 
• St. George’s Respiratory Questionnaire (SGRQ) 
• Spirometry, Post Bronchodilator spirometry,  body plethysmography and diffusing capacity for 
carbon monoxide (DLCO) 
• Stored blood for all exploratory endpoints 
• Collection of exhaled breath condensates 
 
Subjects who meet the inclusion criteria will be enrolled in the study and will start taking their medications 
 
Visit 2  
Visit 2 will take place 8 weeks after a subject starts taking study medication and will require about one hour. 
The following testing will be obtained at this visit: 


April 3, 2017 
10 
 
 
• Interval history and adverse event review 
• Physical exam 
• Blood collection as outlined in table 1 
• Urine pregnancy testing 
Visit 3  
Visit 3 will take place 16 weeks after a subject starts taking study medication and will require about one hour. 
The following testing will be obtained at this visit: 
 
• Interval history and adverse event review 
• Physical exam 
• Blood collection as outlined in table 1 
• Urine pregnancy testing 
 
Visit 4 
Visit 4 will take place at 24 weeks after the start of therapy and after this visit the subject will stop taking the 
medication.  This visit will require about 5 hours.  The following testing will be obtained at this visit: 
 
• Medical history 
• Full physical examination 
• Medication reconciliation 
• Blood collection as outlined in table 1 
• Urine pregnancy testing 
• MRI of the abdomen if angiomyolipoma or any other kidney abnormalities are present on 
screening MRI 
• St. George’s Respiratory Questionnaire (SGRQ) 
• Spirometry, Post Bronchodilator spirometry, body plethysmography and diffusing capacity for 
carbon monoxide (DLCO) 
• Collection of exhaled breath condensates 
 
 
Early termination study visit 
If a subject decides to stop taking part in the study for any reason, she will make a final study visit. The final 
study visit will take about 6 hours. The following testing will be obtained at this visit: 
• Medical history 
• Full physical examination 
• Medication reconciliation 
• Blood collection as outlined in table 1 
• Urine pregnancy testing 
• MRI of the abdomen if angiomyolipoma are present on screening MRI 
• St. George’s Respiratory Questionnaire (SGRQ) 
• Spirometry, Post Bronchodilator spirometry, body plethysmography and diffusing capacity for 
carbon monoxide (DLCO) 
• Collect any unused study drug and drug diaries 
• Collection of exhaled breath condensate 
 
Subject study participation may be discontinued by the PI if: 
 


April 3, 2017 
11 
 
- At the PI’s discretion in the best interest of the subject 
- Subject can’t make study visits 
- The sponsor decides to stop the study 
- The subject has intolerable side effects even at the lower dose of Celecoxib 
- Subject becomes pregnant 
- Subject does not follow study requirements 
 
Labs performing evaluations and special precautions 
The clinical laboratory evaluations will be performed in CLIA approved laboratories at BWH and the NIH 
Clinical Center.  
 
Investigational analyses of VEGF-D and circulating LAM cells will be conducted in research laboratories at 
BWH and NIH. 
i- 
VEGF-D analysis will be performed in Dr. Kwiatkowski’s laboratory at BWH 
ii- 
Next generation sequencing (NGS) assay to detect TSC2 mutant LAM cells in FACS sorted 
circulating LAM cells and/or TSC2 mutations in plasma cell-free DNA will be performed at BWH 
in Dr. Kwiatkowski’s laboratory 
 
Transfer of specimens between sites will be necessary to complete the investigational analyses. Samples will be 
shipped overnight on dry ice Monday to Thursday to keep specimens frozen. Transferring site will keep a log of 
all specimens transferred. The receiving site will keep a log of when samples are received and analyzed.  
 
Primary outcome measurements 
We will perform a two-center phase I trial of celecoxib (COX-2 inhibitor) administered at 200mg by mouth 
daily for 6 months.  A study investigator will obtain informed consent after detailed review of the consent form 
with potential study participants. Consenting subjects will be screened at the initial visit based on predefined 
inclusion/exclusion criteria.  Subjects must have mild to moderate LAM (defined as definite LAM not requiring 
rapalog therapy), have no history of chronic non-steroidal anti-inflammatory drug use, have no previous history 
of treatment with rapamycin or everolimus, and have no angiomyolipoma of diameter > 4 cm. Enrolled subjects 
will receive celecoxib at an initial dose of 200 mg orally daily, with a single dose adjustment to 100mg PO QD 
permitted if needed. 
Any subject who receives at least one dose of celecoxib will be included in the analysis for side effects. 
Dose limiting toxicity will be graded according to the National Cancer Institute’s CTCAE v3.0 
(http://ctep.cancer.gov/protocoldevelopment/electronic_applications /docs/ctcaev3.pdf).  During the 6 months 
of the trial, there will be 3 study related visits. Clinical and laboratory evaluation is described below. 
Since this dose of celecoxib (200mg PO QD) is routinely well tolerated in the clinical setting in patients 
with arthritis and other inflammatory disorders, we expect that it will be well-tolerated in LAM patients. 
However, we will carefully monitor GI tolerance and renal function, since celecoxib can lead to reversible renal 
impairment. 
We will perform assessments of clinical, functional and subject self-reported outcomes to assess 
potential benefit of celecoxib. Since lung function decline is a hallmark of LAM, we will perform spirometry, 
which will be interpreted in accordance with standard guidelines (Anonymous 1991). We will utilize the St. 
George’s Respiratory Questionnaire (SGRQ) (Barr et al. 2000), as an assessment of the impact of treatment on 
dyspnea levels. Finally, we will record the incidence of hospitalization, complications from the disease, and 
other medical problems occurring during the study period 
 
Stopping rules 
The study will be monitored for the occurrence of a specified set of treatment related serious adverse events 
(TRSAE). The following two types of TRSAEs will be considered for early stopping of the study: 
- Death considered to be probably or definitely related to Celecoxib 


April 3, 2017 
12 
 
- Any grade IV toxicity considered to be probably or definitely related to Celecoxib 
 
Subjects will be followed for 24 weeks while on therapy. During the 24 weeks of the trial, there will 4 study 
visits (screening visit and 3 visits while on therapy). Clinical and laboratory evaluations will be performed as 
outlined in Table 1.  
 
Secondary outcome measurements  
We will be performing several correlative studies as part of this protocol. 
a. VEGF-D levels.  VEGF-D is well-known as a biomarker of LAM, and disease extent correlates 
with VEGF-D levels.  Therefore we will measure VEGF-D prior to treatment and at serial time 
points during treatment.  As this is a pilot study, formal statistical analysis is not planned. 
b. Circulating LAM cells.  Circulating LAM cells may be isolated from peripheral blood of subjects 
on this trial, using density gradient and FACS methods.  TSC2 mutations will be identified in 
these cells, if possible, and then the prevalence of LAM cells will be measured serially over time 
in subjects on this study.  If no TSC2 mutation is identified, then mutations in other genes in this 
pathway (RHEB, MTOR) will be sought. However, we will not scan the entire genome in these 
analyses, but rather focus on just a few genes.  We will test the number of circulating LAM cells 
over time to help assess whether the treatment, celecoxib, reduces the number of LAM cells. 
c. Plasma biomarker analyses. Prostaglandin metabolites (PGE2 and 6-keto-PGF1-alpha), matrix 
metalloproteinases MMP2 and MMP9, cathepsin K, and a cytokine array will be measured on 
serial plasma samples (weeks 0, 8, 16, 24) from subjects on this trial. Marked changes in these 
metabolites are expected when subjects are on celecoxib, and this will serve as a potential 
biomarker of drug effect and compliance. 
d. Exhaled breath condensates (EBCs). EBCs will be obtained from subjects on this trial at the time 
of pulmonary function testing (week 0, 24).  Subjects will be required to withhold food and 
carbonated beverages for one hour prior to EBC collection. An Rtube (Respiratory Research, 
Austin, TX) will be used to collect EBCs by tidal breathing through the mouth for 10 minutes 
without a nose clip. The metal sleeve surrounding the collection chamber will be chilled to -20°C 
for at least one hour prior to EBC collection. The EBC will then be stored at -80°C under N2 gas 
to prevent non-enzymatic oxidation of the parent fatty acids and lipid mediators. Samples will be 
analyzed for prostaglandin metabolites (PGE2 and 6-keto-PGF1-alpha).  
e. Plasma cell-free DNA (cfDNA) will be isolated from each subject on this trial at each time point 
a sample is drawn.  The plasma will be separated, and cfDNA isolated.   The cfDNA will be 
subject to targeted next generation sequencing to search for mutations in TSC2. 
 
 
VI- 
Biostatistical analysis 
b- Data variables 
We will collect information about the safety of Celecoxib in women with LAM and TSC. In particular, 
we will collect data on the incidence of adverse events and severe adverse events.  In addition, we will 
collect information about lung function at the screening visist and at the end of the study period. If 
subject has a renal angiomyolipoma at the screening visit, we will collect information about 
angiomyolipoma size at the end of the 6 months of therapy.  
c- Statistical Methods 
Sample Size Considerations  
Common adverse event rate with Celecoxib provided in the package insert are between 0.2% and 6.0%. 
The below table gives the probability of seeing at least one case of common adverse event associated 
with Celecoxib, using different event rates (Table 2).  
  
Table 2. Probability of observing at least one case of an adverse event 


April 3, 2017 
13 
 
Adverse event rate 
Sample size 
6% 
2.5% 
0.2% 
12 
52% 
26% 
2.3% 
  
With the proposed sample size of 12 subjects will allow us to detect at least one case of common 
adverse event with probability of 2.3% to 52%.   Rates of side effects related to celecoxib in the LAM 
population are unknown. 
 
For lung function parameters (FEV1, FVC, DLCO) and angiomyolipoma size (when applicable), the 
difference between baseline and 24 weeks of therapy will be assessed using the Wilcoxon signed-rank 
test, with a two sided alpha at 0.05.  
For the exploratory endpoints, difference between baseline and 24 weeks of therapy will be assessed 
using parametric and non-parametric tests as indicated. Two-sided alpha of 0.05 will be used to 
determine statistical significance.  
 
VII-    Risks and Discomforts 
a- Risks of Celecoxib 
 
Cardiovascular Risk 
Celecoxib may cause an increased risk of serious cardiovascular thrombotic events, myocardial 
infarction and stroke which can be fatal. All NSAIDs may have similar risk. This risk may increase with 
duration of use. Patients with cardiovascular disease or risk factors for cardiovascular disease may be at greater 
risk. The cumulative risk is 2.5% over 3 years at the Celecoxib dose that we are using in this study.  
 
Hypertension 
Celecoxib can lead to new onset hypertension or worsening of exisiting hypertension. The rate of 
hypertension in one trial was 2.4% 
 
Gastrointestinal Risk 
Celecoxib cause an increased risk of serious gastrointestinal adverse events including bleeding, 
ulceration, and perforation of the stomach or intestines, which can be fatal. These events can occur at any time 
during use and without warning symptoms. Elderly patients are at greater risk for serious gastrointestinal 
events. The incidence after 9 months of therapy of complicated and symptomatic ulcers was 0.78% in one 
study, but the risk was 1.4% in patients older than the age of 65. Concomitant use of aspirin increased the risk to 
3.06%.  
 
Hepatic Risk 
The incidence of borderline elevation (greater than 1.2 times and less than 3 times the upper limit of 
normal) of liver enzymes was 6% in one trial. The incidence of significant elevation in liver enzymes was 0.2% 
 
Renal Risk 
Renal toxicity can be seen especially in patients with pre-existing impairment of kidney function, heart 
failure or liver disease taking diuretics. No percentage on the occurrence of renal toxicity with Celecoxib is 
provided in the package insert.  
 
Skin reactions 
Celecoxib is a sulfonamide and can cause serious skin adverse events such as exfoliative dermatitis, 
Stevens-Johnson syndrome and toxic epidermal necrolysis which can be fatal. These serious adverse events can 
occur without warning and in patients without prior history of sulfa allergy.  
 


April 3, 2017 
14 
 
Pregnancy 
Use of NSAID late in pregnancy can lead to early closure of the ductus arteriosus.  
 
b- Risk of procedures 
 
Risk of Lung Function Tests: 
- Cough 
- Lightheadedness 
- Discomfort due to technique 
- Shortness of breath 
- Fatigue 
Lung function tests will be performed by a qualified respiratory therapist and will be done according to 
ATS guidelines 
 
Risk of questionnaires: 
- Fatigue 
- Distress related to questions 
 
Risk of blood draws: 
- Bruising 
- Pain 
- Infection  
- Lightheadedness and/or fainting 
Specimens will be collected by a qualified member of the research team at the time of study visit and 
drawn according to institutional standards.  
 
Risk of MRI: 
There have been no ill effects reported from exposure to the magnetism or radio waves used in the MRI. A 
known risk of MRI is that magnet could attract certain types of metal. Metal within the subject’s body is 
evaluated (this includes certain dyes found in tattoos, pacemakers, implanted defibrillator, metallic particles in 
the eye, non-fixed metallic particles, vascular clips in the head, prosthetic heart valves, etc) in order to acquire 
the image data.  
 
Risk of exhaled breath condensate: 
It is possible that subject may become lighheaded while breathing into the tube to collect breath condensate.  
 
Genetic Risks 
There is a potential psychosocial risk to subjects related to learning genetic information.  However, we will not 
be looking across the entire genome in these subjects, and will be focusing on genetic changes in TSC2, the 
known genetic cause of LAM.  In addition, our findings will be somatic mutations, which are very common in 
neoplasms in general. Last, the information is safeguarded and not provided to anyone outside of our laboratory 
in a manner that identifiers may be used to identify the specific subject. 
 
 
c- Celecoxib drug interactions: 
- Celecoxib metabolosm is predemoninantly mediated via cytochrome P450 (CYP)2C9 in the liver. 
Co-administration of celecoxib with drugs that are known to inhibit CYP2C9 should be done with 
caution.  
- Warfarin 


April 3, 2017 
15 
 
- Lithium 
- Aspirin 
- Angiotensin converting enzyme inhibitors and angiotensin II antagonists. 
- Fluconazole 
- Furosemide 
- Concomittant NSAID use 
- Methotrexate 
 
 
Celecoxib formulation, instruction and storage 
Celecoxib 200mg capsules are white, with reverse printed on gold band with markings of 7767 on the cap and 
200 on the body. Celecoxib should be stored at 25°C (77°F); excursions permitted to 15-30°C (59-86°F). 
Celecoxib will be taken once daily with meals.   
 
Dose interruption, modification or discontinuation due to toxicities related to study drug 
Single dose adjustement to 100mg daily is allowed if subjects develop adverse events related to study drug. If 
symptoms persist at the reduced dose of Celecoxib then subject is withdrawn from the study. 
 
Protection against general subject risks 
Highly trained physician and nursing personnel familiar with clinical care and research protocols will conduct 
the study procedures. Experienced personnel following established guidelines will perform venipuncture. To 
maintain confidentiality, all laboratory specimens, evaluation forms, and reports will be identified only by a 
coded number. A computer will generate the coded number at random and only the study investigators will have 
access to the codes. All records will be kept in a locked, password protected computer. The case report forms 
for the protocols will be maintained on line under the supervision of the investigator. Subjects will be provided 
with contact information for trial investigators, who will be accessible to subjects at all times through the phone, 
paging or email.  
 
All testing will be reviewed with the potential subjects during the consent process.  
 
Celecoxib is a category C drug before 30 weeks of gestation and Category D afterwards out of concern of early 
closure of the ductus arteriosus. Since LAM is a disease that affects women in their childbearing years, this 
population cannot be excluded from the trial. Urine pregnancy testing will be performed at each study visit. 
Women will be required to use two forms of contraception during and for at least 8 weeks after the last dose of 
Celecoxib.  
 
To monitor for liver, kidney impairements laboratory analysis will be perforemd at each visit. Subjects will be 
instructed to contact study staff if they develop fluid retention, elevated blood pressure, chest pain or any 
symptoms.  
 
The Principle Investigator will be responsible for monitoring the safety of subjects who have entered this study 
and for providing appropriate medical  
Care. The investigators will be responsible for alerting the sponsor to any event that seems unusual, even if the 
event may be considered an unanticipated benefit to the subject. By exercising appropriate health-care options, 
the investigator remains responsible for managing AEs that are serious or that cause the subject to discontinue 
before completing the study. Frequency of follow-up beyond that mandated in the protocol is left to the 
discretion of the investigator. 
 
Confidentiality and HIPAA considerations 


April 3, 2017 
16 
 
All subjects must provide written informed consent and signed HIPAA authorization prior to the performance 
of any screening or main study procedures. Subject confidentiality will be protected throughout the study and 
no subject-identifying information will be released to anyone outside the project. Confidentiality will be secured 
through several mechanisms. Each subject will be assigned an anonymous study ID, which be used on all study 
forms. Any study forms and paper records containing personal identifier information (e.g., address, phone 
number) will be kept secured and locked at each clinical site. No personal identifiers will be placed on 
biological samples and other documents forwarded to central labs and reading centers.  
Access to all subject data and information at the clinical sites, including biological samples, will be restricted to 
authorized personnel. Only authorized personnel at the coordinating center will have access to study data files. 
Authorized personnel will be assigned user logon IDs, passwords and appropriate access privileges to study 
data. Study subjects will be identified only by their initials and subject ID number. No personal identifiers such 
as name, address, social security number will be entered into the study database. Any subject-specific data 
reported to any study committees will only be identified by subject ID number.  
Finally, subjects will not be identified by name in any reports or publications, nor will the data be presented in 
such a way that the identity of individual subjects can be inferred. Analysis files created for further study by the 
scientific community will have no subject identifiers.  
 
 
Protection against subject risks related to the study medication 
Subjects will be provided with written, detailed instruction for calling the study coordinator or physician, 
including but not limited to infections requiring antibiotics, planeed or emergent procedures, or addition of 
drugs known to interact with Celecoxib. The risk associated with Celecoxib will be explained to the subject in 
detail. A plan for dose interruption and reduction is included in the experiemntal plan. Subjects may withdraw 
from the trial at any time, or be withdrawn by the site PI for safety reasons at his/her discretion. The Data and 
Safety Monitor will review routine adverse events and trial results every six months, and serious adverse events 
within 72 hours. Dr. Kwiatkowski will review urgent, persistent or life-threatening adverse events at all times 
during the trial. An on-call clinician at each site will be avaialbel for questions and consultation.  
 
Due to the experimental use of celecoxib in LAM and TSC the trial has been designed with a focus on 
protecting subjects against risk from the medication including: 
1- Small sample size consistent with the exploratory nature of the trial. 
2- Specific exclusion criteria to prevent enrollment of subjects with significant co-morbidities that might 
place them at excess risk (see exclusion criteria) 
3- Specific defined dose and holding criteria related to study adverse events.  
 
VIII- Potential benefits 
Participation in the proposed research will not guarantee direct benefit to participants, although it remains 
possible that a previously undiagnosed clinical abnormality will be brought to the clinician’s attention. 
Information obtained from this research will provide information about the relationship between LAM 
treatment and patient outcomes. Sporadic LAM and Tuberous sclerosis affect more than 1.5 million people 
worldwide. Lung manifestations of TSC, including LAM are its most life threatening manifestations. 
Participants may also benefit from the effects of the drug in the trial, as well as frequent follow-up and 
monitoring. Information learned from this research may benefit women with LAM in the future. Understanding 
the interplay between mTOR dysregulation and COX 2 inhibition may have implications for patients suffering 
from other diseases as well.  
 
IX-Data management  
Data management overview 


April 3, 2017 
17 
 
This study will use REDCap for electronic data capture. REDCap is a secure, web-based data management tool. 
Data will be entered into REDCap directly from the information collected on the source documents.  All staff 
will be trained on REDCap.  Traning modules can be accessed via the website https://redcap.partners.org/redcap 
Data entry in to Redcap should be done within 5 business days from the time the information is collected.  
 
Data Querries and Corrections 
Data will be routinely reviewed for any missing data or discrepancies. Data discrepancies that are identified will 
be resolved within 5 business days. Each site must correct or update the database to resolve the data 
discrepancies.  
 
Investigator Electronic Signature 
The investigator or designee indicates their review of the data accuracy on all eCRFs pages for a subject with 
their e-signature within the REDCap system.  
 
Maintaining the Clinical Center Subject File 
Keep the following documents in the clinical center subject study file for each subject entered in the trial: 
 
Copy of Confidential Subject Information page 
 
Original signed consent form 
 
Copies of all submitted source documents 
 
Confidentiality Data Security Procedures and HIPAA considerations 
All subjects must provide written informed consent and signed HIPAA authorization prior to the performance 
of any screening or main study procedures. Subject confidentiality will be protected throughout the study and 
no subject-identifying information will be released to anyone outside the project. Confidentiality will be secured 
through several mechanisms. Each subject will be assigned an anonymous study ID, which be used on all study 
forms. Any study forms and paper records containing personal identifier information (e.g., address, phone 
number) will be kept secured and locked at each clinical site. No personal identifiers will be placed on 
biological samples and other documents forwarded to central labs and reading centers.  
Access to all subject data and information at the clinical sites, including biological samples, will be restricted to 
authorized personnel. Only authorized personnel at the coordinating center will have access to study data files. 
Authorized personnel will be assigned user logon IDs, passwords and appropriate access privileges to study 
data. Study subjects will be identified only by their initials and subject ID number. No personal identifiers such 
as name, address, social security number will be entered into the study database. Any subject-specific data 
reported to any study committees will only be identified by subject ID number.  
Finally, subjects will not be identified by name in any reports or publications, nor will the data be presented in 
such a way that the identity of individual subjects can be inferred. Analysis files created for further study by the 
scientific community will have no subject identifiers.  
 
 
IX- Monitoring and Quality Assurance 
 
a- Safety Monitoring 
We will institute an independent Data Safety Monitoring Board to review procedures and adverse events that 
occur during the clinical study. Board members will not be involved in the active recruitment and daily routine 
of the study. The principal investigator will present study progress on a routine basis to the board members. 
Board members will review new cases and decide whether the study goals should be altered or the study should 
be stopped. 
 
b- Monitoring of Subjects and Criteria for Withdrawal of Subjects 
Subjects will be monitored throughout the clinical trial for new symptoms or complaints. 


April 3, 2017 
18 
 
 
c- Outcome Monitoring 
The clinical outcome of our participants will be continuously monitored by the study staff and the Data Safety 
Monitoring Board.  
 
d- Adverse event reporting guidelines 
 
Definitions 
 
Adverse event means any untoward or unfavorable medical occurrence in a human subject, including any 
abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally 
associated wit 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).  
  
Serious adverse event means any event temporally associated with the subject’s participation in the research 
that meets any of the following criteria: 
- Results in death 
- Is life threatening (places the subject at immediate risk of death from the event as it occurred) 
- Requires inpatient hospitalization or prolongation of existing hospitalization 
- Results in a persistent or significant disability/incapacity 
- Results in a congenital anomaly/birth defect or 
- Any other adverse event that, based upon appropriate medical judgment, may jeopardize the 
subject’s health and may require medical or surgical intervention to prevent one of the outcomes 
listed above.  
 
Important medical events that may not result in death, be life-threatening, or require inpatient hospitalization 
may be considered an SAE when, based on appropriate medical judgment, they may jeopardize the subject and 
may require medical or surgical intervention to prevent on of the outcomes listed above.  
 
An Unexpected Adverse Event is an adverse reaction, the nature or severity of which is not consistent with the 
applicable product information per the Investigator Brochure.  
Life-threatening means that the subject was, in the view of the investigator, at immediate risk of death from the 
AE as it occurred. It does not include an AE that, had it occurred in a more severe form might have caused 
death.  
Persistent or significant disability/incapacity means that the event resulted in permanent or significant and 
substantial disruption of the subject’s ability to carry out normal life functions.  
Associated with the use of the drug means there is reasonable possibility that the AE may have been caused by 
the drug.  
 
Adverse event reporting 
All AEs (serious and non-serious) will be recorded from start of study treatment (enrollment) through final 
study visit on the AE case report form.  
 
All adverse events will be reported to the Partners Human Research Committee (PHRC) in accordance with the 
accepted guidelines. A 24-hour/7-day telephone number will be provided for contacting the licensed physician 
investigator for this purpose.  
 


April 3, 2017 
19 
 
The research staff will monitor all subjects during their study involvement for evidence of any adverse events. 
Local study site personnel will report all serious adverse events (SAEs) to a designated medical monitor who 
will collect initial reports and oversee medical record retrieval for determination of final diagnosis.  
 
Serious Adverse Events Reporting 
All deaths and all SAEs require reporting form the start of treatment through 28 days after discontinuation of 
the study. The site is requried to call the coordinating center to inform them of the SAE within the 24 hours of 
knowledge of the event. The site should also submit an SAE form via fax to the coordinating center and enter 
the SAE information onto the CRF within 24hours of knowledge of the event.  
 
An updated full written report will be filed as additional information becomes available within 10 working days 
(14 calendar days). The report will include a complete description of the event, use of all concomitant 
medications, and the local investigator’s assessment of causality of the SAE. Non-serious adverse events will be 
reported at routine visits, documented on a separate adverse event form, and transmitted to the central 
coordinating center. All serious and unexpected adverse events will be reported to the overall study PI’s.  
 
Expedited Event Reporting 
Adverse events that meet the definition of serious, study drug-related and unexpected (per the Investigator’s 
Drug Brochure) qualify for expedited reporting to the regulatory authorities. The DSMB will perform a medical 
review of all deaths and study drug-related serious adverse events and evaluate for “unexpectedness”.  
The coordinating center will prepare Med Watch repots for those events identified as serious, study drug-related 
and unexpected as determined by the DSMB. The coordinating center will submit all unexpected study drug 
related SAEs as per 21 CRF 32.  
 
Safety events requiring expedite reporting will be sent to the DSMB chair, with forwarding by the chair to the 
full board as needed, and DOD program office within 1-2 business days. DSMB chair will respond to the 
overall principal investigator and the coordinating center within 48 hours. Reporting framework and timelines 
are to be documented in Data and Safety Monitoring Plan after consultation with board members.  
 
 
Coding for Adverse Events 
All reported adverse events will be classified using the Common Terminology Criteria for Adverse Events 
(CTCAE), version 4.  
 
 
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