Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

Transcription

1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Townsley DM, Dumitriu B, Liu D, et al. Danazol treatment for telomere diseases. N Engl J Med 2016;374: DOI: /NEJMoa

2 Protocol Versions for NHLBI protocol number 11-H-0209 Protocol title: Male hormones for telomere related diseases Contents: This document includes the current approved protocol dated 10/27/2015 and the initial approved version of the protocol dated 6/22/2011. In addition, included at the end is a summary of changes to the protocol since the initial approval. A majority of the changes made to the protocol since its initial approval do not directly affect the objectives and endpoints, and are administrative in nature. Table of Contents Current Approved Protocol dated 10/27/2015, approved by the IRB on 10/30/ First approved version of the protocol, dated 6/22/2011, approved by the IRB on 6/28/ Summary of all changes to the protocol

3 Current Approved Protocol dated 10/27/2015, approved by the IRB on 10/30/2015 2

4 CLINICAL RESEARCH PROJECT Protocol # 11-H-0209 Date: October 27, 2015 To: Richard Cannon, M.D., Chair, NHLBI, IRB Title: Male hormones for telomere related diseases Other Identifying Words: Telomeres, aplastic anemia, androgens Protocol Principal Investigator: * H.B., NHLBI (E) Bldg 10,CRC 3E-5132 Medically and Scientifically Responsible Investigator: * Neal S. Young, M.D., Chief, HB, NHLBI (E) Bldg 10, CRC Associate Investigators: Bernadette Gochuico, M.D., NHGRI (E) Bldg 10, MSC 1851 Joel Moss, M.D., Ph.D., CPB, NHLBI (E) Bldg. 10, CRC, 6D03 * Bogdan Dumitriu, M.D., NCI (E) Bldg 10, RM 12N226 Olga Rios, RN, Research Nurse, H.B., NHLBI (E) Bldg 10, CRC Barbara Weinstein, RN, Research Nurse, HB, NHLBI (E) Bldg 10, CRC Colin Wu, PhD, Biostatistician, OBR, NHLBI (E) RKL2, Room 8218 Xin Tian, PhD, Biostatistician, OBR/NHLBI (E) RKL2, Room 8217 Kinneret Broder, RN, HB, NHLBI (E) Bldg 10, CRC Theo Heller, M.D., NIDDK (E) Bldg 10, 9C432B *Janet Valdez, M.S., PAC, HB, NHLBI (E) Bldg 10-CRC Phillip Scheinberg, M.D., HB, NHLBI (V) / Rua Conto Popular 101, Sao Paulo, SP, Brazil The * indicates investigators allowed to obtain informed consent for this protocol. Subjects of Study: Number Sex Age-range 35(25 complete study) Either > 2 years and weight > 12 kg Project Involves Ionizing Radiation? Off-Site Project? Multi center trial? DSMB Involvement? Tech Transfer? 11-H-0209 October 27, 2015 Yes 3 1

5 PRECIS Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder characterized by pancytopenia and a hypocellular bone marrow. Allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) are the main treatments of SAA with comparable longterm outcome achieved with either therapy. 1 AA can be inherited or acquired. Inherited AA is usually diagnosed in childhood, when cytopenias and/or characteristic physical findings manifest. Short stature, hyperpigmentation (café au lait spots), abnormal thumbs and microcephaly occur in Fanconi anemia; hyperpigmentation, nail dystrophy and oral leukoplakia in dyskeratosis congenita (DKC); and exocrine pancreatic insufficiency, malnourishment, malabsorption and short stature in Shwachman-Diamond. However, physical abnormalities may not always be present in patients with an inherited form of SAA and the identification of specific mutations in genes of the telomerase complex in patients with acquired SAA has blurred the distinction between the inherited and acquired forms of AA. While immune destruction of hematopoietic cells is the proximate cause of SAA, target cell abnormalities have been identified as risk factors in bone marrow failure. Mutations in genes of the telomere repair complex are etiologic in DKC, and they are present also in some patients with apparently acquired SAA. 2,3 Telomeres are nucleotide repeats of non-coding DNA at the end of the chromosomes which function as protective caps to prevent erosion of genomic DNA during cell division. Telomeric DNA can be elongated by the telomerase complex, which is comprised of a reverse transcriptase catalytic subunit (encoded by TERT), an RNA template (encoded by TERC) and associated proteins. 2 Telomerase activity is crucial in maintaining telomere length in cells with a high proliferative capacity, such as hematopoietic stem cells (HSCs) and lymphocytes. Telomeres were reported to be short in up to one-third of patients with SAA. 4,5 Initially this occurrence was presumed to be secondary to hematopoietic stress. 2 However, the discovery of loss-of-function mutations in genes of the telomerase complex (TERC, TERT) established a genetic etiology for telomere attrition in some patients with marrow failure who did not have the stigmata associated to an inherited bone marrow failure syndrome. 3,6 These findings implicated telomerase dysfunction in failed hematopoiesis. 7 In family members of probands with SAA, telomerase mutations have been observed which were associated to varying degrees of cytopenias, idiopathic pulmonary fibrosis (IPF) and/or cirrhosis. 8 Telomere length has been associated with human cancer. In a recently completed analysis, shorter telomeres in SAA patients at disease presentation were associated to a greater risk of relapse, clonal evolution to myelodysplasia, and mortality. 9 In acute myelogenous leukemia (AML) patients with no underlying telomerase disorder, hypomorphic mutations in TERT were three-fold more frequent in patients than in controls. 10 Telomere attrition has been implicated in a variety of solid organ malignancies including esophageal and colon adenocarcinoma. 6,11,12 In a longitudinal population based study, shorter telomere length associated to a higher cancer mortality risk overtime. 13 It is plausible that a shorter telomere length is not just a biomarker associated to development of cancer, but involved in its pathogenesis. Ample experimental data supports an important role of critically short telomere length in genomic instability. 14 Furthermore, our laboratory data (unpublished) shows that similar chromosome instability occurs in bone marrow cells of mutant patients, confirming the experimental data. Thus, a common molecular mechanism appears to underlie risk for cancer and a range of clinical entities H-0209 October 27,

6 In vitro studies suggest that telomere length could, in theory, be modulated with sex hormones. 15 Exposure of normal peripheral blood lymphocytes and human bone marrow derived CD34 + cells to androgens increased telomerase activity in vitro and androgens increased low baseline telomerase activity in individuals carrying a loss-of-function TERT mutation to normal levels In retrospect, the beneficial effects of sex hormones on telomerase activity may be the mechanism by which SAA patients treated over 40 years ago with male hormones showed hematologic improvement in some cases. 18 In recent years we have seen patients referred to our clinic with varying degree of cytopenia(s) who had significant family history for cytopenia(s), idiopathic pulmonary fibrosis (IPF) and/or cirrhosis. We have identified very short telomeres in these patients and in some mutations in TERC and TERT. We hypothesize that male hormone therapy might modulate telomere attrition in vivo and ameliorate progression or reverse the clinical consequences of accelerated telomere attrition. Therefore, we propose male hormone therapy in patients with cytopenia(s) and/or IPF who show evidence of telomere dysfunction by a short age adjusted telomere length associated to telomerase gene mutations. The primary biologic endpoint will be delay of telomere attrition over time compared to known rates of telomere erosion in normal individuals and in those who carry mutation in the telomerase genes. The main clinical endpoint will be tolerability of oral danazol over two years. Secondary endpoints will be improvement in blood counts and/or pulmonary function. The small sample size, lack of control groups, and variable clinical course among those with marrow failure and IPF, will not allow for definitive assessment of clinical benefit. Nevertheless, we believe this protocol will provide insight into the possible effects of androgen therapy on telomere attrition in humans and of possible clinical benefit in telomere related disorders, and serve as hypothesis generating for further larger controlled studies. 11-H-0209 October 27,

7 TABLE OF CONTENTS 1. OBJECTIVES BACKGROUND AND SCIENTIFIC JUSTIFICATION STUDY DESIGN ELIGIBILITY ASSESSMENT TREATMENT PLAN CLINICAL MONITORING CRITERIA FOR RESPONSE EXPLORATORY LABORATORY RESEARCH STUDIES BIOSTATISTICAL CONSIDERATIONS DATA AND SAFETY MONITORING HUMAN SUBJECT PROTECTION PHARMACEUTICALS REFERENCES APPENDIX A: HEMATOLOGY BRANCH LABORATORY RESEARCH STUDIES H-0209 October 27,

8 1. OBJECTIVES a. Assess the rate of telomere attrition with androgen therapy in subjects with short telomeres b. Assess the efficacy of androgens in ameliorating cytopenia(s) related to telomere disorder c. Assess the efficacy of androgens in slowing the progression of idiopathic pulmonary fibrosis d. Assess tolerability of long-term androgen use 2. BACKGROUND AND SCIENTIFIC JUSTIFICATION 2.1 Telomeres Telomeres are structural elements that cap the ends of chromosomes, protecting them from recombination, end-to-end fusion, and recognition as damaged DNA. In human somatic cells, telomeres typically consist of more than 1000 tandem repeats of nucleotides (CCCTAA in one strand of DNA and TTAGGG in the other) and associated proteins. These repeats are gradually lost with cellular replication. The attrition of repeats eventually shortens telomeres critically; the result is arrested proliferation and senescence, shortened life span, apoptosis, and genomic instability of the cell. Maintenance of the integrity of telomeres requires the telomerase ribonucleoprotein complex, which consists of telomerase reverse transcriptase (TERT) and its integral RNA template (TERC), in addition to other proteins. TERT copies a short region of TERC into telomeric DNA to extend the 3' end of the chromosome. Telomeres have been implicated in physiologic aging, certain fibrotic disorders, marrow failure and in the development of cancer. 7 The direct link between telomeres and especially telomere repair mechanisms and human disease came from family studies of patients with X-linked dyskeratosis congenita (DKC). Analysis of large pedigrees in the United Kingdom identified a location containing the DKC1 gene, which encodes dyskerin, a small nucleolar ribonucleoprotein that is physically associated with the telomerase complex. 19 Subsequently, telomere length was measured as extremely short in patients with dyskeratosis congenita. 20 This initial discovery led to the purposeful investigation of another gene product, the RNA template encoded by the gene TERC, which was found to be mutated in patients with autosomal dominant dyskeratosis congenita Telomeres and marrow failure About a decade ago, telomeres were first measured by Southern hybridization to be short in about one-third of acquired aplastic anemia cases by investigators at St George's Hospital in London and patients with the shortest telomeres appeared more likely to develop late and 11-H-0209 October 27,

9 malignant clonal complications. We also measured telomere length by a flow cytometric method and found about 1/3 of patients with aplastic anemia had short telomeres for age. 4 We later identified mutations in TERC and also in TERT, the gene encoding the reverse transcriptase telomerase, in some patients with apparently acquired anemia. 2,3,6 Affected patients and also their family members carrying the mutation have short telomeres and evidence of a much diminished hematopoietic compartment. 8 For example, TERC and TERT mutations correlate with decreased bone marrow cellularity, low CD34 cell number, diminished functional hematopoietic progenitor cells in vitro, and increased levels of hematopoietic growth factors. Recently, we have identified a third gene as abnormal in patients with apparently acquired aplastic anaemia. 22 This gene, termed the Schwachman-Bodian-Diamond syndrome gene (SBDS) encodes a protein, which is, like dyskerin, associated with RNA processing. In the Schwachman syndrome, another constitutional marrow failure disease, neutropenia is associated with pancreatic exocrine insufficiency, and presentation of disease manifestation occurs in early childhood. Schwachman syndrome patients also have very short telomeres. We have recently identified four patients with apparently acquired aplastic anemia, without evidence of pancreatic disease or malnutrition, who presented to medical attention in adult life and who have mutations in the SBDS gene. These patients have short telomeres and also diminished telomerase activity, as measured in vitro. For all three genes, family members who also have mutated telomere repair complex genes appear normal, or they have only slight hematologic abnormalities, such as modest anemia, neutropenia, or thrombocytopenia, alone or in combination. The clinical consequences of cytopenia(s) derive from low blood counts. Anemia leads to fatigue, weakness, lassitude, headaches, and in older patients dyspnea and chest pain, and these manifestations are most commonly responsible for the clinical presentation. Thrombocytopenia produces mucosal bleeding: petechiae of the skin and mucous membranes, epistaxis, and gum bleeding are frequent and early complaints. Bleeding can be brisk in the presence of accompanying physical lesions, as in gastritis and fungal infection of the lungs. The most feared complication of thrombocytopenia is intracranial hemorrhage. Bacterial and fungal infections in the setting of neutropenia are a major cause of morbidity and mortality, most often the cause of death in refractory cytopenias. In patients with severe aplastic anemia (SAA), current standard of care is transplant or immunosuppressive therapy (IST), regardless of telomere length. In a recent analysis, a short for age-adjusted telomere length was not sufficient to preclude hematologic recovery, but higher rates of relapse, clonal evolution and mortality were observed in this group. 9 In those with cytopenia(s) associated to short telomeres and/or mutations in genes of the telomere repair complex, management is uncertain. Supportive care with growth factors and transfusional support is sometimes employed depending on symptoms, degree of cytopenia(s) and patient preference. Standard immunosuppression regimens used for marrow failure without known mutations are sometimes considered. Androgens have been administered in this setting with anecdotal success, but systematic studies to assess the activity of this treatment are lacking. Hematopoietic stem cell transplantation is almost never pursued when cytopenia(s) is not severe and patients who are receiving no or only minimal transfusional support. 11-H-0209 October 27,

10 2.3 Telomeres and idiopathic pulmonary fibrosis (IPF) IPF is a chronic progressive fibrotic lung disorder usually affecting adults over the age of 40. Common symptoms are cough and dyspnea and pulmonary studies often reveal impaired gas exchange and reduced lung volumes. Histopathology reveals patchy fibrosis of the lungs, interstitial inflammation, disorganized deposition of collagen and extracellular matrix with distortion of pulmonary architecture. In about 20% of patients with dyskeratosis congenita, lung complications ultimately diagnosed as pulmonary fibrosis develop. 23 This association led to the discovery of telomerase mutations in about 15% of patients with familial IPF. 24 Patients with IPF have a very poor prognosis and treatment options other than lung transplantation for IPF are ineffective. Immunosuppression with antioxidant therapy has been of possible benefit with no difference in mortality observed with the addition of N-acetylcestine to azathioprine/glucocorticoid therapy. 25 Antifibrotic agents are being investigated and may slow the rate of progression. 26,27 However, drugs which halt or reverse disease progression are unavailable, and eventually patients will require supplemental oxygen and, in selected cases, will undergo lung transplantation Scientific and clinical justification of the protocol The exact mechanism by which telomere repair complex gene mutations contribute to the development of bone marrow failure and pulmonary fibrosis is not completely understood. Most obviously, these genes are correlated with decreased hematopoiesis in marrow failure, as described above. However, in our experience of multiple pedigrees, the gene itself is insufficient to produce aplastic anemia by stem cell depletion, and an immunological component appears to be required. One simple possibility is that immune destruction of a very reduced number of hematopoietic stem cells and progenitor cells produces aplastic anemia. Alternatively cells carrying telomere repair complex gene mutations may be qualitatively unable to regenerate in the face of immune-mediated destruction. In dyskeratosis congenita (DKC) kindreds, multi-organ abnormalities have been described, including not only the characteristic ectodermal dysplasia manifestations of nail, skin, and mucous membrane changes but also pulmonary, hepatic, and other organ system involvement. The pathophysiology of aplastic anemia is immune-mediated destruction of the hematopoietic compartment. Such T-cell attack on a specific organ also characterizes other human autoimmune diseases. In many respects, aplastic anemia, multiple sclerosis, type I diabetes, uveitis, and inflammatory bowel disease are similar in demographic features, T H1 /T C1 predominance as measured by secretion of cytokines such as gamma-interferon and tumor necrosis factor, mainly cellular rather than humoral immunity, and responsiveness to immunosuppressive drugs. These diseases can be familial, and some genetic factors, mainly histocompatibility antigens, have been identified. Tissue repair obviously is important for longterm organ function maintenance and may be crucial to the disease s clinical manifestation over time. 11-H-0209 October 27,

11 That telomere attrition can be modulated in vitro and the historically low rate of clonal evolution in AA patients who recovered with androgens 29 suggests therapeutic opportunity to decrease or possibly reverse the rate of telomere shortening in diseases of telomere dysfunction, by increasing telomerase activity, slowing telomere attrition, assisting cell regeneration, and ultimately lead to tissue regeneration and/or improved haematopoiesis. 15 Independent of clinical benefits, blood samples collected prospectively will allow the opportunity to follow the rate of telomere attrition overtime and observe if modulating agents can improve telomerase function in humans. Therefore, we propose a regimen of oral androgen therapy in patients with telomere related diseases with a goal to slow the rate of telomere erosion in mutant patients and avert the clinical consequences of impaired marrow function and/or progressive pulmonary fibrosis. Furthermore, in vivo prospective monitoring of telomere length in patients with telomere diseases who are receiving androgens will help determine if telomere attrition can be modulated, which could have significant implications for cancer prevention, especially in the setting of chronic inflammatory conditions such as Barrett s esophagus and inflammatory bowel disease. 11,30,31 Data on longitudinal rates of attrition in the same individual are insufficient to base a research protocol, but such information will soon become available from our own studies and others. Therefore, we have designed the current protocol to primarily assess the feasibility of long-term use of androgen therapy, and potential benefits on telomere attrition and clinic benefit are secondary endpoints and intended to be hypothesis generating. Androgen therapy has been employed in hematology for many decades to treat marrow failure, with long-term use (years) common. 18,29,32-51 Therefore, vast historical experience suggests that most patients will tolerate danazol in our study. Furthermore, danazol at the route and dose used in this study is FDA approved to treat other conditions such as endometriosis and fibrocystic breast disease. Finally, almost all anticipated toxicity from hormone use should be detected by routine clinical and laboratory monitoring and be reversible with reduction of dose or discontinuation of therapy. 3. STUDY DESIGN This is a phase I/II study designed to assess the safety and activity in attrition rate of a male hormone (danazol) in conditions that are related to telomere dysfunction. Subjects with a short age-adjusted telomere length (first percentile) with or without mutations in telomerase genes will receive danazol 800 mg/d for 2 years. 11-H-0209 October 27,

12 4 ELIGIBILITY ASSESSMENT 4.1 Inclusion criteria Short age-adjusted telomere length in the first percentile and/or a mutation in telomerase genes One or more of the following cytopenia(s). OR Anemia Symptomatic anemia with a hemoglobin < 9.5 g/dl or red cell transfusion requirements > 2 units/month for at least 2 months Reticulocyte count < 60,000 /µl Thrombocytopenia Platelet count < 30,000 /µl or < 50,000 /µl associated with bleeding Decreased megakaryocytic precursors in the bone marrow Neutropenia Absolute neutrophil count < 1,000 /µl Idiopathic pulmonary fibrosis diagnosed by either a lung biopsy of high resolution computed tomography scan of the chest according to guidelines from the American Thoracic Society and European Respiratory Society Age > 2 years Weight > 12 kg 4.2 Exclusion criteria Moribund status or concurrent hepatic, renal, cardiac, neurologic, pulmonary, infectious, or metabolic disease of such severity that it would preclude the patient s ability to tolerate protocol therapy, or that death within 30 days is likely Potential subjects with cancer who are on active chemotherapeutic treatment Current pregnancy, or unwillingness to avoid pregnancy if of childbearing potential 11-H-0209 October 27,

13 4.2.4 t able to understand the investigational nature of the study or give informed consent or does not have a legally authorized representative or surrogate that can provide informed consent per section TREATMENT PLAN 5.1 Danazol Administration Danazol will be administered orally at 400 mg twice a day. The 800 mg dose was chosen since it is known to have activity in marrow failure. The drug will be administered in divided doses. Children less than 50 kg will be dosed at 16 mg/kg not to exceed 800 mg/day. LFTs will be monitored for one year routinely and the danazol will be adjusted accordingly. In subjects who have difficulty tolerating danazol at 800 mg/d, the dose will be reduced by 200 mg/d and side effects will be reassessed. If symptoms related to the drug persist, subsequent 200 mg/d dose reductions will be done until a tolerated dose is achieved. If the subject cannot tolerate danazol 200 mg/d, the drug will be discontinued. Danazol will be continued for 2 years if tolerated. Other events that would cause a dose reduction are liver function abnormalities, irritability, emotional lability, and other labeled events. Subjects may also require dose interruptions due labeled events or for other clinically indicated reasons. Dose interruptions will typically not be for more than 7 days and will be monitored. The treatment period is two-years, so the rare need to interrupt dosing will not affect the study objectives or compromise subject safety. There is no standard of care for patients with telomeropathy. Organ dysfunctions secondary to telomere disorders are treated symptomatically. For marrow failure, immunosuppression is usually ineffective, and for pulmonary fibrosis, there are no effective therapies. For those patients who do not meet the inclusion criteria, the standard treatments, depending on age or disease status, may include immunosuppression for aplastic anemia patients. In severe cases, immunosuppression with antithymocyte globulin may be an option. If the counts are in the non-severe range, other immunosuppression with cyclosporine may be used. Supportive care with transfusions or growth factors may be administered to possibly help with improving blood counts. 6. CLINICAL MONITORING Bone marrow aspirate will be read by a pathologist and used for diagnostic purposes. Samples will be ordered and tracked through the CRIS Screens. Should a CRIS screen not be available, the NIH form will be completed and will accompany the specimen and be filed in the medical record. 11-H-0209 October 27,

14 6.1 Pre-study Evaluation Baseline status will be evaluated as follows: Medical history and physical examination Baseline assessments (done at screening or diagnostic workup, not repeated on study) Pregnancy test (urine HCG in women of child bearing potential) Baseline clinical laboratory studies Complete blood count with differential Reticulocyte count Type and screen Acute Care (Na, K, Cl, CO2, Creatinine, Glucose, and Urea Nitrogen), Mineral (Phosphorus, Magnesium, Albumin, and Calcium), Hepatic (Alk Phosphatase, ALT, AST, Total Bilirubin, and Direct Bilirubin), and Other (Total Protein, CK, Uric Acid, and LD) panels. Lipid profile Baseline Endocrine laboratory studies Vitamin D2 & D3 level Free T4 and TSH Thyroid binding globulin Insulin Glucose Hemoglobin A1C Estradiol Progesterone LH FSH Testosterone DHEAS DHEA Androstenedione Cortisol 17-hydroxyprogesterone ACTH Bone marrow aspirate and biopsy with cytogenetic analysis as appropriate to stage and classify underlying disease Flow cytometry of the peripheral blood for glycosylphosphatidylinisotol anchored proteins HLA typing (if not already available), including those patients with pulmonary fibrosis Chest X-ray Regular and high resolution chest CT scan Pulmonary function test 6-minute walk test Bone density (starting at age 6) In males, testicular ultrasound In females, ovarian ultrasound Inhibin B and anti-mullerian 11-H-0209 October 27,

15 Bone age x-ray once per year (for subjects between the ages of 3 and 18, both inclusive) Ferriman-Gallwey score (every 6 months) Tanner staging (every 6 months) Liver ultrasound Pre-study evaluation will be conducted on another Hematology Branch protocol (97-H-0041) to determine eligibility and baseline status. 6.2 Study drug through 12 months After initiation of drug, subjects will be followed by their home physician or at the Clinical Center and have blood work done as detailed below. Progress notes and laboratory results from home physicians will be faxed to the Research nurse, Barbara Weinstein, RN Subjects must be evaluated at the NIH Clinical Center at the 6-month (+/-7 days) time point, after which they will continue routine follow-up at home. CBC with differential weekly (+/- 3 days), every other week (+/- 3 days) or less frequently depending on severity of cytopenia(s) and/or transfusion requirements Acute Care, Mineral, Hepatic and Other panels(every other week +/- 3 days) (Home MDs: electrolytes, transaminases, urea nitrogen (BUN), serum creatinine, total bilirubin). Monthly (+/- 7 days) from 6-12 months. Lipid profile Reticulocyte count at 6 months only (home MDs only if available) Pregnancy test at 6 months Flow cytometry of the peripheral blood for GPI-cells (6 and 12 months +/- 1 month only) Intermittent chest x-ray, ultrasounds, CT scans, MRI or appropriate laboratorial, imaging or other diagnostic studies as medically indicated Chest x-ray (6 and 12 months +/- 1 month only) if clinically indicated Regular and high resolution chest CT scan (6 and 12 months +/- 1 month only) if clinically indicated 6-minute walk test (6 and 12 months +/- 1 month only) Pulmonary function test (6 and 12 months +/- 1 month only) Ferriman-Gallwey score (every 6 months) Tanner staging (every 6 months) Liver ultrasound (6 and 12 months+/- 1 month only) 6.3 Long term follow up (12 months to 24 months) Subjects must be evaluated at the Clinical Center at 12 months and then yearly thereafter to 2 years and have blood work done as detailed below. Subjects will be seen by their home physician as clinically indicated and the Clinical Center and home physician will continue to communicate clinically significant updates as needed. Complete blood counts with differential (annually +/- 2 months) Acute Care, Mineral, Hepatic and Other panels (annually +/- 2 months) Lipid profile 11-H-0209 October 27,

16 Pregnancy test at 12 and 24 months Reticulocyte counts (annually +/- 2 months) Flow cytometry of the peripheral blood for GPI-cells (annually +/- 2 months) Bone marrow biopsy and aspiration with cytogenetics (yearly +/- 2 months) (For pulmonary fibrosis subjects without cytopenias and normal baseline bone marrow evaluation, no bone marrow biopsy follow up is required. However, if they develop cytopenias, bone marrow biopsy may be clinically indicated.) Chest x-ray (yearly +/- 2 months only) Regular and high resolution chest CT scan (yearly +/- 2 months only) 6-minute walk test (yearly +/- 2 months only) Pulmonary function test (yearly +/- 2 months only) Ferriman-Gallwey score (every 6 months) Tanner staging (every 6 months) Bone density every 2 years (starting at age 6) Liver ultrasound In males, Testicular ultrasound every 2 years In females, ovarian ultrasound every 2 years Inhibin B and anti-mullerian hormone every year Bone age x-ray every year (for subjects between the ages of 3 and 18, both inclusive) Endocrine laboratory studies Vitamin D2 & D3 level Free T4 and TSH Thyroid binding globulin Insulin Glucose Hemoglobin A1C Estradiol Progesterone LH FSH Testosterone DHEAS DHEA Androstenedione Cortisol 17-hydroxyprogesterone ACTH 6.4 Subject Compensation Reimbursement for travel expenses will be provided in accordance with NIH guidelines. In determining reimbursement, the following factors are considered applicable to this protocol: the subjects are diagnosed with a rare disease; the protocol offers the potential for direct benefit; the protocol regimen is demanding; and in order to complete accrual in a reasonable timeframe a 11-H-0209 October 27,

17 geographically dispersed participant population is required in addition to local participant population. Payment for participation: $0 Travel (air/train/bus): Travel from home for the first NIH visit will not be reimbursable. If the patient consents to the research protocol, travel home following the first visit will be reimbursable. Subjects will be reimbursed 100% of government rate for travel once the subject has been determined eligible to participate and signs consent. Local Travel (< 50 miles) (car/taxi/shuttle/train/bus): Subjects will be reimbursed for local train/bus and/or shuttle costs. Reimbursement for car mileage less than 50 miles from home is not provided. Taxi will be paid only when medically necessary and authorized by the PI. Long distance travel (> 50 miles) (car/taxi/shuttle/train/bus): Subjects will be reimbursed for long distance car, train or bus costs. Car mileage will be reimbursed $0.41/mile when the distance from home is greater than 50 miles. Subjects will not be reimbursed for rental car cost beyond the car mileage rate. Taxi will be paid only when medically necessary and authorized by the PI. Meals: Subjects will not be reimbursed for meals. Lodging: Subjects will be reimbursed for hotel/motel lodging at a rate of $60/night for a maximum of 7 nights after which the reimbursement is $30/night. If space is available, the Children s Inn or the Family Lodge will be paid directly. Guardian coverage: Subjects will be reimbursed for guardian travel (100% of government rate) if guardian is pre-approved and it is medically indicated and lodging ($15.00/night) provided the services of a guardian are medically indicated and pre-approved. 5 CRITERIA FOR RESPONSE Hematologic response will be determined as improvement in one or more of the listed parameters: a) ANC Increase in ANC > 500 /µl above baseline b) Platelets Increase > 20,000 /µl above baseline c) Hemoglobin Increase in hemoglobin 1.5 g/dl OR 11-H-0209 October 27,

18 Transfusion independence in transfusion-dependent subjects (> 2 months from last transfusion) OR Reduction in transfusion requirements >50% in transfusion-dependent subjects Changes in pulmonary status will be evaluated by pulmonary function tests (forced vital capacity) and high-resolution chest CT. Improvement in counts should be sustained for at least one month and demonstrated by at least 2 serial measurements. Subjects that are dependent upon exogenously administered growth factors will not be considered as fulfilling response criteria. 8. EXPLORATORY LABORATORY RESEARCH STUDIES 8.1 Collecting, Tracking and Disposition of Samples Intended use: During the course of participating on this study, 60 cc of blood (at baseline) and 5 cc of bone marrow aspirate (baseline, 6 months, 12 months, and annually thereafter) may be requested for the following correlative laboratory research studies. These studies are not used in assessing the primary endpoint but are undertaken for descriptive or exploratory ancillary studies which may or may not be done and if done, may be correlated with response. In pediatric subjects, 3 ml/kg of blood not to exceed 60 ml may be requested for laboratory research studies. These specimens will not be examined by a pathologist. Flow cytometric analysis of lymphocyte subsets Measurement of lymphocyte function and immune responses directed to EBV and CMV. Chromosome analysis using standard cytogenetics and FISH at the initial evaluation and at the 6 month follow-up visit. Analysis of the T cell receptor V-beta profile in the marrow and peripheral blood of responders and non-responders Evaluation for the presence of abnormalities of the telomere repair complex Telomere length assay at baseline, 6, 12, and 24 months Assay for cytokines/chemokines and their receptors Serum (or plasma) and cells for viral analysis Hematopoietic progenitor colony formation and long term-culture-initiating cell assays Flow cytometry for PNH Serum (or plasma) and cells for DNA/RNA extraction and PCR for nucleic acids 11-H-0209 October 27,

19 In the event there is any extra sample, these will be stored with the subject s permission for other exploratory laboratory research studies reviewed and approved by the IRB and listed in Appendix A. Storage: Research samples will be stored coded in the secure laboratory of the principal investigator. Research samples will be stored and tracked in accordance with the NHLBI DIR BSI Policy. Tracking: Samples will be ordered and tracked through the CRIS Research Screens. Should a CRIS screen not be available, the NIH form will be completed and will accompany the specimen and be filed in the medical record. In order to ensure proper tracking across several labs, the progress note detailing the consent process will also detail the laboratory to which the sample was initially sent. Specimens will be entered in the NHLBI Biospecimen Inventory System (BSI). Samples will not be sent outside NIH without IRB notification and an executed MTA. End of study procedures: Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. Loss or destruction of samples: Should we become aware that a major breech in our plan for tracking and storage of samples has occurred, the IRB will be notified. 9. BIOSTATISTICAL CONSIDERATIONS 9.1 Sample sizes In comparison to the normal attrition rate of about 60 bp/year, patients with telomerase gene mutations have an increased attrition rate of about 120 bp/year (our unpublished data). We anticipate that the androgen therapy could reduce the attrition rate in 2 years in subjects with very short telomere length (first percentile) and/or those with telomerase gene mutations to approximate 96 bp/year (a 20% reduction from 120 bp/year) or less, and define the subject to be a responder if his/her attrition rate is on average 96 bp/year or less (i.e., 192 bp or less for 2 years). We hypothesize that the proportion of subjects who respond to the androgen treatment (i.e., response probability) could reach 30% or more at the end of the two-year treatment period. If the response probability is 10% or less it would warrant discontinuing the treatment on this subject population. Let p be the response probability at 24 months for this subject population. Our sample size is determined by testing the null hypothesis, H 0 : p 10%, versus the alternative, H 1 : p 30%, at 0.05 significance level and 0.80 of the power. We would like to test this treatment using a small number of subjects, and terminate the study if early evidence suggests that the response probability falls below the benchmark rate of 10%. We determine the sample size using the Two-Stage Minimax Design outlined in Table 1 of Simon (1989), since it requires a smaller total number of subjects (n=25) compared to the Two-Stage Optimal Design (n=29). At the first 11-H-0209 October 27,

20 stage, 15 subjects will be accrued and the null hypothesis will be accepted (i.e., the treatment will be terminated) if no more than 1 subject has telomere attrition rate reduced to >30% on the treatment in 24 months. If 2 or more subjects have improved attrition rate at 24 months at the first stage, additional 10 subjects will be accrued. The first subject was enrolled on August 10, 2011 and 15 subjects were enrolled by April subject had reached the 24 month time point to evaluate response. The study is designed to have 5% significance level and 80% power based on the Simon s Two-Stage Minimax design, which requires accrual of 15 subjects at the first stage and 10 subjects at the second stage if 2 or more subjects of the first 15 subjects show improved telomere attrition rate at 24 months. As of April 12, 2012, no study stopping rules have been met as no subjects have died and no subjects have suffered a grade IV toxicity considered to be probably or definitely related to danazol. If a single-stage design is used, i.e., continue accruing the remaining 10 subjects, a total of 25 subjects will still yield 80% power at 5% significance level. Therefore, the total sample size, power and significance level will not be affected if we change the Simon s Two-Stage Minimax design to the single-stage one sample test for proportions. Because of the observed hematologic improvements, safety profile and the practical need of offering adequate treatment to our patients, we believe the scientific integrity and statistical accuracy of the study will not be affected if we continue to accrue the remaining 10 subjects without waiting 24 months to evaluate the attrition rate in the first 15 subjects. Therefore, with permission from the IRB (IRB approval obtained June 11, 2012), the remaining 10 subjects will be accrued. As of February 5, 2013, 22 subjects have been enrolled but 8 subjects are off-study. With permission from the IRB, the accrual ceiling will be increased to 35 subjects. By increasing the accrual to 35 subjects we would like have at least 25 fully evaluable subjects at 24 months after taking into account the 8 subjects who are off study. Using the intent-to-treat principle, those subjects who drop out of the study prior to 24 months will be counted as non-responders. The study will still be designed like the original study to have 5% significance level but will have greater than 80% power (86% power) for the primary endpoint. The increase in accrual will increase the number of subjects who will be evaluable for secondary endpoints. The rationale for the request to increase the accrual ceiling to 35 subjects is based on the following: a) the treatment has an acceptable safety and good hematological response profile; b) subjects may be off study prior to evaluation for the primary endpoint and/or secondary endpoints; c) the statistical power of the original design is maintained; d) there is a population of patients with telomere diseases in need of treatment for whom there is currently no standard of care available. 9.2 Primary Endpoints The primary objective of the study is to evaluate safety and activity of danazol in slowing telomere attrition rates in subjects with telomere related diseases. The primary safety endpoint will be toxicity profile in the 24 months following danazol therapy. The biologic endpoint is reduction in telomere attrition rate yearly. 9.3 Secondary Endpoints 11-H-0209 October 27,

21 Secondary endpoints will also be evaluated for the study to include: (a) hematologic response at 3 and 6 months, and yearly thereafter; (b) relapse (c) clonal evolution to PNH, myelodysplasia or acute leukemia; (d) survival; (e) progression of PFTs and CT scans at 6 months, and yearly thereafter (f) 6-minute walk test at 6 months and yearly thereafter. 9.4 Statistical Methods The planned analyses will include descriptive statistics on the incidence and severity of adverse events, such as the percentage of subjects with toxicities. Sample proportion and its standard deviations and 95% confidence intervals will be used to estimate the response probability of the primary endpoint and evaluate its statistical inferences. The testing statistic described in Simon (1989) will be used to test the null and alternative hypotheses. When it is deemed appropriate, sample proportions and their inferences, such as standard deviations, confidence intervals, and t- tests, for the response probabilities under subgroups of patients will also be evaluated. Such subgroup analysis will be useful to evaluate the similarities and differences between different types of patients. Potential subject subgroups may be stratified by age, gender or ethnicity, among others. The proportions of adverse events, serious adverse events (SAE) and treatment related SAEs would be estimated using the sample proportions, and 95% confidence intervals given. The time to adverse events will be analyzed using appropriate survival analysis methods such as Kaplan-Meier estimates. 9.5 Stopping Rules The drug treatment will be discontinued in subjects experiencing side effects from danazol who cannot tolerate lower doses (200 mg/day). In these cases the subject can pursue alternative approaches which will likely involve other experimental therapies (likely elsewhere) as there are no standard therapies for telomeropathies. The study will be monitored to ensure that the occurrence of a specified set of treatment related serious adverse events (TRSAEs) within 6 months of treatment in the trial does not substantially exceed an anticipated rate. The following two types of TRSAEs will be considered for early stopping of the study: Death considered to be probably or definitely related to danazol Any grade IV toxicity considered to be probably or definitely related to danazol The study will be monitored using the stopping rules as outlined below and will be seriously considered for early stopping if the number of subjects in the study who have developed one or more of the above specified TRSAEs is over a pre-specified threshold value. TRSAEs are those attributed as definitely or probably related to the research. From our experience using this agent in other clinical settings, we anticipate the rate of developing at least one of the above specified TRSAEs for this subject population to be 10% or less. Following Geller et al. (2004, Design of Early Trials in Stem Cell Transplantation: A Hybrid Frequentist-Bayesian Approach ), our stopping rule is determined by a Bayesian approach. The stopping boundary for the study is reached if the Bayesian posterior probability H-0209 October 27,

22 that the true probability of developing one or more of the above specified TRSAEs exceeds this benchmark rate of 10% is at least 90%. We take our prior distribution to be a beta distribution with the sum of the two beta parameters to be 3, i.e. the parameters of the beta prior distribution are 0.30 and Since we have seen in the past that the first few subjects to be accrued are possibly sicker than the rest of the subjects in the sample, we will start safety monitoring the study when 3 or more subjects have developed specified TRSAEs. The following tables summarize the threshold numbers for stopping the study: Number of Subjects in the trial Stop if the number of subjects who develop any of the specified TRSAEs reaches: For the stopping rule we generated a study with 25 independent Bernoulli trials, each had a probability p for having the above TRSAE and q=1-p for not having such TRSAE and compared the TRSAE outcomes with the above stopping boundary to determine whether the study was stopped. We repeated the simulation 100,000 times and computed the proportion of stopped studies (i.e. number of stopped studies /100,000), which were stopped using the above stopping rule. The following table summarizes the proportions of stopped studies under a number of scenarios for p: Probability of Monitored TRSAE= p Proportion of Stopped Studies Average Number of Subjects Average Number of Subjects Suffering a specified TRSAE 5% 10% 15% 20% 25% 30% 2.29% 16.06% 40.13% 65.06% 83.01% 92.92% These results suggest that the above stopping rules have a low probability of stopping a study when the proportion of the corresponding specified TRSAE is below the benchmark value of 10%, and the probability of stopping a study is high when the true proportion of the above-specified TRSAE exceeds this benchmark value. Based on these results, we believe that our Bayesian stopping rules have satisfactory statistical properties. 9.6 Off Study Criteria Subject choice: Subjects may withdraw from study at any time. The risks of withdrawing will be discussed, as will alternative treatment options. 11-H-0209 October 27,

23 Principal investigator decision: Should any of the following events occur, study drug administration will be discontinued. The subject will be followed until resolution of the event and laboratory values monitored through 6 months or until initiation of alternative SAA therapy: Intractable intolerance to danazol Pregnancy or unwillingness to refrain from pregnancy Initiation of alternative specific therapy for marrow failure or pulmonary fibrosis (not including supportive or symptomatic treatment) Completion of protocol participation: After 2 years, protocol participation will be deemed complete. Once off study, subjects will be referred back to the referring physician or consented to the Hematology Branch evaluation and treatment protocol (94- H-0010) or evaluated for eligibility for another Branch protocol, depending on the best interest of the subject. 9.7 Data management The Principal Investigator will be responsible for overseeing entry of data into an in-house password protected electronic system and ensuring data accuracy, consistency and timeliness. The Principal Investigator, associate investigators, Hematology Branch fellows, research nurses and/or a contracted data manager will assist with the data management efforts. Data will be abstracted from Clinical Center progress notes as well as from progress notes forwarded from home physician. Laboratory data from NIH will be imported electronically from CRIS into an in-house clinical trial database. Laboratory values from referring home physicians will be entered into the system. Data will not be sent outside NIH without IRB notification and an executed MTA or CTA. All human subjects personally identifiable information (PII) as defined in accordance to the Health Insurance Portability and Accountability Act, eligibility and consent verification will be recorded. Primary data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant, e.g., unique code or minimum PII required for subject identification. Study data will be housed on the Hematology Branch P drive, a secure, limited access drive. End of study procedures: Data will be stored in locked cabinets and in a password protected database until it is no longer of scientific value. Loss or destruction of data: Should we become aware that a major breech in our plan to protect patient confidentiality and trial data has occurred, the IRB will be notified. Publication Policy: Given the research mandate of the NIH, patient data including the results of testing and responses to treatment will be entered into an NIH-authorized and controlled research database. Any future research use will occur only after appropriate human subject protection institutional approval such as prospective NIH IRB review and approval or an exemption from H-0209 October 27,

24 the NIH Office of Human Subjects Research Protections (OHSRP). 10. DATA AND SAFETY MONITORING 10.1 Safety Monitoring Principal Investigator: The protocol oversight monitoring, including accrual, efficacy and safety data, will be monitored by the Principal Investigator, NHLBI IRB. Accrual and safety data will be reviewed annually by the Institutional Review Board (IRB). Prior to implementation of this study, the protocol and the proposed patient consent and assent forms will be reviewed and approved by the properly constituted Institutional Review Board (IRB) operating according to Title 45 CFR 46. This committee will also approve all amendments to the protocol or informed consent, and conduct continuing annual review so long as the protocol is open to accrual or follow up of subjects Event Characterization and Reporting Events include adverse events (AE), serious adverse events (SAE), protocol deviations (PD), unanticipated problems (UP), and non-compliance. The principal investigator will review all events (AEs, protocol deviations, UPs, SAEs) to determine the seriousness, expectedness, and reportability of the event. As required and/or needed, the principal investigator will review the events with the Sponsor to make the final determination of seriousness and reportability Definitions Adverse Event (AE): Any untoward or unfavorable medical occurrence in a human subject, including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the subject s participation in the research, whether or not considered related to the research. Serious Adverse Event (SAE): A serious adverse event that: results in death; is life-threatening (places the subject at immediate risk of death from the event as it occurred); results in in-patient hospitalization or prolongation of existing hospitalization; results in a persistent or significant incapacity; 11-H-0209 October 27,