A Phase II Single Center Single Arm Pilot Study Administering Danazol for Treatment of Cytopenias in Patients With Cirrhosis
Phase: PHASE2
Conditions: Cirrhosis, Liver, Cytopenia
Sponsor: University of Southern California

PROTOCOL NUMBER: 
 
 
 
TITLE: Danazol for Treatment of Cytopenias in Patients with Cirrhosis  
 
 
Study phase: I/II 
 
 
 
STUDY ARMS: Single Arm Pilot Study 
 
 
 
IND OR IDE #: Not applicable 
 
 
PRINCIPAL INVESTIGATOR(S): 
Casey O’Connell, MD FACP 
 
Address 1441 Eastlake Ave NOR 3460 
 
Telephone (323)865-3950 
 
Fax (323)865-0060 
 
 
 
CO-INVESTIGATOR(S): Linda Sher, MD; Brian Kim, MD; Wendy Mack, PhD; Howard Liebman, MD; 
Caroline Piatek, MD; Ilene Weitz, MD; Caitlin 
O’Neill, MD; Karen Haiber, MD.  
 
 
 
Sponsor: N/A 
 
 
PARTICIPANTS/Locations: Single center (LAC+USC Medical Center, Norris Comprehensive  
Cancer Center, Keck Medical Center of USC) 
 
 
 
IRB Study Approval Date: October 27, 2020 
 
Study Expiration Date: October 26, 2021 
 
 
 
 
 
 
 

TABLE OF CONTENTS 
 
 
 
 
 
1.0 
Background and Hypothesis 
 
 
2.0 
Objectives 
 
3.0 
Study Design 
 
4.0 
Drug Information  
 
 
5.0 
Eligibility Assessment 
 
 
6.0  
Study Agent Administration 
 
7.0 
Study Assessments and Calendar 
 
 
8.0 
Assessment of Efficacy and Safety  
 
 
 
 
 
9.0 
Criteria For Evaluation and Endpoint Definitions 
 
10.0      Data Collection and Monitoring 
 
 
 
 
 
 
 
11.0 
Statistical Considerations 
 
12.0 
Registration Guideline 
 
13.0 
Biohazard Containment 
 
14.0 
Ethical and Regulatory Considerations 
 
 
15.0 
References 
 
 
 
 
 
 
 
 
 
 
APPENDICES 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

1.0 
Background and Hypothesis 
 
 
1.1 
Cirrhosis and Cytopenias 
 
Most studies estimate that between 6 and 77% of all patients with cirrhosis have 
abnormal hematologic indices (AHI), including anemia, thrombocytopenia and 
leukopenia. In a homogenous population of patients with compensated Child-Pugh Class 
(CPC) A/B cirrhosis, as many as 84% have AHI.18,19 The presence of AHI contributes to 
increased morbidity and mortality in a large proportion of cirrhotic patients.  For 
example, thrombocytopenia can be a limiting factor when considering invasive surgical 
procedures due to the increased risk for bleeding.  Leukopenia increases the risk for 
infections and chronic anemia contributes to worse outcomes after hemorrhagic 
episodes.18 Thrombocytopenia and leukopenia have been shown to be associated with 
death, transplant, clinical decompensation and hepatocellular carcinoma (HCC).19   
 
The pathogenesis of AHI in patients with cirrhosis is often multifactorial, with splenic 
sequestration, portal hypertension, bone marrow suppression, and changes in 
hematopoietic stimulating factors contributing to the etiology.18 The severity of 
cytopenias does not consistently correlate with the degree of cirrhosis and may not 
correct after liver transplant.  Current therapies have variable efficacy in improving 
cytopenias and management focuses primarily on supportive care with transfusions and 
growth factors.   
 
 
1.2 
Cirrhosis and Telomeres 
 
Telomeres are repetitive DNA sequences located at the natural ends of linear 
chromosomes. They function to cap and protect chromosome ends from being recognized 
as damaged or infected DNA.3 During cell division, the “end-replication problem” arises 
as telomeres continually shorten because DNA polymerase cannot fully replicate the 3’ 
end of chromosomes. The telomerase complex counters telomere attrition by elongating 
the telomere DNA after each cell division. Germline genetic defects in telomere 
maintenance and repair can cause dyskeratosis congenita, bone marrow failure, liver 
cirrhosis, pulmonary fibrosis, as well as increased susceptibility to various cancers. 4-5, 24-25 
 
As a major complication of liver disease, cirrhosis is the main risk factor for progressive 
liver failure and HCC.  To better understand the pathogenesis of cirrhosis, the connection 
between telomere attrition and cirrhosis has been examined in preclinical studies.  
Rudolph et al. found that telomerase reverse transcriptase (TERT)-deficient mice 
displayed reduced liver regeneration after partial hepatectomy and increased hepatic 
fibrosis after carbon tetrachloride exposure. After restoration of telomerase activity, there 
was improved liver function and cirrhosis reduction.21 Similarly, studies in humans found 
significantly accelerated telomere shortening and more telomere mutations in livers with 
cirrhosis and chronic hepatitis compared to normal livers.15 Patients with dyskeratosis 
congenita, pulmonary fibrosis, aplastic anemia, and short telomeres also showed an 
increased frequency of liver fibrosis and cirrhosis. 
 
In an analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare 
database from 1992 through 2009, the development of liver disease was compared 
between 82,938 men with prostate cancer who did and did not undergo androgen 
deprivation therapy (ADT).12 Exposure to ADT was significantly associated with an 
increased subsequent risk of non-alcoholic fatty liver disease (54%), cirrhosis (35%) and 

any liver disease 47%).  These data support a relationship between androgens and liver 
health, the mechanism of which is likely multifactorial.  
 
 
1.3 
Summary of Danazol Efficacy Data  
 
Androgens have historically been used to treat bone marrow failure syndromes, including 
aplastic anemia and myelodysplastic syndrome.8, 17, 14, 22 Evidence in both tissue culture 
and animal models suggest sex hormones play a role in regulating telomerase.  Calado et 
al. showed that the addition of sex hormones resulted in the up regulation of TERT gene 
expression and telomerase enzymatic activity in hematopoietic stem cells in vitro.6 In a 
mouse model of telomere dysfunction, male hormone therapy also resulted in telomere 
elongation and hematological improvement.2 Danazol is a derivative of the synthetic 
steroid ethisterone, which is a modified testosterone. Although the drug’s mechanism of 
action is unclear, it is speculated that danazol is potentially able to modify antiplatelet 
levels (in immune-mediated thrombocytopenia), inhibit the mononuclear phagocyte 
system, and increase telomerase activity. 
 
In 2016, Townsley et al. conducted a phase I/II prospective study in which 27 patients 
with congenital telomere disorders and bone marrow failure were treated with danazol 
800 milligrams daily for a total of 24 months.23 The primary efficacy endpoint was a 20% 
reduction in the annual rate of telomere attrition.  The primary endpoint was achieved in 
92% of patients and 83% had a hematologic response (HR) at 24 months.  Of four 
patients with cirrhosis liver disease who had baseline Fibroscan data available for 
comparison, three had significant alleviation of liver fibrosis at 24 months.  The majority 
of patients in this study had TERT or TERC gene mutations and one patient had a RTEL1 
mutation.  While 79% of patients were noted to have HR as early as 3 months, the RTEL1 
patient did not achieve HR until 24 months. 
 
Danazol has been evaluated for its effect on cytopenias in patients with cirrhosis.  In a 
study of 49 patients with chronic hepatitis C virus (HCV), 90% of whom had cirrhosis, 
danazol was administered to mitigate thrombocytopenia in patients undergoing HCV 
treatment with peginterferon (PEG-INF) and ribavirin.1 Danazol was used concurrently 
with HCV therapy over a duration of 2 to 11 months. Patients received danazol with 
doses ranging from 300 to 600 mg daily, which was titrated based on platelet counts.  
Patients showed a tendency for an average platelet loss of 5,217/mm3/month prior to the 
study which trend reversed upon treatment with danazol.  Platelet counts increased to 
above 100,000/mm3 in 10.6 % of patients, by 10,000-100,000 in 53% of patients and were 
maintained at baseline in 20.4% of patients.  Fourteen percent (14.2%) of patients were 
considered unresponsive after 11 months of treatment. While platelet response was 
delayed in patients without sustained virological response (SVR), their platelet levels 
were similar to those with SVR at the end of the study period. 
 
 
1.4 
Summary of Danazol Safety Data  
 
In the study by Townsley et al, which included nine patients with cirrhosis (6 overt and 3 
subclinical), the most common side effects were elevation in liver-enzyme levels (41% of 
patients), muscle cramps (33%), edema (26%), and lipid abnormalities (26%).  Two 
patients (4%) discontinued danazol due to grade 3/4 adverse events (AE).  One patient 
with preexisting cardiovascular risk factors had a thromboembolic cerebrovascular 
accident prompting discontinuation of the drug.  One patient developed a hemangioma in 

the lower extremity during danazol treatment causing pain and swelling.  Symptoms 
resolved with discontinuation of danazol although the hemangioma persisted on imaging.  
  
In the study by Alvarez et al, all patients had at least one AE.  Five patients withdrew 
prematurely from therapy: 3 discontinued due to intolerance of Peg-INF and ribavirin, 1 
required splenectomy, and 1 died (cause of death not stated). Most treatment-related AEs 
were mild to moderate and consistent with AEs associated with PEG-INF and ribavirin 
treatment.  The most common AEs included anemia (40%), headache (38%), arthralgia 
(31%), myalgia (31%) and malaise (29%).  No patients discontinued therapy due to AEs. 
There were no reports of liver toxicity or withdrawal due to decompensation of liver 
disease.  
 
The most frequent adverse events seen in patients treated with danazol for other 
conditions include modest elevation in liver enzymes and weight gain. One study 
compared side effects of danazol in treated and untreated hereditary angioedema 
patients.11 The most common AEs included menstrual abnormalities, weight gain, arterial 
hypertension and acne. In the setting of myelofibrosis, WHO grade 1-2 transaminitis 
occurred in 8/50 (16%) patients during the first two months of therapy and improved 
following reduction of danazol from 600mg to 400mg daily.9 Two additional patients 
developed cholestasis that resolved after discontinuation of danazol and 4 patients 
stopped therapy, 1 developed prostate adenocarcinoma, 1 developed liver peliosis, 1 died 
due to splenic rupture (a known complication of myelofibrosis), and 1 withdrew despite 
anemia response.  
 
There have been case reports of HCC occurring in patients with and without cirrhosis 
who are treated with danazol although this is rare.  In one survey study of physicians 
treating patients with hereditary angioedema, 1/886 patients (0.1%) developed HCC 
while on danazol.22   
 
 
1.5 
Study Rationale  
 
In our previous study, approximately 20% of patients with cirrhosis presenting for 
evaluation for liver transplantation were found to have a telomere gene variant.  Patients 
with these gene variants tended to have lower blood cell counts and longer post-
transplant hospital stays compared to patients without mutations.  It is possible that 
cirrhotic patients with low blood cell counts may have bone marrow failure related to 
telomere dysfunction as fibrosis and bone marrow failure are both found in patients with 
other inherited telomeropathies.  Given that danazol has been effective in decreasing 
telomere attrition and improving blood cell counts in patients with inherited telomere 
disorders, danazol may have similar benefit in cirrhotic patients found to have telomere 
mutations or accelerated telomere shortening.  There is a suggestion, from one small 
study, that danazol may also improve liver fibrosis.  As leukopenia and thrombocytopenia 
are associated with increased morbidity and mortality in cirrhotic patients, treatment of 
these cytopenias may improve clinical outcomes. 
 
 
1.6 
Hypothesis  
 
We hypothesize that danazol treatment may improve cytopenias in patients with 
compensated Child-Pugh Class (CPC) A/B cirrhosis.  We also hypothesize that danazol 
may reduce progression or decompensation of cirrhosis among patients with telomere 
dysfunction.  

 2.0 
Objectives 
 
2.1 Primary Objectives 
 
• 
Determine the prevalence of telomere gene mutations and/or shortened telomere lengths 
in patients with compensated CPC A/B cirrhosis and leukopenia and/or 
thrombocytopenia. 
• 
Evaluate the safety of danazol in patients with compensated CPC A/B cirrhosis and 
leukopenia and/or thrombocytopenia, with or without telomere gene mutations and/or 
shortened telomere lengths. 
• 
Evaluate the efficacy of danazol in improving cytopenias in patients with compensated 
CPC A/B cirrhosis, with or without telomere gene mutations and/or shortened telomere 
lengths. 
 
2.2 Secondary Objectives 
 
• 
Measure the change in peripheral blood telomere lengths after treatment with danazol. 
• 
Measure the change in liver fibrosis and liver function parameters after treatment with 
danazol. 
• 
Evaluate the outcomes of decompensation, death and transplant in patients treated with 
danazol. 
 3.0 
Study Design  
 
This is a phase II pilot study designed to assess the safety and efficacy of danazol for treatment of 
cytopenias in patients with CPC A/B cirrhosis.  Subjects with telomere mutations and/or 
shortened telomeres will be treated with danazol 600 mg per day by mouth for a duration of 24 
months.  The goal will be to treat a total of 10 patients with telomere gene mutations and/or 
shortened telomere lengths. 
 
 4.0 
Drug Information 
 
 
Danazol is a synthetic steroid derived from ethisterone, which is a modified testosterone that has 
antigonadotropic and anti-estrogenic activities. Danazol suppresses the pituitary-ovarian axis by 
inhibiting the pituitary output of gonadotropins. This suppression is thought to be through a 
combination of depressed hypothalamic-pituitary response to lowered estrogen production, the 
alteration of sex steroid metabolism, and interaction of danazol with sex hormone receptors. The 
only other demonstrable hormonal effect is weak androgenic activity. The pituitary-suppressive 
action of danazol is reversible. Danazol has been approved in treating endometriosis, fibrocystic 
breast disease, hereditary angioedema, thrombocytopenic purpura, and other conditions. 
 
 
Danazol is lipophilic and can partition into cell membranes, indicating the likelihood of 
distribution into deep tissue compartments. Danazol appears to be metabolized and eliminated by 
renal and fecal pathways. The reported elimination half-life of danazol is variable across studies. 
The mean half-life of danazol in healthy males is 9.7 hours. After 6 months of 200 mg three times 
a day dosing in endometriosis patients, the half-life of danazol was reported as 23.7 hours. 
 
 
Adverse reactions from danazol include androgen like effects (i.e. weight gain, acne, mild 
hirsutism, edema, hair loss, voice change) and menstrual disturbances. The use of danazol in 

pregnancy is contraindicated. Other common side effects also include elevations in liver-enzyme 
levels and lipid abnormalities. 
 
5.0 
Eligibility Assessment 
 
 
5.1 
Patient Eligibility  
 
 
Eligibility waivers are not permitted. Subjects must meet all of the inclusion and exclusion 
criteria to be registered to the study. Study treatment may not begin until a subject is registered.  
 
Accrual time frame will be 18 months.  
 
5.2 
Inclusion Criteria 
 
• 
Age 18 years or older and able to provide informed consent 
• 
ECOG 0-2 
• 
Compensated Child-Pugh class A of any etiology with the exception of chronic hepatitis 
B with one or more of the following cytopenias  
1. Leukopenia defined as white blood cell count <2000/mm3  or absolute neutrophil 
count <1000/mm3 along with thrombocytopenia <150,000/mm3 measured on two 
separate occasions at least 3 months apart within 6 months of enrollment 
2. Thrombocytopenia defined as platelet count <50,000/mm3 along with white blood 
cell count <4000/mm3 measured on two separate occasions at least 3 months apart 
within 6 months of enrollment 
 
• 
Compensated Child-Pugh class B cirrhosis of any etiology with the exception of chronic 
hepatitis B with one or more of the following cytopenias: 
1. Leukopenia defined as white blood cell count ≤ 3500/mm3 measured on two separate 
occasions at least 3 months apart within 6 months of enrollment 
3. Thrombocytopenia defined as platelet count ≤ 100,000/mm3 measured on two 
separate occasions at least 3 months apart within 6 months of enrollment 
 
• 
Enrolled patients must have one or more of the following: 
o Presence of a genetic variant (defined as a known mutation, variant likely to be 
pathogenic or variant of undetermined significance with likely deleterious effect 
on transcription or translation) in at least one of the following genes: TERT, 
TERC, RTEL1, DKC, NOP10, NHP2, TINF2, WRAP53  
o Shortened telomere length in peripheral blood mononuclear cells (defined as age-
adjusted telomere length at or below the 5th percentile) 
o Of note, patient’s found to have telomere mutations know to confer a gain of 
function will be excluded  
• 
For women of childbearing potential: agreement to remain abstinent (refrain from 
heterosexual intercourse) or use contraceptive methods that result in a failure rate of < 
1% per year during the treatment period  

o A woman is considered to be of childbearing potential if she is postmenarcheal, 
has not reached a postmenopausal state (>= 12 continuous months of amenorrhea 
with no identified cause other than menopause), and has not undergone surgical 
sterilization (removal of ovaries and/or uterus) 
o Examples of contraceptive methods with a failure rate of < 1% per year include 
bilateral tubal ligation, male sterilization, proper use of hormonal contraceptives 
that inhibit ovulation, hormone-releasing intrauterine devices, and copper 
intrauterine devices 
o The reliability of sexual abstinence should be evaluated in relation to the duration 
of the clinical trial and the preferred and usual lifestyle of the patient; periodic 
abstinence (e.g., calendar, ovulation, symptothermal, or postovulation methods) 
and withdrawal are not acceptable methods of contraception 
• 
Women of childbearing potential (WOCBP) must have a negative serum test (minimum 
sensitivity 25 IU/L or equivalent units of human chorionic gonadotropin [HCG]) within 
72 hours prior to the start of treatment 
 
5.3 Exclusion Criteria 
 
• 
Cirrhosis secondary to chronic hepatitis B or any history of hepatitis B 
• 
Patients with telomere related mutations know to confer gain of function will be excluded 
• 
Patients known to be infected with HIV  
• 
History of any hormone sensitive malignancy, including breast cancer, prostate cancer, 
hepatocellular carcinoma or liver adenoma as well as any patient considered high risk for 
developing malignancy (i.e. history of familial cancers including a first degree relative)  
• 
Patients who are actively receiving anti-cancer therapy 
• 
Liver decompensation event within the last 6 months (i.e. variceal bleed, ascites requiring 
paracentesis, hepatic encephalopathy) 
• 
Active thrombosis or history of unprovoked thromboembolic disease, including 
cardiovascular events. If a patient has received and completed adequate anticoagulation 
for a provoked thrombosis, they can be included in the study.  
• 
Pregnant or planning to become pregnant 
• 
Females patients who are breast feeding  
• 
Any contraindication to danazol use 
• 
Uncontrolled co-morbid condition which would make the administration of danazol 
unsafe, including decompensated heart failure or known EF less than 40%, unstable 
angina pectoris, uncontrolled cardiac arrhythmia, decompensated liver failure, renal 
failure defined as creatinine greater than >1.6 or psychiatric illness that would limit 
compliance with study requirements  
• 
Alanine aminotransferase and/or aspartate aminotransferase >3x upper limit of normal 
• 
Alkaline phosphatase >2.5 x upper limit of normal 
• 
Total bilirubin or direct bilirubin >2.5 x upper limit of normal 
• 
Patients with known alcohol or drug abuse within the last year 
• 
Concomitant use of hormone stimulants or hormone blocking agents. 
• 
Concomitant use of other bone marrow stimulating agents that may affect white blood 
cell and platelet counts (i.e. G-CSF, romiplostim, eltrombopag, corticosteroids). Short 
term use of growth factors per standard of care in preparation for procedure or for other 
medical indications is acceptable. Patients taking corticosteroids above 5 mg of 

prednisone or the equivalent who are on a stable dose for at least 8 weeks prior to 
enrollment can be included. 
• 
Concomitant treatment with systemic immunosuppressive medications (including but not 
limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumor 
necrosis factor [anti-TNF] agents)  
 
 
5.4 Withdrawal Criteria 
 
Patients will be removed from therapy when any of the criteria listed below apply. Notify the 
Principal Investigator, and document the reason for study removal and the date the patient was 
removed from treatment in the Case Report Form. The patient should be followed-up per protocol.  
Patients can be taken off the study treatment and/or study at any time at their own request, or they 
may be withdrawn at the discretion of the investigator for safety, behavioral or administrative 
reasons. The reason(s) for discontinuation of treatment will be documented and may include:  
• 
Any decompensation event (i.e variceal bleed, ascites requiring paracentesis, hepatic 
encephalopathy) 
• 
Any grade 4 toxicity that is considered likely related to study drug 
• 
Development of liver enzyme or bilirubin elevation to grade 3 that does not resolve 
to baseline grade with cessation or reduction of study drug 
• 
Any new diagnosis of malignancy 
• 
Any new thrombotic event 
• 
Patient becomes pregnant  
• 
Worsening of cardiac, hepatic or renal function from baseline defined as, known EF 
less than 40% or creatinine greater than >1.6 
• 
Patient withdraws consent (follow up)  
• 
Patient is unable to comply with protocol requirements 
• 
Treating physician determines continuation on the study would not be in the patient’s 
best interest 
• 
Lost to follow-up: If a research subject cannot be located to document survival after a 
period of 2 years, the subject may be considered “lost to follow-up.” All attempts to 
contact the subject during the two years must be documented in CRF’s. 
 
 
5.5 Replacement Criteria 
• 
Any patient who discontinues danazol therapy prior to receiving at least 3 months 
duration  
• 
Any patient who cannot tolerate danazol at a minimum dose of 200 mg/day 
6.0 
Study Agent Administration 
 
 
6.1 
Treatment Dosages and Administration 
 
AGENT 
DOSE 
ROUTE 
FREQUENCY 
DURATION 
Danazol  
600 mg 
Oral 
Daily 
24 months 
 

Danazol will be administered orally in a dose of 600 milligrams daily. Danazol treatment 
will be for a total duration of 24 months.  The treatment duration is chosen based on the 
same study showing that patients with RTEL1 mutations, which we found to have a high 
prevalence in cirrhotic patients, may require as long as 24 months to demonstrate a 
hematologic response to danazol.23     
Danazol will be discontinued for treatment-related serious adverse events or if the patient 
decides to withdraw from the study.  As response will be assessed at the conclusion of the 
study, response will not be used as a criterion to determine if danazol should be held 
during the study period.  
 
6.2 
Monitoring 
 
Patients will have routine blood draws every 2 weeks for the first month, then every 4 
weeks for the following 3 months, then every 3 months thereafter for toxicity monitoring 
including assessment of renal and liver function.   
Patients will be asked about symptoms at each study visit with particular attention to 
symptoms known to be associated with androgen therapy including but not limited to 
fatigue, arthralgias, acne, weight gain, hirsutism and mood changes. 
Physical exam will be performed at each 3-month study visit to assess for signs of liver 
decompensation (i.e. ascites, edema, encephalopathy) or treatment-related effects. 
During the study period, patients will continue to follow with their primary care 
physician for standard of care evaluations typical for patients with cirrhosis and low 
blood cell counts. This should include abdominal ultrasound every 6 months or per local 
standard of care (no less frequently than yearly) for HCC surveillance.  
 
After the study is completed, patients will continued to be followed for an additional 24 
months with abdominal ultrasounds as a part of the standard of care surveillance by chart 
review and phone calls made to the patient.  
Safety will be evaluated in this study through the monitoring of all serious and non-
serious AEs, defined and graded according to NCI CTCAE v4.0. Patients will be assessed 
for safety (including laboratory values) according to the schedule in Appendix 
I. General safety assessments will include serial interval histories, physical 
examinations, and specific laboratory studies, including serum chemistries and blood 
counts (see study calendar for the list and timing of study assessments). All serious 
adverse events (SAEs) will be reported in an expedited fashion. In addition, the 
investigators will review and evaluate observed AEs on a regular basis. Patients who 
have an ongoing study treatment−related AE upon study completion or at discontinuation 
from the study will be followed until the event has resolved to baseline grade, the event is 
assessed by the investigator as stable, new anticancer treatment is initiated, the patient is 
lost to follow-up, the patient withdraws consent, or until it has been determined that study 
treatment or participation is not the cause of the AE.  
Some people may find it upsetting to learn that they have certain mutations or errors in genes that 
could lead to future health problems for themselves or their children. A referral to a genetic 
counselor will be made available to the participant for the purpose of answering questions about 
the implications of the genetic testing results. If a genetic counselor is not available, hematology is 
accustomed to discussing the implications of testing results. 

 
6.3 
Toxicities and Dosing Delays/Dose Modifications   
 
 
Any patient who receives treatment on this protocol will be evaluable for toxicity. Each 
patient will be assessed for the development of toxicity according to the Study Calendar 
(Appendix 2). Toxicity will be assessed according to the NCI Common Terminology 
Criteria for Adverse Events (CTCAE), version 4.0. Dose adjustments should be made 
according to the system showing the greatest degree of toxicity (Appendix 1).  
 
 
6.4 
Concomitant Medications/Treatments    
 
 
Concomitant therapy includes any prescription medications or over-the-counter 
preparations used by a patient between the 7 days preceding the screening evaluation and 
the treatment discontinuation visit. These are recorded during each study visit as dictated 
by study calendar (Section 7.0). Males and females of reproductive potential should use 
highly effective means of contraception. 
 
 
It is strongly recommended that: Traditional or herbal medicines not be administered 
because the ingredients of many herbal medicines are not fully studied and their use may 
result in unanticipated drug-drug interactions that may cause, or confound assessment of, 
toxicity.  
 
 
6.5 
General Plan to Manage Safety Concerns    
 
 
Measures will be taken to ensure the safety of patients participating in this trial, including 
the use of stringent inclusion and exclusion criteria (see Section 5.0) and close monitoring 
(as indicated above). See Section 8.4 for complete details regarding safety reporting for 
this study. 
 
 
7.0 Study Assessments and Calendar 
 
7.1 
Response Criteria  
 
 
 
Hematologic Response (HR) defined as:  
• 
Normalization of WBC to ≥ 4000/µL or doubling of WBC from baseline AND 
• 
Normalization of platelet count to ≥150,000/µL or doubling of platelet count from 
baseline 
 
Patients dependent on exogenous hematopoietic growth factors or requiring transfusions 
will not be considered as fulfilling response criteria. 
 
   
7.2 
Screening/Baseline Procedures  
 
Assessments performed exclusively to determine eligibility for this study will be done 
only after obtaining written informed consent. Assessments performed for clinical 
indications (not exclusively to determine study eligibility) may be used for baseline 
values even if the studies were done before informed consent was obtained. All screening 
procedures must be performed within 14 days prior to registration unless otherwise 
stated.  
 
  
7.3 
Medical History  

 
 Complete medical and surgical history, history of infections, prior cancer history and 
treatment, transfusion requirements will be performed.  
 
 Medical history includes clinically significant diseases within the previous 5 years, 
smoking history, cancer history (including tumor characteristics such as hormone 
receptor status), prior cancer therapies and procedures, and all medications used by the 
patient within 7 days before the screening visit (including prescription, over-the-counter, 
and herbal/homeopathic remedies and therapies).  
 
  
7.4 
Physical Examination  
 
 A complete physical examination will be performed at screening and at the treatment 
discontinuation visit and should include the evaluation of head, eye, ear, nose, and throat 
and cardiovascular, dermatologic, musculoskeletal, respiratory, gastrointestinal, and 
neurologic systems.  
 
 A limited physical examination will be performed at other visits to assess changes from 
baseline abnormalities and any new abnormalities and to evaluate patient-reported 
symptoms. New or worsened abnormalities should be recorded as AEs if appropriate.  
 
  
7.5 
Telomere gene mutation analysis 
 
 All eligible patients who have signed informed consent will be screened at baseline for 
mutations in known telomere genes using a CLIA-certified panel, which includes the 
following genes: TERT, TERC, RTEL1, DKC, NOP10, NHP2, TINF2, and WRAP53.  
Three milliliters of whole blood from each patient will be sent to Fulgent Diagnostics 
Laboratory (Temple City, CA).  Each sample will be tested for the presence of telomere 
complex variants.  Genomic DNA is first isolated from peripheral blood mononuclear 
cells, then enriched for the coding exons of targeted genes using hybrid capture 
technology.  Prepared DNA libraries are then sequenced using next-generation 
sequencing technology. Following alignment, variants are detected in regions of at least 
10x coverage. 
 
  
7.6 
Telomere length measurement 
 
 Telomere lengths of peripheral blood mononuclear cells will be measured at baseline and 
24 months using a CLIA-certified test performed by Repeat Dx. 
 
  
7.7 
Blood draws 
 
 Blood draws will be performed at baseline and every 2 weeks for the first 4 weeks, then 
every 4 weeks for 3 months, then every 3 months thereafter as part of the standard of care 
for monitoring patients with low blood cell counts. 
 
  
7.8 
Transient Elastography 
 
FibroScan is a non-invasive tool to measure the fibrosis of the liver. FibroScan or 
transient elastography creates vibration of the skin that passes through the liver. The tool 
uses ultrasound to track the vibration and measure the stiffness of the liver which is then 
reported. The method can indirectly measure the degree of fibrosis in the liver tissue 

without means of an invasive procedure like liver biopsy. The entire procedure is 
performed through a hand-held probe that is placed on the upper right lateral wall of the 
abdomen. On average, the procedure takes 5 minutes. The technique has no risks 
associated with the procedure. 
 
This will be performed at baseline and 24 months by Dr. Brian Kim at Keck Hospital of 
the University of Southern California. Fibroscan is not necessarily required as part of 
standard of care work up for patients with cirrhosis and cytopenias, but it will be a more 
sensitive test to measure changes in degree of fibrosis for the purposes of this study. 
 
  
7.9 
Bone Marrow Biopsy 
 
 A bone marrow biopsy will be performed at baseline (per standard of care for evaluation 
of unexplained low blood cell counts).  If indicated to reassess cytopenias, a bone marrow 
biopsy will be performed again at 24 months.  Additional biopsy will be done only if 
there are changes in clinical condition and it is required for standard of care assessment.  
 
  
7.10 
Study Calendar - See Appendix 2 
 
 The flowchart of scheduled study assessments is provided in Appendix 2. Patients will be 
closely monitored for safety and tolerability throughout the study. All assessments must 
be performed and documented for each patient.  
 
 Patients should be assessed for toxicity prior to each dose; dosing will occur only if the 
clinical assessment and local laboratory test values are acceptable.  
 
 If the timing of a protocol-mandated study visit coincides with a holiday and/or weekend 
that precludes the visit, the visit should be scheduled on the nearest following feasible 
date, with subsequent visits rescheduled accordingly. A +/- 4 day window is allowed for 
scheduling of study visits that must be moved for other reasons.  
 
  
7.11 
Duration of Follow Up 
 
 After the study is completed, patients will continued to be followed for an additional 24 
months with abdominal ultrasounds as a part of the standard of care surveillance by chart 
review and phone calls made to the patient.  
 
8.0 
Assessment of Safety and Efficacy 
 
Baseline adverse events will be assessed. See Section 8.4 for Adverse Event monitoring and 
reporting. 
Analyses of safety/ toxicity will be performed for all patients having received at least one dose of 
study drug. The study will use the CTCAE version 4 (See Appendix 1) for reporting of adverse 
events (http://ctep.cancer.gov/reporting/ctc.html). 
Safety assessments will consist of monitoring and reporting AEs and SAEs that are considered 
related to Danazol, all events of death, and any study-specific issue of concern.  
8.1 
Side effects/toxicities to be monitored at each visit:  

• 
Fatigue 
• 
Weight gain  
• 
Acne or oily skin 
• 
Hair changes (hirsutism or hair loss)  
• 
Edema or fluid retention  
• 
Voice changes 
• 
Menstrual disturbances 
• 
Breast atrophy or decreased breast size  
• 
Flushing or sweating 
• 
Mood changes 
• 
Elevation of liver enzymes  
• 
Muscle cramps  
• 
Hypertension  
• 
Headache  
• 
Thrombotic events 
 
8.2 
Long-term toxicities to be monitored after completion of therapy: 
• 
Development of ovarian cancer  
• 
Development of liver tumors (i.e. hepatic adenoma, hepatocellular carcinoma)  
• 
Development of peliosis hepatitis  
• 
Development of other hepatic toxicity  
 
8.3  
Dosage change based on toxicity: 
 
• 
Danazol will be initiated at a dose of 600 mg/d. 
• 
Danazol will be dose reduced to 400 mg/d if not tolerating based on side effects and 
toxicity assessment in 8.1 – see Table 1.  
• 
If patient achieves hematologic response, danazol dose may be reduced to 200mg/day; 
patients who are unable to tolerate Danazol dose of 400 mg/d prior to achieving 
hematologic response will be removed from study 
• 
Patients who develop grade 4 toxicity thought to be possibly or definitely related to 
danazol, will be removed from the study. 
• 
Danazol will be held for imaging findings suspicious for tumors until work up has been 
completed. If negative, patient can resume danazol at previous dose. 
• 
Danazol will be discontinued for any acute decompensation event; new diagnosis of 
arterial or venous thrombosis; new malignancy; pregnancy. 
• 
Toxicity grading will be determined per CTAE version 5.0 guidelines.  
 
 
 
 
 
Table 1. Management of Danazol Toxicities 
 
NCI CTAE Grade 
Management  
Any Grade 1 Toxicity 
Continue Danazol at current dose with continued 
monitoring  

Grade 2 Toxicity at 600 
mg/d dose  
Danazol should be dose reduced to 400 mg/d  
Grade 2 Toxicity at 400 
mg/d dose  
Discontinue Danazol and patient will be removed from the 
study  
Grade 3 Toxicity at 600 
mg/d dose 
Danazol should be held for at least 2 weeks then resumed at 
reduced dose of 400 mg/d if labs have returned to baseline  
Grade 3 Toxicity 
at 400 mg/d dose  
Discontinue Danazol and patient will be removed from the 
study  
Any Grade 4 Toxicity  
Discontinue Danazol and patient will be removed from the 
study  
 
 
Table 2. Edema Management 
 
 
 
 
NCI CTAE Grade  
Management  
Danazol Dosage 
Grade 1 Edema  
Consider starting low dose diuretics or 
increasing current diuretic dose 
Continue Danazol at current dose  
Grade 2 Edema  
Start low dose diuretic or increase current 
diuretic dose  
Resume Danazol at dose reduced to 400 
mg/d  
Grade 3 Edema  
Start low dose diuretic or increase current 
diuretic dose  
Danazol should be held for at least 2 weeks 
then resumed at reduced dose of 400 mg/d 
if edema improves  
 
 
8.4 Adverse Event Reporting: 
 
8.4.1 
Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a patient receiving study 
treatment and which does not necessarily have a causal relationship with this treatment. 
An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of an 
experimental intervention, whether or not related to the intervention.   An AE is any 
unfavorable and unintended sign, symptom, or disease temporally associated with the use 
of an investigational medicinal product (IMP) or other protocol-imposed intervention, 
regardless of attribution.   
 
This includes the following: 
 
• 
AEs not previously observed in the patient that emerge during the protocol-specified 
AE reporting period, including signs or symptoms that were not present prior to the 
AE reporting period  
• 
Complications that occur as a result of protocol-mandated interventions (e.g., 
invasive procedures such as cardiac catheterizations)  
• 
If applicable, AEs that occur prior to assignment of study treatment associated with 
medication washout, no treatment run-in, or other protocol-mandated interventions 

• 
Pre-existing medical conditions (other than the condition being studied) judged by 
the investigator to have worsened in severity or frequency or changed in character 
during the protocol-specified AE reporting period  
 
 
 
The following events meet the definition of unanticipated problem (UPR): 
• 
Any serious event (injuries, side effects, deaths or other problems), which in the 
opinion of the Principal Investigator was unanticipated, involved risk to subjects or 
others, and was possibly related to the research procedures.  
• 
Any serious accidental or unintentional change to the IRB-approved protocol that 
alters the level of risk.  
• 
Any deviation from the protocol taken without prior IRB review to eliminate 
apparent immediate hazard to a research subject.  
• 
Any new information (e.g., publication, safety monitoring report, updated safety 
report), interim result or other finding that indicates an unexpected change to the 
risk/benefit ratio for the research.  
• 
Any breach in confidentiality that may involve risk to the subject or others.  
• 
Any complaint of a subject that indicates an unanticipated risk or that cannot be 
resolved by the Principal Investigator.  
 
8.4.2 
Serious Adverse Events 
 
A “serious” adverse event (SAE) is defined in regulatory terminology as any untoward 
medical occurrence that:  
 
• 
Results in death (If death results from progression of the disease, the disease 
should be reported as event SAE itself);  
 
• 
Is life-threatening (the patient was at risk of death at the time of the event; it does 
not refer to an event that hypothetically might have caused death if it were more 
severe);  
 
• 
Requires in-patient hospitalization or prolongation of existing hospitalization for 
≥ 24 hours;  
 
• 
Results in persistent or significant disability or incapacity;  
 
• 
Is an important medical event; or  
 
• 
Any event that does not meet the above criteria, but that in the judgment of the 
investigator jeopardizes the patient, may be considered for reporting as a serious 
adverse event. The event may require medical or surgical intervention to prevent 
one of the outcomes listed in the definition of “Serious Adverse Event“ (for 
example: allergic bronchospasm requiring intensive treatment in an emergency 
room or at home; convulsions that may not result in hospitalization; development 
of drug abuse or drug dependency).  
 
The Principal Investigator must be notified within 24 hours of learning of any serious 
adverse events (SAE), regardless of attribution, occurring during the study or within 30 
days of the last administration of the study drug.  

 
SAE occurring after consent but before first dose will not require expedited reporting and 
for which there will be no protocol-mandated intervention; management should be per 
investigator or treating physician and in accordance with standard of care. 
 
8.4.3 
Reporting of Adverse Events and Serious Adverse Events  
 
The USC NCCC Data and Safety Monitoring Committee (DSMC) must be notified 
within 24 hours of submission of such reportable event to the IRB. The patient ID and the 
study number as well as identifier of the AE or SAE report should be submitted to the 
DSMC Coordinator via email or Fax to the attention of the DSMC Coordinator at 323-
865-0089.  
 
The USC NCCC Data and Safety Monitoring Committee (DSMC) must be notified 
within 24 hours of submission of such reportable event to the IRB. The patient ID and the 
study number as well as identifier of the SAE report should be submitted to the DSMC 
Coordinator via email or Fax to the attention of the DSMC Coordinator at 323-865-0089. 
The study period during which all AEs and SAEs must be reported begins after informed 
consent is obtained and initiation of study treatment and ends 60 days following the last 
administration of study treatment or study discontinuation/termination, whichever is 
earlier. After this period, investigators should only report SAEs that are attributed to prior 
study treatment.  
 
The Institutional IRB (US) must be notified of “any unanticipated problems involving 
risk to subjects or others” in accordance with the Institutional policy. Such policies will 
be provided to the USC Clinical Investigation Support Office (CISO) QA prior to 
enrolling 1st patient. (for USC refer to HSPP Policies and Procedures chapter 14 
available at https://oprs.usc.edu/files/2017/04/2017-Policies-and-Procedures-PDF-
1JUN2017.pdf). 
 
8.5 Adverse Event Monitoring    
Adverse event data collection and reporting, which are required as part of every clinical trial, are done to 
ensure the safety of subjects enrolled in the studies as well as those who will enroll in future studies using 
similar agents. Adverse events are reported in a routine manner at scheduled times during a trial. 
Additionally, certain adverse events must be reported in an expedited manner to allow for optimal 
monitoring of patient safety and care.    
All patients experiencing an adverse event, regardless of its relationship to study drug, will be monitored 
until the adverse event resolves or the symptoms or signs that constitute the adverse event return to 
baseline; any abnormal laboratory values have returned to baseline; or death.    
All AEs and SAEs, whether volunteered by the patient, discovered by study personnel during questioning, 
or detected through physical examination, laboratory test, or other means, will be reported appropriately. 
Each reported AE or SAE will be described by its duration (i.e., start and end dates), regulatory 
seriousness criteria if applicable, suspected relationship to the study drug (see following guidance), and 
actions taken.    
To ensure consistency of AE and SAE causality assessments, investigators should apply the following 
general guideline:    

Yes  There is a plausible temporal relationship between the onset of the AE and administration of 
Danazol, and the AE cannot be readily explained by the patient’s clinical state, inter-current illness, or 
concomitant therapies; and/or the AE follows a known pattern of response to Danazol; and/or the AE 
abates or resolves upon discontinuation of Danazol or dose reduction and, if applicable, reappears upon 
re-challenge.   
No  Evidence exists that the AE has an etiology other than Danazol (e.g., pre-existing medical 
condition, underlying disease, inter-current illness, or concomitant medication); and/or the AE has no 
plausible temporal relationship to Danazol administration (e.g., cancer diagnosed 2 days after first dose of 
study drug).   Expected AEs are those AEs that are listed or characterized in the Package Insert (PI) or 
current Investigator’s Brochure.   Unexpected AEs are those not listed in the PI or current 
Investigator’s Brochure or not identified. This includes AEs for which the specificity or severity is not 
consistent with the description in the PI or Investigator’s Brochure. For example, under this definition, 
hepatic necrosis would be unexpected if the PI or Investigator’s Brochure only referred to elevated 
hepatic enzymes or hepatitis.  
8.6 Procedures for Eliciting, Recording and Reporting Adverse Events   
 8.6.1 Eliciting Adverse Events   
A consistent methodology for eliciting AEs at all patient evaluation time-points should be adopted. 
Examples of non-directive questions include:  
 • 
“How have you felt since your last clinical visit?”  
 • 
“Have you had any new or changed health problems since you were last here?” 
8.6.2 
 Routine Reporting   
All other adverse events - such as those that are expected, or are unlikely or definitely not related to the 
study participation- are to be reported annually as part of regular data submission. This report will be 
forwarded to the USC DSMC Coordinator. All toxicities will be included in the IND annual report.  
8.6.3 
Monitoring Rules for Safety  
A USC Data Safety Monitoring Board will be notified of all SAEs reported during the course of this trial. 
8.6.4 
Deaths 
All deaths that occur during the protocol-specified AE reporting period, regardless of attribution, will be 
reported to the appropriate parties. When recording a death, the event or condition that caused or 
contributed to the fatal outcome should be reported as the single medical concept. If the cause of death is 
unknown and cannot be ascertained at the time of reporting, report “Unexplained Death.” 
8.6.5 
Pre-existing Medical Conditions  
A pre-existing medical condition is one that is present at the start of the study. Such conditions should be 
reported as medical and surgical history. A pre-existing medical condition should be re-assessed 
throughout the trial and reported as an AE or SAE only if the frequency, severity, or character of the 
condition worsens during the study. When reporting such events, it is important to convey the concept 

that the pre-existing condition has changed by including applicable descriptors (e.g., “more frequent 
headaches”).  
8.6.6 
Hospitalizations for Medical or Surgical Procedures   Any AE that results in 
hospitalization or prolonged hospitalization should be documented and reported as an SAE. If a patient is 
hospitalized to undergo a medical or surgical procedure as a result of an AE, the event responsible for the 
procedure, not the procedure itself, should be reported as the SAE. For example, if a patient is 
hospitalized to undergo coronary bypass surgery, record the heart condition that necessitated the bypass 
as the SAE.   Hospitalizations for the following reasons do not require reporting:   1. 
Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for pre-existing 
conditions;   2. Hospitalization or prolonged hospitalization required to allow efficacy measurement 
for the study; or   3. Hospitalization or prolonged hospitalization for scheduled therapy of the target 
disease of the study.  
8.6.7 
Pregnancies in Female Patients   Female patients of childbearing potential should utilize 
contraception and take active measures to avoid pregnancy while undergoing Danazol treatment and for at 
least 90 days after the last dose of Danazol.   Female patients of childbearing potential will be 
instructed to immediately inform the investigator if they become pregnant during the study or within 90 
days after the last dose of study drug. A Pregnancy Report CRF should be completed by the investigator 
immediately (i.e., no more than 24 hours after learning of the pregnancy) and submitted via fax. 
Pregnancy should not be recorded on the Adverse Event CRF. The investigator should discontinue study 
drug and counsel the patient, discussing the risks of the pregnancy and the possible effects on the fetus. 
  Monitoring of the patient should continue until conclusion of the pregnancy. Any SAEs associated 
with the pregnancy (e.g., an event in the fetus, an event in the mother during or after the pregnancy, or a 
congenital anomaly/birth defect in the child) should be reported on the Adverse Event CRF.  
 
 
9.0 
Criteria for Evaluation and Endpoint Definitions  
 
 
 
The outcome status (in terms of toxicity, response, reason off study, progression, and survival) of 
all eligible patients will be reported. All eligible patients who begin treatment will be included in 
the analysis of survival and time-to-failure.  
 
 
 
Evaluable for toxicity: All patients who receive one dose of danazol are evaluable for safety and 
toxicity.  
 
 
 
Evaluable for response: All patients who receive danazol for at least 3 months will be evaluable 
for response. In instances of drug interruptions, patients who receive danazol for at least 3 months 
with no more than 4 weeks of interruption will be assessed for hematologic response. Liver and 
telomere response will be assessed in patients who receive at least 12 months of danazol with no 
more than 8 weeks of interruption. Patients who do not receive danazol for at least 3 months will 
be replaced.  
 
 
Endpoint Definitions 
 
Primary endpoints:  
1) 
Hematologic response, defined as normalization of WBC to ≥ 4000/µL or doubling of WBC from 
baseline, AND/OR normalization of platelet count to ≥150,000/µL or doubling of platelet 
count from baseline, from study entry to three months. 
2) 
Occurrence of grade 3+ CTCAE 5 adverse events. 
 
Secondary endpoints include:  

1) 
Change in blood cell counts baseline to three months. 
2) 
Change in peripheral blood telomere length from baseline to 24 months. 
3) 
Change in liver fibrosis as measured by Fibroscan from baseline to 24 months 
4) 
Change in liver function parameters from baseline to 24 months;  
5) 
Transplant-free survival, defined as time from study entry until liver transplant.  Patients who 
have not undergone transplant will be censored at the time of last contact. 
6) 
Overall survival defined as the time from study entry until death.  Patients who are alive at last 
follow-up will be censored.  
7) 
Occurrence of clinical decompensation events (variceal hemorrhage, ascites requiring 
intervention, and hepatic encephalopathy).  
 
10.0      Data Collection and Monitoring  
 
10.1 
Active Monitoring Program Details  
10.1.1 Adherence to Protocol/Per Patient: It is the responsibility of the USC Principal Investigator 
(PI) to ensure that patient recruitment and enrollment, treatment, follow-up for toxicities and response, 
and documentation and reporting at USC are all performed as specified in the protocol.  
10.1.2 Day-to-Day Monitoring – Eligibility: At USC, the Study Coordinator will assist the Investigator 
in reviewing eligibility and will assemble the required source documents, and do a final review by 
completing an Eligibility Registration Worksheet.  
10.1.3 Day-to-Day Monitoring – Informed Consent: Prior to registering the patient on study, the 
Study Coordinator will review the informed consent, to ensure that the patient has signed and dated the 
most current IRB-approved form, and that the form has been signed and dated by the person obtaining the 
consent as well as appropriate witnesses. A copy of the ICF will also be provided to CISO QA for review. 
CISO SOP 3.3 will be followed.  
10.1.4 Day-to-Day Monitoring – Treatment: The PI and co-investigators are responsible for ensuring 
that treatment is given per protocol. The Study Coordinator will review the treatment orders and calendar-
specified diagnostic/monitoring items with the treating investigator.  
10.1.5 Data Management – Patient Charts: At USC, all written source documents not associated with 
the study research are maintained in the patient chart, which is stored in the Department of Medical 
Records at the appropriate hospital. At the Norris Hospital, the official medical record is on Cerner. 
Radiographical images are stored in the Department of Radiology and in an electronic system called 
Synapse. At Los Angeles County General Hospital the official medical record is on Orchid. These are the 
permanent, official documents for each patient on-study. A copy of the signed informed consent, 
physician’s notes, orders, test results and pathology notes are maintained in the patients’ hospital charts. It 
is the responsibility of the research staff to ensure that the patient chart contains the required documents 
and work closely with treating investigators to ensure all protocol-related assessments are carefully 
documented.  
10.1.6 Data Management – Research Charts: At USC, to facilitate adherence to the protocol schedule 
and data management, research charts are created to collect copies of the relevant notes, orders and results 
that are in the Patient Chart. In addition, all source documents related to the research, such as original 
informed consent forms, HIPAA Forms, AE assessment worksheets and disease response worksheets are 
maintained in the Research Charts. Protocol calendars, worksheets, and checklists, are also kept in the 
research chart. These are maintained in the locked offices of the clinical trial research unit until the study 
is completed and the results are published and no further need is anticipated. These are then stored off-

site. It is the responsibility of the Data Manager to ensure that the research chart contains all the required 
documents.  
10.1.7 Data Management – Case Report Forms: It is the responsibility of the Data Manager to 
complete the required case report forms. For in-house trials, case report forms are developed for each 
trial; these are used to finalize the data entry screens in the Cancer Center clinical trials database. It is the 
responsibility of the PI to review the Off-Study Summary form which summarizes pertinent toxicity, 
response and adherence information, once the patient has completed treatment.  
10.2 
Quality Assurance Monitoring Committee (QAMC) Oversight: The QAMC will be provided 
by CTSI at the University of Southern California Keck School of Medicine.  
10.2.1 Data and Safety Monitoring Committee (DSMC) Oversight: The data safety and monitoring 
group for this study will be comprised of at least 3 individuals from different specialties (at least 1 MD 
and 1 representative of CTSI) who will be asked by the principal investigator to review toxicity data at 6 
months and then every 12 months until study completion.   
10.2.2 Adherence to the Protocol: Except for an emergency situation in which proper care for the 
protection, safety, and well-being of the study patient requires alternative treatment, the study shall be 
conducted exactly as described in the approved protocol.  
10.2.3 Emergency Modifications: Investigators may implement a deviation from, or a change of, the 
protocol to eliminate an immediate hazard(s) to trial subjects without prior IRB approval. For any such 
emergency modification implemented, an IRB modification form must be completed within five (5) 
business days of making the change.  
10.2.4 Non-Emergency departures from protocol: A protocol deviation is any variance from an IRB 
approved protocol.  If the deviation meets all of the following criteria, it is considered a minor 
protocol deviation that:  
• 
Is generally noted or recognized only after it occurs;  
• 
Has no substantive effect on the risks to research participants;  
• 
Has no substantive effect on the scientific integrity of the research plan or the value of the data 
collected; or  
• 
Did not result from willful or knowing misconduct on the part of the investigator(s).  
If the deviation meets any of the following criteria, it is considered a protocol violation:  
• 
Has harmed or increased the risk of harm to one or more research participants;  
• 
Has damaged the scientific integrity of the data collected for the study;  
• 
Results from willful or knowing misconduct on the part of the investigator(s); or  
• 
Demonstrates serious noncompliance with federal regulations, State laws, or University policies.  
Protocol Deviations: personnel will report to the Study team data and safety monitoring committee in 
accordance with their policies.  
Protocol Violations: All protocol violations will be entered in the clinical trial database by the Research 
Coordinator. In addition, Research Coordinator and Investigator should report all protocol violations 
within one (1) week of the knowledge of the event using iStar.  

10.2.8 Amendments to the Protocol: Should amendments to the protocol be required, the amendments 
will be originated and documented by the Principal Investigator. It should also be noted that when an 
amendment to the protocol substantially alters the study design or the potential risk to the patient, a 
revised consent form might be required.  
The written amendment, and if required the amended consent form, must be sent to the IRB as well as to 
all the sponsoring agencies (FDA, NCI, etc.) for review and for approval prior to implementation. It is the 
responsibility of the study PI to ensure that the appropriate agencies have been informed of the proposed 
amendments and that these have been reviewed and approved.  
10.2.9 Retention of Records: Study documentation includes all Case Report Forms, data correction 
forms or queries, source documents, Study team correspondence, monitoring logs/letters, and regulatory 
documents (e.g., protocol and amendments, IRB correspondence and approval, signed patient consent 
forms).  
Source documents include all recordings of observations or notations of clinical activities and all reports 
and records necessary for the evaluation and reconstruction of the clinical research study.  
Government agency regulations and directives require that the study investigator must retain all study 
documentation pertaining to the conduct of a clinical trial. In the case of a study with a drug seeking 
regulatory approval and marketing, these documents shall be retained for at least two years after the last 
approval of marketing application in an International Conference on Harmonization (ICH) region. In all 
other cases, study documents should be kept on file until three years after the completion and final study 
report of this investigational study.  
11.0 
Statistical Considerations 
 
11.1    Sample Size   
 
In this pilot study, 10 patients meeting trial inclusion and exclusion criteria will be enrolled to receive 
danazol 600 mg per day for 24 months. Based on the previous study by Chiu et al, we expect to screen 
around 50 patients to find 10 patients for our study 10. The sample size of 10 was based on feasibility and 
available study resources, including study product.  
 
For estimated proportions of telomere shortening/mutation ranging from 0.2-0.8, a sample size of 50 will 
provide 95% binomial confidence intervals with a width (from estimate to upper or lower confidence 
limit) of 0.237-0.289.   
 
The sample size of 10 who will be treated with danazol will allow estimation of 95% binomial 
(specifically, exact Clopper-Pearson) confidence intervals with a width (from estimated proportion to 
upper or lower limit) of ±0.53-0.63 for estimated proportions ranging from 0.20-0.80.   For the secondary 
continuous outcomes of change from baseline, the sample will also allow estimation of 95% confidence 
intervals on a one-sample mean with a width (from estimated mean to upper or lower limit) of ±0.71 of a 
standard deviation unit 
 
11.2     Statistical Analysis 
 
Primary Aim 1: Determine the prevalence of telomere gene mutations and/or shortened telomere lengths 
in the study population.  Among consenting patients evaluated for telomere mutations and shortening, the 
proportion (with binomial 95% CI) of each telomere mutation, and any telomere mutation will be 
computed.    

 
Primary Aim 2: Evaluate the efficacy of danazol in improving cytopenias in the study population.  The 
proportion (with binomial 95% CI) of patients showing a Hematologic Response will be computed.   
 
Primary Aim 3: Evaluate the safety of danazol in the study population.  Proportions (with 95% CI) of 
patients experiencing danazol-targeted AEs/SAEs, other AEs/SAEs, and toxicity-based danazol dose 
reduction will be computed.   
 
Secondary Aims: Continuous measures assessed at baseline and at 24 months include peripheral blood 
telomere length and liver fibrosis.  Mean (SD) (median, IQR) change will be computed; difference of the 
mean change from a null value of 0 will be evaluated with a paired t-test (or non-parametric Wilcoxon 
signed rank test).  Blood counts (WBC, ANC, platelets) and liver function tests will be completed 
throughout the trial (every 2 weeks for the first month, every 4 weeks for the next 3 months, every 3 
months thereafter).   Mean (95% CI; median, IQR) of values at each time point and changes from the 
baseline pre-treatment values will be summarized; changes from baseline will be tested with a linear 
mixed effects model for longitudinal data, specifying a patient-level random effect. Survival distributions 
(median, 25th and 75th percentiles) for death and transplant will be computed with 95% confidence 
intervals.     
 
12.0 
Registration Guideline  
 
12.1 
Patient Informed Consent Process   Before recruitment and enrollment onto this study, the 
patient will be given a full explanation of the study and will be given the opportunity to review 
the consent form. Each consent form must include all the relevant elements currently required by 
the FDA Regulations and local or state regulations. Once this essential information has been 
provided to the patient and the investigator is assured that the patient understands the 
implications of participating in the study, the patient will be asked to give consent to participate 
in the study by signing a dated IRB approved consent form.   In obtaining and documenting 
informed consent, the investigator should comply with the applicable regulatory requirement(s), 
and should adhere to Good Clinical Practice (GCP) and to ethical principles that have their 
origin in the Declaration of Helsinki. Patients enrolling at USC will receive the California 
Experimental Research Subject’s Bill of Rights.   At the time of registration, signed and 
dated copies of the patient Informed Consent document with the California Experimental 
Research Subject’s Bill of Rights (California patients only) and the HIPAA authorization must 
be given to the patient. Institutional policy regarding distribution and location of original consent 
documents should be followed. When a study is opened at two or more institutions, a copy of the 
signed consent and HIPAA should be sent to USC Clinical Investigation Support Office (CISO) 
QA team as soon as possible, and not later than within 5 business days of obtaining consent. For 
patients consented at USC/LAC, institutional policy should be followed: a copy of ICF and 
HIPAA should be uploaded through True to USC CRO and to CISO QA Team. The original will 
be kept in the patient research chart maintained by the study assigned Data Manager.   The 
informed consent document must be signed by the subject or the subject’s legally authorized 
representative before his or her participation in the study. The case history for each subject shall 
document that informed consent was obtained prior to participation in the study. A copy of the 
informed consent document must be provided to the subject or the subject's legally authorized 
representative. If applicable, it will be provided in a certified translation of the local language. 
  Signed consent forms must remain in each subject’s study file and must be available for 
verification by study monitors at any time.   
 
12.2  
Registration Eligibility Worksheet   At the time of registration, the completed Eligibility 
Worksheet will be submitted to the QA Monitor at CISO for review of eligibility compliance.   

 
12.3 
Institutional Review Board (IRB) Approval   It is expected that the IRB will have the 
proper representation and function in accordance with federally mandated regulations. This 
protocol, the informed consent document, and relevant supporting information must be 
submitted to the IRB for review and must be approved before the study is initiated. The study 
will be conducted in accordance with FDA, applicable national and local health authorities, and 
IRB requirements. The Principal Investigator is responsible for keeping the IRB apprised of the 
progress of the study and of any changes made to the protocol as deemed appropriate, but in any 
case, the IRB must be updated at least once a year. The Principal Investigator must also keep the 
IRB informed of any significant AEs. Investigators are required to promptly notify their 
respective IRB of all adverse drug reactions that are both serious and unexpected. This generally 
refers to SAEs that are not already identified in the Investigator’s Brochure and that are 
considered possibly or probably related to the molecule or study drug by the investigator. Some 
IRBs may have other specific AE requirements to which investigators are expected to adhere. 
   
 
12.4 
USC Registration: For patients enrolled at USC, the Research Coordinator must complete the 
protocol eligibility form to ensure that the patient is eligible. The PI will review the patient 
eligibility (with assistance from the Research Coordinator - who will assemble the required 
source documents, and do an initial review) prior to registering the patient on study.   The 
Research Coordinator or data manager will then register the patient into the Cancer Center 
database, Café, by accessing the Registration forms. Likewise, after the patient has completed the 
study, the Off Study forms in cafe will need to be completed, for Off Treatment and Off Study.  
 
12.5 
Confidentiality of Records: The original data collection forms will be kept in secure file 
cabinets, for USC patient’s forms will be kept in the Clinical Investigations Support Office 
(CISO).   Patient medical information obtained by this study is confidential and may be 
disclosed to third parties only as permitted by the ICF (or separate authorization to use and 
disclose personal health information) signed by the patient or unless permitted or required by 
law. Medical information may be given to a patient’s personal physician or other appropriate 
medical personnel responsible for the patient’s welfare for treatment purposes.   Data 
generated by this study must be available for inspection upon request by representatives of the 
FDA and other regulatory agencies, national and local health authorities, and the IRB/Ethics 
Committee (EC) for each study site, if appropriate.  
 
13.0 
Biohazard Containment  
 
 
All blood samples drawn for study testing will be obtained handled and discarded through CLIA-certified 
laboratory according their standard operating protocol. Bone marrow aspiration and biopsies obtained for 
the study will be performed per protocol in the LAC+USC hematology outpatient clinic (A4A) by 
medical personnel and sent to the LAC+USC pathology department for processing and reporting. Any 
biohazard material obtained as a part of this study will be handled according to the guidelines outlines in 
the USC Biosafety Manual (https://ehs.usc.edu/files/BSM-2020.pdf).  
 
 
 
14.0 
Ethical and Regulatory Considerations 
 
All institutional and Federal regulations concerning the Informed Consent form will be fulfilled.  The 
study will be conducted in adherence to ICH Good Clinical Practice.  The Principal Investigator is 
responsible for the conduct of the clinical trial at the site in accordance with Title 21 of the Code of 

Federal Regulations and/or the Declaration of Helsinki. The Principal Investigator is responsible for 
personally overseeing the treatment of all study patients. The Principal Investigator must assure that all 
study site personnel, including sub- investigators and other study staff members, adhere to the study 
protocol and all FDA/GCP/NCI regulations and guidelines regarding clinical trials both during and after 
study completion. This clinical research study will be monitored both internally by the PI and externally 
by the USC IRB. In terms of internal review, the PI will continuously monitor and tabulate AEs. 
Appropriate reporting to the USC IRB will be made. The PI of this study will also continuously monitor 
the conduct, data, and safety of this study to ensure that:   
• 
Interim analyses occur as scheduled,  
• 
Stopping rules for toxicity and/or response are met,  
• 
Risk/benefit ratio is not altered to the detriment of the subjects,  
• 
Appropriate internal monitoring of AEs and outcomes is done,  
• 
Over-accrual does not occur,  
• 
Under-accrual is addressed with appropriate amendments or actions, and  
• 
Data are being appropriately collected in a reasonably timely manner.  
Routine monitoring will be carried out via a periodic team conference among investigators during which 
toxicity data, including all SAEs, will be reviewed and other issues relevant to the study such as interim 
assessment of accrual, outcome, and compliance with study guidelines, will be discussed. Monitoring will 
be carried out on an ongoing basis. The severity, relatedness, and whether or not the event is expected 
will be reviewed. 
 
15.0 
References 
 
1. Álvarez, G. C., Gómez-Galicia, D., Rodríguez-Fragoso, L., Marina, V. M., Dorantes, L. C., Sánchez-
Alemán, M., … Esparza, J. R. (2011). Danazol improves thrombocytopenia in HCV patients 
treated with peginterferon and ribavirin. Annals of Hepatology, 10(4), 458–468. 
2. Bär C, Huber N, Beier F, Blasco MA. Therapeutic effect of androgen therapy in a mouse model of 
aplastic anemia produced by short telomeres. Haematologica 2015; 100: 1267-74. 
3. Blackburn EH. Switching and signaling at the telomere. Cell 2001; 106:661-673. 
4. Calado RT, Brudno J, Mehta P, Kovacs JJ, Wu C, Zago MA, Chanock SJ, Boyer TD, Young NS. 
Constitutional telomerase mutations are genetic risk factors for cirrhosis. Hepatology 2011; 53: 
1600-1607 [PMID: 21520173 DOI: 10.1002/ hep.24173]. 
5. Calado RT, Regal JA, Hills M, Yewdell WT, Dalmazzo LF, Zago MA, et al. Constitutional 
hypomorphic telomerase mutations in patients with acute myeloid leukemia. Proc Nat Acad Sci 
USA 2009;106: 1187-1192. 
6. Calado RT, Yewdell WT, Wilkerson KL, et al. Sex hormones, acting on the TERT gene, increase 
telomerase activity in human primary hematopoietic cells. Blood 2009; 114: 2236-43. 
7. Calado RT, Young NS. Telomere diseases. N Engl J Med 2009; 361: 2353-2365 [PMID: 20007561 
DOI: 10.1056/ NEJMra0903373]. 
8. Camitta BM, Thomas ED, Nathan DG, et al. A prospective study of androgens and bone marrow 
transplantation for treatment of severe aplastic anemia. Blood 1979; 53: 504-14. 

9. Cervantes F, Alvarez-Larran A, Hernandez-Boluda J-C, Correa J-G, Pereira A. Annals of 
Hematology 2015;94:1791-96. 
10. Chiu, V. , Hogen, R. , Sher, L. , Wadé, N. , Conti, D. , Martynova, A. , Li, H. , Liang, G. and 
O'Connell, C. (2019), Telomerase Variants in Patients with Cirrhosis Awaiting Liver 
Transplantation. Hepatology, 69: 2652-2663. doi:10.1002/hep.30557 
11. Cicardi M., Castelli R., Zingale L.C., Agostoni A. Side effects of long-term prophylaxis with 
attenuated androgens in hereditary angioedema: comparison of treated and untreated patients. 
J Allergy Clin Immunol 1997;99:194-196. 
12. Gild P, Cole AP, Krasnova A, Dickerman BA, von Landenberg N, Sun M, Mucci LA, Lipsitz SR, 
Chun FKH, Nguyen PL, Kibel AS, Choueiri TK, Basaria S, Trinh Q-D. Liver disease in men 
undergoing androgen deprivation therapy for prostate cancer.  The Journal of Urology 
2018;200:573-81.  
13. Heiss NS, Knight SW, Vulliamy TJ, et al. X-linked dyskeratosis congenita is caused by mutations in 
a highly conserved gene with putative nucleolar functions. Nat Genet 1998; 19: 32-8. 
14. Khincha PP, Wentzensen IM, Giri N, Alter BP, Savage SA. Response to androgen therapy in patients 
with dyskeratosis congenita. Br J Haematol 2014; 165: 349- 57. 
15. Kitada T, Seki S, Kawakita N, Kuroki T, Monna T. Telomere shortening in chronic liver diseases. 
Biochem Biophys Res Commun 1995; 211: 33-39 [PMID: 7779103 DOI: 10.1006/ 
bbrc.1995.1774]. 
16. McKay JD, Hung RJ, Gaborieau V, Boffetta P, Chabrier A, Byrnes G, et al. Lung cancer 
susceptibility locus at 5p15.33. Nat Genet 2008;40: 1404-1406. 
17. Najean Y. Long-term follow-up in patients with aplastic anemia: a study of 137 androgen-treated 
patients surviving more than two years. Am J Med 1981; 71: 543-51. 
18. Qamar AA, Grace ND. Abnormal hematological indices in cirrhosis. Can J Gastroenterol 
2009;23(6):441-445. 
19. Qamar AA, Grace ND, Groszmann RJ, et al. The incidence, prevalence and clinical significance of 
abnormal hematological indices in patients with compensated cirrhosis. Clin Gastroenterol 
Hepatol 2009; 7(6): 689–695. 
20. Rafnar T, Sulem P, Stacey SN, Geller F, Gudmundsson J, Sigurdsson A, et al. Sequence variants at 
the TERT-CLPTM1L locus associate with many cancer types. Nat Genet 2009;41:221-227. 
21. Rudolph KL, Chang S, Millard M, Schreiber-Agus N, DePinho RA. Inhibition of experimental liver 
cirrhosis in mice by telomerase gene delivery. Science 2000; 287: 1253-1258 [PMID: 10678830 
DOI: 10.1126/science.287.5456.125]. 
22. Sánchez-Medal L, Gomez-Leal A, Duarte L, Guadalupe Rico M. Anabolic androgenic steroids in the 
treatment of acquired aplastic anemia. Blood 1969; 34: 283-300. 
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(2016). Danazol Treatment for Telomere Diseases. New England Journal of Medicine, 374(20), 
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24. Tsakiri KD, Cronkhite JT, Kuan PJ, Xing C, Raghu G, Weissler JC, et al. Adult-onset pulmonary 
fibrosis caused by mutations in telomerase. Proc Nat Acad Sci U S A 2007;104:7552-7557. 
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1424. 
 
 
APPENDICES  
 
 
Appendix 1: 
Current National Cancer Institute Common Terminology Criteria For Adverse 
Events  
 
Appendix 2:  
Study Calendar  
 
Appendix 3: 
ECOG Performance Scale  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
APPENDIX 1 – CURRENT NATIONAL CANCER INSTITUTE COMMON TERMINOLOGY 
CRITERIA FOR ADVERSE EVENTS (NCI CTCAE)  
Please use the following link to the NCI CTCAE website:  

http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm  
 
 
 
 
 
 
 
 
 
 
 
 
Grade 1 
Grade 2  
Grade 3 
Grade 4 
ALT 
> ULN - 3.0 x ULN if 
baseline was normal or 1.5 
- 3.0 x baseline if baseline 
was abnormal  
>3.0 - 5.0 x ULN if 
baseline was normal or 3.0 
- 5.0 x baseline if baseline 
was abnormal  
>5.0 - 20.0 x ULN if 
baseline was normal or 5.0 
- 20.0 x baseline if baseline 
was abnormal  
>20.0 x ULN if baseline 
was normal or >20.0 x 
baseline if baseline was 
abnormal  
AST 
> ULN - 3.0 x ULN if 
baseline was normal or 1.5 
- 3.0 x baseline if baseline 
was abnormal  
>3.0 - 5.0 x ULN if 
baseline was normal or 3.0 
- 5.0 x baseline if baseline 
was abnormal  
>5.0 - 20.0 x ULN if 
baseline was normal or 5.0 
- 20.0 x baseline if baseline 
was abnormal  
>20.0 x ULN if baseline 
was normal or >20.0 x 
baseline if baseline was 
abnormal  
Alkaline  
phosphata
se 
> ULN - 2.5 x ULN if 
baseline was normal or 2.0 
- 2.5 x baseline if baseline 
was abnormal  
>2.5 - 5.0 x ULN if 
baseline was normal or 2.5 
- 5.0 x baseline if baseline 
was abnormal  
>5.0 - 20.0 x ULN if 
baseline was normal or 5.0 
- 20.0 x baseline if baseline 
was abnormal  
>20.0 x ULN if baseline 
was normal or >20.0 x 
baseline if baseline was 
abnormal  
Bilirubin  
> ULN - 1.5 x ULN if 
baseline was normal or 1.0 
- 1.5 x baseline if baseline 
was abnormal  
> 1.5 - 3.0 x ULN if 
baseline was normal or 1.5 
- 3.0 x baseline if baseline 
was abnormal  
>3.0 - 10.0 x ULN if 
baseline was normal or 3.0 
- 10.0 x baseline if baseline 
was abnormal  
>10.0 x ULN if baseline 
was normal or >10.0 x 
baseline if baseline was 
abnormal  
Creatinine 
> ULN - 1.5 x ULN 
>1.5 - 3.0 x baseline or 
>1.5 - 3.0 x ULN  
>3.0 x baseline or >3.0 - 
6.0 x ULN  
>6.0 x ULN  
Edema  
5 - 10% inter-limb 
discrepancy in volume or 
circumference at point of 
greatest visible difference; 
swelling or obscuration of 
anatomic architecture on 
close inspection 
>10 - 30% inter-limb 
discrepancy in volume or 
circumference at point of 
greatest visible difference; 
readily apparent 
obscuration of anatomic 
architecture; obliteration 
of skin folds; readily 
apparent deviation from 
normal anatomic contour; 
limiting instrumental ADL 
>30% inter-limb 
discrepancy - - in volume; 
gross deviation 
from normal anatomic 
contour; limiting self care 
ADL 
 

APPENDIX 2 – Study Calendar 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Event/Visit 
Pre- 
Treatment 
Week 0 
(Start of 
 Treatment) 
( 
Week 
 2  
Week 
 4 
Week 
 8 
Written informed consent 
X 
Eligibility assessment  
X 
X 
Demographics  
X 
 
 
 
 
Comprehensive History & Physical Examination 
X 
 
 
 
 
Targeted History &  
Physical Examination  
X 
X 
X 
X 
Complete Blood Count  
X 
X 
X 
X 
X 
Complete Metabolic Panel 
X 
X 
X 
X 
X 
Reticulocyte Count 
X 
X 
X 
X 
X 
PT/INR 
X 
Alpha fetoprotein 
X 
Abdominal Ultrasound 
X 
Fibroscan 
X 
Telomere Gene Panel  
Testing 
X 
Telomere Length 
Measurement 
X 
Bone Marrow Biopsy* 
X 
 
 
 
 
Danazol administration  
X 
X 
X 
X 
Side effect and toxicity 
Assessment 
X 
X 
X 
Current medication list  
Review 
X 
X 
X 
X 
X 

APPENDIX 3 – ECOG Performance Scale