Danazol Treatment for Telomere Biology Disorders: Long-Term Results of a Phase I/II Study

Introduction: Telomere biology disorders (TBDs) are characterized by abnormal telomere maintenance and a clinically heterogenous phenotype, including marrow failure, pulmonary fibrosis, cirrhosis, and malignancy predisposition. Treatment options are limited, including supportive care, androgens, and/or hematopoietic cell transplant (HCT). We have reported the results from a prospective phase I/II study for danazol therapy in a cohort of TBD patients (NCT01441037; Townsley et al, 2016). Here we present long-term follow-up.

Methods: Patients were treated with 800mg danazol, daily, for two years, with a primary biologic endpoint of telomere attrition (determined by qPCR). Inclusion criteria required age-adjusted telomere length (TL) ≤ 1^st percentile and/or identified mutations in telomere maintenance plus one cytopenia and/or pulmonary fibrosis. Hematologic relapse, overall survival, and event free survival (requiring HCT, lung transplant, liver transplant, or death) were determined using Kaplan-Meier curves. In a subset of patients, error-corrected sequencing (ECS) with a customized panel was retrospectively performed to evaluate clonal hematopoiesis (CH at minimum VAF of 0.5%) in myeloid cancer and telomere related genes. Flow-FISH (RepeatDx) was performed in serial samples post-trial to measure TL during follow-up.

Results: Of 26 evaluable patients, most had identifiable mutations in known TBD genes, including TERT (n=10), TERC (n=7), DKC1 (n=3), or RTEL1 (n=1); 6 had no germline variants identified. Pulmonary fibrosis was present in 13 cases, liver fibrosis in 6, and previously diagnosed solid cancers in 2 (not active at time of enrollment). Median follow-up time to last clinical evaluation was 3.3 years, with a range from 0.3 to 12.6 years. Half the cohort (n=13) had at least one post-trial follow-up visit after stopping danazol. After drug discontinuation, either per protocol at two years or when the study was halted for efficacy, the median time to hematologic relapse for responders (n=12) from time of danazol cessation was 1.6 years. Nine patients restarted danazol post-trial at a median dose of 200mg (range: 75-600mg) with hematologic response in 86% (n=7). Median EFS was 5.7 years. Median overall survival was 7.7 years with 62% of participants (n=16) known to have died at last follow-up; the most common known cause of death (n=10) was infection in 60% (n=6).

No new solid malignancies developed on trial. One patient developed MDS with multilineage dysplasia (MLD) with monosomy 7 at 24 months, and one TERT-mutated patient, with baseline MDS-MLD, progressed to AML 3 years after drug discontinuation. The patient who developed monosomy 7 on trial had no identified germline variant, a small PNH clone, and later underwent ECS detecting BCOR and PIGA mutations, suggestive of immune AA. Two patients who developed isolated cytogenetic abnormalities on trial, trisomy 21 and +1q, remained without morphologic MDS at last follow-up.

A major concern with danazol treatment is the selection/expansion of pre-malignant clones in the marrow. At baseline, 15/24 (63%) patients had CH, most commonly in TERT promoter (n=7, 5 patients), PPM1D (n=6/5), and U2AF1 (n=4/4), and other MDS-related genes (n=8). However, longitudinal analysis of CH in 10 patients (median follow up [range] = 4.5 [2-6.5]) showed that mutations associated with myeloid malignancy (splicing factors, ASXL1, RUNX1, ETV6, SEPBP, and TP53) were largely stable during danazol treatment and follow-up.

Serial TL by flow-FISH was available for 9 patients. TL was stable in 5/9 patients, four of whom re-initiated danazol, with median follow up of 4 years (average loss of 7bp per year [SD ± 28 bp/year]); a telomere attrition rate of 51bp/yr is typically seen in heathy individuals. Three individuals, 2 of whom re-initiated danazol, had an average telomere attrition rate of 110bp/yr (median follow-up of 3 years), similar to what is reported in TBD patients. A single patient had telomere elongation over 8 years of follow-up (from 4.5 to 5.5kb).

Conclusion: Most patients required continuous administration of danazol to improve hematologic parameters, which worked at lower doses than mandated by protocol. Clonal dynamics were stable on danazol treatment over time. Telomere length, assessed by flow-FISH, was stable over time in a subset patients.