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

2 Table of contents Methods... 2 Telomere Measurement and Gene Sequencing... 2 Figure S1. Patient flow diagram... 3 Figure S2. Leukocyte telomere length by qpcr at enrollment... 4 Figure S3. Changes in lung diffusing capacity in telomeropathy patients receiving danazol... 5 Figure S4. Telomere attrition in blood lymphocytes measured by qpcr and flow-fish... 6 Table S1. Estimated telomere attrition rates in healthy and telomeropathy patients... 7 Table S2. Baseline clinical features... 8 Table S3. Telomere length and genomic characteristics Table S4. Lymphocyte telomere length by flow-fish Table S5. Summary statistics for changes in peripheral blood counts according to cytopenia satisfying entry criteria Table S6. Summary statistics for changes in peripheral blood counts for all patients Table S7. Adverse events related to danazol administration References

3 Methods Telomere Measurement and Gene Sequencing Genomic DNA was purified from peripheral blood leukocytes within 24h from collection using the automated Maxwell 16 Instrument (AS2000, Promega, Madison, WI). Telomere length was determined by a semi-automated CLIAapproved real time quantitative PCR (RT-qPCR) performed in triplicate and validated for human cells, as previously described. 1,2 A limit for intra-assay coefficient of variation of 2% was determined; measurements exceeding this limit were discarded. Serial samples from an individual patient were batched in single runs. For each sample, the average telomere length was calculated as a ratio between telomere (T) and single-copy gene 36B4 (S) amplifications (Ct), T/S ratio, then normalized to a standard control (average telomere length by Southern blot, 8.6 kb; 2 ΔΔ Ct). Peripheral blood leukocytes from healthy volunteers ages 0 to 99 were used for controls and to generate age-adjusted telomere lengths. 3 To convert T/S ratios to base pairs, telomere length of healthy subjects with short and long telomeres also measured by Southern blot, according to the equation: telomere length kb = (5.5969*T/S ratio) , rendering high linearity (r 2 =0.86). Nucleotide sequencing for telomere maintenance genes was performed in a CLIA laboratory by Sanger or next generation sequencing. 2

4 Figure S1. Patient flow diagram Enrollment began in June There are no data in 15 subjects at 24 months: 10 withdrew from the study for various reasons depicted above and 5 subjects had not reached the primary endpoint of 2 years due to the study being halted early in April *Two subjects were unable to tolerate side effects of danazol despite offered dose reduction. ^Two subjects had danazol discontinued due to grade 3 adverse events. 3

5 Figure S2. Leukocyte telomere length by qpcr at enrollment Individual mean telomere lengths of peripheral blood leukocytes as measured by qpcr in all 27 patients is shown in comparison with 300 healthy volunteers (grey dots) ages 0 (newborn) to 100 years. Danazol cohort patients are shown in various colors based on their telomere maintenance gene mutation (DKC1-red, TERC yellow, TERT-light green, RTEL1 light blue, and no mutation identified-dark blue). 4

6 Figure S3. Changes in lung diffusing capacity in telomeropathy patients receiving danazol 100 *" Adjusted DLCO (%ref) Before 0 mo 6 mo 12 mo 24 mo 36 mo Patients Carbon monoxide diffusing capacity, adjusted for hemoglobin (DLadj) is shown as percentage of the age-adjusted predicted value at various time points before, during, and after 2 years of danazol administration. Paired t-test showed a statistically significant (* p=0.012) 10% decrease before starting danazol and no change during the first 36 months after starting study drug. Grey symbols depict all other patients with PFT available for enrollment in the trial. Seven subjects had PFT results available at least 6 months prior to starting danazol (range 9-58 months, median 16 months), and 5 subjects had PFTs available after discontinuing danazol. 5

7 Figure S4. Telomere attrition in blood lymphocytes measured by qpcr and flow-fish A' B' Q"PCR& Flow"FISH& 0'mo' Average'telomere'content'' 1.2$ 1.15$ 1.1$ 1.05$ 1$ 0.95$ 0.9$ *'p<0.05' *' *' *' 0$mo$ 6$mo$ 12$mo$ 24$mo$ Pa:ents' 11' 11' 8' 7' 6/12'mo' (A). Lymphocyte sorting by flow cytometry (left panel) and patient weighted telomere length by qpcr in DNA extracted from 11 patients (right panel). (B). Flow-FISH gating strategy for comparison of telomere length at 0 and 6/12 months on danazol (left panel). 6

8 Table S1. Estimated telomere attrition rates in healthy and telomeropathy patients Institution Population N Method Study design NHLBI 3,4 Healthy 175 qpcr NHLBI* Healthy 298 qpcr Univ. of British Columbia 5 Healthy 400 flow-fish Univ. of Sao Crosssectional Crosssectional Crosssectional Paulo 6 Healthy 261 qpcr Crosssectional Univ. of Sao Paulo 6 Healthy 180 flow-fish Crosssectional Dyskeratosis NCI 7 Attrition rate, bp loss per year (error) 60 (95% CI, 45-71) 54 (95% CI, 47-60) 51 (NR) 40 (95% CI, 48-33) 47 (95% CI, 53-41) 9 flow-fish Longitudinal 170 (±100 SD) congenita NHLBI* Telomere disease 25 qpcr Longitudinal 147 (±281 SD) NHLBI, National Heart, Lung, and Blood Institute; NCI, National Cancer Institute; NR, none reported *Our unpublished data 7

9 UPN # Table S2. Baseline clinical features Age Sex Dx Bone marrow pathology 1 42 F MAA 2 23 M MAA 3 41 F SAA 4 40 M MAA IPF 5 27 F MAA 6 33 F MAA 7 47 M MDS (RC MD) 8 66 F MAA 9 66 F IPF M MDS (RCU D) M MAA F IPF F MAA M MAA F MAA M MAA M MAA F MAA F MAA F SAA M MAA Markedly variably cellular, trilineage hematopoiesis. Hypocellular, trilineage hypoplasia Mildly hypocellular, markedly decreased megakaryocytes Hypocellular, trilineage hypoplasia Hypocellular, trilineage hypoplasia Hypocellular, without morphologic evidence of significant dysplasia Hypercellular, atypical megakaryocytes, mild erythroid hyperplasia with dyserythropoiesis Markedly hypocellular, trilineage hypoplasia Normocellular, trilineage hematopoiesis, mild megakaryocytic atypia Normocellular, mildly atypical megakaryocytes, erythroid hyperplasia, mild myeloid hypoplasia. Variably hypocellular, erythroid predominance, decreased megakaryocytes Variably and mildly hypocellular, mildly decreased megakaryocytes, mild megaloblastoid erythropoiesis Hypocellular, trilineage hypoplasia, mild left shift in myeloid maturation, megaloblastic changes Variably hypocellular, trilineage hypoplasia Mildly hypocellular, mild erythroid predominance, mild megakaryocytic atypia Variably hypocellular, myeloid hypoplasia, decreased megakaryocytes Variably hypocellular, trilineage hypoplasia Moderately hypocellular, focal erythroid hyperplasia, mild dyserythropoiesis Markedly hypocellular marrow demonstrating trilineage hypoplasia Marrow containing only fat, stromal cells, no hematopoietic elements present Variably cellular, trilineage hematopoiesis, decreased Liver cirrhosis Lung fibrosis Early gray hair Transfusions Mucocutaneous features RBCs Absent Overt No No - Subclinical Subclinical No No RBCs Absent Subclinical No No - Absent Overt No Rash Family history Anemia Liver cirrhosis Anemia Lung disease Aplastic anemia Acute leukemia Early graying Acute leukemia Early graying RBCs Subclinical Subclinical No No No - Absent Subclinical No No RBCs Subclinical Overt Yes Nails and rash Anemia Liver cirrhosis Early graying Oral cancer - Absent Absent No No No - Absent Overt No No Anemia Lung fibrosis RBCs Absent Subclinical No No Anemia - Overt Subclinical Yes No - Absent Overt No SCC Myelodysplasia Liver cirrhosis Aplastic anemia Lung fibrosis - Absent Subclinical No SCC Aplastic anemia - Absent Subclinical No No Aplastic anemia Oral cancer RBCs Overt Overt No No Lung fibrosis - Overt Subclinical Yes No Early graying RBCs Overt Overt No No Lung fibrosis RBCs Absent Subclinical No No Anemia RBCs Absent Subclinical No No Anemia RBCs Absent Overt Yes No Aplastic anemia Liver cirrhosis Lung fibrosis Early graying - Absent Subclinical No No No 8

10 megakaryocytes M MAA Mildly hypocellular, mild megakaryocytic hypoplasia - Overt Overt Yes Nails and rash Aplastic anemia Liver cirrhosis Skin changes F SAA M SAA M MAA F MAA F MAA Hypocellular, trilineage hypoplasia Hypocellular, markedly decreased megakaryocytes, relative erythroid predominance with mild dyserythropoiesis, focal increase in CD34-positive cells (overall <4%), Markedly hypocellular, trilineage hypoplasia Markedly hypocellular, trilineage hypoplasia, atypical megakaryocytes Variably hypocellular, trilineage hypoplasia, mild megakaryocytic atypia, erythroid predominance RBCs, plts RBCs, plts Absent Subclinical No No Anemia Liver cirrhosis Absent Absent No No No - Overt Overt Yes Rash Early graying RBCs Absent Subclinical No No Aplastic anemia - Absent Subclinical No No Liver cirrhosis was categorized as overt if subjects entered the study with a preexisting diagnosis of liver cirrhosis and subclinical if liver cirrhosis was identified on baseline evaluation. Pulmonary fibrosis was categorized as overt if symptoms were present in addition to supportive imaging and pulmonary function testing at baseline, and subclinical if symptoms were absent. MAA, moderate aplastic anemia; SAA, severe aplastic anemia; MDS, myelodysplastic syndrome; RCMD, refractory cytopenia with multilineage dysplasia; RCUD, refractory cytopenia with unilineage dysplasia; IPF, idiopathic pulmonary fibrosis; RBCs, red blood cells; plts, platelets; SCC, squamous cell carcinoma Anemia Early graying 9

11 Table S3. Telomere length and genomic characteristics UPN# Age-matched percentile at diagnosis 1 < 1st 2 < 1st Mutations TERC n del TERT c.994g>a (p.ala1062thr)* + c.844t>c (p.ser795pro) Telomere change (bp) at 24 months (primary endpoint 96 bp/yr loss) 189 gain 255 gain 3 < 1st TERC n.-58c>g 198 gain 4 < 1st not identified - 5 < 1st not identified 981 gain 6 < 1st not identified - 7 < 1st DKC1 c.1178 T>A (p.ile393asn) 373 gain 8 5th to 10th TERT c.833 G>A (p.glu280lys)* - 9 < 1st TERT c.175 C>T (p.pro59ser) st to 5th TERT c.1760 T>C (p.ile587thr) 951 gain 11 < 1st RTEL1 c.2227g>a (p.asp734asn) + c.2684c>t (p.pro895leu) 395 gain 12 < 1st TERC n.119 C>G - 13 < 1st TERC n.128 A>G - 14 < 1st TERC n.128 A>G st to 5th TERT c.3029 C>A (p.ala1009ile) - 16 < 1st 17 < 1st TERT c.389 C>T (p.ala130val) TERC n.433 G>C 610 gain 58 gain 18 < 1st DKC1 c.1512_1514del 213 gain 19 < 1st TERT c.1895 C>T (p.pro632leu) - 20 < 1st TERC n.114_115del 482 gain 21 < 1st not identified - 22 < 1st DKC1 c.160g>c (p.leu54val) 76 loss 23 1st to 5th TERT c.3184 G>A (p.ala1062thr)* - 24 < 1st not identified - 25 < 1st TERT c.2318 T>C (p.met773thr) - 26 < 1st TERT c.2591 T>C (p.leu864pro) - 27 < 1st not identified - All mutations were heterozygous. *Pathogenicity ambiguous; the p.ala1062thr and Glu280Lys variant has an allele frequency of 1.3% and 0.05% in healthy controls respectively ( 10

12 Table S4. Lymphocyte telomere length by flow-fish Time point (months) telomere length (kb) qpcr Δ telomere length (bp) telomere length (kb) flow- FISH Δ telomere length (bp) UPN# UPN# UPN# UPN# UPN# UPN# UPN# Mean (±SD) 313 (±372) 799 (±1113) Lymphocyte telomere length was measured by flow-fish for seven patients. Corresponding leukocyte telomere length measured by qpcr is shown for comparison. In all but one case, UPN#10, both methods were in agreement and showed telomere elongation. As expected, both methods correlated well, but the variance of differences is high. 6 11

13 Table S5. Summary statistics for changes in peripheral blood counts according to cytopenia satisfying entry criteria Mean 95% CI p-value N Δ Hemoglobin (g/dl) 0 to 6 months , 3.06 < to 12 months , 4.42 < to 24 months , 4.12 < Δ Reticulocytes (k/ul) 0 to 6 months , to 12 months , < to 24 months , Δ Platelets (k/ul) 0 to 6 months , to 12 months , to 24 months , Δ Neutrophils (/ul) 0 to 6 months , to 12 months , to 24 months , Two-sided paired t-tests for the null hypothesis of true Δ=0 are shown only for subjects with an abnormally low value in that lineage as defined by the protocol s entry criteria. Anemia (n=14): hemoglobin < 9.5gm/dL or significant red cell transfusion requirements; Thrombocytopenia (n=13): platelet count < 30,000/µL, or platelet count < 50,000/µL and bleeding; Neutropenia (n=10): absolute neutrophil count < 1,000/µL. 12

14 Table S6. Summary statistics for changes in peripheral blood counts for all patients Mean 95% CI p-value N Δ Hemoglobin (g/dl) 0 to 6 months , 2.54 < to 12 months , 3.70 < to 24 months , Δ Reticulocytes (k/ul) 0 to 6 months , to 12 months , < to 24 months , Δ Platelets (k/ul) 0 to 6 months , < to 12 months , to 24 months , Δ Neutrophils (/ul) 0 to 6 months , to 12 months , to 24 months , Two-sided paired t-tests for the null hypothesis of true Δ=0 are shown for all subjects. 13

15 Table S7. Adverse events related to danazol administration <Grade 3 Grade 3 Alanine aminotransferase increase 11 (41%) Muscle spasm or cramps 9 (33%) Aspartate aminotransferase increase 8 (30%) Edema 7 (26%) High cholesterol 7 (26%) Headaches 4 (15%) Weight gain 4 (15%) Arthralgia 3 (11%) Hair thinning 3 (11%) Dizziness 2 (7%) Hirsutism 2 (7%) Nasal congestion 2 (7%) Rash 2 (7%) Testicular atrophy 5 (19%) Alkaline phosphatase increase 1 (4%) Acne 1 (4%) Agitation + 1 (4%) Cognitive disturbance 1 (4%) Depression 1 (4%) Nausea 1 (4%) Reduced appetite 1 (4%) Vomiting 1 (4%) Voice alteration 1 (4%) Hot flashes 1 (4%) Thromboembolic cerebrovascular accident* 1 (4%) Hemangioma* 1 (4%) Suspected treatment-related adverse events in all 27 patients. Alterations in serum lipid profiles occurred in all patients, namely increase in serum low-density lipoprotein and decrease in high-density lipoprotein, and changes met criteria for an adverse event in seven cases (grade 1 or 2 hypercholesterolemia). Testicular size decreased in all male subjects at 14 months (n=5). Bone densitometry and uterine/ovarian ultrasounds were unchanged while on danazol. Three patients chose to discontinue drug due to adverse events (Figure S1). *Severe adverse event that prompted discontinuation of danazol by investigator due to possible relationship to danazol. In one subject, UPN#8, with preexisting cardiovascular risk factors a thromboembolic cerebrovascular accident causing dysphasia and extremity weakness prompted discontinuation of the drug; the subject had a prolonged hospital course but fully recovered. A hemangioma was diagnosed in another subject, UPN#6, who developed swelling and pain in the 14

16 lower extremity while on danazol; symptoms resolved with discontinuation of danazol but the hemangioma persisted on repeat imaging. + Resolved with dose-reduction of danazol. 15

17 References 1. Winkler T, Hong SG, Decker JE, et al. Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia ipscs. J Clin Invest 2013;123: Peffault de Latour R, Calado RT, Busson M, et al. Age-adjusted recipient pretransplantation telomere length and treatment-related mortality after hematopoietic stem cell transplantation. Blood 2012;120: Calado RT, Cooper JN, Padilla-Nash HM, et al. Short telomeres result in chromosomal instability in hematopoietic cells and precede malignant evolution in human aplastic anemia. Leukemia 2012;26: Scheinberg P, Cooper JN, Sloand EM, Wu CO, Calado RT, Young NS. Association of telomere length of peripheral blood leukocytes with hematopoietic relapse, malignant transformation, and survival in severe aplastic anemia. JAMA 2010;304: Yamaguchi H, Calado RT, Ly H, et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med 2005;352: Gutierrez-Rodrigues F, Santana-Lemos BA, Scheucher PS, Alves- Paiva RM, Calado RT. Direct comparison of flow-fish and qpcr as diagnostic tests for telomere length measurement in humans. PLoS One 2014;9:e Alter BP, Rosenberg PS, Giri N, Baerlocher GM, Lansdorp PM, Savage SA. Telomere length is associated with disease severity and declines with age in dyskeratosis congenita. Haematologica 2012;97:

Danazol Treatment for Telomere Diseases

Danazol Treatment for Telomere Diseases The new england journal of medicine Original Article Treatment for Telomere Diseases Danielle M. Townsley, M.D., Bogdan Dumitriu, M.D., Delong Liu, Ph.D., Angélique Biancotto, Ph.D., Barbara Weinstein,