REGULAR ARTICLE Metformin for treatment of cytopenias in children and young adults with Fanconi anemia Jessica A. Pollard,1-3,* Elissa Furutani,1-3,* Shanshan Liu,4 Erica Esrick,1,3 Laurie E. Cohen,3,5 Jacob Bledsoe,6 Chih-Wei Liu,7 Kun Lu,7 Maria Jose Ramirez de Haro,8,25 Jordi Surralle s,8,25 Maggie Malsch,1,9 Ashley Kuniholm,9 Ashley Galvin,9 Myriam Armant,10 Annette S. Kim, Kaitlyn Ballotti, Lisa Moreau, Yu Zhou,1 Daria Babushok,13 Farid Boulad,14 Clint Carroll,15 Helge Hartung,16 11 1 12 Amy Hont,17 Taizo Nakano,18 Tim Olson,16 Sei-Gyung Sze,19 Alexis A. Thompson,20 Marcin W. Wlodarski,21 Xuesong Gu,22 Towia A. Libermann,22 Alan D’Andrea,23 Markus Grompe,24 Edie Weller,3,4 and Akiko Shimamura1-3 1 Pediatric Hematology-Oncology, Boston Children’s Hospital, Boston, MA; 2Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA; 3Department of Pediatrics, Harvard Medical School, Boston, MA; 4Biostatistics and Research Design Center, Institutional Centers for Clinical and Translational Research, Harvard Medical School, Boston, MA; 5 Department of Endocrinology, and 6Department of Pathology, Boston Children’s Hospital, Boston, MA; 7Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC; 8Joint Research Unit UAB-Sant Pau Biomedical Research Institute,Institut de Recerca Hospital de la Santa Creu i Sant Pau-IIB onoma de Barcelona, Barcelona Spain; 9Clinical Research Operations Center, Institutional Centers for Clinical and Translational Research, Boston Sant Pau, Universitat Aut Children’s Hospital, Boston, MA; 10Trans Laboratory, Boston Children’s Hospital, Boston, MA; 11Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 12Comprehensive Center for Fanconi Anemia, Dana-Farber Cancer Institute, Boston, MA; 13Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA; 14Pediatric Hematology-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY; 15Pediatric Hematology-Oncology, The Children's Hospital at TriStar Centennial, Nashville, TN; 16Pediatric Hematology-Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA; 17Pediatric Hematology-Oncology, Children’s National Medical Center, Washington, DC; 18Pediatric Hematology-Oncology, Children’s Hospital Colorado, Denver, CO; 19Department of Pediatrics, Maine Medical Center, Tufts University School of Medicine, Portland, ME; 20Pediatric Hematology-Oncology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL; 21Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN; 22Beth Israel Deaconess Medical Center Genomics, Proteomics, Bioinformatics and Systems Biology Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 23Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 24Oregon Stem Cell Center, Department of Pediatrics, Pap e Family Institute, Oregon Health and Science University, Portland, OR; and 25Center for Biomedical Network Research on Rare Diseases, Madrid, Spain Key Points Fanconi anemia (FA), a genetic DNA repair disorder characterized by marrow failure and cancer susceptibility. In FA mice, metformin improves blood counts and delays  Metformin was safe tumor development. We conducted a single institution study of metformin in nondiabetic and well tolerated in patients with FA to determine feasibility and tolerability of metformin treatment and to nondiabetic patients assess for improvement in blood counts. Fourteen of 15 patients with at least 1 cytopenia with FA. (hemoglobin , 10 g/dL; platelet count , 100 000 cells/mL; or an absolute neutrophil count  Hematologic , 1000 cells/mL) were eligible to receive metformin for 6 months. Median patient age was response was 9.4 years (range 6.0-26.5 ). Thirteen of 14 subjects (93%) tolerated maximal dosing for observed in 4 of age; 1 subject had dose reduction for grade 2 gastrointestinal symptoms. No subjects 13 (30.8%; 90% CI, developed hypoglycemia or metabolic acidosis. No subjects had dose interruptions 11.3-57.3) evaluable caused by toxicity, and no grade 3 or higher adverse events attributed to metformin patients with FA treated with were observed. Hematologic response based on modified Myelodysplastic Syndrome metformin. International Working Group criteria was observed in 4 of 13 evaluable patients (30.8%; 90% confidence interval, 11.3-57.3). Median time to response was 84.5 days (range 71-128 days). Responses were noted in neutrophils (n 5 3), platelets (n 5 1), and red blood cells (n 5 1). No subjects met criteria for disease progression or relapse during treatment. Submitted 29 October 2021; accepted 15 April 2022; prepublished online on Blood The current affiliation for E.F. is Novartis Institutes for Biomedical Research, Cam- Advances First Edition 2 May 2022; final version published online 27 June 2022. DOI bridge, MA. 10.1182/bloodadvances.2021006490. The full-text version of this article contains a data supplement. *J.A.P. and E.F. contributed equally to this study. © 2022 by The American Society of Hematology. Licensed under Creative Presented in abstract form at the 63rd Annual Meeting of the American Society of Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC- Hematology, Atlanta, GA, 11-14 December 2021. ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other Individual participant data will not be shared. For original data from correlative analyses, rights reserved. please contact Akiko Shimamura at akiko.shimamura@childrens.harvard.edu. 28 JUNE 2022 • VOLUME 6, NUMBER 12 3803 Correlative studies explored potential mechanisms of metformin activity in FA. Plasma prote- omics showed reduction in inflammatory pathways with metformin. Metformin is safe and tolerable in nondiabetic patients with FA and may provide therapeutic benefit. This trial was registered at as #NCT03398824. Introduction appropriate. A medical monitor reviewed data on a quarterly basis for assessment of toxicities and/or futility. This trial was registered at https://clinicaltrials.gov/ct2/show/NCT03398824. Fanconi anemia (FA) is a genetic bone marrow failure disorder char- acterized by DNA repair defects and propensity to develop hemato- Patients logic malignancies and solid tumors. Bone marrow failure is a major Patients with FA 6 to 35 years of age were eligible for enrollment at cause of early morbidity/mortality in FA, with a cumulative incidence Boston Children’s Hospital. All patients had baseline increased chro- of 90% by age 40.1,2 Although allogeneic hematopoietic stem cell mosomal breakage with mitomycin C (MMC) and diepoxybutane transplantation (HSCT) offers curative therapy for hematologic com- (DEB). Patients met at least 1 of the following cytopenia requirements plications of FA, graft-versus-host disease and other therapy-related for study enrollment: hemoglobin (Hb) , 10 g/dL, platelet count adverse effects remain challenging. Exposure to genotoxic agents in , 100,000 cells/mL, or an absolute neutrophil count (ANC) , 1000 transplant conditioning regimens also raise concern given the cells/mL. Exclusion criteria included, but were not limited to, prior increased risk of solid tumors with FA.3 Androgens can improve HSCT, history of myeloid malignancy, use of concurrent medications blood counts in a subset of patients with FA, although patients may with the intent to improve hematopoiesis, history of type I diabetes or relapse and remain at risk for hematologic malignancies.4-7 Long- prior symptomatic hypoglycemia, G6PD deficiency, vitamin B12 defi- term treatment with androgens is limited by side effects such as ciency, or other significant health comorbidities. transaminitis, virilization, and risk of hepatic adenomas and carcino- mas.8,9 Thus, the identification of safe and effective oral agents that Study design aid hematopoiesis and reduce the risk of malignant transformation This was an investigator-initiated single institution, open-label, sin- are of clinical interest. gle-arm pilot study of metformin in patients with FA. Patient Previous studies of resveratrol in a Fancd22/2 murine model of FA examinations were conducted in person before initiation of met- demonstrated that the drug improved hematopoietic function but formin and after 6 months of treatment with remote interim weekly had no effect on tumor formation.10 Resveratrol is a stilbenoid poly- assessments for the first month and monthly assessments there- phenol with multiple bioactivities including activation of AMP- after. Patients underwent bone marrow (BM) aspiration and activated protein kinase (AMPK). AMPK plays an important role in biopsy evaluations at study entry and at end of protocol therapy. hematopoietic stem cell maintenance.11,12 Metformin (N,N-dimethyl- Metformin immediate-release tablets (Ascend Laboratories) were guanide), an oral drug used to safely treat type 2 diabetes and insu- administered orally for 6 months using dosing prescribed for type lin resistance, is known to activate AMPK.13 In addition, metformin 2 diabetes. Starting dose was 500 mg once daily for all subjects, may also scavenge aldehydes through the Mannich reaction.14 FA and the dose was increased by 500 mg/day until goal dosing mice with mutations in the aldehyde detoxifying enzymes Aldh2 or was reached (500 mg twice daily for patients , 10 years of age Aldh5 manifest accelerated bone marrow failure.15-17 An increased and 1000 mg twice daily for patients 10 years or older). For risk of severe bone marrow failure was also observed in patients patients exhibiting metformin intolerance, as defined by gastroin- with FA with impaired aldehyde detoxification from constitutional testinal toxicity, renal toxicity, hypoglycemia, lactic acidosis, or mutations in ALDH2.18 Treatment of Fancd22/2 mice with metfor- sustained grade 2/onset of grade 3 toxicity, the dose was min improved hematopoiesis and delayed tumor formation.19 Based reduced by 500 mg/day until a tolerated dose was achieved. If on these findings, we hypothesized that metformin may provide ther- an individual was already on the lowest dosing of drug (500 mg/ apeutic benefit in patients with FA. As insulin resistance is also fre- day), treatment was discontinued. Blood counts were assessed quent in patients with FA, metformin treatment may also afford a monthly. Liver function and renal function were assessed at 1, 3, secondary benefit on glucose homeostasis.20,21 To investigate this and 6 months. Data on adverse events (AEs) were collected in hypothesis, we developed a pilot study of metformin for nondiabetic accordance with the National Cancer Institute Common Terminol- children and young adults with FA to assess safety, tolerability, and ogy for Adverse Events, Version 4.03. AEs are reported for impact of the drug on hematologic parameters. Exploratory biologi- patients that received metformin for any duration of time. cal correlative assays were also assessed. The primary end point was the proportion of subjects with a hemato- logic response (HR) after initiation of metformin. Patients were Methods deemed evaluable for HR if they received metformin for at least 1 month. Hematologic improvement criteria were based on modified Human subjects research Myelodysplastic Syndrome International Working Group (IWG) criteria The study was conducted in accordance with the principles of the (supplemental Appendix 1).22 To be classified as a HR, protocol- Declaration of Helsinki and Good Clinical Practice guidelines. The defined metrics of response needed to be sustained for at least 2 protocol was approved by the institutional review board at Boston months and demonstrated on 2 consecutive measurements in at least Children’s Hospital. All patients/legal guardians provided written 1 hematopoietic lineage. BM cellularity was evaluated by blinded cen- informed consent, and patient assent was also obtained where tral pathology review. 3804 POLLARD et al 28 JUNE 2022 • VOLUME 6, NUMBER 12 Statistical analysis DPCs based on calibration curve were then performed as previously Predefined stopping rules were developed in the event that an described.24 unacceptable frequency of severe AEs was observed. Any patient Methylcellulose colony-forming cell assays who received at least 1 dose of metformin on study was eligible for safety analysis. The primary end point of the study was to assess CD341 cells were isolated from mononuclear cells by magnetic cell the proportion of subjects with a HR during 6 months of metformin sorting using the EasySep human CD34-positive selection kit II (Stem treatment; sample size was calculated assuming that a HR rate $ Cell Technologies, Vancouver, Canada) following the manufacturer’s 20% suggests preliminary efficacy of treatment that warranted addi- recommendations. CD341 cells were plated at low-density in 2 to 4 tional investigation and, conversely, that the study should be wells of a SmartDish (Stem Cell Technologies) in MethoCult H4034 deemed futile if the rate of HR was #5%. Accrual goal for the study Optimum Methylcellulose-based media containing Stem Cell Factor, was 22 evaluable subjects; therefore, the treatment would warrant GM-CSF, G-CSF, interleukin-3, and EPO (Stem Cell Technologies). further investigational studies if at least 3 of 22 evaluable subjects Cultures were placed in an incubator set at 37 C, 5% CO2, and had HR. The probability of observing at least 3 of 22 responders is 90% humidity for 14 days. At the end of the incubation, colonies at least 0.84 assuming the true response is $20% and is ,10% if were scored (CFU-E, BFU-E, CFU-GM) in a blinded manner by 2 the true response is ,5%. Study accrual was stopped early on 9 independent investigators, and photographs were documented with October 2020 given the COVID-19 pandemic’s impact on clinical the STEMvision automated colony counter (Stem Cell Technologies). research infrastructure and evidence that the primary end point had Fluorescent in situ hybridization been achieved after 14 patients received protocol therapy. Fluorescent in situ hybridization (FISH) was performed by Integrated Patient demographic and baseline characteristics, including age, Oncology. Four applications of FISH were performed on interphase sex, medical history, FA subtype, prior therapy, and baseline hemato- nuclei using the following commercial probes: (1) a dual-color probe logic values (Hb, platelet count, ANC, transfusion requirements) targeting either side of BCL6 on chromosome 3q27, (2) 5q33 were summarized using descriptive statistics. Responses were (CSF1R/RPS14)/5p12(FGF10) probes, (3) 7q31 (MDFIC)/SE7 reported for Hb, platelet count, and ANC along with 90% exact (D7Z1), and (4) SE8 (D8Z1)/8q24(MYC). binomial Clopper-Pearson confidence interval (CI). For secondary objectives, change from baseline in continuous variables (Hb, plate- Somatic mutation analysis let count, ANC) was reported as proportion of subjects with no The RHPv3 assay assessed targeted regions in 88 genes that are improvement and maximum improvement. For assessment of safety recurrently mutated in myeloid and lymphoid neoplasms. DNA from and tolerability, the proportion of patients with each AE was peripheral blood was submitted to NEBNext Direct chemistry (New reported. Maximal dosing and dose interruptions/modifications were England Biolabs, Ipswich, MA) with dual indices and unique molecular recorded for patients enrolled. Change in exploratory end points identifiers allowing sub-1% variant calling at many loci. Samples were before and after treatment was assessed using a paired t test or the pooled before massively parallel sequencing on a NextSeq 550Dx Wilcoxon signed-rank test. (150 base pair paired-end sequencing, Illumina, San Diego, CA). The Chromosomal breakage assessment bioinformatic pipeline integrated several publicly available modules and internally developed algorithms for the detection of copy number Chromosomal breakage was quantitated before and after treatment variants and FLT3 internal tandems duplications.25 with MMC or DEB in the cytogenetics laboratory in the Comprehen- sive Center for Fanconi Anemia at the Dana-Farber Cancer Institute SOMAscan analysis as previously described.23 Peripheral blood stimulated with phytohe- SOMAscan (SomaLogic, Boulder, CO) analysis of plasma samples magglutinin was cultured and subsequently exposed to MMC or was performed at the Beth Israel Deaconess Medical Center Geno- DEB. Metaphase cells were scored and analyzed for chromosomal mics, Proteomics, Bioinformatics, and Systems Biology Center breakage, including formation of radials. Results were compared according to standard protocols for biologic fluids from SomaLogic with healthy subjects (negative controls) and FANCD12/2 cell lines that have been described elsewhere.26,27 Plasma samples (50 mL) (positive control). were run on the SOMAscan Assay 1.3k for human plasma, which Nanoscale liquid chromatography coupled to measures the expression of 1305 human proteins with the use of highly selective single-stranded modified slow off-rate modified DNA tandem mass spectrometry quantification of aptamers. Five pooled human plasma samples and a no-protein endogenous DNA-protein cross-link buffer control were run in parallel with the plasma test samples. Flash-frozen BM aspirates were homogenized with 1 mL ice-cold Sample-to-sample variability was controlled by several hybridization homogenization buffer (20 mM sodium phosphate buffer, 250 mM spike-in control cases. Data quality control, calibration, plate scaling, sucrose, 5 mM MgCl2, 25 mM KCl, pH 7.2) and centrifuged at hybridization normalization, and median normalization were done 4000g at 4 C for 10 minutes before resuspension in 1 mL ice-cold according to the manufacturer’s protocol.28,29 SOMAscan data 20 mM sodium phosphate buffer (pH 7.2). The sample was again were analyzed using methods previously described.30,31 centrifuged at 4000g at 4 C for 10 minutes, and the resulting pellet was resuspended in 1 mL DNAzol reagent. Proteinase K (20 mL, Oral glucose tolerance test 20 mg/mL) was added for overnight digestion. DNA-protein cross- An oral glucose tolerance test was performed before and after link (DPC) isolation, further enzymatic digestions, high-performance completion of study treatment. Patients fasted overnight before the liquid chromatography purification of target DPCs, nano-LC-MS/MS fasting glucose and insulin levels were drawn, and 5 mg/kg Trutol detection of purified DPCs fraction, and accurate quantification of (Permula) (100 g glucose/295 mL bottle, maximum dose of 28 JUNE 2022 • VOLUME 6, NUMBER 12 METFORMIN FOR FANCONI ANEMIA 3805 220 mL) was administered. Repeat glucose levels were drawn cells/mL (range, 0.6-1.6K cells/mL). One patient was transfusion 2 hours after administration of Trutol. dependent for red blood cells (Table 1). Micronucleus assay in buccal cells Treatment Buccal swabs were obtained at different time points before and Twelve of 14 patients (86%) completed 6 months of metformin after metformin treatment from each cheek. After washing, fixation, treatment. One patient was lost to follow-up after 2 weeks of metfor- and staining with 49 ,6-diamidino-2-phenylindole, the frequency of min treatment and another withdrew from the study after 3 months micronuclei (MN) was studied as previously described.32 To com- of treatment due to a desire to proceed to HSCT. Thirteen of 14 pare means of the frequency of MN before and after treatment, the patients treated (93%) achieved maximal age-based dosing of the Wilcoxon matched-pairs signed rank test was used. study drug. One subject underwent dose reduction from 2000 to 1500 mg/day for sustained grade 2 gastrointestinal symptoms, a Results known side effect of metformin that often improves with time. With dose reduction, the subject completed the remainder of protocol Patients treatment. Eligible patients were recruited from April 2018 through February 2020. Safety and tolerability Fifteen subjects consented to study; 1 patient was deemed ineligible AEs felt to be possibly related to metformin are summarized in after screening BM evaluation demonstrated an increasing del(7q) Table 2. The most common side effect was diarrhea (12 of 14, clone. Fourteen subjects received protocol therapy (Figure 1). 85.7%). The worst grade recorded for a given AE was grade 1 in 9 subjects and grade 2 in 4 subjects. No grade 3 or higher adverse Clinical characteristics of study population events attributed to study drug were observed. No subjects devel- oped symptomatic hypoglycemia or metabolic acidosis. No serious The median age of treated patients was 9.4 years (range, 6.0-26.5 years), and 8 subjects were male (57.1%). All patients had chromo- AEs were reported. somal breakage studies and genetic studies consistent with FA Because vitamin B12 depletion has been reported with metformin (supplemental Appendix 2). Median blood counts at study entry treatment because of malabsorption, vitamin B12 levels were moni- were Hb of 11 g/dL (range, 8.4-14.2 g/dL), platelet count of tored. Ten of 11 evaluable patients (90.9%) had vitamin B12 levels 54 000 cells/mL (range, 16 000-78 000 cells/mL), ANC of 1.1K in the normal range at initiation and completion of metformin treat- ment; in 1 instance (9.1%), the vitamin B12 level fell from a normal level of 355 pg/mL at treatment start to 168 pg/mL (lower limit of Ineligible normal 5 190 pg/mL) at the end of therapy. Given metformin’s Screened for eligibility (n=60) impact on glucose control, we also assessed the effects of metfor- Post-transplant, lack of min on glucose metabolism, particularly given the inherent risk for (n=75) cytopenias, age, concurrent medications, G6PD deficiency insulin resistance and higher rates of diabetes seen with FA. The oral glucose tolerance test was assessed at the start and end of treatment in 11 evaluable patients. None of the study subjects had frank diabetes, defined as fasting blood glucose $ 126 mg/dL or Enrolled 2-hour blood glucose $ 200 mg/dL. The mean change in fasting (n=15) insulin level was 22.0 mcIU/mL (95% CI, 25.1 to1.1; P 5 .17), which was not significantly different before and after metformin ther- Ineligible post-enrollment apy. Three subjects had mildly elevated insulin levels consistent with (n=1) MDS diagnosed in pre-treatment mild insulin resistance prior to study start. Two of 3 normalized their bone marrow insulin levels after metformin treatment. Growth parameters were also assessed before and after metformin treatment. No significant Initiated treatment (n=14) Table 1. Baseline characteristics of study subjects Characteristic Median (range) or n (%) Discontinued treatment after 3 months (n=1) Age, y 9.4 (6.0-26.5) Sex Lost to follow up after Female 6 (42.9%) 2 weeks (n=1) Male 8 (57.1%) FA subtype Completed 6 months of treatment FANCA 12 (85.7%) (n=12) FANCC 2 (14.3%) Red blood cell transfusion dependent 1 (7.1%) Figure 1. Flow diagram of study population. Includes patients screened, Total number of study subjects 5 14. enrolled, and determined to be eligible for protocol treatment. 3806 POLLARD et al 28 JUNE 2022 • VOLUME 6, NUMBER 12 Table 2. Toxicities possibly, probably, or definitely related to Targeted deep sequencing analysis of somatic mutations was con- metformin ducted on blood samples collected from 11 subjects before and Worst grade after metformin treatment. No new mutations were detected after Toxicity type 1 2 Total Percentage* metformin treatment. Nine patients lacked somatic mutations in either sample. Two patients with a variant of unknown significance at study Diarrhea 11 1 12 27.9 entry (CUX1 and EP300, respectively) had no change in these Nausea 6 1 7 16.3 clones following metformin treatment (supplemental Appendix 2). Abdominal pain, intermittent 4 0 4 9.3 Sufficient CD341 cells were collected from paired marrow samples Increased alanine aminotransferase (ALT) 2 1 3 7 before and after metformin treatment for 4 patients, none of whom Increased aspartate aminotransferase (AST) 2 1 3 7 had achieved HR. Despite not meeting HR criteria, increased mye- Fatigue 2 0 2 4.7 loid and erythroid colony-forming cell numbers were noted in 2 of Headache 2 0 2 4.7 the 4 patient samples performed in replicate plates (supplemental Papulopustular rash, localized 2 0 2 4.7 Appendix 4). Anorexia, intermittent 1 0 1 2.3 Chromosomal breakage after exposure to MMC or DEB is a hallmark Anxiety, intermittent 1 0 1 2.3 of cells from patients with FA. We therefore determined whether met- Dyspepsia 1 0 1 2.3 formin impacts chromosomal breakage. Chromosomal aberrations, radial forms, and number of metaphase cells with radials after MMC Eye disorders, other-pruritis 1 0 1 2.3 or DEB were quantitated in blood samples drawn before or after met- Eye disorders, other-redness/edema 1 0 1 2.3 formin (supplemental Appendix 5). Following metformin treatment, a Hyperkalemia 1 0 1 2.3 decline in breakage was observed in 8 of 10 patient samples Vomiting, intermittent 1 0 1 2.3 exposed to MMC and 7 of 10 patient samples exposed to DEB, Watering eyes 1 0 1 2.3 although the absolute change in breaks for the entire cohort was not Worst grade across all toxicities (no. of patients) 9 4 13 statistically significantly different before and after treatment. Specifi- cally, with MMC treatment, the mean of change before vs after met- *Percentage of total toxicities. formin for chromosomal aberrations was 2114.8 (95% CI, 2325.5 to 95.9; P 5 .10), for radial forms was 231.6 (95% CI, 2116 to 53.5; P 5 .23), and for cells with radials was 28.0 (95% CI, 225.5 change in height, weight, or body mass index was noted after met- to 9.5; P 5 .38). With DEB treatment, the mean of change for chro- formin treatment. Specifically, the mean change before and after mosomal aberrations was 229.2 (95% CI, 2144.3 to 86; P 5 .56), metformin in height z-scores was 0.02 (95% CI, 20.17 to 0.13; for radial forms was 24.6 (95% CI, 244.9 to 35.7; P 5 .51), and P 5 .77), weight Z-scores was 20.03 (95% CI, 20.21 to 0.15; for cells with radials was 23.0 (95% CI, 216.6 to 10.6; P 5 .38). P 5 .72), and body mass index Z-scores was 0 (95% CI, 20.2 to 0.2; P 5 .92). The frequency of MN is a biomarker of chromosomal fragility. FA has been associated with an increase in the frequency of MN in Treatment response buccal cells.32 A reduction in radiation-induced MN formation has been reported with metformin treatment of cultured human lympho- Four patients (30.8%; 90% CI, 11.3-57.3) achieved HR on metfor- cytes.33 We therefore quantitated MN present in buccal epithelial min therapy. The median time to first response was 84.5 days cells before and after metformin treatment for 14 evaluable subjects. (range, 71-128 days). One subject had a bilineage (Hb and ANC) No significant mean change was noted in numbers of MN per 1000 response, 2 subjects had a neutrophil response, and 1 subject had buccal epithelial cells (mean change, 1.2; 95% CI, 20.7 to 3.1; a platelet response. Age of the patient was not associated with P for generalized estimation equation 5 .22) or in cells with MN per response. The patient with HR based on Hb improvement had been 2000 buccal epithelial cells (mean change, 1.2; 95% CI, 21.2 to red cell transfusion dependent at the time of study entry and 3.6; P for generalized estimation equation 5 .34; supplemental became transfusion independent after 1 month of therapy. Overall Appendix 6). changes in blood counts are summarized in Table 3 and Figure 2. Maximum improvement in Hb was 2.3 g/dL (median, 0.7 g/dL), pla- DPCs are considered the primary DNA damage induced by alde- telets was 49 000 cells/mL (median, 13.8K cells/mL), and ANC 1.4K hyde exposure. To explore the potential effects of metformin on cells/mL (median, 0.8K cells/mL) . No subjects met criteria for dis- aldehyde-mediated DNA damage, we quantified DPCs before and ease progression or relapse after response during the study period. after metformin therapy. Baseline DPCs levels were low, and analy- sis was limited by inadequate DNA yields from 5 of the 10 paired All subjects had low BM cellularity for age. For evaluable patients, marrow samples. For the 5 evaluable subjects, none of whom there was no significant change in marrow cellularity at the end achieved HR with metformin therapy, levels of DPCs/108dG are of metformin treatment (P 5 .8164; supplemental Appendices 2 shown in supplemental Appendix 7. Analysis was limited by the and 3). FISH studies were negative for all patients evaluable both small sample numbers and low levels of adducts at baseline, so before and after metformin. All 12 patients with normal cytogenetics these observations are included descriptively. at study entry retained normal karyotype at study completion. Two patients had cytogenetic abnormalities noted at study entry: 1 had a We conducted quantitative proteomic assessments of 1305 plasma stable clone and 1 had emergence of a new clone and resolution proteins before and after metformin treatment using the aptamer-based of the previously identified clone at time of study completion SOMAscan multiplexed platform. We observed a reduction of 32 (supplemental Appendix 2). proteins and an increase in 67 proteins (jlog2(FCmean)j . log2(1.2); 28 JUNE 2022 • VOLUME 6, NUMBER 12 METFORMIN FOR FANCONI ANEMIA 3807 Table 3. Magnitude of hematologic improvement with metformin Maximum percentage Maximum improvement over improvement over baseline Hematologic parameter Number (N) Baseline studies median (range) baseline median (range) median (range) Hemoglobin* g/dL 13 11 g/dL (8.4-14.2) 0.7 g/dL (0-2.3) 6.2% (0-27.4) Reticulocyte count (cells/mL) 14 60,000 cells/mL (0.02-0.1) 2,000 cells/mL (0-0.022) 4.5% (0-91.7) Mean corpuscular volume (fL) 14 105.3 fL (92.1-111.2) 1.2 fL (0-6.2) 1.2% (0-5.6) Platelets (cells/mL) 14 53,800 cells/mL (16-78) 11,500 cells/mL (0-49) 31.8% (0-87.5) Neutrophils (cells/mL) 14 1,100 cells/mL (0.6-1.6) 800 cells/mL (0-1.4) 55.4% (0-166.7) *The patient who was transfusion dependent for red blood cells at baseline before metformin initiation was not included in this hemoglobin analysis. P , .05) after metformin treatment (supplemental Appendices effects.13,35-37 These also warrant further investigation as potential 8 and 9). mechanisms for metformin benefit. Corresponding KEGG and GSEA analyses identified downregulation Recent studies in non-FA populations have reported decreased of cytokine and cytokine receptor interaction pathways, inflammatory incidence of solid tumors in patients taking metformin, although pathways, and type 1 diabetes mellitus pathways (supplemental causality and effects in nondiabetic populations remain to be investi- Appendices 10 and 11). gated.38-41 Patients with FA have increased rates of both hemato- logic malignancies and solid tumors, particularly squamous cell Discussion carcinomas of the head, neck, and gastrointestinal tract, as well as hepatocellular carcinomas, and therefore strategies to reduce can- In this prospective pilot study, we found that metformin, adminis- cer risk in FA are also of great clinical interest. As metformin has tered at dosing used for diabetes, is safe and well tolerated in non- been previously shown to delay tumor formation in Fancd22/2 diabetic patients with FA. The most common AEs were mild mice,19 further study regarding the drug’s impact on risk for malig- transient gastrointestinal side effects, as have been previously nant transformation would also be beneficial. reported in patients without FA. No grade 3 or higher treatment- related AEs were identified in our study cohort. Importantly, we did This study has several limitations. FA is a rare disease, and recruit- not observe weight loss or growth reduction with treatment. Despite ment to our study was further impacted by a halt on enrollment metformin being an insulin-sensitizing agent, we found no significant because of the COVID-19 pandemic. Pandemic-related restrictions difference in markers of glucose homeostasis before and after tre- also curtailed sample acquisition for correlative biology studies. atment. Further assessment is needed to determine whether me- Importantly, the assessment of metformin treatment was limited to tformin can protect against end-organ toxicity secondary to 6 months in this pilot trial. Future studies should consider a longer- hyperglycemia and insulin resistance in patients with FA, particularly term exposure to metformin to determine whether this enhances after HSCT.34 clinical benefit and a possible escalation in dosing from that used We observed HR in 4 of 13 patients with FA treated with metformin for diabetes to determine whether that might afford greater thera- for 6 months. No patients had worsening cytopenias or relapse. peutic benefit in the context of FA. Because of the rarity of FA, a Although many of our correlative analyses were limited by sample randomized design with a control group was not feasible to rigor- availability and assay limitations, interestingly, improvement in hema- ously study metformin effects. As only one-third of our patients topoiesis with metformin was observed with in vitro erythroid and responded to treatment, correlative assays that facilitate our ability myeloid hematopoietic colony assay after metformin treatment in to predict early in treatment those patients most likely to respond 3 of 4 patients who did not meet HR criteria. These results sup- would be advantageous in future studies. Moreover, a study design port further investigation of the impact of metformin on hema- that entails longer duration of metformin treatment may improve the topoiesis in FA. response rate in a larger subset of patients and aid our identification of prognostic correlative assays. Biomarkers for genomic instability The mechanisms by which metformin improves hematopoiesis in FA before and after metformin treatment in this study were limited by remain to be determined. Despite the observed improvement in low baseline levels and low cellularity of marrow and blood samples blood counts when Fancd22/2 mice are treated with metformin in this small FA cohort. Development of more robust correlative in vivo, the addition of metformin in vitro to isolated hematopoietic assays could further clarify metformin’s potential for mitigation of stem cell precursors from Fancd22/2 mice19 or to marrow samples such instability. from patients with FA (unpublished observation) fails to increase hematopoietic colony numbers. These observations raise the possi- Despite these limitations, this pilot study suggests that metformin is bility that the hematopoietic stem/progenitor cells are not directly safe and well tolerated in FA and may afford clinical benefit in a sub- targeted by metformin. Exploratory proteomic studies of plasma set of patients. Development of a subsequent multicenter efficacy from patients with FA treated with metformin suggest that metformin study may provide greater clarity regarding metformin’s role in the might reduce inflammatory pathways, although further studies are treatment of FA. Given the historical experience of treating FA- needed to address this question. Metformin is also known to inhibit associated cytopenias with androgens, combination treatment of mitochondrial complex 1, activate AMPK with resultant downstream androgens with metformin could also be explored. Future efficacy effects including inhibition of mTOR, and cause other metabolic end points should study the utility of metformin as both a prophylactic 3808 POLLARD et al 28 JUNE 2022 • VOLUME 6, NUMBER 12 A Hemoglobin at baseline and maximum B Platelets at baseline and maximum 125 000 14 100 000 Hemoglobin (g/dL) Platelets (cells/uL) 12 75 000 10 50 000 8 25 000 Baseline Maximum Baseline Maximum C ANC at baseline and maximum D MCV at baseline and minimum 2 500 110 2 000 105 ANC (cells/uL) MCV fL 1 500 100 1 000 95 500 90 Baseline Maximum Baseline Minimum E Absolute retic at baseline and maximum 100 000 Absolute retic count (cells/uL) 80 000 60 000 40 000 20 000 Baseline Maximum Figure 2. Hematologic parameters at baseline and maximum response following metformin treatment. (A) Hemoglobin. (B) Platelet count. (C) Absolute neutrophil count. (D) Mean corpuscular volume. (E) Absolute reticulocyte count. 28 JUNE 2022 • VOLUME 6, NUMBER 12 METFORMIN FOR FANCONI ANEMIA 3809 agent aimed at curtailing progression of bone marrow failure and as a regulatory support; M.A. processed samples and conducted and ana- chemo preventive agent aimed at reducing cancer risk in FA. lyzed the hematopoietic colony assays; A.S.K. analyzed the somatic mutations and contributed to the manuscript; K.B. processed samples Acknowledgments and conducted and analyzed the hematopoietic colony assays; L.M. conducted and analyzed the chromosomal breakage assays; Y.Z., The authors gratefully acknowledge the patients, families, and X.G., and T.A.L. analyzed the proteomics data and contributed to the referring providers who participated in this study. David Williams manuscript; D.B., F.B., C.C., H.H., A.H., T.N., T.O., S.-G.-S., A.A.T., provided advice for this study. and M.W.W. collected data and contributed to the manuscript; This work was supported in part by National Institutes of A.D.A. and M.G, contributed to the study concept and development; Health/National Institute for Diabetes and Digestive and Kidney E.W. was responsible for statistical design, analysis of clinical char- Diseases (NIDDK) grant P01 HL048546-21A (to A.S.), Boston acteristic, treatment, response, adverse events, and bone marrow Children’s Hospital Mooney Family Fund (to E.F.), and the Fanconi cellularity, and contributed to the manuscript; and A.S. was responsi- Anemia Research Fund (to E.F. and A.S.). The authors thank the ble for study design, data analysis, and wrote the manuscript. mass spectrometry support from the Chemistry and Analytical Core of the University of North Carolina (UNC) Superfund Conflict-of-interest disclosure: J.A.P. was on the advisory board Research Program (grant P42ES031007). for Syndax, Inc. and Kura Oncology. E.F. was employed by Keros Therapeutics (following completion of all protocol enrollment and after all patients had completed protocol therapy). E.E. was on the Authorship advisory board for Bluebird Bio. All remaining authors declare no Contribution: J.A.P. analyzed the data and wrote the manuscript; E.F. financial competing interests. wrote the study protocol, performed clinical research, and edited the ORCID profiles: L.E.C., 0000-0002-8196-8800; J.B., 0000- manuscript; S.L. was responsible for statistical design, analysis of clini- 0002-2387-5462; C-W.L., 0000-0002-0823-0252; A.G., 0000- cal characteristic, treatment, response, adverse events, and bone mar- 0002-6036-1591; A.S.K., 0000-0002-8699-2439; Y.Z., 0000- row cellularity and contributed to the manuscript; E.E. performed 0001-7557-3186; D.B., 0000-0002-1745-0353; H.H., 0000- clinical research and contributed to the manuscript; L.E.C. designed 0003-2725-0635; A.A.T., 0000-0003-4961-8103; M.W.W., 0000- and analyzed the endocrine studies and contributed to the manu- 0001-6638-9643; T.A.L., 0000-0002-4006-8179; A.D’Andrea, script; J.B. conducted the central pathology review and contributed to 0000-0001-6168-6294; A.S., 0000-0002-4683-9958. the manuscript; C.-W.L. and K.L. assayed and analyzed the DNA- protein adducts and contributed to the manuscript; M.J.R.d.H. and J.S. Correspondence: Akiko Shimamura, Boston Children’s Hospital, conducted and analyzed the micronucleus assay and contributed to 1 Blackfan Circle, Karp 8210, Boston, MA 02115; e-mail: akiko. the manuscript; M.M., A.A., A.K., and A.G. collected data and provided shimamura@childrens.harvard.edu. References 1. Kutler DI, Singh B, Satagopan J, et al. A 20-year perspective on the International Fanconi Anemia Registry (IFAR). Blood. 2003;101(4):1249-1256. 2. Rosenberg PS, Huang Y, Alter BP. Individualized risks of first adverse events in patients with Fanconi anemia. Blood. 2004;104(2):350-355. 3. Ebens CL, MacMillan ML, Wagner JE. Hematopoietic cell transplantation in Fanconi anemia: current evidence, challenges and recommendations. Expert Rev Hematol. 2017;10(1):81-97. 4. Scheckenbach K, Morgan M, Filger-Brillinger J, et al. Treatment of the bone marrow failure in Fanconi anemia patients with danazol. Blood Cells Mol Dis. 2012;48(2):128-131. 5. Rose SR, Kim MO, Korbee L, et al. Oxandrolone for the treatment of bone marrow failure in Fanconi anemia. Pediatr Blood Cancer. 2014;61(1):11-19. 6. Paustian L, Chao MM, Hanenberg H, et al. Androgen therapy in Fanconi anemia: a retrospective analysis of 30 years in Germany. Pediatr Hematol Oncol. 2016;33(1):5-12. 7. Calado RT, Cl e DV. Treatment of inherited bone marrow failure syndromes beyond transplantation. Hematology (Am Soc Hematol Educ Program). 2017;2017(1):96-101. 8. Velazquez I, Alter BP. Androgens and liver tumors: Fanconi’s anemia and non-Fanconi’s conditions. Am J Hematol. 2004;77(3):257-267. 9. Catala A, Ali SS, Cuvelier GDE, et al. Androgen therapy in inherited bone marrow failure syndromes: analysis from the Canadian Inherited Marrow Failure Registry. Br J Haematol. 2020;189(5):976-981. 10. Zhang QS, Marquez-Loza L, Eaton L, et al. Fancd2-/- mice have hematopoietic defects that can be partially corrected by resveratrol. Blood. 2010; 116(24):5140-5148. 11. Nakada D, Saunders TL, Morrison SJ. Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells. Nature. 2010;468(7324): 653-658. 12. Saito Y, Nakada D. The role of the Lkb1/AMPK pathway in hematopoietic stem cells and leukemia. Crit Rev Oncog. 2014;19(5):383-397. 13. He, L. Metformin ,and systemic metabolism. Trends Pharmacol Sci. 2020;41:868-881. 14. Kazachkov M, Chen K, Babiy S, Yu PH. Evidence for in vivo scavenging by aminoguanidine of formaldehyde produced via semicarbazide-sensitive amine oxidase-mediated deamination. J Pharmacol Exp Ther. 2007;322(3):1201-1207. 3810 POLLARD et al 28 JUNE 2022 • VOLUME 6, NUMBER 12 15. Langevin F, Crossan GP, Rosado IV, Arends MJ, Patel KJ. Fancd2 counteracts the toxic effects of naturally produced aldehydes in mice. Nature. 2011;475(7354):53-58. 16. Garaycoechea JI, Crossan GP, Langevin F, Daly M, Arends MJ, Patel KJ. Genotoxic consequences of endogenous aldehydes on mouse haematopoietic stem cell function. Nature. 2012;489(7417):571-575. 17. Pontel LB, Rosado IV, Burgos-Barragan G, et al. Endogenous formaldehyde is a hematopoietic stem cell genotoxin and metabolic carcinogen. Mol Cell. 2015;60(1):177-188. 18. Hira A, Yabe H, Yoshida K, et al. Variant ALDH2 is associated with accelerated progression of bone marrow failure in Japanese Fanconi anemia patients. Blood. 2013;122(18):3206-3209. 19. Zhang QS, Tang W, Deater M, et al. Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice. Blood. 2016;128(24):2774-2784. 20. Elder DA, D’Alessio DA, Eyal O, et al. Abnormalities in glucose tolerance are common in children with fanconi anemia and associated with impaired insulin secretion. Pediatr Blood Cancer. 2008;51(2):256-260. 21. Petryk A, Kanakatti Shankar R, Giri N, et al. Endocrine disorders in Fanconi anemia: recommendations for screening and treatment. J Clin Endocrinol Metab. 2015;100(3):803-811. 22. Cheson BD, Greenberg PL, Bennett JM, et al. Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood. 2006;108(2):419-425. 23. Auerbach AD. Diagnosis of Fanconi anemia by diepoxybutane analysis. Curr Protoc Hum Genet. 2015;85:87–1-87-17. 24. Liu CW, Tian X, Hartwell HJ, et al. Accurate measurement of formaldehyde-induced DNA-protein cross-links by high-resolution orbitrap mass spectrometry. Chem Res Toxicol. 2018;31(5):350-357. 25. Tsai HK, Brackett DG, Szeto D, et al. Targeted informatics for optimal detection, characterization, and quantification of FLT3 internal tandem duplications across multiple next-generation sequencing platforms. J Mol Diagn. 2020;22(9):1162-1178. 26. Gold L, Ayers D, Bertino J, et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS One. 2010;5(12):e15004. 27. Mehan MR, Ostroff R, Wilcox SK, et al. Highly multiplexed proteomic platform for biomarker discovery, diagnostics, and therapeutics. Adv Exp Med Biol. 2013;735:283-300. 28. Biberoglu E, Kirbas A, Daglar K, et al. Serum angiogenic profile in abnormal placentation. J Matern Fetal Neonatal Med. 2016;29(19):3193-3197. uke B, Akkaya H, Demir S, et al. Relationship between first trimester aneuploidy screening test serum analytes and placenta accreta. J Matern 29. B€ Fetal Neonatal Med. 2018;31(1):59-62. 30. Vasunilashorn SM, Dillon ST, Chan NY, et al. Proteome-wide analysis using SOMAscan identifies and validates chitinase-3-like protein 1 as a risk and disease marker of delirium among older adults undergoing major elective surgery. J Gerontol A Biol Sci Med Sci. 2022 ;77(3):484–493 31. Fong TG, Chan NY, Dillon ST, et al. Identification of plasma proteome signatures associated with surgery using SOMAscan. Ann Surg. 2021; 273(4):732-742. 32. Ramırez MJ, Minguill on J, Loveless S, et al. Chromosome fragility in the buccal epithelium in patients with Fanconi anemia. Cancer Lett. 2020;472:1-7. 33. Cheki M, Shirazi A, Mahmoudzadeh A, Bazzaz JT, Hosseinimehr SJ. The radioprotective effect of metformin against cytotoxicity and genotoxicity induced by ionizing radiation in cultured human blood lymphocytes. Mutat Res Genet Toxicol Environ Mutagen. 2016;809:24-32. 34. Anur P, Friedman DN, Sklar C, et al. Late effects in patients with Fanconi anemia following allogeneic hematopoietic stem cell transplantation from alternative donors. Bone Marrow Transplant. 2016;51(7):938-944. 35. Mallik R, Chowdhury TA. Metformin in cancer. Diabetes Res Clin Pract. 2018;143:409-419. 36. Kulkarni AS, Gubbi S, Barzilai N. Benefits of metformin in attenuating the hallmarks of aging. Cell Metab. 2020;32(1):15-30. 37. Marcucci F, Romeo E, Caserta CA, Rumio C, Lefoulon F. Context-dependent pharmacological effects of metformin on the immune system. Trends Pharmacol Sci. 2020;41(3):162-171. 38. Noto H, Goto A, Tsujimoto T, Noda M. Cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis. PLoS One. 2012;7(3):e33411. 39. Zhang P, Li H, Tan X, Chen L, Wang S. Association of metformin use with cancer incidence and mortality: a meta-analysis. Cancer Epidemiol. 2013;37(3):207-218. 40. Lin HC, Kachingwe BH, Lin HL, Cheng HW, Uang YS, Wang LH. Effects of metformin dose on cancer risk reduction in patients with type 2 diabetes mellitus: a 6-year follow-up study. Pharmacotherapy. 2014;34(1):36-45. 41. Gandini S, Puntoni M, Heckman-Stoddard BM, et al. Metformin and cancer risk and mortality: a systematic review and meta-analysis taking into account biases and confounders. Cancer Prev Res (Phila). 2014;7(9):867-885. 28 JUNE 2022 • VOLUME 6, NUMBER 12 METFORMIN FOR FANCONI ANEMIA 3811