For Fanconi Anemia Research Articles

Outcomes of hematopoietic stem cell transplantation in 813 pediatric patients with Fanconi anemia

AbstractAllogeneic hematopoietic stem cell transplantation (HSCT) is the only established curative option for Fanconi anemia (FA)–associated bone marrow failure (BMF)/aplastic anemia (AA) and acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS). We performed a retrospective multicenter study on 813 children with FA undergoing first HSCT between 2010 and 2018. Median duration of follow-up was 3.7 years. Median age at transplant was 8.8 years (IQR, 6.5-18.1). Five-year overall survival (OS), event-free survival (EFS), and graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS) were 83% (95% confidence interval [CI], 80-86), 78% (95% CI, 75-81), and 70% (95% CI, 67-74), respectively. OS was comparable between matched family donor (MFD; n = 441, 88%) and matched unrelated donor (MUD; n = 162, 86%) and was superior to that of mismatched family donor (MMFD) or mismatched unrelated donor (MMUD; n = 144, 72%) and haploidentical donor (HID; n = 66, 70%; P < .001). In multivariable analysis, a transplant indication of AML/MDS (vs AA/BMF), use of MMFD/MMUD and HID (vs MFD), and fludarabine-cyclophosphamide (FluCy) plus other conditioning (vs FluCy) independently predicted inferior OS, whereas alemtuzumab vs antithymocyte globulin was associated with better OS. Age ≥10 years was associated with worse EFS and GRFS. Cumulative incidences (CINs) of primary and secondary graft failure were 2% and 3% respectively. CINs of grade 3 to 4 acute GVHD and chronic GVHD were 12% and 8% respectively. The 5-year CIN of secondary malignancy was 2%. These data suggest that HSCT should be offered to patients with FA with AA/BMF at a younger age in the presence of a well-matched donor.

Umbilical Cord Blood Transplantation for Fanconi Anemia With a Special Focus on Late Complications: a Study on Behalf of Eurocord and SAAWP-EBMT

Hematopoietic cell transplantation (HCT) remains the sole available curative treatment for Fanconi anemia (FA), with particularly favorable outcomes reported after matched sibling donor (MSD) HCT. This study aimed to describe outcomes, with a special focus on late complications, of FA patients who underwent umbilical cord blood transplantation (UCBT). In this retrospective analysis of allogeneic UCBT for FA performed between 1988 and 2021 in European Society for Blood and Marrow Transplantation (EBMT)-affiliated centers, a total of 205 FA patients underwent UCBT (55 related and 150 unrelated) across 77 transplant centers. Indications for UCBT were bone marrow failure in 190 patients and acute leukemia/myelodysplasia in 15 patients. The median age at transplantation was 9 years (range, 1.2 to 43 years), with only 20 patients aged >18 years. Among the donor-recipient pairs, 56% (n = 116) had a 0 to 1/6 HLA mismatch. Limited-field radiotherapy was administered to 28% (n = 58) and 78% (n = 160) received a fludarabine (Flu)-based conditioning regimen. Serotherapy consisted of antithymocyte globulin (n = 159; 78%) or alemtuzumab (n = 12; 6%). The median follow-up was 10 years for related UCBT and 7 years for unrelated UCBT. Excellent outcomes were observed in the setting of related UCBT, including a 60-day cumulative incidence (CuI) of neutrophil recovery of 98.1% (95% confidence interval [CI], 93.9% to 100%), a 100-day CuI of grade II-IV acute graft-versus-host disease (GVHD) of 17.3% (95% CI, 9.5% to 31.6%), and a 5-year CuI of chronic GVHD (cGVHD) of 22.7% (95% CI, 13.3% to 38.7%; 13% extensive). Five-year overall survival (OS) was 88%. In multivariate analysis, none of the factors included in the model predicted a better OS. In unrelated UCBT, the 60-day CuI of neutrophil recovery was 78.7% (95% CI, 71.9% to 86.3%), the 100-day CuI of grade II-IV aGVHD was 31.4% (95% CI, 24.6% to 40.2%), and the 5-year CuI of cGVHD was 24.3% (95% CI, 17.8% to 32.2%; 12% extensive). Five-year OS was 44%. In multivariate analysis, negative recipient cytomegalovirus serology, Flu-based conditioning, age <9 years at UCBT, and 0 to 1/6 HLA mismatch were associated with improved OS. A total of 106 patients, including 5 with acute leukemia/myelodysplasia, survived for >2 years after UCBT. Nine of these patients developed subsequent neoplasms (SNs), including 1 donor-derived acute myelogenous leukemia and 8 solid tumors, at a median of 9.7 years (range, 2.3 to 21.8 years) post-UCBT (1 related and 8 unrelated UCBT). In a subset of 49 patients with available data, late nonmalignant complications affecting various organ systems were observed at a median of 8.7 years (range, 2.7 to 28.8 years) post-UCBT. UCB is a valid source of stem cells for transplantation in patients with FA, with the best results observed after related UCBT. After unrelated UCBT, improved survival was observed in patients who underwent transplantation at a younger age, with Flu-based conditioning, and with better HLA parity. The incidence of organ-specific complications and SNs was relatively low. The incidence of SNs, mostly squamous cell carcinoma, increases with time. Rigorous follow-up and lifelong screening are crucial in survivors of UCBT for FA.

FLUDARABINE-BASED LOW-INTENSITY CONDITIONING FOR FANCONI ANEMIA IS ASSOCIATED WITH GOOD OUTCOMES IN APLASTIC ANEMIA BUT NOT IN MDS - A SINGLE-CENTER EXPERIENCE

Background: Hematopoietic stem cell transplantation [HSCT] is the only curative option for Fanconi Anemia (FA) patients with hematological abnormalities.&#x0D; Materials and Methods: This is a retrospective analysis of patients with FA who underwent a matched-related donor HSCT.&#x0D; Results: Sixty patients underwent 65 transplants between 1999-2021 using a fludarabine-based low-intensity conditioning regimen. The median age at transplant was 11 years (range: 3-37). Aplastic anemia (AA) was the underlying diagnosis in 55 (84.6%) while 8 (12.4%) had myelodysplastic syndrome (MDS) and 2 (3%) had acute myeloid leukemia (AML). The conditioning regimen used was fludarabine with low-dose cyclophosphamide for aplastic anemia and Fludarabine with low-dose busulfan for MDS/AML. Graft versus host disease (GVHD)prophylaxis consisted of cyclosporine and methotrexate. Peripheral blood stem cells was the predominant graft source (86.2%). Engraftment occurred in all but 1 patient. The median time to neutrophil and platelet engraftment was 13 days (range: 9-29) &amp; 13 days (range: 5-31), respectively. Day 28 chimerism analysis showed complete chimerism in 75.4 % and mixed chimerism in 18.5%. Secondary graft failure was encountered in 7.7%. Grade II–IV acute GVHD occurred in 29.2% while Grade III-IV acute GVHD occurred in 9.2%. Chronic GVHD was seen in 58.5% and was limited in most patients. The median follow-up is 55 months [range: 2-144] &amp; the 5-year estimated overall survival (OS) is 80.2 ± 5.1%. Secondary malignancies were noted in 4 patients. The 5-year OS was significantly higher in patients undergoing HSCT for AA (86.6 + 4.7%) as compared to MDS/AML (45.7+16.6%) (p= 0.001).&#x0D; Conclusion: SCT using a fully matched donor provides good outcomes with low-intensity conditioning regimens in patients with FA who have an aplastic marrow.

Haplo-identical or mismatched unrelated donor hematopoietic cell transplantation for Fanconi anemia: Results from the Severe Aplastic Anemia Working Party of the EBMT.

Allogeneic hematopoietic cell transplantation (HCT) is the only curative option for bone marrow failure or hematopoietic malignant diseases for Fanconi anemia (FA) patients. Although results have improved over the last decades, reaching more than 90% survival when a human leukocyte antigen (HLA)-identical donor is available, alternative HCT donors are still less reported. We compared HCT outcomes using HLA-mismatched unrelated donors (MMUD; n = 123) or haplo-identical donors (HDs), either using only in vivo T cell depletion (n = 33) or T cells depleted in vivo with some type of graft manipulation ex vivo (n = 59) performed for FA between 2000 and 2018. Overall survival (OS) by 24 months was 62% (53-71%) for MMUD, versus 80% (66-95%) for HDs with only in vivo T cell depletion and 60% (47-73%) for HDs with in vivo and ex vivo T cell depletion (p = .22). Event-free survival (EFS) was better for HD-transplanted FA patients with only in vivo T cell depletion 86% (73-99%) than for those transplanted from a MMUD 58% (48-68%) or those with graft manipulation 56% (42-69%) (p = .046). Grade II-IV acute graft-versus-host disease (GVHD) was 41% (MMUD) versus 40% (HDs with no graft manipulation) versus 17% (HDs with T cell depleted graft), (p = .005). No differences were found for the other transplant related outcomes. These data suggest that HDs might be considered as an alternative option for FA patients with better EFS using unmanipulated grafts.

Novel diagnostic approaches for Fanconi anemia (FA) by single-cell sequencing and capillary nano-immunoassay.

Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia (FA). However, mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes. Herein, we introduced two novel diagnostic methods, including single-cell sequencing and capillary nano-immunoassay. One FA case with FANCM c.4931G>A p.R1644Q and FANCD1 c.6325G>A p.V2109I was studied. The DNA of 28 cells was amplified and eight types of cells were observed after Sanger sequencing. There were two homozygous mutations (FANCM/FANCD1). Furthermore, the capillary nano-immunoassay was conducted to analyze the expression profile of FA-associated proteins. Abnormal FANCM and FANCD1 expressions simultaneously existed. This case was thus diagnosed as FA-D1/FA-M dual subtype. Compared with mixed cell sequencing, single-cell sequencing data shows more accuracy for the FA subtype evaluation, while the capillary nano-immunoassay is a good method to detect the expression profile of abnormal or modified FA protein.

HLA-Haploidentical TCR αβ/CD19-Depleted Hematopoietic Stem Cell Transplantation in Children with Fanconi Anemia

Introduction. Allogeneic HSCT currently represents the only consolidated curative approach for Fanconi anemia (FA) patients, with best results observed in the HLA-identical sibling setting. For patients lacking an HLA-matched related or unrelated donor, haploidentical HSCT virtually assures the opportunity for nearly all patients to benefit from HSCT, offering the advantage of immediate accessibility to the transplant procedure. In order to overcome the limitation of delayed immune recovery, historically associated with this type of allograft, in the last few years we developed a novel method of ex vivo graft manipulation, consisting of the negative depletion of T-cell receptor (TCR) αβ+ T-lymphocytes and CD19 B-cells from peripheral blood stem cells (PBSC) grafts (ClinicalTrial.gov identifier: NCT01810120) (Bertaina el al., Blood 2014). Here we report our analysis in a subgroup of FA patients given TCRαβ/CD19-depleted haploidentical HSCT at our Institution.Patients and methods. Ten consecutive FA patients (6 girls and 4 boys) underwent a TCRαβ/CD19-depleted HSCT from an HLA-haploidentical relative between September 2011 and July 2015. Median age at diagnosis was 6.6 (range 2.7-22.0) years and median age at time of transplantation was 8.1 (range 4.4-22.2) years. The conditioning regimen included Cyclophosphamide 300 mg/m2/day and Fludarabine 30 mg/m2/day for 4 consecutive days (days -6 to -3), with 200 cGy single-dose TBI. Pretransplantation Fresenius® ATG was administered at a dose of 4 mg/kg/day for 3 consecutive days (days -5 to -3) in order to prevent both graft failure and graft-versus-host disease (GVHD). All patients received Rituximab 200 mg/m2 to reduce the risk of Epstein-Barr virus-related post-transplant lymphoproliferative disorders. Selective removal of TCRαβ+ and B-cells was performed on G-CSF-mobilized donor PBSC through labeling with biotinylated anti-TCRαβ antibodies and anti-CD19 antibodies, followed by incubation with anti-biotin antibodies conjugated to paramagnetic beads (CliniMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). No immunosuppressive therapy was administered as post-transplantation prophylaxis against GVHD.Results. The TCRαβ/CD19-depletedgrafts contained a median of 20.40 x106/kg (range 15.80-33.40) CD34+ cells, 5.60 x106/kg (range 1.78-69.60) CD3+ lymphocytes, 0.021 x106/kg (range 0.002-0.043) TCRαβ+ lymphocytes, 5.60 x106/kg (range 1.78-69.60) TCRγδ+ lymphocytes, 0.036 x106/kg (range 0.013-0.079) CD20+ lymphocytes, and 45.30 x106/kg (range 16.2-177.0) NK cells. Engraftment with sustained full donor chimerism was achieved in 9 out of 10 patients, the cumulative incidence of graft rejection being 10% (95% CI, 0-26.8). The patient who rejected his first allograft achieved a complete engraftment after a second HSCT from one-antigen mismatched unrelated donor. No secondary graft failures were observed. The median time for neutrophil and platelet engraftment was 12 days (range, 9-15) and 9 days (range, 8-12), respectively. No patient experienced acute or chronic GVHD in the follow-up period. No transplant-related deaths occurred in our cohort. With a median follow-up of 28 months (range 13.2-39.1), the Kaplan-Meier estimates of OS and DFS were both 100%, while the EFS probability was 90% (95% CI, 47.3-98.5).Discussion. These data suggest that haploidentical HSCT after removal of TCRαβ+ and CD19+ lymphocytes is able to guarantee engraftment with excellent OS and DFS in patients affected by FA. Moreover, given the very low incidence of both acute and chronic GVHD, which has been shown to contribute to the increased risk of developing late post-transplantation malignancies in FA patients, this approach can be considered a very attractive option for FA patients in need of an allograft and lacking an HLA-identical family donor. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of interstrand DNA crosslink repair and human disorders.

Interstrand DNA crosslinks (ICLs) are the link between Watson-Crick strands of DNAs with the covalent bond and prevent separation of DNA strands. Since the ICL lesion affects both strands of the DNA, the ICL repair is not simple. So far, nucleotide excision repair (NER), structure-specific endonucleases, translesion DNA synthesis (TLS), homologous recombination (HR), and factors responsible for Fanconi anemia (FA) are identified to be involved in ICL repair. Since the presence of ICL lesions causes severe defects in transcription and DNA replication, mutations in these DNA repair pathways give rise to a various hereditary disorders. NER plays an important role for the ICL recognition and removal in quiescent cells, and defects of NER causes congential progeria syndrome, such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. On the other hand, the ICL repair in S phase requires more complicated orchestration of multiple factors, including structure-specific endonucleases, and TLS, and HR. Disturbed this ICL repair orchestration in S phase causes genome instability resulting a cancer prone disease, Fanconi anemia. So far more than 30 factors in ICL repair have already identified. Recently, a new factor, UHRF1, was discovered as a sensor of ICLs. In addition to this, numbers of nucleases that are involved in the first incision, also called unhooking, of ICL lesions have also been identified. Here we summarize the recent studies of ICL associated disorders and repair mechanism, with emphasis in the first incision of ICLs.

Alternative donor hematopoietic cell transplantation for Fanconi anemia

AbstractHistorically, alternative donor hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) patients resulted in excessive morbidity and mortality. To improve outcomes, we made sequential changes to the HCT conditioning regimen. A total of 130 FA patients (median age, 9.0 years; range, 1-48) underwent alternative donor HCT at the University of Minnesota between 1995 and 2012. All patients received cyclophosphamide (CY), single fraction total body irradiation (TBI), and antithymocyte globulin (ATG) with or without fludarabine (FLU), followed by T-cell–depleted bone marrow or unmanipulated umbilical cord blood transplantation. The addition of FLU enhanced engraftment 3-fold. The incidence of grades 2-4 acute and chronic graft-versus-host disease was 20% and 10%, respectively. Severe toxicity was highest in patients >10 years of age or those with a history of opportunistic infections or transfusions before HCT. Mortality was lowest in patients without a history of opportunistic infection or transfusions and who received conditioning with TBI 300 cGy, CY, FLU, and ATG. These patients had a probability of survival of 94% at 5 years. Alternative donor HCT is now associated with excellent survival for patients without prior opportunistic infections or transfusions and should be considered for all FA patients after the onset of marrow failure. These studies were registered at http://www.clinicaltrials.gov as NCT00005898, NCT00167206, and NCT00352976.

Cytogenetic Assessment of Fanconi Anemia in Children With Aplastic Anemia in Tunisia

Chromosome breakage hypersensitivity to alkylating agents is the gold standard test for Fanconi anemia (FA) diagnosis. The aim of the present study was to assess the proportion of FA cases among aplastic anemia (AA) in Tunisian pediatric patients. Investigation of mitomycin C-induced chromosomal breakage was carried out in 163 pediatric patients with AA and siblings of the cases where diagnosis of FA was confirmed. We identified 31 patients with FA whose percentage of unstable mitoses ranges from 65% to 100%. Among 18 siblings who were investigated for chromosomal instability, 3 were incidentally found to be affected. FA is an important cause of AA in Tunisia. Our report is the first study in North Africa that explored cytogenetic and phenotypic findings in FA children. It also showed the importance of mitomycin C sensitivity screening in all FA siblings.

Alemtuzumab As Therapy for Mixed Chimerism After Hematopoietic Stem Cell Transplantation for Fanconi Anemia (FA)

s / Biol Blood Marrow Transplant 19 (2013) S233eS256 S243 unrelated donor HSCT ALL patients supports a graft vs leukemia effect translating into better outcomes.

Analysis of the Novel Fanconi Anemia GeneSLX4/FANCPin Familial Breast Cancer Cases

SLX4/FANCP is a recently discovered novel disease gene for Fanconi anemia (FA), a rare recessive disorder characterized by chromosomal instability and increased cancer susceptibility. Three of the 15 FA genes are breast cancer susceptibility genes in heterozygous mutation carriers--BRCA2, PALB2, and BRIP1. To investigate if defects in SLX4 also predispose to breast cancer, the gene was sequenced in a cohort of 729 BRCA1/BRCA2-negative familial breast cancer cases. We identified a single splice site mutation (c.2013+2T>A), which causes a frameshift by skipping of exon 8. We also identified 39 missense variants, four of which were selected for functional testing in a Mitomycin C-induced growth inhibition assay, and appeared indistinguishable from wild type. Although this is the first study that describes a truncating SLX4 mutation in breast cancer patients, our data indicate that germline mutations in SLX4 are very rare and are unlikely to make a significant contribution to familial breast cancer.

Outcome of Busulfan and Fludarabine-Based Reduced Intensity Conditioning Regimen for Related and Unrelated HSCT in Fanconi Anemia Patients

Abstract 1964 Rationale:HSCT is the only curative treatment for Fanconi Anemia (FA) pts with either bone marrow failure or MDS/Leukemia. Due to characteristic chromosomal instability, the poor outcome of FA pts transplanted after so-called conventional myelo-ablative conditioning regimen has been proved. Then, reduced-intensity conditionning regimen (RIC) has been considered for years as a model for allogeneic HSCT in FA pts. The use of fludarabine-based cond' regimen from about 2000 increased dramatically the overall survival of FA pts. Different Flu-based RIC were developed.Patients and method: Between Feb'02 and Dec'10, 17 FA pts from 3 academic French centers were included: 11 from R. Debre hospital, 5 from St Louis hospital and 1 from J. de Flandre hospital. All pts underwent HSCT because of bone marrow failure. They presented no MDS or leukemia. All time-to-event outcomes were counted from the date of HSCT to the date of event or date of last follow-up, except acute GVHD that was arbitrarily censored at 200 days. Death was considered as a competing risk in analyses of neutrophil and platelet recovery and chronic and acute GVHD. Overall survival was estimated using Kaplan-Meier product-limit estimator. For competing risks analyses, cumulative incidences were estimated using usual methodology. Results:Median age at diagnosis and at HSCT were 4.7 years (range 1,8-9,3) and 7,4 years (range 4,4-15,2), respectively. 12 pts presented with less than 3 FA-related malformations and 5 with more than 3 malformations. 2 patients received more than 20 transfusions before HSCT, whereas 8 pts received less than 20 transfusions and 7 patients did not received any. 2 patients received androgen therapy before HSCT.All patients received the same RIC i.e. fludarabine 30mg/m2/d × 3d, cyclophosphamide 10mg/kg/d × 4d and IV busulfan (Bu) 0.75 of body weight- adjusted recommended dose (equivalent to 6.4mg/kg total dose of oral Bu). This RIC did not contain any irradiation. Graft versus-host disease (GvHD) prophylaxis consisted of CSA associated with MMF or corticosteroids. Donors were either matched related (sibling, n= 6; other, n=2) or unrelated (10/10, n= 6; 9/10, n= 3). Stem cell sources were BM (n=10), UCB (n=4) and PBSC (n=2). 9 out of 17 pts had a donor from the same gender whereas 4 male recipients received transplant from female donor. CMV status were −/−, −/+, +/− and +/+ for 8, 4, 1 and 4 D/R pairs, respectively.Median follow-up was 32 months (range 3–102).Successful engraftment was obtained in all patients with a median time for neutrophil recovery of 17 days (range 10–42). All patients presented with 100% donor chimerism. One patient experimented secondary graft failure and died at D291 from infection and renal failure. During transplant procedure, 13 pts experimented at least one severe infectious complication (staphylococcus n=2; pseudomonas n=1; aspergillus n= 2; candida n=2; viral reactivation n= 13). 1 pt presented with moderate hepatic veno-occlusive disease. Five pts died from TRM and 12 pts remained alive in a good health condition. 36 month OS was 69% (95%CI 50 to 96). Cumulative incidence of grade 2 to 4 acute GVHD was 71% (95%CI: 41–87). 5 pts presented with either limited (n=4) or extensive (n=1) chronic GvHD and 36 month cumulative incidence of chronic GVHD was 33% (95%CI 11 to 58). To date, no pt had secondary malignancy. Discussion:Our study confirms the good results obtained by other groups when using flu-based RIC in FA pts. Indeed, satisfying engraftment and long-time survival rates were obtained without any TBI, irrespective of the stem cell source and the donor type. However, we have a concern regarding the cGVHD rate we obtained. As demonstrated by an on-going study of EBMT registry, the risk of secondary malignancy in these pts is statistically correlated to cGvHD. Then, this rate still remains probably too high in our study, even though only one pt presented with extensive cGvHD and no pts developed any secondary malignancy. But this could be explained by the short follow-up. Suppression of one alkalyting agent may reduce both cGVHD incidence and other tissue injuries leading to secondary malignancies. Then, we claim for suppression of Bu for related donor HSCT. In FA pts receiving transplant from unrelated donor, the relative impact of either low-dose IV-Bu – as we used here - or low-dose irradiation on toxicity and especially development of secondary malignancies remains to be evaluated. Disclosures:No relevant conflicts of interest to declare.

Pulmonary Glial Heterotopia in a Child Diagnosed With Fanconi Anemia and Epilepsy

Pulmonary glial heterotopia is rare and its pathogenesis is still obscure as for Fanconi anemia (FA). This study describes a very rare case of an incidental finding of pulmonary glial heterotopia in a girl diagnosed with FA, epilepsy, and mental retardation. Before this report, the association of pulmonary glial heterotopia and FA had not been described. The unique finding in this patient could be a link between FA and abnormal cell migration, but it certainly teaches us that there is still much to be learnt of the molecular mechanisms underlying the clinical manifestations in FA.

Fanconi anemia‐like presentation in an infant with constitutional deletion of 21q including the RUNX1 gene

We describe a newborn female with a de novo interstitial deletion of chromosome 21q21.1-22.12 including the RUNX1 gene who had developmental delay, multiple congenital anomalies, tetralogy of Fallot, anemia, and chronic thromobocytopenia requiring frequent platelet transfusions from birth. Because of her physical and hematologic abnormalities, she was tested for Fanconi anemia (FA). Lymphocytes and fibroblasts from this patient demonstrated increased chromosome breakage with exposure to the clastogen mitomycin C, but not, in contrast to most FA patients, to diepoxybutane. Further testing by Western analysis and complementation testing did not show a defect in the function of known Fanconi proteins. Her constitutional deletion was later found to span 13.2 Mb by chromosome microarray analysis, encompassing the RUNX1 gene that has been implicated in thrombocytopenia and predisposition to acute myelogenous leukemia (AML) when in the haploinsufficient state. We compare her phenotype to other individuals with similar 21q deletions and thrombocytopenia, as well as those with FA. We suggest that deletion of RUNX1 or another critical gene within the deleted region may result in chromosomal instability similar to that seen in FA.

How Rare Is Rare? Carrier Frequencies for Fanconi Anemia In the United States and Israel

AbstractAbstract 2229 Background:Although carrier frequencies for Fanconi anemia (FA) have been estimated for several founder populations, carrier frequencies in different countries remain unclear. One exception is Israel. In Jewish populations, founder mutations have been identified, e.g. the FANCC IVS+4 A>T mutation in Ashkenazi Jews, and the FANCA 2172–2173insG mutation in Moroccan Jews. The ability to test for specific mutations has enabled screening studies: the carrier frequency in Israeli Ashkenazi Jews is around 1:85; limited available data suggest that carrier frequencies in other Israeli Jewish subgroups may be similar (Tamary et al. BJH 2000). FA also occurs in Israeli Arab populations but carrier frequencies have not been determined. In contrast, there are much less data for the United States (US). Swift (Nature 1971) estimated the US FA carrier frequency as 1:300, but this estimate was based on surprisingly limited data - in total, the 12 persons with FA born in New York State from 1956 until 1967 who were known to the author among the corresponding total of 4.2 million live births. Nonetheless, this figure remains widely cited and has not been updated even after 40 years. We sought to update this estimate given the biological importance of the FA pathway. Methods:We applied Swift's approach (knowledge of the number of FA cases born during a given period with a known overall birth rate) to contemporary data for the United States and Israel. Specifically, we used the Hardy-Weinberg Law and demographic data from the Fanconi Anemia Research Fund (FARF, 488 FA) and the Israeli Fanconi Anemia Registry (ISFAR, 66 FA). Results:On average during the 1990s, 15 persons with FA were born each year in the US who eventually became known to the FARF, amongst the 4.0 million persons born each year in the US during that period. The corresponding Hardy-Weinberg carrier frequency is 1:257 (95% Confidence Interval: 1:240 – 1:277). This range describes a lower bound because ascertainment in FARF must be less than 100%. Even so, the lower confidence limit is significantly greater that 1:300. The true ascertainment in FARF is unknown. We propose that values between 40% – 60% are plausible. Using this range to adjust the observed birth incidence upwards, we obtained a plausible range for the carrier frequency of 1:156 – 1:209 [midpoint 1:182]. We applied the same approach to the ISFAR where 2.6 births per year were observed. We assumed 50% – 100% ascertainment by ISFAR since cases were identified through a country-wide hospital network. For the entire country of Israel (Jews and non-Jews combined), we obtained a plausible range for the carrier frequency of 1:66 – 1:128 [midpoint 1:93]. Hence, the range for Israel derived using Swift's indirect approach (estimated from birth rates) is broadly consistent with direct surveys. Conclusions:The FA carrier frequency in the US may be higher than previously thought, around 1:200 or perhaps even higher. From the perspective of population genetics, our results suggest there is less difference between the average carrier frequency in the US and higher carrier frequencies of around 1:100 reported for a number of ethnic groups including Ashkenazi Jews. This is consistent with the facts that the general US population is heterogeneous mixture of descendents of many ancestral groups, and FA is found world-wide. Our results also suggest that some European populations may have higher carrier frequencies than currently recognized. Our findings are sensitive to a number of assumptions. Going forwards, large scale re-sequencing studies could more precisely determine how many persons in the general population carry causative alleles for FA and other rare recessive syndromes. Disclosures:No relevant conflicts of interest to declare.

A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

Fanconi Anemia (FA) is a cancer-susceptibility syndrome characterized by cellular sensitivity to DNA inter-strand cross-link (ICL)-inducing agents. The Fanconia Anemia D2 (FANCD2) protein is implicated in repair of various forms of DNA damage including ICLs. Studies with replicating extracts from Xenopus eggs indicate a role for FANCD2 in processing and repair of DNA replication-associated double stranded breaks (DSB). We have investigated the role of FANCD2 in cell cycle progression of cultured human cells. Similar to Xenopus cell-free extracts, we show that chromatin association of FANCD2 in human cells is coupled to ongoing DNA replication. siRNA depletion experiments demonstrate that FANCD2 is necessary for efficient DNA synthesis. However, in contrast with Xenopus extracts, FANCD2-deficiency does not elicit a DNA damage response, and does not affect the elongation phase of DNA synthesis, suggesting that FANCD2 is dispensable for repair of replication-associated DNA damage. Using synchronized cultures of primary untransformed human dermal fibroblasts we demonstrate that FANCD2 is necessary for efficient initiation of DNA synthesis. Taken together, our results suggest a novel role for the FA pathway in regulation of DNA synthesis and cell cycle progression. Inefficient DNA replication may contribute to the genome instability and cancer-propensity of FA patients.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34+ cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor (SCF), and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC exhibited increased colony formation compared to 21% oxygen without NAC (P < 0.03), demonstrated increased resistance to mitomycin C compared to green fluorescent protein (GFP )-transduced controls (P < 0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Frequency and Natural History of Inherited Bone Marrow Failure Syndromes: The Israeli Inherited Bone Marrow Failure Registry

Inherited bone marrow failures syndromes (IBMFS) are rare genetic disorders usually characterized by congenital anomalies, development of bone marrow failure and a tendency to develop malignant diseases. Although single disease registries for Fanconi anemia (FA), Diamond-Blackfan Anemia (DBA), Severe Congenital Neutropenia (SCN) and Dyskeratois Congenita (DC) have been established the true incidence of many of the IBMFS is still unknown. To investigate the prevalence of each of the IBMFS and to the types and frequencies of complications we set up a retrospective national registry of these disorders in Israel. We reviewed charts of 127 patients with IBMFS diagnosed between 1964 and 2005. This represents the majority of such patients in the country. Consanguinity was recorded in 50 (40%) patients. Median time since diagnosis was 6 years (range: 1 month-39 years). Genetic analysis was available in 60 (47%) patients. The number of patients within each disease category is presented in the Table. The majority of patients who succumbed to their disease had FA. Of the 29 patients who developed malignancies (hematological and solid) the majority 23 (79%) had FA. All of the 6 patients who developed solid tumors had FA. The solid tumors were squamous cell carcinoma of head and neck, esophagus, cervix and vulva.Table: Israeli Inherited Bone Marrow Failure Cohort - Diagnosis, Complications and SurvivalNo. of pts.FADBASCNCAMTDCSDSTARNOSAllTotal (%)66 (52)18 (14)21 (16.5)8 (6)6 (5)3 (2)3 (2)2 (1.5)127 (100)Per 106 people5.32.62.11.10.860.430.29Deceased (%)24 (36)-6 (29)-2 (33)1 (33)--33 (26)Molecular diagnosis (%)34 (51)6 (33)7 (33)8 (100)4 (67)1 (33)--60 (47)MDS/AML/ALL (%)19 (29)-4 (19)2 (25)----25 (20)Solid tumor (%)6 (9)-------6 (5)CAMT, Congenital Amegakaryocytic Thrombocytopenia; SDS-Shwachman-Diamond Syndrome; TAR, Thrombocytopenia Absent Radii; NOS-not otherwise specified. This is the largest population-based study which has examined the relative frequency of each of the IBMFS. In this cohort, FA was by far the most common form of an IBMFS (52% of pts), followed by DBA (16.5%) and SCN (14%), while CAMT, DC, SDS and TAR were far less common. These findings agree with the frequencies of FA, DBA and SCN reported by individual disease registries, and contrast with the results from the Canadian inherited marrow failure registry (Pediatr Blood Cancer 47:918, 2006), where among a smaller number (39) of patients with an IBMFS the frequencies of FA, SDS, DBA and SCN were similar (12% each). One caveat is that the Israeli cohort has a large proportion of consanguineous families, which differs from most cohorts in other countries. The number of undiagnosed patients with IBMFS in our study was very small. SDS and DC are either rare in our region or under-diagnosed. The implementation of new diagnostic tests will help to resolve this issue. These data provide a rational basis for longitudinal surveillance and prevention of complications in the severe forms of IBMFS.

Favourable Long-Term Outcome after Matched Sibling Transplantation for Fanconi-Anemia (FA) and in vivo T-Cell Depletion

Hematopoietic stem cell transplantation is the only permanent treatment for the hematological manifestations in Fanconi anemia (FA). As FA patients have a dramatically increased intrinsic propensity to develop malignancies later in life and the genotoxic stress afflicted during conditioning advances the manifestation age especially of squamous cell carcinomas, choosing an optimally suited treatment regimen appears critical for long-term, tumor-free survival after stem cell transplantation. Here, we report our experiences in 6 consecutive FA patients transplanted with HLA-matched sibling donors where we combined an established pre-transplantation treatment consisting of thoraco-abdominal irradiation (TAI), cyclophosphamide (CYC) and cyclosporine A graft-versus-host prophylaxis with antibody-mediated IN VIVO T-cell depletion strategies after infusion of the graft. This approach has ensured sustained engraftment with long-term survival and an excellent post transplant performance status without any evidence of secondary malignancies in all six patients after a median follow-up of more than 10 years.

Alternate Donor HSCT for Fanconi Anemia (FA): Results of a Total Body Irradiation (TBI) Dose De-Escalation Study.

Major advances have been made that have improved survival in FA patients undergoing alternate donor hematopoietic stem cell transplantation (AD-HSCT). While addition of fludarabine (FLU) to cyclophosphamide (CY) and single dose total body irradiation (450 cGy TBI) has been the single most important factor improving engraftment rates in FA patients, risks of infection and late effects remain important challenges. Seeking to reduce these risks, we hypothesized that FA patients undergoing AD-HSCT could reliably achieve neutrophil engraftment with less or no irradiation due to the profound immunosuppressive effects of FLU. Therefore, we conducted a single center, single arm, TBI dose de-escalation trial designed to determine the lowest possible dose of TBI required for engraftment in recipients of T cell depleted bone marrow (TCD BM) or umbilical cord blood (UCB) from AD. All patients received CY 10 mg/kg x 4 days, FLU 35 mg/m2 x 4 days, ATG 30 mg/kg x 5 days and a single fraction of TBI with CT guided thymic shielding. TBI dose de-escalation strata were: TBI 300 cGy (cohort 1); TBI 150 cGy (cohort 2); no TBI (cohort 3). All patients received CSA and methylprednisolone as GVHD prophylaxis and G-CSF 5 ug/kg/day until engraftment. The decision to proceed with each stepwise decrease in TBI is based upon achieving primary neutrophil engraftment in 10 of 10 patients at each dose level or 14 of 15 patients if one graft failure is observed in the first 10 patients. More than 1 graft failure in 15 patients was considered unacceptable and the next higher TBI dose level was thereafter to be considered the optimal dose. Between July 2006-July 2007, 13 FA patients were enrolled with 11 in cohort I (as patient 11 was enrolled prior to proven engraftment in patient 10) and 2 thus far in cohort 2. All patients achieved primary engraftment at a median of 11 days after HSCT. One patient (TBI 300) however developed secondary graft failure at day 178 and died at day 253. Two patients developed grade II acute GVHD and none developed chronic GVHD. With a median follow up of 10.2 months, 12 of 13 patients are alive with complete chimerism and no active GVHD.Patient Characteristics and HSCT OutcomesUPNTBI DoseAge, SexDonorDay to ANC>500Acute GVHDComplicationsSurvival (days)441330017.3, M8/8 URD BM10-Aspergillus, secondary GF, MSOFdied day 25344173009.5, M7/8 URD BM16grade 2PRESS403+44253008.0, M8/8 URD BM11-PRESS, hem cystitis, pneumonia392+443830019.4, F8/8 URD BM11--374+44563009.1, M8/8 URD BM10-Aspergillus pneumonia354+446530012.1, M5/6 URD UCB11grade 2Pulmonary cytolytic thrombi335+44803008.1, M7/8 URD BM10-Hem cystitis312+453830012.9, F5/6 URD UCB30--207+460130011.2, M8/8 URD BM10--124+46503005.2, F8/8 URD BM9--45+46553006.1, F7/8 RD BM12-Bacteremia34+46601507.3, F8/8 URD BM10-Bacteremia27+466215022.3, M8/8 URD BM12--24+The results demonstrate that TBI 450 cGy is not required for engraftment in recipients of CY-FLU-ATG and HLA matched or mismatched TCD BM or UCB from AD. While longer followup is needed to quantitate the impact of lower dose radiation on risks of infection and therapy-related late effects, TBI 450 cGy should no longer be considered the standard of care.