Fanconi Anemia Mutations Research Articles

The enrichment of Fanconi anemia/homologous recombination pathway aberrations in ATM/ATR-mutated NSCLC was accompanied by unique molecular features and poor prognosis

BackgroundATM and ATR are two critical factors to regulate DNA damage response (DDR), and their mutations were frequently observed in different types of cancer, including non-small cell lung cancer (NSCLC). Given that the majority of identified ATM/ATR mutations were variants of uncertain significance, the clinical/molecular features of pathogenic ATM/ATR aberrations have not been comprehensively investigated in NSCLC.MethodsNext-generation sequencing (NGS) analyses were conducted to investigate the molecular features in 191 NSCLC patients who harbored pathogenic/likely pathogenic ATM/ATR mutations and 308 NSCLC patients who did not have any types of ATM/ATR variants. The results were validated using an external cohort of 2727 NSCLC patients (including 48 with ATM/ATR pathogenic mutations).ResultsMost pathogenic ATM/ATR genetic alterations were frameshift and nonsense mutations that disrupt critical domains of the two proteins. ATM/ATR-mutated patients had significantly higher tumor mutational burdens (TMB; P < 0.001) and microsatellite instabilities (MSI; P = 0.023), but not chromosomal instabilities, than those without any ATM/ATR variations. In particular, KRAS mutations were significantly enriched in ATM-mutated patients (P = 0.014), whereas BRCA2 mutations (P = 0.014), TP53 mutations (P = 0.014), and ZNF703 amplification (P = 0.008) were enriched in ATR-mutated patients. Notably, patients with ATM/ATR pathogenic genetic alterations were likely to be accompanied by mutations in Fanconi anemia (FA) and homologous recombination (HR) pathways, which were confirmed using both the study (P < 0.001) and validation (P < 0.001) cohorts. Furthermore, the co-occurrence of FA/HR aberrations could contribute to increased TMB and MSI, and patients with both ATM/ATR and FA/HR mutations tended to have worse overall survival.ConclusionsOur results demonstrated the unique clinical and molecular features of pathogenic ATM/ATR mutations in NSCLC, which helps better understand the cancerous involvement of these DDR regulators, as well as directing targeted therapies and/or immunotherapies to treat ATM/ATR-mutated NSCLC, especially those with co-existing FA/HR aberrations.

Mutation spectrum, expression profiling, and prognosis evaluation of Fanconi anemia signaling pathway genes for 4259 patients with myelodysplastic syndromes or acute myeloid leukemia

BackgroundIndividuals diagnosed with Fanconi anemia (FA), an uncommon disorder characterized by chromosomal instability affecting the FA signaling pathway, exhibit heightened vulnerability to the onset of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML).MethodsHerein, we employed diverse bioinformatics and statistical analyses to investigate the potential associations between the expression/mutation patterns of FA pathway genes and MDS/AML.ResultsThe study included 4295 samples, comprising 3235 AML and 1024 MDS from our and nine other online cohorts. We investigated the distinct proportion of race, age, French-American-British, and gender factors. Compared to the FA wild-type group, we observed a decrease in the expression of FNACD2, FANCI, and RAD51C in the FA mutation group. The FA mutation group exhibited a more favorable clinical overall survival prognosis. We developed a random forest classifier and a decision tree based on FA gene expression for cytogenetic risk assessment. Furthermore, we created an FA-related Nomogram to predict survival rates in AML patients.ConclusionsThis investigation facilitates a deeper understanding of the functional links between FA and MDS/AML.

Landscape of DNA damage response gene alterations in breast cancer: A comprehensive investigation.

DNA damage response (DDR) gene alterations are prevalent in breast cancer (BC) and important for treatment decisions. Intensive studies on DDR alterations in BC are still needed. The authors included 438 patients with metastatic breast cancer from their next-generation sequencing database and 1091 patients with early-stage breast cancer from The Cancer Genome Atlas (TCGA) database in the analysis to characterize molecular alterations in the DDR pathway. Germline DDR mutations were more prevalent in younger patients and those with HER2-negative cancers. Tumors with germline DDR mutations more commonly had somatic DDR mutations, especially those with germline Fanconi anemia (FA) pathway mutations. Notably, 66.67% (four of six) of patients with germline PALB2 mutations had tumors that harbored somatic PALB2 mutations. No differences in prognosis were observed in patients with germline or tumor somatic DDR mutations compared to patients and tumors that were wild-type. Compared to early BC, the frequency of somatic DDR mutations in metastatic cancers was significantly higher (24.89% vs. 16.02%, p<.001). Higher tumor mutation burdens were observed in cancers with somatic DDR mutations, but not in cancers with germline DDR mutations. Furthermore, tumors with somatic DDR mutations showed an abundance of anticancer immunological phenotypes. Somatic FA and mismatch repair pathway mutations were associated with increased expression of immune checkpoint molecules. Although most DDR genes were significantly positively associated with expression of proliferation-related genes, PARP3 expression was negatively correlated with MKI67 expression. Lower PARP3 expression was associated with a worse prognosis in TCGA database by multivariate Cox analysis. Patients with germline FA mutations more frequently have tumors with somatic DDR mutations. Somatic DDR mutations lead to anticancer immunological phenotypes in BC. No differences in prognosis according to germline or somatic DDR mutations were found.

Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction FA is the most common inherited bone marrow failure syndrome caused by a mutation in one of 23 genes involved in DNA repair; the disease manifests as hematologic abnormalities by age seven with near uniform bone marrow failure in adult patients. The gold standard therapy is bone marrow transplantation (BMT), which portends best outcomes when performed at a young age from Human Leukocyte Antigen (HLA)-matched sibling donors. However, due to the underlying pathophysiology of the disease, FA patients are especially sensitive to the chemoradiation conditioning regimens used with BMT, leading to high short-term morbidity and an increased long-term cancer risk. Due to these challenges of conventional BMT in FA, IUHCT is an attractive non-myeloablative and non-immunosuppressive option capable of inducing donor-specific tolerance when in utero testing has identified pathogenic FA mutations. Here we analyzed whether non-conditioned IUHCT confers fetal engraftment as a potential therapy in the FA mouse disease model. Methods Fanconi anemia type C homozygous mice (Fancc-/-) underwent IUHCT on gestation day 14 (E14) via vitelline vein injections of 1x107 T-cell depleted bone marrow cells from green fluorescent protein (GFP)-expressing congenic donor mice. Littermate wild-type mice served as controls. Donor cell engraftment in peripheral blood and bone marrow was assessed by flow cytometry. To confirm long-term donor HSC engraftment, we performed primary and secondary transplantation of an equivalent number of BM mononuclear cells from either Fancc -/- or wild-type mice that had undergone IUHCT. Statistical analysis was performed via Welch's t-test. P values <0.05 were considered statistically significant; for graphical purposes ***p<0.001; **p<0.002; *p<0.033; nonsignificant, ns. Results Fancc-/- homozygous mice had significantly higher donor GFP+ cell engraftment than wild-type controls at one, three, and six months post-transplantation (Figure 1). This higher engraftment was maintained across cell types in peripheral blood including CD3+ T cells (0.7% vs 3.4%), B220+ B cells (1.2% vs 6.9%), GR1+ granulocytes (1.7% vs 10.6%), and CD11B+ myeloid cells (1.9% vs 11.5%). Analysis of the bone marrow of these mice at 6 months post-transplant similarly showed a trend toward increased donor cell engraftment of progenitor cell populations including LSK, HSPC, LTHSC, STHSC, MPP2, MPP3, MPP4, CLP, CMP, GMP, and MEP cells in Fancc -/- recipients compared to wild-type mice. At all timepoints after primary and secondary transplants, recipient mice engrafted with BM mononuclear cells from prenatally transplanted Fancc -/- mice had higher peripheral blood GFP+ chimerism than those engrafted with BM mononuclear cells from prenatally transplanted wild-type mice (Figure 2). Conclusion Fancc-/- homozygous mice have higher sustained donor cell engraftment compared with wild-type littermates, indicating successful engraftment and possible early selection for wild-type donor cells in this preclinical model. These initial studies support the potential of IUHCT as a therapy for FA and highlight the need for additional studies to identify approaches to enhance donor cell engraftment to more clinically relevant levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

P814: CLINICAL CHARACTERISTICS AND GENE MUTATION ANALYSIS OF 148 CHILDREN WITH FANCONI ANEMIA IN CHINA

Background: Fanconi anemia (FA) is a rare autosomal recessive, X-linked (FANCB) or autosomal dominant (FANCR/RAD51) disease of bone marrow failure. Pathogenesis is mainly caused by gene mutation related to the FA pathway. At present, at least 23 genes have been discovered to play a role in the FA pathway. The gene mutations of FA have been reported in more and more countries. In China, there no large-scale cases have been reported. Aims: In this study, we analyzed the clinical characteristics of 148 FA children from 20 provinces in China from October 2003 to October 2021. We explored the relationship between genotype and phenotype in 105 children with FA genotyping results. This is the largest series of subtyped Chinese Fanconi anemia patients to date, and the results will be helpful for future clinical management. Methods: A total of 148 Fanconi anemia patients from 140 families were diagnosed by clinical phenotype, family history, chromosome breakage test induced by mitomycin C, Single cell gel electrophoresis experiment, targeted - sequence, whole exon sequencing, and MLPA method from October 2003 to October 2021. Since there were no genetic test results for the children before 2010, only the 105 patients after 2010 had gene sequencing results, which allowed genotyping. DNA damage repair defects were detected by MMC-induced Single cell gel electrophoresis and chromosome breakage experiments. Targeted-seq was used to identify the FA mutations of the patients. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large fragment deletions in patients with negative results in the targeted-seq but conforming to the FA by MMC-induced chromosome breakage experiments test and clinical manifestations. We performed WES of DNA samples from five patients and their parents. Results: 1) The most common subtype of FA in China is FA-A, followed by FA-D2 and FA-P。2) The most common deformities in FA-A patients are finger deformities and skin pigmentation, while heart deformities are more common in FA-D2/B/G/I/P subtypes. 3) The common mutations of FANCA are exon23c.2101A>G, exon28c.2778 + 1G>A, exon34c.3348 + 1G>A, exon21c.1844delC, and exon30 C2941T>G, which are different from Korea and Japan. 4) Homozygous nonsense mutation of exon32 c.3188G>A and exon29 c.2851C>T in FA-A likely benign, but the homozygous splicing mutation in exon29c.2852 + 1G>T is harmful. 4) Children with splicing and Del mutations of FANCD1 gene had poor clinical prognosis, while children with homozygous mutations of exon10c. 1792A>G is likely benign. 5) Cases of initial diagnosis of MDS or poor disease progression tend to occur in FA-B/C/E/G/J/L/M and FA-S subtypes. 6)Chromosome 1, 3, 7, and 8 abnormalities and mutations of SF3B1 P53 are related to disease progression. Summary/Conclusion: We made a comprehensive description and analysis of the clinical characteristics and mutant genes of Children with FA in China. We proposed the mutation sites related to clinical prognosis and the cytogenetic abnormalities of disease progression, providing new data for our comprehensive understanding of FA. Our data will be useful for FA future menagement.

Prevalence of Fanconi anemia gene mutations among men undergoing multigene germline testing for prostate cancer: Interim results from the EMPOWeR study.

188 Background: Germline testing for prostate cancer (PCA) is now central to treatment, screening, and hereditary cancer management. The Fanconi anemia (FA) pathway is a key DNA repair pathway involved in PCA biology and treatment. Prevalence of FA genes BRCA2, PALB2, and BRIP1 is well-described; however, multiple other FA genes are not routinely tested, with limited prevalence data. Here we report mutation prevalence of a spectrum of FA genes among men undergoing PCA multigene testing on the Evaluation and Management for Prostate Oncology, Wellness, and Risk (EMPOWeR) study. Methods: Eligibility includes any male with PCA or at-risk for PCA. Multigene testing includes 51 genes; FA pathway genes include BRCA2, PALB2, BRIP1, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, and FANCM. Multiple additional cancer risk genes were tested. Fisher’s exact tests were conducted to compare the prevalence of FA gene mutations between participants in the EMPOWeR study vs population prevalence reported in the literature. Statistical significance level of all tests was set a priori to 0.05. Results: The current cohort includes 235 participants. Characteristics are White (83.3%), Black (13.7%), PCA diagnosis (83.4%), mean age of PCA diagnosis 61.7 + 7.69 years, Gleason score &gt;=7 (66.2%), and T3 or higher (29.4%). Genetic results were available for 179 participants. Overall, 11.1% of participants (n=20) had a pathogenic/likely pathogenic mutation identified. Among mutation carriers, 45% (n=9) had mutations in FA genes, including FANCA (n=3), BRCA2 (n=2), FANCM (n=1), FANCD2 (n=1), PALB2 (n=1), and BRIP1 (n=1). Table shows clinical characteristics of participants with mutations in FANCA, FANCM, and FANCD2. Further mutation spectrum included: CHEK2 (n=3), NBN (n=2), MUTYH (n=2), BRCA (n=1), ATM (n=1), HOXB13 (n=1), APC (n=1). Compared to population prevalence, FA mutation prevalence was significantly higher overall (5.0% vs. 0.6%, p = 0.010) and among mutation carriers (45% vs. 0.6%, p&lt;0.001). Conclusions: While prevalence of FA genes BRCA2, PALB2, and BRIP1 is well-described, our study supports testing a broader range of FA genes given the prevalence rates, potential implications for clinical trials, targeted therapy, inherited syndromes, and reproductive implications.[Table: see text]

The neglected members of the family: non-BRCA mutations in the Fanconi anemia/BRCA pathway and reproduction.

BReast CAncer (BRCA) genes are extensively studied in the context of fertility and reproductive aging. BRCA proteins are part of the DNA repair Fanconi anemia (FA)/BRCA pathway, in which more than 20 proteins are implicated. According to which gene is mutated and which interactions are lost owing to the mutation, carriers and patients with monoallelic or biallelic FA/BRCA mutations exhibit very different phenotypes, from overt FA to cancer predisposition or no pathological implications. The effect of the so far neglected non-BRCA FA mutations on fertility also deserves consideration. As improved treatments allow a longer life expectancy in patients with biallelic FA mutations and overt FA, infertility is emerging as a predominant feature. We thus reviewed the mechanisms for such a manifestation, as well as whether they also occur in monoallelic carriers of FA non-BRCA mutations. Electronic databases PUBMED, EMBASE and CENTRAL were searched using the following term: 'fanconi' OR 'FANC' OR 'AND' 'fertility' OR 'pregnancy' OR 'ovarian reserve' OR 'spermatogenesis' OR 'hypogonadism'. All pertinent reports in the English-language literature were retrieved until May 2021 and the reference lists were systematically searched in order to identify any potential additional studies. Biallelic FA mutations causing overt FA disease are associated with premature ovarian insufficiency (POI) occurring in the fourth decade in women and with primary non-obstructive azoospermia (NOA) in men. Hypogonadism in FA patients seems mainly associated with a defect in primordial germ cell proliferation in fetal life. In recent small, exploratory whole-exome sequencing studies, biallelic clinically occult mutations in the FA complementation group A (Fanca) and M (Fancm) genes were found in otherwise healthy patients with isolated NOA or POI, and also monoallelic carrier status for a loss-of-function mutation in Fanca has been implicated as a possible cause for POI. In those patients with known monoallelic FA mutations undergoing pre-implantation genetic testing, poor assisted reproduction outcomes are reported. However, the mechanisms underlying the repeated failures and the high miscarriage rates observed are not fully known. The so far 'neglected' members of the FA/BRCA family will likely emerge as a relevant focus of investigation in the genetics of reproduction. Several (rather than a single) non-BRCA genes might be implicated. State-of-the-art methods, such as whole-genome/exome sequencing, and further exploratory studies are required to understand the prevalence and mechanisms for occult FA mutations in infertility and recurrent miscarriage.

Abstract 2041: Genetic profiling of DNA damage repair genes in hepatobiliary cancer

Abstract Background: Hepatobiliary cancers are genetically and clinically heterogenous. Current precision therapy for hepatobiliary cancers is limited and only benefits a minority. New biomarkers and precision therapy are needed to improve clinical outcome of hepatobiliary cancer patients. Method: Tumor samples of 379 hepatocellular cancer (HCC) patients and 490 biliary tract cancer (BTC) patients from Chinese population were genetically profiled using the targeted next-generation sequencing. Nonsynonymous mutations and copy number variations (CNV) of tumor samples were analyzed. The R package ReactomePA was used for pathway enrichment analysis. Tumor mutation burden was calculated by counting all nonsynonymous mutations per megabase of coding sequence. Subgroup analyses were performed to test the correlation between clinical, pathological, and genetic covariates. Results: The pathway analysis of somatic mutations in HCC and BTC showed mutation enrichment in the DNA damage repair (DDR) pathway (including TP53), of which mutations were found over 70% patients (78% in HCC and 73% in BTC). The most prevalent DDR mutations occurred in TP53 (56% in BTC and 65% in HCC) and ATM (8.2% in BTC and 4.5% in HCC), which was followed by ATR in HCC (4.2%) and BRCA2 in BTC (4.1%). A co-occurrence of DDR wild type and low TMB was found. In DDR-mutant and DDR-wild-type groups, the percentage of TMB-high patients was 15% vs. 4% for HCC, and 15% vs. 0% for BTC, which suggests a negative predictive value of DDR wild type for high TMB. In both HCC and BTC, mutations in homologous recombination repair (HRR), mismatch repair (MMR) and base excision repair (BER) pathways were significantly correlated with high TMB (p-value &amp;lt; 0.001). Interestingly, compared to HCC, BTC showed additional associations of the mutations in Fanconi anemia and cell-cycle checkpoint pathways with high TMB (p-value &amp;lt; 0.001). Among the four BTC subtypes, TMB-high patients were relatively enriched in gall bladder cancer and peri-hilar cholangiocarcinoma (13% vs. 9% in intra-hepatic and distal cholangiocarcinoma). Furthermore, gall bladder cancer showed higher than average abundance of BRCA2 mutations (6.3%), which was recently proposed as an actionable target for PARP inhibitor treatment. All CNVs of DDR genes were copy number loss and they were much more frequent in HCC than BTC (18% vs. 6%). However, CNVs of DDR genes were found not significantly associated with high TMB. Conclusions: Our genetic profiling of hepatobiliary cancer mutations demonstrated that DDR mutations in specific pathways could serve as useful biomarkers for rational patient selection for targeted therapy or immunotherapy. Citation Format: Zhenhao Fang, Qiuxiang Ou, Rui Liu, Hua Bao, Xue Wu, Yang Shao. Genetic profiling of DNA damage repair genes in hepatobiliary cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2041.

Increased PD-L1 Expression in Acute Myeloid Leukemia with Fanconi Anemia/BRCA Mutations

Introduction: Loss of DNA repair fidelity is a common feature of tumors and can drive genomic instability, tumor evolution, and therapy response. 1 Fanconi anemia (FA) is a DNA damage response (DDR) pathway and biallelic germline mutations in the pathway lead to a bone marrow failure syndrome with a predisposition for cancer, especially acute myeloid leukemia (AML). Unfortunately, FA-AML is a secondary AML with very poor prognosis, with an increased risk for relapse even after initial cure with bone marrow transplantation. Therefore FA patients need further options for therapy. DNA damage response (DDR) deficiency has emerged as a predictive biomarker of response to immune checkpoint inhibition. 2 In some solid tumors, defective DDR leads to genomic instability, increased mutational burden, increased neo antigen expression and increased PD-L1 expression; and respond robustly to PD-1 checkpoint inhibitor therapy. The mutational burden and PD-L1 expression in FA-mutated tumors remain to be discovered. Therefore, we hypothesize that FA mutations in AML increase mutational burden and PD-L1 expression. This work can set the stage for future studies involving PD-1 blocking therapies for FA-mutated tumors. Methods/Results: To determine the mutational burden in FA-mutated tumors, we didwhole exome sequencing (WES)of three AML cell lines obtained from pediatric FA patients, all were FANCD1/BRCA2 mutated.3,4 The three AML cell lines also represent a progression of AML disease- an early clone, a late clone and a relapsed AML after bone marrow transplantation. There was an average of 180,000 somatic mutations/case, pared down to 1,000 mutations after limiting calls with &amp;gt;10 reads and allele frequency &amp;gt;0.1%. Mutational burden of pediatric AML in non-FA patients has been reported to be ~10 mutations/case.5Therefore there is about a 100-fold increase in mutations in AML from FA patients compared to AML from non-FA patients.Then we determined PD-L1 mRNA and protein expression of the three FANCD1/BRCA2 mutated AML cells with quantitative PCR and flow cytometry respectively. We found increasing PD-L1 mRNA expression from early to late to relapsed clones (2^-ΔCt values 1.21x10-5, 1.54x10-5, and 7.02x10-5 respectively; p-value&amp;lt;0.05). Interestingly, interferon-gamma (IFN-g), a known inducer and regulator of PD-L1 expression, is detected at high levels in the bone marrow of FA patients.5 We find that PD-L1 expression significantly increases in FANCD1/BRCA2 mutated AML cells after treatment with physiologic doses of IFN-g (fold change in 2^-ΔΔCt before and after treatment with 5ng IFN-g for early clone (x16.55 fold change) late clone (x38.46 fold change) and relapsed clone (x17.57 fold change), p-value&amp;lt;0.05. We also found stable PD-L1 protein expression on the cell surface (% PD-L1-positive cells for early clone (15.2%), late clone (17.7%), and relapsed AML (18.9%). Conclusions: Our data show there is a high mutational load in FA-AML cell lines, which also express PD-L1. Both of these tumor characteristics are predictors of response to PD-1 checkpoint inhibitor immunotherapy. This will lead to future work on PD-1 blockade as a potential therapeutic option for FA-AML. Disclosures No relevant conflicts of interest to declare.

Alterations of DNA damage repair genes in Chinese colorectal cancer patients.

e16121 Background: Colorectal cancer (CRC) is the second most common cancer in women and third in men. DNA damage repair (DDR) deficiency has emerged as a predictive biomarker for chemotherapy, PARP and immune checkpoint inhibitors. However, comprehensive molecular characteristics of DDR variants in Chinese CRC patients is lacking. Methods: Formalin fixed, paraffin embedded (FFPE) tumor tissues and matched blood samples were collected for targeted next-generation sequencing (NGS) assay. The testing was carried out in a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Alterations of six functional gene sets involved in DDR pathways: homologous recombination (HR), mismatch repair (MMR), base excision repair (BER), nonhomologous end-joining (NHEJ), checkpoint factors (CPF) and Fanconi anemia (FA) were analyzed. The association of DDR gene mutations with TMB/MSI was assessed. Results: In total, 319 CRC patients were recruited, 127 female and 192 male with a median age of 59 (range 23-92). Over one third (36%,115/319) patients had at least one mutation in DDR genes. Mutation rates varied in different DDR pathway: HR (20.7%), CPF (15.1%), FA (11.3%), MMR (10.3%), BER (8.8%), and NHEJ (5.3%). The most frequently mutated DDR genes were ARID1A (13.4%), ATM (10.2%), BRCA2 (6.8%), MLH1 (5.6%), MSH6 (5.6%), POLE (5.0%). Germline mutations in MLH1 (2.5%), BRCA2 (1.2%), MSH2 (0.9%), MSH6 (0.9%), FANCA (0.9%), ATM (0.6%), BRCA1 (0.3%), and CHEK2 (0.3%) were detected in 24 patients. DDR variations were enriched in the right-side CRC compared to the left side CRC (50% vs. 32.8%, p= 0.024). Early stage (I-II) harbored more DDR variations. 20.1% of patients had high TMB (≥10 muts/Mb) with a median of 51 muts/Mb (10-326.7 mus/Mb). Patients with DDR mutations had a significantly higher TMB than patients with wild type DDR (8.5 vs. 4.6 muts/Mb, p&lt; 0.001). All CRC tumors with high MSI harbored DDR mutations. Importantly, the mutations in “HM” (HR/MMR), but not BER/CPF/NHEJ/FA mutations, were significantly correlated with high MSI ( p&lt; 0.001). Conclusions: DDR gene alterations occurred in 36% of Chinese CRC patients and were enriched in right sided tumors. DDR pathway alterations are relatively frequent in CRC and consideration for biomarker-enriched clinical trials with PARP, immune checkpoint inhibitors, and novel combinations are warranted.

Emerging functions of Fanconi anemia genes in replication fork protection pathways.

Germline mutations in Fanconi anemia (FA) genes predispose to chromosome instability syndromes, such as FA and cancers. FA gene products have traditionally been studied for their role in interstrand cross link (ICL) repair. A fraction of FA gene products are classical homologous recombination (HR) factors that are involved in repairing DNA double-strand breaks (DSBs) in an error-free manner. Emerging evidence suggests that, independent of ICL and HR repair, FA genes protect DNA replication forks in the presence of replication stress. Therefore, understanding the precise function of FA genes and their role in promoting genome stability in response to DNA replication stress is crucial for diagnosing FA and FA-associated cancers. Moreover, molecular understanding of the FA pathway will greatly help to establish proper functional assays for variants of unknown significance (VUS), often encountered in clinics. In this short review, we discuss the recently uncovered molecular details of FA genes in replication fork protection pathways. Finally, we examine how novel FA variants predispose to FA and cancer, due to defective replication fork protection activity.

NHEJ-Mediated Gene Editing, a Versatile Approach to Correct a Variety of Fanconi Anemia Genes in HSCs

Allogeneic hematopoietic stem cell (HSC) transplantation is currently the only curative treatment for the bone marrow failure in Fanconi anemia (FA) patients. However, recent advances in lentiviral-mediated gene therapy have shown that corrected FA HSCs develop an in vivo proliferation advantage, facilitating the engraftment of corrected HSCs in non-conditioned FA patients. Based on these observations, we proposed that gene editing might constitute a promising alternative to correct patients' hematopoietic stem and progenitor cells (HSPCs) in this disorder. Since non-homologous end joining (NHEJ) is the most frequent repair pathway in HSCs, particularly in FA-HSCs, we aimed at exploiting this DNA repair mechanism to remove/compensate specific mutations in different FANC genes by the use of CRISPR/Cas9 system, thus mimicking spontaneous genetic reversions observed in FA mosaic patients. Our results in lymphoblastic cell lines from five different complementation groups (FANCA, FANCB, FANCC, FANCD2 and FANCD1/BRCA2) demonstrated the efficiency of this approach to generate potentially corrective events in all the different complementation groups studied. Importantly, corrected cells showed a marked proliferative advantage after in vitro culture and the analysis by next generation sequencing confirmed the expansion of cells harboring therapeutic events. Functional studies showing the reversion of mitomycin C sensitivity, FANCD2 foci formation and chromosomal instability supported the phenotypic correction of different mutations by NHEJ-mediated gene editing. Moving towards the clinical application of NHEJ-mediated repair we focused on improving the gene editing efficiency in HSCs. To this aim, chemically modified small guide RNAs (MS-sgRNAs) enabled us to increase the editing efficacy 8-fold compared to efficacies obtained with in vitro transcribed sgRNAs, reaching up to 89% indels in healthy donor hematopoietic stem/progenitor cells. Moreover, the CRISPR/Cas9 system demonstrated high editing capacity in the primitive HSCs capable of engrafting immunodeficient NSG mice, confirming the efficacy of NHEJ-editing to correct the phenotype of long-term repopulating HSCs. Finally, studies conducted in mobilized peripheral blood and bone marrow CD34+ cells from FA patients demonstrated the feasibility to correct FA HSCs by NHEJ-mediated gene editing and confirmed the proliferative advantage of NHEJ-mediated corrected cells both in vitro and in vivo. Our results suggest that NHEJ-mediated gene editing should constitute a versatile and simple therapeutic approach to efficiently correct specific mutations in FA and other monogenic disorders of the hematopoietic system. Disclosures Sevilla: Rocket Pharmaceuticals, Inc.: Honoraria, Patents &amp; Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents &amp; Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Rio:Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents &amp; Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

PF354 HOW TELOMERE LENGTH SCREENING CAN BE HELPFUL FOR THE DIAGNOSIS OF CYTOPENIC PATIENTS?

Background: Telomere-related disorders are a clinically and genetically heterogeneous group of diseases characterized by premature telomere shortening and proliferative failure of a variety of tissues.The main organs subject to failure of function and fatty or fibrotic replacement are the bone marrow, the lung, and the liver. All 3 may be affected in an individual or a pedigree, with variable severity.Screening for telomeropathies is of particular importance in cytopenic young patients as the disorders encompass a wide spectrum and clinical presentation can therefore be subtle. Aims: To evaluate lymphocyte and granulocyte telomere length (TL) in cytopenic patients and its correlation withphenotypical and hematological features. Methods: TL in lymphocytes and granulocytes was measured using flow fluorescence in situ hybridization (flow FISH) and compared to percentiles (p) of reference ranges. All patients underwent bone marrow evaluation and for some patients genetic tests were performed (NGS or Sanger sequencing). Furthermore, we recorded for all patients somatic and hematological features (see table). Somatic abnormalities and grade of cytopenia were scored according to scores published in other genetic marrow failure syndromes (Fanconi Anemia, from Svahn et al. Am J Hematol 2016). Dyskeratosis congenita (DC) phenotype was considered as established when one of the typical symptoms/signs was present (skin,nails, leuko/erythrtoplakia, lung, liver fibrosis). Results: During the timespan from april 2015 to February 2019 we studied 36 cytopenic patients, M/F 1.25, median age 12.5 years (range 1–51 years). Based on the evaluation of telomere lenght, we divided our patients into 3 groups - Group 1: no telomere lenght abnormalities: 25 patients - Group 2: telomere lenght between 1st and 5thp in at least one of cells subsets analyzed (lymphocytes or granulocytes): 3 patients - Group 3: telomere lenght <1st p in at least one of cells subsets analyzed (lymphocytes or granulocytes): 8 patients Patients hematological and phenotypic features are summarized in table 1 Patients with phenotypic abnormalities were 20/36, including 4 with established DC features. Patients with cytopenia of central origin were 23/36, 17 with Bone Marrow Failure, 5 with Myelodysplasia and 1 with Juvenile Myelomonocytic Leukemia. In 20 patients we performed genetic tests (NGS or Sanger sequencing) and we identified in 14/20 patients a pathogenic mutation (3 Fanconi Anemia mutations, 1 Blackfan Diamond Anemia, 1 LIG4 mutation, 2 TACI mutations, 1 PI3K mutation, 1 NFKB mutation, 1 GATA mutation, 1Ohdo syndrome, 3 DC mutations −1 DKC1, 1 TERT,1 TERC-). In patients with TL <5°p (group 2 and 3) we found that established DC phenotype was more frequent (p = 0.0011).According to the data available in the literature, all patients with genetically confirmed DC had a telomere <1st centile. Furthermore, the following combinations: phenotypic abnormalities+cytopenia and cytopenia+ established DC phenotype (with or without genetic confirmation) had excellent concordance with telomere shortening (Cohen's kappa coefficient >0.81).Summary/Conclusion: Our findings suggest that in patients with cytopenia, telomere shortening is more likely to be detected in those who have established DC phenotype and/ or other somatic abnormalities. Based on this it might be advisable to test telomere length in these categories in order to detect telomere disease subject that might be otherwise missed.

Generation of Fanconi Anemia iPSC Clones By Addition of a Small Molecule Inhibitor of p53 during Reprogramming

Fanconi anemia (FA) is an inherited bone marrow failure syndrome characterized by loss of function mutations in any of at least 22 FANC genes that are critical for DNA repair. Progressive loss of hematopoietic stem/progenitor cells (HSPCs) in FA patients has been attributed in part to a heightened p53/p21 axis that restricts the cell cycle to G0/G1 in response to accumulation of unresolved DNA damage and replicative stress. Silencing of p53 was previously shown to rescue HSPC defects in FA models in vitro and in vivo. Due to insufficient numbers of primary HSPCs in FA for disease modeling and cell therapies, the concept of generating induced pluripotent stem cells (iPSCs) from an individual patient, and differentiating the FA-iPSCs into a theoretically infinite supply of autologous HSPCs arose. However, reprogramming FA cells has proven to be challenging and mostly dependent on hypoxic culture conditions or genetic complementation of the defect by integrating lentiviral vectors expressing a normal FANC gene. A recent study reported low-efficiency generation of integration-free FA-iPSCs by electroporation of fibroblasts with episomal plasmid vectors expressing both reprogramming factors and p53 shRNA. Here we sought to develop an improved and streamlined reprogramming strategy for FA by transduction of readily accessible FA mononuclear cells (MNCs) with commercially available non-integrating reprogramming Sendai Viruses (SeV2), combined with a more adaptable inhibition of the p53 pathway by addition of variable concentrations of a small molecule during reprogramming. Approximately 100,000 MNCs from an FA patient with confirmed biallelic mutations in FANCA were cultured for 10 days under hypoxic conditions (5% O2) favoring either myeloid or erythroid expansion. While myeloid cultures led to minimal proliferation of FA MNCs, we observed a 6-fold increase in total cell numbers in the presence of EPO. Erythroid-expanded FA cells were transduced with SeV2 harboring four standard reprogramming transcription factors (SOX2, OCT4, KLF4 and c-MYC). Cells were subsequently cultured under hypoxia for at least 30 days in a reprogramming cocktail supplemented or not with 0.3uM, 0.6µM or 1.2µM cyclic pifthrin-α (cPFT), a reversible small molecule inhibitor of p53-dependent gene transcription. In the absence of cPFT, no FA-iPSC clones were obtained, consistent with reported reprogramming barriers in FA cells. In contrast, several iPSC colonies were generated with cPFT-treated FA cells, albeit at much slower kinetics (~30 days) compared to MNCs without FA mutations (~20 days). The overall efficiency of reprogramming was ~0.01%, with a trend toward enhanced efficiencies in cultures containing the highest concentration of cPFT (1.2µM). To investigate the need for p53 inhibition beyond the reprogramming period, half of the FA-iPSC clones were passaged and further cultured in 5% O2 with cPFT 1.2µM, whereas the remaining clones were similarly treated but without p53 inhibitor. None of the FA-iPSC clones that were maintained in cPFT-containing cultures following reprogramming survived further passaging. In contrast, when cPFT was removed after reprogramming, all iPSC clones could stably undergo repeated passages provided that hypoxia (5% O2) was maintained. The FA-iPSC clones expressed high levels alkaline phosphatase and were TRA 1-60- and NANOG-positive, preliminarily validating their pluripotent nature. Karyotyping, trilineage differentiation, and in vivo teratoma assays are ongoing. Overall, this study provides a practical approach for successful reprogramming of FA peripheral blood cells based on commercially available non-integrating SeV2 reprogramming strategies supplemented with p53 inhibition. Regulated addition of variable concentrations of a small molecule inhibitor of p53 at various steps of the reprogramming process will allow further enhancement of reprogramming efficiencies in FA. DisclosuresNo relevant conflicts of interest to declare.

Functional analysis of Fanconi anemia mutations in China

Fanconi anemia (FA) is a rare recessive disease characterized by progressive bone marrow failure, congenital abnormalities, and increased incidence of cancers. To date, mutations in 22 genes can cause FA or an FA-like phenotype. In China, in addition to clinical information, FA diagnosis primarily relies on genetic sequencing because the chromosome breakage test is rarely performed. Here, we employed multiple genetic diagnostic tools (DNA sequencing, multiplex ligation-dependent probe amplification, and chromosome microarray) and a variant-based functional assay platform to investigate the genetic cause in 25 Chinese suspected FA patients. A total of 45 distinct candidate variants were detected in six FA genes (FA-A, FA-B, FA-C, FA-D2, FA-G, and FA-J), of which 36 were novel. Eight missense variants and one indel variant were unable to restore FANCD2 mono-ubiquitination and mitomycin C resistance in a panel of FA indicator cell lines, indicating that these mutations are deleterious. Three missense variants (FANCA-L424V, FANCC-E273K, and FANCG-A153G) were harmless. Finally, 23 patients were molecularly diagnosed with FA, consistent with their clinical phenotype. In the FA-A subgroup, large deletions accounted for 14% of the disease-causing variants. We have established a comprehensive molecular diagnostic workflow for Chinese FA patients that can substitute for standard FA cytogenetic analysis.

Lipidomic Profiling Links the Fanconi Anemia Pathway to Glycosphingolipid Metabolism in Head and Neck Cancer Cells.

Purpose: Mutations in Fanconi anemia (FA) genes are common in sporadic squamous cell carcinoma of the head and neck (HNSCC), and we have previously demonstrated that FA pathway depletion in HNSCC cell lines stimulates invasion. The goal of our studies was to use a systems approach in order to define FA pathway-dependent lipid metabolism and to extract lipid-based signatures and effectors of invasion in FA-deficient cells.Experimental Design: We subjected FA-isogenic HNSCC keratinocyte cell lines to untargeted and targeted lipidomics analyses to discover novel biomarkers and candidate therapeutic targets in FA-deficient cells. Cellular invasion assays were carried out in the presence and absence of N-butyldeoxynojirimycin (NB-DNJ), a biosynthetic inhibitor of the newly identified class of gangliosides, to investigate the requirement of ganglioside upregulation in FA-deficient HNSCC cells.Results: The most notable element of the lipid profiling results was a consistent elevation of glycosphingolipids, and particularly the accumulation of gangliosides. Conversely, repression of this same class of lipids was observed upon genetic correction of FA patient-derived HNSCC cells. Functional studies demonstrate that ganglioside upregulation is required for HNSCC cell invasion driven by FA pathway loss. The motility of nontransformed keratinocytes in response to FA loss displayed a similar dependence, thus supporting early and late roles for the FA pathway in controlling keratinocyte invasion through lipid regulation.Conclusions: Elevation of glycosphingolipids including the ganglioside GM3 in response to FA loss stimulates invasive characteristics of immortalized and transformed keratinocytes. An inhibitor of glycosphingolipid biosynthesis NB-DNJ attenuates invasive characteristics of FA-deficient HNSCC cells. Clin Cancer Res; 24(11); 2700-9. ©2018 AACR.

Fanconi anemia and homologous recombination gene variants are associated with functional DNA repair defects in vitro and poor outcome in patients with advanced head and neck squamous cell carcinoma.

Mutations in Fanconi Anemia or Homologous Recombination (FA/HR) genes can cause DNA repair defects and could therefore impact cancer treatment response and patient outcome. Their functional impact and clinical relevance in head and neck squamous cell carcinoma (HNSCC) is unknown. We therefore questioned whether functional FA/HR defects occurred in HNSCC and whether they are associated with FA/HR variants. We assayed a panel of 29 patient-derived HNSCC cell lines and found that a considerable fraction is hypersensitive to the crosslinker Mitomycin C and PARP inhibitors, a functional measure of FA/HR defects. DNA sequencing showed that these hypersensitivities are associated with the presence of bi-allelic rare germline and somatic FA/HR gene variants. We next questioned whether such variants are associated with prognosis and treatment response in HNSCC patients. DNA sequencing of 77 advanced stage HNSCC tumors revealed a 19% incidence of such variants. Importantly, these variants were associated with a poor prognosis (p = 0.027; HR = 2.6, 1.1–6.0) but favorable response to high cumulative cisplatin dose. We show how an integrated in vitro functional repair and genomic analysis can improve the prognostic value of genetic biomarkers. We conclude that repair defects are marked and frequent in HNSCC and are associated with clinical outcome.

LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining.

The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.

Rare Variants Affecting the Fanconi Anaemia DNA Repair Genes Associate with Increased Risk for AML

While there have been extensive studies to define the roles of recurrent somatic mutations in AML, the contribution of germline variants to AML initiation and progression is less well established. DNA repair disorders often predispose patients to developing myeloid malignancies. In particular, biallelic mutations affecting FANC genes cause the recessive heritable bone marrow failure syndrome Fanconi Anemia (FA), which is associated with >800-fold increased risk of progression to AML. A recent explosion of cancer predisposition studies has also revealed the importance of germlineFANC variants in elevated cancer risk (Cancer Treat Rev 2012; 38:89). To investigate the role of FANC gene variants in AML we have performed a case-control study, analyzing rare, deleterious somatic and germline variants for the 19 FANC genes in adult AML and healthy controls cohorts.Whole exome sequencing was performed on diagnosis samples from 131 adult Caucasian AML patients from two major Australian centers, and a cohort of 329 healthy females. We identified rare Tier 1 variants using a minor allele frequency (MAF) < 0.001, as reported in common dbSNP137, 1000 Genome and NHLBI-ESP project databases. Combined Annotation Dependent Depletion algorithm (CADD, Nat Genet 2014; 46: 310) >10 was used to filter for FANC gene variants with high probability of pathogenicity. Sanger sequencing of matched tumour/non-tumour DNA showed the large majority of variants tested to be germline (90%), consistent with previous studies reporting that somatic FANC genes variants are extremely rare in AML (< 1%). Overall, we identified 52 FANC gene variants in 44 cases with 34% of AML cases carrying one or more variant.For independent validation we determined the presence of somatic and germline FANC variants in the TCGA AML cohort using an identical pipeline and filtering analysis. In line with our results, we found that 36% of TCGA AML patients carry at least one germline FANC variant. We investigated known disease-causing (D-C) variants in these two AML cohorts using the FA (FAMutdb) and breast cancer (kConFab and BIC) mutation databases. We found 8 D-C FANC variants in the Australian AML cohort and 5 in the TCGA cohort, with 1 variant present in both cohorts. Moreover, the frequency of D-C variants in our cohort of females with AML (n=51) is 13.7%, while the frequency in the healthy female cohort is 4.5%, comparable to that reported in the ESP database for female European-Americans (2.1%, Hum Mol Genet 2014; 23: 6815). Accordingly, we determined that deleterious FANC germline variants confer a significant increased risk of AML (P=0.018, OR=3.3 for the Australian AML cohort).Finally, we performed mutational burden analysis to investigate enrichment of variants associated with particular FANC genes across the AML cohort. This revealed a significant enrichment of FANCL variants in AML vs healthy controls (P=0.008, Figure 1). FANCL is the enzymatic component of the FA core complex that monoubiquitinates the FANCD2/I heterodimer initiating DNA repair, and its down-regulation has been linked to AML (Oncogene 2016; doi:10.1038). Several FANCL variants, found in our AML cohort, affect the catalytic RING domain and are of particular interest. These include a D-C null variant present in 2 patients, a frame shift variant in 2 patients who presented with AML at a very early age (27 and 46 years old), and a variant affecting a critical conserved residue required for monoubiquitination of FANCD2/I.In conclusion, we show enrichment of rare potentially deleterious FANC gene mutations in AML, associated with a 3-fold increased risk of developing the disease. We hypothesise that, in hematopoietic stem/progenitor cells, these variants confer a subtle defect in interstrand cross-link repair leading to an increased accumulation of mutations and subsequent development of AML. Consistent with this there have been several reports of defective DNA damage repair and increased sensitivity to DNA damaging agents in cells from FANC carriers compared to normal controls (Nat Commun 2014; 5:5496; Mutagenesis 2009; 24:67). Importantly, it is possible to target defects in several DNA repair pathways, and our finding identifies a group of AML patients who may benefit from approaches that target defective FA and homologous recombination pathways. [Display omitted] DisclosuresGill:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.