Najim Ameziane

Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors

Ovarian tumor cells are often genomically unstable and hypersensitive to cisplatin. To understand the molecular basis for this phenotype, we examined the integrity of the Fanconi anemia–BRCA (FANC-BRCA) pathway in those cells. This pathway regulates cisplatin sensitivity and is governed by the coordinate activity of six genes associated with Fanconi anemia (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) as well as BRCA1 and BRCA2 (FANCD1). Here we show that the FANC-BRCA pathway is disrupted in a subset of ovarian tumor lines. Mono-ubiquitination of FANCD2, a measure of the function of this pathway, and cisplatin resistance were restored by functional complementation with FANCF, a gene that is upstream in this pathway. FANCF inactivation in ovarian tumors resulted from methylation of its CpG island, and acquired cisplatin resistance correlated with demethylation of FANCF. We propose a model for ovarian tumor progression in which the initial methylation of FANCF is followed by FANCF demethylation and ultimately results in cisplatin resistance.

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.

RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics

Summary Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”.

Bi-allelic silencing of the Fanconi anaemia gene FANCF in acute myeloid leukaemia

Summary. Fanconi anaemia (FA) is a chromosomal instability disorder associated with a high risk of acute myeloid leukaemia (AML). Previous work has shown that the AML cell line CHRF‐288, derived from a sporadic AML‐M7 patient, does not express FANCF protein and exhibits a cellular FA phenotype. We show that this phenotype is corrected by a FANCF‐expressing plasmid and that the absence of FANCF protein is explained by hypermethylation of the promoter region of the FANCF gene. As FANCF is localized in a hot‐spot region for somatic hypermethylation (11p15), FANCF silencing might be an early step in sporadic carcinogenesis, including leukaemogenesis.

Functional Ex Vivo Assay to Select Homologous Recombination–Deficient Breast Tumors for PARP Inhibitor Treatment

Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors are promising targeted treatment options for hereditary breast tumors with a homologous recombination (HR) deficiency caused by BRCA1 or BRCA2 mutations. However, the functional consequence of BRCA gene mutations is not always known and tumors can be HR deficient for other reasons than BRCA gene mutations. Therefore, we aimed to develop a functional test to determine HR activity in tumor samples to facilitate selection of patients eligible for PARP inhibitor treatment. Experimental design: We obtained 54 fresh primary breast tumor samples from patients undergoing surgery. We determined their HR capacity by studying the formation of ionizing radiation induced foci (IRIF) of the HR protein RAD51 after ex vivo irradiation of these organotypic breast tumor samples. Tumors showing impaired RAD51 IRIF formation were subjected to genetic and epigenetic analysis. Results: Five of 45 primary breast tumors with sufficient numbers of proliferating tumor cells were RAD51 IRIF formation deficient (11%, 95% CI, 5%–24%). This HR defect was significantly associated with triple-negative breast cancer (OR, 57; 95% CI, 3.9–825; P = 0.003). Two of five HR-deficient tumors were not caused by mutations in the BRCA genes, but by BRCA1 promoter hypermethylation. Conclusion: The functional RAD51 IRIF assay faithfully identifies HR-deficient tumors and has clear advantages over gene sequencing. It is a relatively easy assay that can be performed on biopsy material, making it a powerful tool to select patients with an HR-deficient cancer for PARP inhibitor treatment in the clinic. Clin Cancer Res; 20(18); 4816–26. ©2014 AACR.

Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

Hypermethylation of the FANCC and FANCL promoter regions in sporadic acute leukaemia

Objective: Inactivation of the FA-BRCA pathway results in chromosomal instability. Fanconi anaemia (FA) patients have an inherited defect in this pathway and are strongly predisposed to the development of acute myeloid leukaemia (AML). Studies in sporadic cancers have shown promoter methylation of the FANCF gene in a significant proportion of various solid tumours. However, only a single leukaemic case with methylation of one of the FA-BRCA genes has been described to date, i.e. methylation of FANCF in cell line CHRF-288. We investigated the presence of aberrant methylation in 11 FA-BRCA genes in sporadic cases of leukaemia. Methods: We analyzed promoter methylation in 143 AML bone marrow samples and 97 acute lymphoblastic leukaemia (ALL) samples using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Samples with aberrant methylation were further analyzed by bisulphite sequencing and tested for mitomycin C sensitivity using Colony Forming Units assays. Results: MS-MLPA showed promoter methylation of FANCC in one AML and three ALL samples, while FANCL was found methylated in one ALL sample. Bisulphite sequencing of promoter regions confirmed hypermethylation in all cases. In addition, samples with hypermethylation of either FANCC or FANCL appeared more sensitive towards mitomycin C in Colony Forming Units assays, compared to controls. Conclusion: Hypermethylation of promoter regions from FA-BRCA genes does occur in sporadic leukaemia, albeit infrequently. Hypermethylation was found to result in hypersensitivity towards DNA cross-linking agents, a hallmark of the FA cellular phenotype, suggesting that these samples displayed chromosomal instability. This instability may have contributed to the occurrence of the leukaemia. In addition, this is the first report to describe hypermethylation of FANCC and FANCL. This warrants the investigation of multiple FA-BRCA genes in other malignancies.

Diagnosis of Fanconi Anemia: Mutation Analysis by Next-Generation Sequencing

Fanconi anemia (FA) is a rare genetic instability syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. Fifteen genetic subtypes have been distinguished. The majority of patients (≈85%) belong to the subtypes A (≈60%), C (≈15%) or G (≈10%), while a minority (≈15%) is distributed over the remaining 12 subtypes. All subtypes seem to fit within the “classical” FA phenotype, except for D1 and N patients, who have more severe clinical symptoms. Since FA patients need special clinical management, the diagnosis should be firmly established, to exclude conditions with overlapping phenotypes. A valid FA diagnosis requires the detection of pathogenic mutations in a FA gene and/or a positive result from a chromosomal breakage test. Identification of the pathogenic mutations is also important for adequate genetic counselling and to facilitate prenatal or preimplantation genetic diagnosis. Here we describe and validate a comprehensive protocol for the molecular diagnosis of FA, based on massively parallel sequencing. We used this approach to identify BRCA2, FANCD2, FANCI and FANCL mutations in novel unclassified FA patients.

Genotyping of Fanconi Anemia Patients by Whole Exome Sequencing: Advantages and Challenges

Fanconi anemia (FA) is a rare genomic instability syndrome. Disease-causing are biallelic mutations in any one of at least 15 genes encoding members of the FA/BRCA pathway of DNA-interstrand crosslink repair. Patients are diagnosed based upon phenotypical manifestationsand the diagnosis of FA is confirmed by the hypersensitivity of cells to DNA interstrand crosslinking agents. Customary molecular diagnostics has become increasingly cumbersome, time-consuming and expensive the more FA genes have been identified. We performed Whole Exome Sequencing (WES) in four FA patients in order to investigate the potential of this method for FA genotyping. In search of an optimal WES methodology we explored different enrichment and sequencing techniques. In each case we were able to identify the pathogenic mutations so that WES provided both, complementation group assignment and mutation detection in a single approach. The mutations included homozygous and heterozygous single base pair substitutions and a two-base-pair duplication in FANCJ, -D1, or -D2. Different WES strategies had no critical influence on the individual outcome. However, database errors and in particular pseudogenes impose obstacles that may prevent correct data perception and interpretation, and thus cause pitfalls. With these difficulties in mind, our results show that WES is a valuable tool for the molecular diagnosis of FA and a sufficiently safe technique, capable of engaging increasingly in competition with classical genetic approaches.

Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed.