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Background In the majority of familial breast cancer (BC) families, the etiology of the disease remains unresolved. To identify missing BC heritability resulting from relatively rare variants (minor allele frequency ≤ 1%), we have performed whole exome sequencing followed by variant analysis in a virtual panel of 492 cancer-associated genes on BC patients from BRCA1 and BRCA2 negative families with elevated BC risk. Methods BC patients from 54 BRCA1 and BRCA2 -negative families with elevated BC risk and 120 matched controls were considered for germline DNA whole exome sequencing. Rare variants identified in the exome and in a virtual panel of cancer-associated genes [492 genes associated with different types of (hereditary) cancer] were compared between BC patients and controls. Nonsense, frame-shift indels and splice-site variants (strong protein-damaging variants, called PDAVs later on) observed in BC patients within the genes of the panel, which we estimated to possess the highest probability to predispose to BC, were further validated using an alternative sequencing procedure. Results Exome- and cancer-associated gene panel-wide variant analysis show that there is no significant difference in the average number of rare variants found in BC patients compared to controls. However, the genes in the cancer-associated gene panel with nonsense variants were more than two-fold over-represented in women with BC and commonly involved in the DNA double-strand break repair process. Approximately 44% (24 of 54) of BC patients harbored 31 PDAVs, of which 11 were novel. These variants were found in genes associated with known or suspected BC predisposition ( PALB2, BARD1, CHEK2, RAD51C and FANCA ) or in predisposing genes linked to other cancer types but not well-studied in the context of familial BC ( EXO1, RECQL4, CCNH, MUS81, TDP1, DCLRE1A, DCLRE1C, PDE11A and RINT1 ) and genes associated with different hereditary syndromes but not yet clearly associated with familial cancer syndromes ( ABCC11, BBS10, CD96, CYP1A1, DHCR7, DNAH11, ESCO2, FLT4, HPS6, MYH8, NME8 and TTC8 ). Exome-wide, only a few genes appeared to be enriched for PDAVs in the familial BC patients compared to controls. Conclusions We have identified a series of novel candidate BC predisposition variants/genes. These variants/genes should be further investigated in larger cohorts/case-control studies. Other studies including co-segregation analyses in affected families, locus-specific loss of heterozygosity and functional studies should shed further light on their relevance for BC risk.

Background Among the 10% of pancreatic cancers that occur in a familial context, around a third carry a pathogenic variant in a cancer predisposition gene. Genetic studies of pancreatic cancer predisposition are limited by high mortality rates amongst index patients and other affected family members. The genetic risk for pancreatic cancer is often shared with breast cancer susceptibility genes, most notably BRCA2, PALB2, ATM and BRCA1 . Therefore, we hypothesized that additional shared genetic etiologies might be uncovered by studying families presenting with both breast and pancreatic cancer. Methods Focusing on a multigene panel of 276 DNA Damage Repair (DDR) genes, we performed next-generation sequencing in a cohort of 41 families with at least three breast cancer cases and one pancreatic cancer. When the index patient with pancreatic cancer was deceased, close relatives (first or second-degree) affected with breast cancer were tested (39 families). Results We identified 27 variants of uncertain significance in DDR genes. A splice site variant (c.1605 + 2T > A) in the RAD17 gene stood out, as a likely loss of function variant. RAD17 is a checkpoint protein that recruits the MRN (MRE11-RAD50-NBS1) complex to initiate DNA signaling, leading to DNA double-strand break repair. Conclusion Within families with breast and pancreatic cancer, we identified RAD17 as a novel candidate predisposition gene. Further genetic studies are warranted to better understand the potential pathogenic effect of RAD17 variants and in other DDR genes.

While environmental factors can greatly increase cancer risk, it is clear that an individual’s genetic constitution has strong impact on tumor formation. Hereby we present an alternative cancer predisposition model built on the assumption that efficiencies of DNA maintenance mechanisms in normal cells are similar but not identical for each person. Small variations in an individual’s genetic constitution may result in slightly increased genomic instability and generate typical mutational signatures in normal cells. With recent and expected advances in the next-generation sequencing field, qualitative and quantitative establishment of such mutational signatures in normal tissue must become feasible, and may meanwhile provide a more accurate estimation of individual cancer risks, even in persons without familial antecedents. An additional advantage of this approach is that cancer risk assessment will not strictly rely on the individual’s genetic identity, but will also consider other factors (e.g., environmental and age) that can affect genomic integrity.

Two out of 41 non-small cell lung cancer patients enrolled in a clinical study were found with a somatic CRAF mutation in their tumor, namely CRAF P261A and CRAF P207S . To our knowledge, both mutations are novel in lung cancer and CRAF P261A has not been previously reported in cancer. Expression of CRAF P261A in HEK293T cells and BEAS-2B lung epithelial cells led to increased ERK pathway activation in a dimer-dependent manner, accompanied with loss of CRAF phosphorylation at the negative regulatory S259 residue. Moreover, stable expression of CRAF P261A in mouse embryonic fibroblasts and BEAS-2B cells led to anchorage-independent growth. Consistent with a previous report, we could not observe a gain-of-function with CRAF P207S . Type II but not type I RAF inhibitors suppressed the CRAF P261A -induced ERK pathway activity in BEAS-2B cells, and combinatorial treatment with type II RAF inhibitors and a MEK inhibitor led to a stronger ERK pathway inhibition and growth arrest. Our findings suggest that the acquisition of a CRAF P261A mutation can provide oncogenic properties to cells, and that such cells are sensitive to combined MEK and type II RAF inhibitors. CRAF mutations should be diagnostically and therapeutically explored in lung and perhaps other cancers.

Linkage analysis suggests that mutations in the BRCA1 and BRCA2 genes are responsible for cancer predisposition in more than 80% of the families with high incidence of breast/ovarian cancer. However, pathogenic mutations in the BRCA1/2 genes are generally identified in much less than half of the families investigated in a diagnostic setting with the currently used PCR‐based screening protocols. Here we report the identification of two different de novo Alu element insertions within the BRCA1/2 coding sequences in three out of the 50 families in which we found a cancer predisposing mutation, suggesting that this type of mutation is much more common than suggested by their occurrence in mutation databases. The Alu insertion in the BRCA2 gene resulted in the removal of the targeted exon from the corresponding mRNA molecule. Unexpectedly the Target Site Duplications generated by both Alu element insertions contained a specific 9 bp long segment, which might eventually serve as a recognition site for the transposition machinery. Finally, in contrast to the disease causing Alu insertions reported to date, the transposon identified in the BRCA1 gene does not belong to a “young” AluY but to an AluS subfamily, indicating that some of these “old” Alu elements, which are supposed to be non‐functional fossil relics, are still able to retrotranspose in vivo. © 2005 Wiley‐Liss, Inc.

Mutation and gene amplification of the epithelial growth factor receptor (EGFR) is one of the most common genetic alterations in glioblastoma (GB). EGFR is, therefore, an attractive molecular target for the treatment of GB. EGFR-targeted therapies however have been largely ineffective in clinical trials. In this study, we investigated the correlation between the EGFR gene amplification status, expression of the EGFR variant III (EGFRvIII) and EGFR variant IV (EGFRvIV) mutations, expression of the phosphatase and tensin homologue gene on chromosome 10 (PTEN) and mutation of the isocitrate dehydrogenase 1 (IDH1) gene and the survival of patients suffering from recurrent glioblastoma who were treated with the EGFR-targeted monoclonal antibody cetuximab in a prospective phase II clinical trial. EGFR amplification was detected in 19 out of 35 GB (54%), EGFRvIII expression in 11 (31.4%) and EGFRvIV expression in 7 (20%). The EGFRvIII and EGFRvIV mutations were exclusively found in GB with EGFR amplification and were almost mutually exclusive with IDH1 mutation (EGFRvIII mutation was found in 1 out of 11 GB with an IDH1 mutation). Patients with an EGFR amplification lacking EGFRvIII expression had a significantly superior progression free survival (PFS) and a numerical better overall survival (OS) following treatment with cetuximab [median PFS 3.03 vs. 1.63 months (p=0.006); median OS 5.57 vs. 3.97 months (p= 0.12)]. Within the subgroup of patients with EGFR amplification, patients with EGFRvIII positive glioblastoma had a worse survival [median PFS 1.63 vs. 3.03 months (p=0.01); median OS 3.27 vs. 5.57 months (p=0.08)]. Our observations indicate that the type of EGFR mutation may determine the outcome of GB patients treated with cetuximab. Prospective investigation of both the EGFR amplification and mutation status in clinical trials with EGFR-targeted therapies for GB is indicated.

To the Editor: Calin et al. (April 21 issue) 1 report on ARLTS1, a novel tumor-suppressor gene with proapoptotic characteristics and a member of the ADP-ribosylation factor family. It is proposed that a protein-truncating mutation (G446A) predisposes patients to cancer on the basis of the higher frequency of this allele among patients with familial cancer than among patients with sporadic tumors. The allele frequency was population-dependent, but when patient groups were compared with the control group, this heterogeneity was not accounted for. Using Fisher's exact test, we arrive at a P value of 0.56 instead of 0.02 when comparing familial and . . .

TO THE EDITOR: [George Adrian Calin] et al. identified the tumorsuppressor gene ARLTS1, a member of the ARF-ARL family, and showed that its G446A (Trp149Stop) variant predisposes patients to familial cancer.1 As a result, we investigated the influence of this nonsense mutation on the risk of familial breast cancer.

Background: Retrotransposons are genetic elements that jump within the genome via an RNA intermediate. Although they had a strong impact on human genome evolution, only a very tiny fraction of them can be reactivated nowadays, most often with neutral or detrimental consequences. The pathological outcomes associated with such genetic alterations are poorly investigated in the clinic, merely due to their difficult detection. Results: We developed a strategy to detect rare retrotransposon mediated insertions in Whole Exome Sequencing data from 65 familial breast cancer patients. When restricting our search to high confidence retrotransposition events occurring in less than 10% of the samples, we identified only ten different Alu elements, two L1 elements, one SVA and two processed pseudogenes. Only two of these insertions occurred within protein coding sequences and interestingly, several of the targeted genes have been previously linked to cancer, in three cases even to increased breast cancer risk (GHR, DMBT1 and NEK10). When investigating the molecular consequences of four Alu insertions at the mRNA level, we found that the element present in the 3′UTR of GHR repressed expression of the corresponding allele. Moreover, the analysis of a near exonic Alu insertion in PTPN14 (a mediator of P53 tumor suppressor activity) revealed that this gene was imprinted and that the presence of an intronic Alu element can lead to loss of imprinting. Conclusions: Our data underline the relevance of incorporating the search for uncommon retrotransposition events in Next Generation Sequencing pipelines when analyzing patients with a suspected genetic disease. Competing Interest Statement The authors have declared no competing interest.