Identification of RAD17 as a candidate cancer predisposition gene in families with histories of pancreatic and breast cancers.

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.

The genomic landscape of nonsmall cell lung carcinoma in never smokers.

Lung cancer is the number one cause of cancer-related death worldwide with cigarette smoking as its major risk factor. Although the incidence of lung cancer in never smokers is rising, this subgroup of patients is underrepresented in genomic studies of lung cancer. Here, we assembled a prospective cohort of 46 never-smoking, nonsmall cell lung cancer (NSCLC) patients and performed whole-exome and low-coverage whole-genome sequencing on tumors and matched germline DNA. We observed fewer somatic mutations, genomic breakpoints and a smaller fraction of the genome with chromosomal instability in lung tumors from never smokers compared to smokers. The lower number of mutations, enabled us to identify TSC22D1 as a potential driver gene in NSCLC. On the other hand, the frequency of mutations in actionable genes such as EGFR and ERBB2 and of amplifications in MET were higher, while the mutation rate of TP53, which is a negative prognostic factor, was lower in never smokers compared to smokers. Together, these observations suggest a more favorable prognosis for never smokers with NSCLC. Classification of somatic mutations into six-substitution type patterns or into 96-substitution type signatures revealed distinct clusters between smokers and never smokers. Particularly, we identified in never smokers signatures related to aging, homologous recombination damage and APOBEC/AID activity as the most important underlying processes of NSCLC. This further indicates that second-hand smoking is not driving NSCLC pathogenesis in never smokers.

Identification of candidate cancer predisposing variants by performing whole-exome sequencing on index patients from BRCA1 and BRCA2-negative breast cancer families.

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.

Systematic detection of pathogenic alu element insertions in NGS-based diagnostic screens: the BRCA1/BRCA2 example.

Pathogenic Alu element insertions are rarely reported, whereas their occurrence is expected to be much higher. Alu containing alleles are usually out-competed during the PCR process and consequently undetectable with the classical screening methods. However, with the introduction of the next generation sequencing (NGS) technology in the diagnostic field, new opportunities are emerging. NGS data for a particular genomic region can be seen as the summation of all the individual sequences (reads) obtained for that region and no longer as the mean of this sum as it is the case for traditional Sanger sequencing. Because each single read covering that region is expected to be generated from a different template molecule, the presence of one single mutant read must theoretically be sufficient to identify the mutation. However, generation and identification of mutant reads bearing Alu insertions remains challenging and several wet/dry bench parameters need to be optimized. Hereby we present the proof of principle of a NGS-based mutation screening procedure allowing the detection of inherited Alu insertions within any predefined sequence by investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2.

Cancer predisposing missense and protein truncating BARD1 mutations in non-BRCA1 or BRCA2 breast cancer families.

Fifteen years ago BRCA1 and BRCA2 were reported as high penetrant breast cancer predisposing genes. However, mutations in these genes are found in only a fraction of high risk families. BARD1 is a candidate breast cancer gene, but only a limited number of missense mutations with rather unclear pathogenic consequences have been reported.We screened 196 high risk breast cancer families for the occurrence of BARD1 variants. All genetic variants were analyzed using clinical information as well as IN SILICO predictive tools, including protein modeling. We found three candidate pathogenic mutations in seven families including a first case of a protein truncating mutation (p.Glu652fs) removing the entire second BRCT domain of BARD1. In conclusion, we provide evidence for an increased breast cancer risk associated to specific BARD1 germline mutations. However, these BARD1 mutations occur in a minority of hereditary breast cancer families.

CRAF mutations in lung cancer can be oncogenic and predict sensitivity to combined type II RAF and MEK inhibition.

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 CRAFP261A and CRAFP207S. To our knowledge, both mutations are novel in lung cancer and CRAFP261A has not been previously reported in cancer. Expression of CRAFP261A 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 CRAFP261A 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 CRAFP207S. Type II but not type I RAF inhibitors suppressed the CRAFP261A-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 CRAFP261A 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.

Hereditary breast cancer: from bench to bedside.

PURPOSE OF REVIEW: The proportion of breast cancers directly attributable to determinant hereditary factors is estimated to be 5-10%. A number of recent findings with regard to hereditary breast cancer should affect the criteria and scope of routine genetic testing and, soon, breast cancer therapy. RECENT FINDINGS: The number of genes causing genetic cancer has expanded, mostly with genes that encode proteins that function in the BRCA1/2 pathways. The risk level associated with some genes is still under investigation, but is high for specific mutations. Some mutant alleles occur frequently, some are rare. High-throughput technologies will progressively allow investigating all genes involved in genetic (breast) cancer risks in all individuals for whom this information could be relevant. This and the emerging novel treatment options specific for cancers in mutation carriers will oblige us to progressively drop all currently used selection criteria such as familial phenotype for genomic testing. A major challenge remains the effective penetration of this knowledge in the professional and lay community, the broad application and financing of this high-throughput technology, and the identification of as yet unknown breast cancer predisposition genes. SUMMARY: The assessment of breast cancer predisposition genes, previously only an optional predictive genetic test, is growing in importance as it also becomes a therapeutic predictive test.

Type II RAF inhibitor causes superior ERK pathway suppression compared to type I RAF inhibitor in cells expressing different BRAF mutant types recurrently found in lung cancer.

A large fraction of somatic driver BRAF mutations in lung cancer are non-V600 and impaired-kinase. Non-V600 BRAF mutations predict sensitivity to combination of a type I RAF inhibitor, Dabrafenib, and a MEK inhibitor, Trametinib. Singly, Dabrafenib only weakly suppresses mutant BRAF-induced ERK signaling and can induce ERK paradoxical activation in CRAF-overexpressing cells. The present study compared the effects of Dabrafenib and a type II RAF inhibitor, AZ628, on ERK activity in HEK293T cells expressing several tumor-derived BRAF mutants, and in a non-V600 and impaired-kinase BRAF-mutant lung cancer cell line (H1666). Unlike Dabrafenib, AZ628 did not induce paradoxical ERK activation in CRAF-overexpressing cells and BRAF-mutant cells overexpressing CRAF were more responsive to AZ628 compared to Dabrafenib in terms of ERK inhibition. AZ628 inhibited ERK more effectively than Dabrafenib in both H1666 cells and HEK293T cells co-expressing several different BRAF-mutants with CRAF. Similarly, AZ628 plus Trametinib had better MEK-inhibitory and pro-apoptotic effects in H1666 cells than Dabrafenib plus Trametinib. Moreover, prolonged treatment of H1666 cells with AZ628 plus Trametinib produced greater inhibition of cell growth than Dabrafenib plus Trametinib. These results indicate that AZ628 has greater potential than Dabrafenib, both as a single agent and combined with Trametinib, for the treatment of non-V600 BRAF mutant lung cancer.

Loss of nuclear BRCA1 protein staining in normal tissue cells derived from BRCA1 and BRCA2 mutation carriers.

Enhanced genomic instability has been recently reported in normal cells derived from BRCA1/2 mutation carriers when placed in vitro in non-physiological stress conditions. We present here original data which help to explain the observed genomic instability. Leucocytes from BRCA1/2 mutation carriers, sporadic breast cancer patients and controls were prepared for BRCA1 immunocytochemistry. We show that BRCA1 containing nuclear dot like structures are detectable in about 80% of the leucocytes from controls and sporadic breast cancer patients, but are absent in the majority of normal cells from BRCA1 as well as BRCA2 mutation carriers (also in their normal breast cells). Our results thus indicate that the genomic instability observed in normal cells from BRCA1 and BRCA2 mutation carriers is associated with a down-regulation of nuclear BRCA1 protein accumulation in the dot like structures. These results suggest in addition that immunocytochemical or alternative molecular screening strategies might help to identify women with a high risk for breast (ovarian) cancer even when the underlying genetic defect remains undetectable.

An alternative model for (breast) cancer predisposition.

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.

Non-V600 BRAF mutations recurrently found in lung cancer predict sensitivity to the combination of Trametinib and Dabrafenib.

Approximately half of BRAF-mutated Non-small cell lung cancers (NSCLCs) harbor a non-V600 BRAF mutation, accounting for ∼40,000 annual deaths worldwide. Recent studies have revealed the benefits of combined targeted therapy with a RAF-inhibitor (Dabrafenib) and a MEK-inhibitor (Trametinib) in treating V600 BRAF mutant cancers, including NSCLC. In contrast, sensitivity of non-V600 BRAF mutations to these inhibitors is not documented. Non-V600 mutations can either increase or impair BRAF kinase activity. However, impaired BRAF kinases can still activate the ERK pathway in a CRAF-dependent manner. Herein, beyond describing a cohort of BRAF mutant NSCLC patients and functionally analyzing 13 tumor-derived BRAF mutations, we demonstrate that both types of non-V600 BRAF mutations can be sensitive to clinically relevant doses of Dabrafenib and Trametinib in HEK293T cells, in lung epithelial cellular model (BEAS-2B) and in human cancer cell lines harboring non-V600 BRAF mutations. ERK activity induced by both types of these mutations is further reduced by combinatorial drug treatment. Moreover, the combination leads to more prolonged ERK inhibition and has anti-proliferative and pro-apoptotic effects in cells harboring both types of non-V600 BRAF mutations. This study provides a basis for the clinical exploration of non-V600 BRAF mutant lung cancers upon treatment with Trametinib and Dabrafenib.

De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes.

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.

Detection of EGFR-TK domain-activating mutations in NSCLC with generic PCR-based methods.

Somatic mutations in the epidermal growth factor receptor-tyrosine kinase (EGFR-TK) domain of non-small cell lung cancer (NSCLC) influence the responsiveness of these tumors to EGFR-TK inhibitors, indicating their usefulness as a predictive molecular marker. However, for mutation analysis, the amount of clinical material available from NSCLC patients is often very limited, suboptimally preserved, and composed of both normal and tumor cells. As a consequence, the total amount of recovered DNA is frequently very limited, with mutant alleles being often strongly underrepresented, and thus requiring highly sensitive methods for the detection of mutations. In the present study, EGFR mutation screening was performed on 210 NSCLC clinical samples by heminested polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE). Candidate mutations were further characterized by sequencing. Seventeen different types of pathogenic EGFR-TK domain mutations were detected in 55 of the 210 samples (26%). We reanalyzed 149 of the 155 samples in which no mutation was found by real-time PCR for the presence of recurrent exon 21 and exon 19 mutations using peptide nucleic acid probes in the PCR mix to increase sensitivity by mutant allele enrichment. Four additional samples with exon 19 mutations were detected. Thus, it is found that the relatively simple and inexpensive PCR-DGGE assay is already very sensitive for the detection of mutations in clinical samples, including samples with low tumor cellularity (10% or higher tumor cell content), although the sensitivity and speed of the assay can be further increased for a restricted panel of mutations by introducing peptide nucleic acid probes in the DGGE or real-time PCR-based assay.

Biomarker Analysis in a Phase II Study of Sunitinib in Patients with Advanced Melanoma.

AIM: To investigate the efficacy of sunitinib in patients with advanced melanoma and to correlate angiogenic biomarkers with response and survival. PATIENTS AND METHODS: We performed a phase II study in patients with advanced pre-treated melanoma. The primary endpoint was tumor response. Blood samples for biomarker analysis including vascular endothelial growth factor (VEGF), and its receptors VEGFR1 and -2, placental growth factor (PlGF) and circulating endothelial cells (CEC) were collected at baseline and during the first cycle. RESULTS: Four out of 39 patients (13%) achieved a partial response and eight (26%) stable disease. Time to progression was at least six months in seven patients. High baseline VEGFR1 levels and high baseline PlGF levels were both associated with a non-significant worse survival (p=0.08 for both). CONCLUSION: Sunitinib demonstrates limited activity in unselected patients with refractory advanced melanoma, but a minority of patients experienced long-term disease control. Identification of these patients remains a challenge.

Correlation of EGFR, IDH1 and PTEN status with the outcome of patients with recurrent glioblastoma treated in a phase II clinical trial with the EGFR-blocking monoclonal antibody cetuximab.

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.