Half of the high-risk colorectal cancer families that fulfill the clinical criteria for Lynch syndrome lack germline mutations in the mismatch repair (MMR) genes and remain unexplained. Genetic ...testing for hereditary cancers is rapidly evolving due to the introduction of multigene panels, which may identify more mutations than the old screening methods. The aim of this study is the use of a Next Generation Sequencing panel in order to find the genes involved in the cancer predisposition of these families. For this study, 98 patients from these unexplained families were tested with a multigene panel targeting 94 genes involved in cancer predisposition. The mutations found were validated by Sanger sequencing and the segregation was studied when possible. We identified 19 likely pathogenic variants in 18 patients. Out of these, 8 were found in MMR genes (5 in MLH1, 1 in MSH6 and 2 in PMS2). In addition, 11 mutations were detected in other genes, including high penetrance genes (APC, SMAD4 and TP53) and moderate penetrance genes (BRIP1, CHEK2, MUTYH, HNF1A and XPC). Mutations c.1194G>A in SMAD4, c.714_720dup in PMS2, c.2050T>G in MLH1 and c.1635_1636del in MSH6 were novel. In conclusion, the detection of new pathogenic mutations in high and moderate penetrance genes could contribute to the explanation of the heritability of colorectal cancer, changing the individual clinical management. Multigene panel testing is a more effective method to identify germline variants in cancer patients compared to single-gene approaches and should be therefore included in clinical laboratories.
The identification of the two most prevalent susceptibility genes in breast cancer, BRCA1 and BRCA2, was the beginning of a sustained effort to uncover new genes explaining the missing heritability ...in this disease. Today, additional high, moderate and low penetrance genes have been identified in breast cancer, such as P53, PTEN, STK11, PALB2 or ATM, globally accounting for around 35 percent of the familial cases. In the present study we used massively parallel sequencing to analyze 7 BRCA1/BRCA2 negative families, each having at least 6 affected women with breast cancer (between 6 and 10) diagnosed under the age of 60 across generations. After extensive filtering, Sanger sequencing validation and co-segregation studies, variants were prioritized through either control-population studies, including up to 750 healthy individuals, or case-control assays comprising approximately 5300 samples. As a result, a known moderate susceptibility indel variant (CHEK2 1100delC) and a catalogue of 11 rare variants presenting signs of association with breast cancer were identified. All the affected genes are involved in important cellular mechanisms like DNA repair, cell proliferation and survival or cell cycle regulation. This study highlights the need to investigate the role of rare variants in familial cancer development by means of novel high throughput analysis strategies optimized for genetically heterogeneous scenarios. Even considering the intrinsic limitations of exome resequencing studies, our findings support the hypothesis that the majority of non-BRCA1/BRCA2 breast cancer families might be explained by the action of moderate and/or low penetrance susceptibility alleles.
Colon cancer patients with the same stage show diverse clinical behavior due to tumor heterogeneity. We aimed to discover distinct classes of tumors based on microarray expression patterns, to ...analyze whether the molecular classification correlated with the histopathological stages or other clinical parameters and to study differences in the survival.
Hierarchical clustering was performed for class discovery in 88 colon tumors (stages I to IV). Pathways analysis and correlations between clinical parameters and our classification were analyzed. Tumor subtypes were validated using an external set of 78 patients. A 167 gene signature associated to the main subtype was generated using the 3-Nearest-Neighbor method. Coincidences with other prognostic predictors were assesed.
Hierarchical clustering identified four robust tumor subtypes with biologically and clinically distinct behavior. Stromal components (p < 0.001), nuclear β-catenin (p = 0.021), mucinous histology (p = 0.001), microsatellite-instability (p = 0.039) and BRAF mutations (p < 0.001) were associated to this classification but it was independent of Dukes stages (p = 0.646). Molecular subtypes were established from stage I. High-stroma-subtype showed increased levels of genes and altered pathways distinctive of tumour-associated-stroma and components of the extracellular matrix in contrast to Low-stroma-subtype. Mucinous-subtype was reflected by the increased expression of trefoil factors and mucins as well as by a higher proportion of MSI and BRAF mutations. Tumor subtypes were validated using an external set of 78 patients. A 167 gene signature associated to the Low-stroma-subtype distinguished low risk patients from high risk patients in the external cohort (Dukes B and C:HR = 8.56(2.53-29.01); Dukes B,C and D:HR = 1.87(1.07-3.25)). Eight different reported survival gene signatures segregated our tumors into two groups the Low-stroma-subtype and the other tumor subtypes.
We have identified novel molecular subtypes in colon cancer with distinct biological and clinical behavior that are established from the initiation of the tumor. Tumor microenvironment is important for the classification and for the malignant power of the tumor. Differential gene sets and biological pathways characterize each tumor subtype reflecting underlying mechanisms of carcinogenesis that may be used for the selection of targeted therapeutic procedures. This classification may contribute to an improvement in the management of the patients with CRC and to a more comprehensive prognosis.
PURPOSE Earlier studies have shown that endogenous gonadal hormones play an important role in the etiology of breast cancer among BRCA1/2 mutation carriers. So far, little is known about the safety ...of exogenous hormonal use in mutation carriers. In this study, we examined the association between oral contraceptive use and risk of breast cancer among BRCA1/2 carriers. PATIENTS AND METHODS In the International BRCA1/2 Carrier Cohort study (IBCCS), a retrospective cohort of 1,593 BRCA1/2 mutation carriers was analyzed with a weighted Cox regression analysis. Results We found an increased risk of breast cancer for BRCA1/2 mutation carriers who ever used oral contraceptives (adjusted hazard ratio HR = 1.47; 95% CI, 1.16 to 1.87). HRs did not vary according to time since stopping use, age at start, or calendar year at start. However, a longer duration of use, especially before first full-term pregnancy, was associated with an increased risk of breast cancer for both BRCA1 and BRCA2 mutation carriers (4 or more years of use before first full-term pregnancy: HR = 1.49 95% CI, 1.05 to 2.11 for BRCA1 carriers and HR = 2.58 95% CI, 1.21 to 5.49 for BRCA2 carriers). CONCLUSION No evidence was found among BRCA1/2 mutation carriers that current use of oral contraceptives is associated with risk of breast cancer more strongly than is past use, as is found in the general population. However, duration of use, especially before first full-term pregnancy, may be associated with an increasing risk of breast cancer among both BRCA1 and BRCA2 mutation carriers.
Anthracyclines are frequently used for the treatment of breast cancer and topoisomerase II alpha ( TOP2A ) is considered to be the molecular target. Numerous studies have evaluated the predictive ...value of TOP2A using different methodological approaches and inconsistent results have been reported. Indeed, the correlation between techniques for the assessment of TOP2A status has not been well evaluated. In this study, we determined TOP2A status in 61 breast tumor samples by real-time PCR, DNA microarrays, immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH), and then evaluated these results with clinical-pathological features and breast cancer intrinsic subtypes. First, we observed a statistical significant correlation of TOP2A gene expression between real-time PCR and microarrays (Pearson coefficient, 0.816; P < 0.001), and both predicted TOP2A IHC results fairly well (area under the curve > 0.74). In contrast, poor agreement between FISH and IHC data was observed (k: 0.134). Secondly, TOP2A expression was found significantly associated with cell proliferation, and with the highly proliferative Luminal B, Her2-enriched and Basal-like intrinsic subtypes. In conclusion, TOP2A expression in breast cancer was associated with high proliferation and aggressive tumor subtypes and appears to be independent of its amplification status. All of these features should be taken into consideration when assessing the predictive value of TOP2A for anthracycline-based chemotherapy.
The present article summarizes recent developments in the characterization of genetic predisposition to colorectal cancer (CRC). The main themes covered include new hereditary CRC and polyposis ...syndromes, non-CRC hereditary cancer genes found mutated in CRC patients, strategies used to identify novel causal genes, and review of candidate genes that have been proposed to predispose to CRC and/or colonic polyposis. We provide an overview of newly described genes and syndromes associated with predisposition to CRC and polyposis, including: polymerase proofreading-associated polyposis, NTHL1-associated polyposis, mismatch repair gene biallelic inactivation-related adenomatous polyposis (including MSH3- and MLH3-associated polyposes), GREM1-associated mixed polyposis, RNF43-associated serrated polyposis, and RPS20 mutations as a rare cause of hereditary nonpolyposis CRC. The implementation of next generation sequencing approaches for genetic testing has exposed the presence of pathogenic germline variants in genes associated with hereditary cancer syndromes not traditionally linked to CRC, which may have an impact on genetic testing, counseling and surveillance. The identification of new hereditary CRC and polyposis genes has not deemed an easy endeavor, even though known CRC-related genes explain a small proportion of the estimated familial risk. Whole-genome sequencing may offer a technology for increasing this proportion, particularly if applied on pedigree data allowing linkage type of analysis. The final section critically surveys the large number of candidate genes that have been recently proposed for CRC predisposition.
Exosome production from cancer-associated fibroblasts seems to be an important driver of tumor progression. We report the first in-depth biotype characterization of ncRNAs, analyzed by Next ...Generation Sequencing and Bioinformatics, expressed in established primary human normal and cancer-associated fibroblasts (CAFs) from cancer and normal mucosa tissues from 9 colorectal cancer patients, and/or packaged in their derived exosomes. Differential representation and enrichment analyses based on these ncRNAs revealed a significant number of differences between the ncRNA content of exosomes and the expression patterns of the normal and cancer-associated fibroblast cells. ncRNA regulatory elements are specifically packaged in CAF-derived exosomes, supporting a specific cross-talk between CAFs and colon cancer cells and/or other stromal cells, mediated by exosomes. These sncRNAs are potential biomarkers present in cancer-associated fibroblast-derived exosomes, which should thereby contribute to developing new non-invasive diagnostic, prognostic and predictive methods for clinical applications in management of cancer patients.
Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family ...with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene ( FAN1 ), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.
A subset of colorectal cancers (CRC) arises in families that, despite fulfilling clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC), do not show evidence of a mismatch repair ...(MMR) deficiency. The main objective of this study was to characterize these tumors at the molecular level.
After comprehensive germ line mutation scanning, microsatellite analysis, and MMR protein expressions, we selected a well-defined cohort of 57 colorectal tumors with no evidence of MMR defects. In this group of tumors, we analyzed KRAS, BRAF, and APC somatic mutations, as well as methylguanine methyltransferase (MGMT) and beta-catenin expression. We correlated these alterations with clinicopathologic data and explored the relationship between KRAS G > A transitions and lack of MGMT expression.
The mutation profile at the RAS/RAF/MAPK pathway mimics sporadic microsatellite-stable CRCs. We found an average age of diagnosis 10 years older in KRAS-mutated patients (P = 0.001). In addition, we show that KRAS G > A transitions are actively selected by tumors, regardless of MGMT status. Similarities with HNPCC high-microsatellite instability tumors are observed when APC data are analyzed. The APC mutation rate was low and small insertions/deletions accounted for 70% of the alterations. In addition, we found a low frequency of beta-catenin nuclear staining. Finally, we did not find evidence of tumors arising in individuals from the same family sharing molecular features.
We show evidence that CRC tumors arising in HNPCC families without MMR alterations have distinctive molecular features. Overall, our work shows that systematic analysis of somatic alterations in a well-defined subset of CRCs is a good approach to provide new insights into the mechanisms of colorectal carcinogenesis.
RAD51C was defined by Meindl et al. in 2010 as a high-risk gene involved in hereditary breast and ovarian cancers. Although this role seems to be clear, nowadays there is controversy about the ...indication of including the gene in routine clinical genetic testing, due to the lower prevalence or the absence of mutations found in subsequent studies. Here, we present the results of a comprehensive mutational screening of the RAD51C gene in a large series of 785 Spanish breast and/or ovarian cancer families, which, in contrast to the various subsequent studies published to date, includes the functional characterization of suspicious missense variants as reported in the initial study. We have detected 1.3% mutations of RAD51C in breast and ovarian cancer families, while mutations in breast cancer only families seem to be very rare. More than half of the deleterious variants detected were of missense type, which highlights their significance in the gene, and suggest that RAD51C mutations may have been so far partially disregarded and their prevalence underestimated due to the lack of functional complementation assays. Our results provide new evidences, suggesting that the genetic testing of RAD51C should be considered for inclusion into the clinical setting, at least for breast and ovarian cancer families, and encourage re-evaluating its role incorporating functional assays.
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