Association of miR-21 and miR-335 to microsatellite instability and prognosis in stage III colorectal cancer.

BACKGROUND: MicroRNAs (miRs) are frequently altered in colorectal cancer (CRC) and can be used as prognostic factors. OBJECTIVE: To confirm in stage III CRC patients a reported miR signature that was associated to the presence of metastatic disease. To correlate miR expression with microsatellite instability (MSI) and mutations in RAS and BRAF. METHODS: miR-21, miR-135a, miR-206, miR-335 and miR-Let-7a expression was analyzed by RT-qPCR in 150 patients out of the 329 patients used to analyze MSI and RAS and BRAF mutations. Association with disease free survival (DFS) and overall survival (OS) was analyzed. Data was confirmed by a multivariate analysis. RESULTS: MiR-21 high expression (p= 0.034) and miR-335 low expression (p= 0.0061) were significantly associated with MSI-H. A positive trend (p= 0.0624) between miR-135a high expression and RAS mutations was found. Lower miR-21 expression levels are associated with DFS (HR = 2.654, 95% CI: 1.066-6.605, p= 0.036) and a trend with OS (HR = 2.419, 95% CI: 0.749-7.815, p= 0.140). MiR-21 high expression significantly improves DFS of the poor prognosis group (T4 or N2) (p= 0.03). CONCLUSIONS: Association of increased expression of miR-21 and better prognosis in the poor prognostic group may be of interest and could be explored in future prospective clinical trials.

BRIP1, a Gene Potentially Implicated in Familial Colorectal Cancer Type X.

Familial colorectal cancer Type X (FCCTX) comprises a heterogeneous group of families with an increased risk of developing colorectal cancer and other related tumors, but with mismatch repair-proficient, microsatellite-stable (MSS) tumors. Unfortunately, the genetic basis underlying their cancer predisposition remains unknown. Although pathogenic germline variants in BRIP1 increase the risk of developing hereditary ovarian cancer, the involvement of BRIP1 in hereditary colorectal cancer is still not well known. In order to identify new BRIP1 variants associated with inherited colorectal cancer, affected and nonaffected individuals from 18 FCCTX or high-risk MSS colorectal cancer families were evaluated by whole-exome sequencing, and another 62 colorectal cancer patients from FCCTX or high-risk MSS colorectal cancer families were screened by a next-generation sequencing (NGS) multigene panel. The families were recruited at the Genetic Counseling Unit of Hospital Clínico San Carlos of Madrid. A total of three different BRIP1 mutations in three unrelated families were identified. Among them, there were two frameshift variants [c.1702_1703del, p.(Asn568TrpfsTer9) and c.903del, p.(Leu301PhefsTer2)] that result in the truncation of the protein and are thus classified as pathogenic (class 5). The remaining was a missense variant [c.2220G>T, p.(Gln740His)] considered a variant of uncertain significance (class 3). The segregation and loss-of-heterozygosity studies provide evidence linking the two BRIP1 frameshift variants to colorectal cancer risk, with suggestive but not definitive evidence that the third variant may be benign. The results here presented suggest that germline BRIP1 pathogenic variants could be associated with hereditary colorectal cancer predisposition.Prevention Relevance: We suggest that BRIP1 pathogenic germline variants may have a causal role in CRC as moderate cancer susceptibility alleles and be associated with hereditary CRC predisposition. A better understanding of hereditary CRC may provide important clues to disease predisposition and could contribute to molecular diagnostics, improved risk stratification, and targeted therapeutic strategies.

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene.

Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T.

Association Between Baseline Circulating Tumor Cells, Molecular Tumor Profiling, and Clinical Characteristics in a Large Cohort of Chemo-naïve Metastatic Colorectal Cancer Patients Prospectively Collected.

BACKGROUND: Clinicopathologic characteristics and prognostic and predictive factors offer valuable guidance when selecting optimal first-line treatment in patients with metastatic colorectal cancer (CRC). The association between baseline circulating tumor cell (bCTC) count, molecular tumor profile, and clinicopathologic features was analyzed in a chemo-naïve metastatic CRC population. PATIENTS AND METHODS: A total of 1202 patients from the Spanish VISNÚ-1 (FOLFIRINOX/bevacizumab vs. FOLFOX/bevacizumab) and VISNÚ-2 (FOLFIRI/bevacizumab vs. FOLFIRI/cetuximab; RAS-wildtype) studies were analyzed for mutational status and bCTC count. The association between clinicopathologic characteristics and bCTC count, mutational status, and microsatellite instability (MSI) was analyzed in 589 eligible patients. RESULTS: Interestingly, 41% of the population studied presented ≥3 bCTC count. bCTC count ≥3 was associated with worse performance status (according Eastern Cooperative Oncology Group scale), stage IV at diagnosis, at least 3 metastatic sites, and elevated carcinoembryonic antigen (CEA) levels; but not with RAS or BRAF mutations or high MSI. BRAFmut (BRAF mutated) tumors were associated with right-sided primary tumors, peritoneum, distant lymph node metastasis, and less frequent liver involvement. RASmut (RAS mutated) was associated with worse performance status; stage IV at diagnosis; right-sided primary tumors; liver, lung, and bone metastases; at least 3 metastatic sites; and elevated CEA, whereas PIK3CAmut (PIK3CA mutated) tumors were associated with right-sided primary tumors, high CEA serum levels, and older age. High MSI was associated with right-sided primary tumors, distant lymph nodes metastasis, and lower CEA levels. CONCLUSIONS: In our study, elevated bCTCs and RASmut were associated with clinicopathologic features known to be associated with poor prognosis; whereas the poor prognosis of BRAFmut tumors in chemo-naïve metastatic CRC is not explained by associations with poor clinicopathologic prognostic factors, except right-sided primary tumors. TRIAL REGISTRATION NUMBER: VISNU 1 ClinicalTrials.gov ID: NCT01640405/ VISNU 2 ClinicalTrials.gov ID: NCT01640444.

Contribution of New Adenomatous Polyposis Predisposition Genes in an Unexplained Attenuated Spanish Cohort by Multigene Panel Testing.

Attenuated adenomatous polyposis (AAP) is a heterogeneous syndrome in terms of clinical manifestations, heritability and etiology of the disease. Genetic heterogeneity and low penetrance alleles are probably the best explanation for this variability. Certainly, it is known that APC and MUTYH are high penetrance predisposition genes for adenomatous polyposis, but they only account for 5-10% of AAP. Other new predisposition genes, such as POLE, POLD1, NTHL1, AXIN2 or MSH3, have been recently described and have been associated with AAP, but their relative contribution is still not well defined. In order to evaluate the genetic predisposition to AAP in a hospital based population, germline DNAs from 158 AAP subjects were screened for genetic variants in the coding regions and intron-exon boundaries of seven associated genes through a next-generation sequencing (NGS) custom gene panel. Splicing, segregation studies, somatic mutational screening and RNA quantitative expression assays were conducted for selected variants. In four of the probands the adenoma susceptibility could be explained by actionable mutations in APC or MUTYH, and one other patient was a double carrier of two truncating variants in both POLE and NTHL1. Furthermore, 16 additional patients harbored uncertain significance variants in the remaining tested genes. This report gives information about the contribution of the newly described adenomatous polyposis predisposition genes in a Spanish attenuated polyposis cohort. Our results highly support the convenience of NGS multigene panels for attenuated polyposis genetic screening and reveals POLE frameshift variants as a plausible susceptibility mechanism for AAP.

Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report.

BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.

Novel genetic mutations detected by multigene panel are associated with hereditary colorectal cancer predisposition.

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.

Role of GALNT12 in the genetic predisposition to attenuated adenomatous polyposis syndrome.

The involvement of GALNT12 in colorectal carcinogenesis has been demonstrated but it is not clear to what extent it is implicated in familial CRC susceptibility. Partially inactivating variant, NM_024642.4:c.907G>A, p.(D303N), has been previously detected in familial CRC and proposed as the causative risk allele. Since phenotypes of the described carrier families showed not only CRC but also a polyp history, we hypothesized that GALNT12 could be involved in adenoma predisposition and consequently, in hereditary polyposis CRC syndromes. For that purpose, we have screened the GALNT12 gene in germline DNA from 183 unrelated attenuated polyposis patients. c.907G>A, p.(D303N) was detected in 4 cases (MAF = 1.1%) and no other candidate variants were found. After segregation studies, LOH analyses, glycosylation pattern tests and case-control studies, our results did not support the role of c.907G>A, p.(D303N) as a high-penetrance risk allele for polyposis CRC.

A novel TP53 germline inframe deletion identified in a Spanish series of Li-fraumeni syndrome suspected families.

Li-Fraumeni syndrome (LFS) is an autosomal dominant, inherited tumor predisposition syndrome associated with heterozygous germline mutations in the TP53 gene. The molecular diagnosis of LFS is important to develop strategies for early detection and access to the genetic counseling. Our study evaluated germline TP53 mutations in Spanish families with a history suggestive of LFS. Germline TP53 alterations in 22 families with a history suggestive of LFS were evaluated by Sanger sequencing and multiplex ligation-dependent probe amplification. Loss of heterozygosity analysis and immunohistochemistry of the protein in the tumor were performed in order to evaluate the pathogenicity of a novel alteration detected. A total of seven TP53 mutations were detected, six point mutations (4 missense and 2 nonsense) and a novel inframe deletion. 93% of mutation carriers developed at least one malignancy (mainly breast cancer and sarcomas), with a mean age at diagnosis of the first tumor of 30.2 years. Two missense mutations acted as dominant-negative. The novel inframe mutation c.437_445del was located in the DNA-binding domain. This mutation segregated with cancer in the family, and both high expression of the protein and loss of the wild-type TP53 allele were detected in the tumor of the carrier. We have found a novel inframe deletion in TP53 that likely results in the loss of p53 function and acts in a non-dominant negative way, although further studies are necessary to clarify this issue. The identification of novel TP53 alterations is crucial for a personalized cancer-risk management of the Li-Fraumeni syndrome.

Prognostic Value of BRAF, PI3K, PTEN, EGFR Copy Number, Amphiregulin and Epiregulin Status in Patients with KRAS Codon 12 Wild-Type Metastatic Colorectal Cancer Receiving First-Line Chemotherapy with Anti-EGFR Therapy.

INTRODUCTION: Mutational analysis of RAS is required for anti-epidermal growth factor receptor (EGFR) treatment for patients with metastatic colorectal cancer (mCRC). However, most patients with KRAS wild-type tumors still do not respond. Other molecules downstream of the EGFR may also play a role in resistance to EGFR therapies. OBJECTIVE: Our objective was to investigate the clinical importance of biomarkers in relation to response, progression-free survival, and overall survival in patients with mCRC receiving first-line treatment with anti-EGFR therapy plus chemotherapy. METHODS: We studied the EGFR pathway [EGFR, NRAS, BRAF, PIK3CA, phosphatase and tensin homolog (PTEN), amphiregulin (AREG), and epiregulin (EREG)] in 105 patients with mCRC KRAS codon 12 wild type. We analysed objective response, progression-free survival, and overall survival in molecularly defined subgroups of the patients receiving anti-EGFR therapy plus chemotherapy as first-line treatment. RESULTS: We found a significant association between RAS wild-type, BRAF wild-type, EREG, and AREG overexpression and response to anti-EGFR therapy (p = 0.003, p = 0.015, p = 0.05, and p = 0.009, respectively). Progression-free survival and overall survival were lower in patients with RAS (p = 0.36 and p ≤ 0.001, respectively) or BRAF (p = 0.003 and p = 0.002, respectively) mutant tumors. Patients with EREG and AREG messenger RNA (mRNA) expression had longer survival than those with low-expression tumors; progression-free survival and overall survival were significant for AREG (p = 0.001 and p = 0.05, respectively). Patients with EGFR amplification tumors responded better to treatment and had better survival rates, although this was not significant. PIK3CA and PTEN were not associated with either response or survival. The multivariate logistic regression model for response showed only BRAF as a significant predictor after adjustment for the other covariates (p = 0.04, odds ratio 8.3, 95 % confidence interval 0.81-86.0). CONCLUSIONS: RAS, BRAF, AREG, and EREG predict for efficacy of first-line anti-EGFR therapy in patients with mCRC.

Cancer risk and overall survival in mismatch repair proficient hereditary non-polyposis colorectal cancer, Lynch syndrome and sporadic colorectal cancer.

Mismatch repair proficient hereditary non-polyposis colorectal cancer (MSS-HNPCC) encloses a heterogeneous group of families consisting of different unknown genetic syndromes and/or aggregations cases. The lack of information about the hereditability of cancer risk in these families makes it difficult to carry out an individualized Genetic Counseling. Therefore, deep description of such families becomes important for a better classification and search for underlying susceptibility causes. The aim of this study is to describe and compare the clinical, morphological features, tumor KRAS status and overall survival in MSS-HNPCC, Lynch and sporadic colorectal cancer. A total of 37 MSS-HNPCC families, 50 Lynch families and 612 sporadic CRC were included. Clinical and morphological data were evaluated by reviewing medical and pathology reports of 55, 69 and 102 tumors respectively. KRAS/BRAF status were detected by allele specific real-time PCR. Standardized incidence ratios (SIR) were calculated among 602 MSS-HNPCC relatives and 668 Lynch relatives. Main features distinguishing MSS-HNPCC were diagnosis age (55.1 ± 12.6), preferential distal location (76%), polyp detection (45%) and familial colorectal cancer incidence (SIR = 6.6). In addition, we found increased incidences rates for kidney, stomach and uterus tumors. KRAS mutation rates were similar in the study populations (48.8 ± 5.8) but higher than those described before by Sanger sequencing. MSS-HNPCC overall survival was similar to Lynch in B Dukes' stage tumors and between Lynch and sporadic in C stage tumors. Anatomical and morphological data of MSS-HNPCC are consistent with other described populations. Our studies disclose an increased HNPCC-extracolonic tumors incidence and improved overall survival in MSS-HNPCC families.

Frequency and variability of genomic rearrangements on MSH2 in Spanish Lynch Syndrome families.

Large genomic rearrangements (LGRs) in DNA-mismatch-repair (MMR) genes, particularly among MSH2 gene, are frequently involved in the etiology of Lynch syndrome (LS). The Multiplex Ligation and Probe Amplification assay (MLPA) is commonly used to identify such alterations. However, in most cases, the MLPA-identified alteration is not characterized at the molecular level, which might be important to identify recurrent alterations and to analyze the molecular mechanisms underlying these mutational events. Probands from a cohort of Lynch Syndrome families were screened for point mutation in MMR genes, subsequently the MLPA assay was used for LGR screening. The identified MLPA alteration was confirmed by cDNA, CGH-microarrays or massive parallel sequencing. In this study, we have delimited the region of 11 LGRs variants on MSH2 locus. Six of them were fully characterized the breakpoints and 9 of them were considered pathogenic. According to our data, LGR on MSH2 locus constituted the 10.8% (9 out of 83) of pathogenic germline alterations found in LS. The frequency of colorectal cancer (CRC) and endometrial cancer (EC) in LGR carriers was 55% and 11% respectively. Analysis of the breakpoint sequences revealed that in 3 cases, deletions appeared to originate from Alu-mediated recombination events. In the remaining cases, sequence alignment failed to detect microhomology around the breakpoints. The present study provides knowledge on the molecular characterization of MSH2 LGRs, which may have important implications in LS diagnosis and Genetic Counseling. In addition, our data suggests that nonhomologous events would be more frequently involved in the etiology of MSH2 LGRs than expected.

Study of KRAS new predictive marker in a clinical laboratory.

BACKGROUND: The presence of somatic mutations in the KRAS gene has been identified as a reliable strong negative predictor for the response to targeting the epidermal growth factor receptor (EGFR), in patients with metastatic colorectal cancer and the use of anti-EGFR monoclonal antibodies such as Cetuximab and Panitumumab is now restricted to patients with no detectable KRAS mutations. Between 30 and 40 % of colorectal cancers contain a mutated KRAS oncogene. The aim of this study was to evaluate concordance between three methods to analyze KRAS mutational status in regard to clinical testing. METHODS: We analyzed KRAS mutations in codons 12 and 13 of exon 2 in one hundred formalin-fixed paraffin-embedded (FFPE) colorectal cancer samples by three different methods: Direct Sequencing and two commercial kits on allele-specific oligonucleotide hybridization (KRAS StripAssay, Vienna Lab.) and Amplification Refractory Mutation System/Scorpions (ARMS/S; TheraScreen KRAS Mutation kit DxS) based on q-PCR. RESULTS: We have found similar frequencies of KRAS mutations by TheraScreen and Strip-Assay (44 and 48 %), with a κ value of 0.90, indicating almost perfect agreement between methods. The frequency by direct sequencing was much lower (26 %) and the κ values were 0.67 (compared to TheraScreen) and 0.57 (compared to Strip-Assay) indicating low sensitivity. CONCLUSIONS: On analyzing KRAS mutation in FFPE tumor samples, direct sequencing sensitivity is too low to be used in a clinical setting. Choosing between ARMS/S; TheraScreen KRAS Mutation kit DxS and KRAS StripAssay, Vienna Lab, will depend on laboratory facilities and expertise.

Analysis of the oxidative damage repair genes NUDT1, OGG1, and MUTYH in patients from mismatch repair proficient HNPCC families (MSS-HNPCC).

PURPOSE: Several studies have described molecular differences between microsatellite stable hereditary nonpolyposis colorectal cancer (MSS-HNPCC) and microsatellite unstable Lynch syndrome tumors (MSI-HNPCC). These differences highlight the possibility that other instability forms could explain cancer susceptibility in this group of families. The base excision repair (BER) pathway is the major DNA repair pathway for oxidative DNA damage. A defect in this pathway can result in DNA transversion mutations and a subsequent increased cancer risk. Mutations in MUTYH have been associated with increased colorectal cancer (CRC) risk while no association has been described for OGG1 or NUDT1. EXPERIMENTAL DESIGN: We performed mutational screening of the three genes involved in defense against oxidative DNA damage in a set of 42 MSS-HNPCC families. RESULTS: Eight rare variants and 5 frequent variants were found in MSS-HNPCC patients. All variants were previously described by other authors except variant c.285C>T in OGG1. Segregation studies were done and in silico programs were used to estimate the level of amino acid conservation, protein damage prediction, and possible splicing alterations. Variants OGG1 c.137G>A; MUTYH c.1187G>A were detected in Amsterdam I families and cosegregate with cancer. Analysis of OGG1 c.137G>A transcripts showed an inactivation of the splicing donor of exon 1. CONCLUSIONS: Two rare variants (OGG1 c.137G>A; MUTYH c.1187G>A) and one common polymorphism (NUDT1 c.426C>T) were associated with CRC risk. We show that the BER pathway can play a significant role in a number of MSS-HNPCC colorectal cancers. More studies could be of interest in order to gain further understanding of yet unexplained CRC susceptibility cases.