Carla Pinto

Comparison of methodologies for KRAS mutation detection in metastatic colorectal cancer

Cetuximab and panitumumab are two monoclonal antibodies targeting the epidermal growth factor receptor that have been approved for treatment of metastatic colorectal cancer. Recent clinical trials found an association between KRAS mutation status and resistance to anti-epidermal growth factor receptor therapy, leading to the recommendation to perform KRAS mutation analysis before cetuximab or panitumumab treatment. This study was designed to compare and evaluate the efficacy of four different methodologies--high resolution melting, Sanger sequencing, DxS kit, and SNaPshot--for KRAS mutation detection in a clinical setting. In total, 372 samples from patients with metastatic colorectal cancer were analyzed by high resolution melting and SNaPshot, with 184 of those being further analyzed by Sanger sequencing and 188 with the DxS kit. Sensitivities were compared after consensus findings were determined by the presence of the same result in two of the three methodologies used in each case. The frequency of KRAS codon 12 and 13 mutations in our population was 43.5%, and a discordant finding was observed in 22 samples. Comparing to Sanger sequencing, significantly more consensus mutations were detected by the DxS kit (P=0.0139), high resolution melting (P=0.0004), and SNaPshot (P=0.00001), but no statistically significant differences were found among the three methodologies with higher sensitivity.

Colorectal carcinomas with microsatellite instability display a different pattern of target gene mutations according to large bowel site of origin

BackgroundOnly a few studies have addressed the molecular pathways specifically involved in carcinogenesis of the distal colon and rectum. We aimed to identify potential differences among genetic alterations in distal colon and rectal carcinomas as compared to cancers arising elsewhere in the large bowel.MethodsConstitutional and tumor DNA from a test series of 37 patients with rectal and 25 patients with sigmoid carcinomas, previously analyzed for microsatellite instability (MSI), was studied for BAX, IGF2R, TGFBR2, MSH3, and MSH6 microsatellite sequence alterations, BRAF and KRAS mutations, and MLH1 promoter methylation. The findings were then compared with those of an independent validation series consisting of 36 MSI-H carcinomas with origin from each of the large bowel regions. Immunohistochemical and germline mutation analyses of the mismatch repair system were performed when appropriate.ResultsIn the test series, IGFR2 and BAX mutations were present in one and two out of the six distal MSI-H carcinomas, respectively, and no mutations were detected in TGFBR2, MSH3, and MSH6. We confirmed these findings in the validation series, with TGFBR2 and MSH3 microsatellite mutations occurring less frequently in MSI-H rectal and sigmoid carcinomas than in MSI-H colon carcinomas elsewhere (P = 0.00005 and P = 0.0000005, respectively, when considering all MSI-carcinomas of both series). No MLH1 promoter methylation was observed in the MSI-H rectal and sigmoid carcinomas of both series, as compared to 53% found in MSI-H carcinomas from other locations (P = 0.004). KRAS and BRAF mutational frequencies were 19% and 43% in proximal carcinomas and 25% and 17% in rectal/sigmoid carcinomas, respectively.ConclusionThe mechanism and the pattern of genetic changes driving MSI-H carcinogenesis in distal colon and rectum appears to differ from that occurring elsewhere in the colon and further investigation is warranted both in patients with sporadic or hereditary disease.

High resolution melting analysis of KRAS, BRAF and PIK3CA in KRAS exon 2 wild-type metastatic colorectal cancer

BackgroundKRAS is an EGFR effector in the RAS/RAF/ERK cascade that is mutated in about 40% of metastatic colorectal cancer (mCRC). Activating mutations in codons 12 and 13 of the KRAS gene are the only established negative predictors of response to anti-EGFR therapy and patients whose tumors harbor such mutations are not candidates for therapy. However, 40 to 60% of wild-type cases do not respond to anti-EGFR therapy, suggesting the involvement of other genes that act downstream of EGFR in the RAS-RAF-MAPK and PI3K-AKT pathways or activating KRAS mutations at other locations of the gene.MethodsDNA was obtained from a consecutive series of 201 mCRC cases (FFPE tissue), wild-type for KRAS exon 2 (codons 12 and 13). Mutational analysis of KRAS (exons 3 and 4), BRAF (exons 11 and 15), and PIK3CA (exons 9 and 20) was performed by high resolution melting (HRM) and positive cases were then sequenced.ResultsOne mutation was present in 23.4% (47/201) of the cases and 3.0% additional cases (6/201) had two concomitant mutations. A total of 53 cases showed 59 mutations, with the following distribution: 44.1% (26/59) in KRAS (13 in exon 3 and 13 in exon 4), 18.6% (11/59) in BRAF (two in exon 11 and nine in exon 15) and 37.3% (22/59) in PIK3CA (16 in exon 9 and six in exon 20). In total, 26.4% (53/201) of the cases had at least one mutation and the remaining 73.6% (148/201) were wild-type for all regions studied. Five of the mutations we report, four in KRAS and one in BRAF, have not previously been described in CRC. BRAF and PIK3CA mutations were more frequent in the colon than in the sigmoid or rectum: 20.8% vs. 1.6% vs. 0.0% (P=0.000) for BRAF and 23.4% vs. 12.1% vs. 5.4% (P=0.011) for PIK3CA mutations.ConclusionsAbout one fourth of mCRC cases wild-type for KRAS codons 12 and 13 present other mutations either in KRAS, BRAF, or PIK3CA, many of which may explain the lack of response to anti-EGFR therapy observed in a significant proportion of these patients.

A novel exonic rearrangement affecting MLH1 and the contiguous LRRFIP2 is a founder mutation in Portuguese Lynch syndrome families

Purpose: Although Lynch syndrome is characterized by marked genetic heterogeneity, some specific mutations are observed at high frequency in well-defined populations or ethnic groups due to founder effects.Methods: Genomic breakpoint identification, haplotype analysis, and mutation age determination were performed in 14 unrelated patients and 95 family members presenting the same MLH1 exonic rearrangement, among a series of 84 Lynch syndrome families with germline mutations in MLH1, MSH2, or MSH6.Results: All 14 probands harbored an identical deletion, comprising exons 17–19 of the MLH1 gene and exons 26–29 of the LRRFIP2 gene, corresponding to the MLH1 mutation c.1896 + 280_oLRRFIP2:c.1750-678del. This mutation represents 17% of all deleterious mismatch repair mutations in our series. Haplotype analysis showed a conserved region of approximately 1 Mb, and the mutation age was estimated to be 283 ± 78 years. All 14 families are originated from the Porto district countryside.Conclusion: We have identified a novel MLH1 exonic rearrangement that is a common founder mutation in Lynch syndrome families, indicating that screening for this rearrangement as a first step may be cost-effective during genetic testing of Lynch syndrome suspects of Portuguese ancestry, especially those originating from the Porto district.

Mitochondrial genome alterations in rectal and sigmoid carcinomas.

The scarce studies on the molecular pathways involved in the pathogenesis of rectal cancer indicate that these may vary, at least in part, from those relevant for colon cancer. Mitochondrial DNA alterations have been described in several human cancers. We aimed to study D310, ND1 and ND5 microsatellite sequence alterations and nuclear microsatellite instability in a series of 38 rectal carcinomas as compared to a series of 25 sigmoid carcinomas. D310 sequence alterations were observed in 34.3% and 37.5% of rectal and sigmoid carcinomas, respectively, whereas ND1 mutations were present in 2.6% in RC and ND5 mutations were detected in 5.3% and 8% of rectal and sigmoid carcinomas, respectively. A trend toward an association between nuclear and mitochondrial microsatellite instability was observed in sigmoid but not in rectal cancers. In conclusion, mitochondrial genome alterations are common in both rectal and sigmoid carcinomas and may contribute to their pathogenesis.

Hereditary gastrointestinal stromal tumors sharing the KIT Exon 17 germline mutation p.Asp820Tyr develop through different cytogenetic progression pathways

Hereditary gastrointestinal stromal tumor (GIST) syndrome is a rare autosomal dominant genetic disorder originated by germline mutations in the KIT or PDGFRA genes. We report the third family with hereditary predisposition to GIST due to the KIT Exon 17 germline mutation p.Asp820Tyr and characterize the cytogenetic progression pathways followed by different GIST sharing the same primary genetic event, using a combination of chromosome banding, comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH) analyses. The missense mutation p.Asp820Tyr was detected in the probands rectal and gastric GIST, as well as in his aunts GIST epiplon metastasis. The mutation p.Asp820Tyr was subsequently also found in the probands peripheral blood DNA, as well as in that of 4 of 10 relatives thus far analyzed. CGH analysis revealed loss of 14q and 15q in the probands gastric lesion, whereas FISH analysis of the probands rectal GIST did not detect loss of 14q and 15q, but instead loss of 4q and 22q and gain of 20q, indicating that the two tumors were independent GIST. Chromosome banding and CGH analyses of the aunts GIST epiplon metastasis revealed multiple cytogenetic alterations, including 1p loss, but none in common with the two probands GIST. We conclude that, although the patients share the same KIT Exon 17 germline mutation, the multiple GIST analyzed followed different pathogenetic progression pathways, each of which did not significantly differ from what has been described in sporadic GIST.

Pathogenicity Evaluation of BRCA1 and BRCA2 Unclassified Variants Identified in Portuguese Breast/Ovarian Cancer Families

Hereditary breast/ovarian cancer syndrome is caused by germline deleterious mutations in BRCA1 and BRCA2. A major problem of genetic testing and counseling is the finding of variants of uncertain significance (VUS). We sought to ascertain the pathogenicity of 25 BRCA1 and BRCA2 VUS identified in Portuguese families during genetic testing. We performed cosegregation analysis of VUS with cancer in families, evaluated their frequency in unaffected controls, and looked for loss of heterozygosity in tumors. In addition, three different bioinformatic algorithms were used (Interactive Biosoftware, ESEfinder, and PolyPhen). Finally, six VUS located in exon-intron boundaries were analyzed by RT-PCR. We found that seven variants segregated with the disease, six variants co-occurred with a pathogenic mutation in the same gene, and four variants co-occurred with a deleterious mutation in the other BRCA gene. By RT-PCR, we observed that four variants (BRCA1 c.4484G>T, BRCA2 c.682-2A>C, BRCA2 c.8488-1G>A, and BRCA2 c.8954-5A>G) disrupted splicing. After the combined analysis, we were able to classify 4 splicing variants as pathogenic mutations, 16 variants as neutral, and 3 variants as polymorphisms; only 2 variants remained classified as VUS. This work highlights the contribution of DNA, RNA, and in silico data to assess the pathogenicity of BRCA1/2 VUS, which, in turn, allows more accurate genetic counseling and clinical management of the families carrying them.

The MSH2 c.388_389del mutation shows a founder effect in Portuguese Lynch syndrome families.

The MSH2 c.388_389del mutation has occasionally been described in Lynch families worldwide. At the Portuguese Oncology Institute in Porto, Portugal, we have identified 16 seemingly unrelated families with this germline mutation. To evaluate if this alteration is a founder or a recurrent mutation we performed haplotype analysis in the 16 Portuguese index cases and 55 relatives, as well as in four index cases and 13 relatives reported from Germany, Scotland, England, and Argentina. In the Portuguese families we observed a shared haplotype of approximately 10 Mb and all were originated from the north of Portugal. These results suggest that this alteration is a founder mutation in Portugal with a relatively recent origin. In the reported families outside Portugal with this mutation different haplotype backgrounds were observed, supporting the hypothesis that it occurred de novo on multiple occasions. We also conclude that the high proportion of families with the MSH2 c.388_389del mutation indicates that screening for this alteration as a first step may be cost‐effective in the genetic testing of Lynch syndrome suspects of Portuguese ancestry, especially those originating from the north of Portugal.

Chromosome copy number changes carry prognostic information independent of KIT/PDGFRA point mutations in gastrointestinal stromal tumors

BackgroundOncogenic point mutations in KIT or PDGFRA are recognized as the primary events responsible for the pathogenesis of most gastrointestinal stromal tumors (GIST), but additional genomic alterations are frequent and presumably required for tumor progression. The relative contribution of such alterations for the biology and clinical behavior of GIST, however, remains elusive.MethodsIn the present study, somatic mutations in KIT and PDGFRA were evaluated by direct sequencing analysis in a consecutive series of 80 GIST patients. For a subset of 29 tumors, comparative genomic hybridization was additionally used to screen for chromosome copy number aberrations. Genotype and genomic findings were cross-tabulated and compared with available clinical and follow-up data.ResultsWe report an overall mutation frequency of 87.5%, with 76.25% of the tumors showing alterations in KIT and 11.25% in PDGFRA. Secondary KIT mutations were additionally found in two of four samples obtained after imatinib treatment. Chromosomal imbalances were detected in 25 out of 29 tumors (86%), namely losses at 14q (88% of abnormal cases), 22q (44%), 1p (44%), and 15q (36%), and gains at 1q (16%) and 12q (20%). In addition to clinico-pathological high-risk groups, patients with KIT mutations, genomic complexity, genomic gains and deletions at either 1p or 22q showed a significantly shorter disease-free survival. Furthermore, genomic complexity was the best predictor of disease progression in multivariate analysis.ConclusionsIn addition to KIT/PDGFRA mutational status, our findings indicate that secondary chromosomal changes contribute significantly to tumor development and progression of GIST and that genomic complexity carries independent prognostic value that complements clinico-pathological and genotype information.

TP53 germline mutations in Portugal and genetic modifiers of age at cancer onset

The Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant disease caused by TP53 germline mutations. This study aimed to characterize the TP53 mutational spectrum in patients suspected to have LFS in Portugal and to evaluate the influence of the MDM2-SNP309 and TP53-72Arg variants and of telomere length on age of tumor onset. Probands were primarily selected using the classical LFS criteria (two cases) or the more sensitive Chompret Li-Fraumeni-like (LFL) criteria (13 cases), but 12 additional patients that did not comply with those LFS or LFL criteria were included in the analysis based on clinical suspicion (LFS suspects). Nine of the 27 probands (33.3%) presented germline TP53 mutations, two of them occurring de novo and two of them being novel. Three of the nine TP53 mutations were found in families that did not comply with any of the commonly used criteria for TP53 testing, leaving room to recommend the use of less stringent criteria. An association was found between the presence of the TP53-72Arg (but not the MDM2-SNP309) variant and earlier age of onset in TP53 carriers. A negative correlation between telomere length and age of cancer onset was found in patients with germline TP53 mutation, whereas no such correlation was found in controls or in patients with wild-type TP53.