A comprehensive characterization of the spectrum of MUTYH germline pathogenic variants in Latin America.

MUTYH-Associated Polyposis (MAP) is caused by biallelic pathogenic germline variants in the MUTYH gene. However, individuals harboring monoallelic MUTYH pathogenic variants in the presence of a positive family history have been reported to have a twofold increased risk of colorectal cancer (CRC) and extra colonic cancers. Our aim was to characterize the spectrum of monoallelic and biallelic germline MUTYH pathogenic variants in Latin American patients and to describe their clinical and genetic characteristics. Patients were identified from eight high-risk genetic cancer centers of five Latin American countries. Statistical analysis was performed using the two-sided P test using the Vassarstats statistical tools. Statistical significance was set at a p value ≤ 0.05. Of the 105 unrelated patients with cancer or colorectal polyposis, 84.8% and 15.2% carried pathogenic monoallelic and biallelic MUTYH variants, respectively. The most common pathogenic variants were p.Gly396Asp and p.Tyr179Cys (55% and 23%, respectively). The mean age at first diagnosis was 48.29 years (range 31-71) and 49.90 years (range 27-87) in biallelic and monoallelic MUTYH patients, respectively. CRC was the only cancer diagnosed in patients with biallelic MUTYH pathogenic variants (75%), while breast cancer (46.1%) was more common than CRC (24.7%) in individuals with monoallelic MUTYH pathogenic variants. We reported a high frequency of European founder variants in our diverse population. Some phenotypic differences from current studies were identified, such as a higher breast cancer burden in monoallelic carriers and a complete absence of extra-colon tumors in biallelic patients.

Dominantly inherited micro-satellite instable cancer - the four Lynch syndromes - an EHTG, PLSD position statement.

The recognition of dominantly inherited micro-satellite instable (MSI) cancers caused by pathogenic variants in one of the four mismatch repair (MMR) genes MSH2, MLH1, MSH6 and PMS2 has modified our understanding of carcinogenesis. Inherited loss of function variants in each of these MMR genes cause four dominantly inherited cancer syndromes with different penetrance and expressivities: the four Lynch syndromes. No person has an "average sex "or a pathogenic variant in an "average Lynch syndrome gene" and results that are not stratified by gene and sex will be valid for no one. Carcinogenesis may be a linear process from increased cellular division to localized cancer to metastasis. In addition, in the Lynch syndromes (LS) we now recognize a dynamic balance between two stochastic processes: MSI producing abnormal cells, and the host's adaptive immune system's ability to remove them. The latter may explain why colonoscopy surveillance does not reduce the incidence of colorectal cancer in LS, while it may improve the prognosis. Most early onset colon, endometrial and ovarian cancers in LS are now cured and most cancer related deaths are after subsequent cancers in other organs. Aspirin reduces the incidence of colorectal and other cancers in LS. Immunotherapy increases the host immune system's capability to destroy MSI cancers. Colonoscopy surveillance, aspirin prevention and immunotherapy represent major steps forward in personalized precision medicine to prevent and cure inherited MSI cancer.

Mortality by age, gene and gender in carriers of pathogenic mismatch repair gene variants receiving surveillance for early cancer diagnosis and treatment: a report from the prospective Lynch syndrome database.

Background: The Prospective Lynch Syndrome Database (PLSD) collates information on carriers of pathogenic or likely pathogenic MMR variants (path_MMR) who are receiving medical follow-up, including colonoscopy surveillance, which aims to the achieve early diagnosis and treatment of cancers. Here we use the most recent PLSD cohort that is larger and has wider geographical representation than previous versions, allowing us to present mortality as an outcome, and median ages at cancer diagnoses for the first time. Methods: The PLSD is a prospective observational study without a control group that was designed in 2012 and updated up to October 2022. Data for 8500 carriers of path_MMR variants from 25 countries were included, providing 71,713 years of follow up. Cumulative cancer incidences at 65 years of age were combined with 10-year crude survival following cancer, to derive estimates of mortality up to 75 years of age by organ, gene, and gender. Findings: Gynaecological cancers were more frequent than colorectal cancers in path_MSH2, path_MSH6 and path_PMS2 carriers [cumulative incidence: 53.3%, 49.6% and 23.3% at 75 years, respectively]. Endometrial, colon and ovarian cancer had low mortality [8%, 13% and 15%, respectively] and prostate cancers were frequent in male path_MSH2 carriers [cumulative incidence: 39.7% at 75 years]. Pancreatic, brain, biliary tract and ureter and kidney and urinary bladder cancers were associated with high mortality [83%, 66%, 58%, 27%, and 29%, respectively]. Among path_MMR carriers undergoing colonoscopy surveillance, particularly path_MSH2 carriers, more deaths followed non-colorectal Lynch syndrome cancers than colorectal cancers. Interpretation: In path_MMR carriers undergoing colonoscopy surveillance, non-colorectal Lynch syndrome cancers were associated with more deaths than were colorectal cancers. Reducing deaths from non-colorectal cancers presents a key challenge in contemporary medical care in Lynch syndrome. Funding: We acknowledge funding from the Norwegian Cancer Society, contract 194751-2017.

Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium.

OBJECTIVE: To compare colorectal cancer (CRC) incidences in carriers of pathogenic variants of the MMR genes in the PLSD and IMRC cohorts, of which only the former included mandatory colonoscopy surveillance for all participants. METHODS: CRC incidences were calculated in an intervention group comprising a cohort of confirmed carriers of pathogenic or likely pathogenic variants in mismatch repair genes (path_MMR) followed prospectively by the Prospective Lynch Syndrome Database (PLSD). All had colonoscopy surveillance, with polypectomy when polyps were identified. Comparison was made with a retrospective cohort reported by the International Mismatch Repair Consortium (IMRC). This comprised confirmed and inferred path_MMR carriers who were first- or second-degree relatives of Lynch syndrome probands. RESULTS: In the PLSD, 8,153 subjects had follow-up colonoscopy surveillance for a total of 67,604 years and 578 carriers had CRC diagnosed. Average cumulative incidences of CRC in path_MLH1 carriers at 70 years of age were 52% in males and 41% in females; for path_MSH2 50% and 39%; for path_MSH6 13% and 17% and for path_PMS2 11% and 8%. In contrast, in the IMRC cohort, corresponding cumulative incidences were 40% and 27%; 34% and 23%; 16% and 8% and 7% and 6%. Comparing just the European carriers in the two series gave similar findings. Numbers in the PLSD series did not allow comparisons of carriers from other continents separately. Cumulative incidences at 25 years were < 1% in all retrospective groups. CONCLUSIONS: Prospectively observed CRC incidences (PLSD) in path_MLH1 and path_MSH2 carriers undergoing colonoscopy surveillance and polypectomy were higher than in the retrospective (IMRC) series, and were not reduced in path_MSH6 carriers. These findings were the opposite to those expected. CRC point incidence before 50 years of age was reduced in path_PMS2 carriers subjected to colonoscopy, but not significantly so.

Breast Cancer Phenotype Associated With Li-Fraumeni Syndrome: A Brazilian Cohort Enriched by TP53 p.R337H Carriers.

Breast cancer (BC) is the most prevalent malignancy in women with Li-Fraumeni syndrome (LFS). The literature on BC in LFS is limited due to its rarity worldwide. A TP53 founder pathogenic variant (c.1010G>A; p.R337H) is responsible for the higher prevalence of this syndrome among women of Brazilian ancestry. Purpose: The aim of the study was to describe the BC phenotype expressed by Brazilian female LFS carriers and compare the data between p.R337H and other TP53 germline pathogenic/likely pathogenic variants (non-p.R337H carriers). Methods: We searched for cases of TP53 germline pathogenic/likely pathogenic variant carriers affected by BC included between 2015 and 2020 in the BLiSS (Brazilian Li-Fraumeni Study) registry at the Sírio-Libanês Hospital. Results: Among 163 adult female carriers from the registry, 91 (56%) had received a BC diagnosis, including 72 p.R337H carriers. BC was the first cancer diagnosed in 90% of cases. Early onset BC (age ≤45 years) was diagnosed in 78.2% of cases (11.5% <31 years; 66.7% 31-45 years; 21.8% >45 years). The median age of BC diagnosis for p.R337H carriers was 39.5 years (range 20-69 years) compared to 34 years (range 21-63 years) for non-p.R337H carriers (p = 0.009). In total, 104 breast tumors were observed in 87 women. Bilateral BC was observed in 29.3% of cases. Histology was available for 96 tumors, comprising 69 invasive breast carcinomas, which were mostly invasive ductal carcinomas (95.6%), 25 ductal in situ carcinomas and 2 soft-tissue sarcomas. Overall, 90.5% of invasive breast carcinomas were hormone receptor (HR)-positive, 39.5% were human epidermal growth factor receptor 2 (HER2)-positive, and 32.8% showed HR and HER2 co-expression. In addition, 55.4% of patients opted for contralateral prophylactic mastectomy after a first BC diagnosis. There were no significant differences in the risk of developing contralateral BC or in the immunohistochemical profile between p.R337H and non-p.R337H groups. Conclusions: The expressed phenotype of p.R337H is similar to that of other TP53 pathogenic/likely pathogenic variants, except for an average older age at the onset of disease; however, this is still younger than the general population.

Expanding the phenotype of E318K (c.952G > A) MITF germline mutation carriers: case series and review of the literature.

BACKGROUND: The microphthalmia-associated transcription factor gene (MITF) belongs to the MYC supergene family and plays an important role in melanocytes' homeostasis. Individuals harboring MITF germline pathogenic variants are at increased risk of developing cancer, most notably melanoma and renal cell carcinoma. CASE PRESENTATION: We describe a cohort of ten individuals who harbor the same MITF c.952G > A (p.Glu 318Lys), or p.E318K, germline pathogenic variant. Six carriers developed at least one malignancy (4 cases of breast cancer; 1 cervical cancer; 1 colon cancer; 1 melanoma; 1 ovarian/fallopian tube cancer). A significant phenotypic heterogeneity was found among these individuals and their relatives. Breast cancer was, overall, the most frequent malignancy observed in this case series, with 13 occurrences of 60 (21.67 %) total cancer cases described among the probands and their relatives. CONCLUSIONS: Our retrospective analysis data raise the hypothesis of a possible association of the MITF p.E318K pathogenic variant with an increased risk of breast cancer.

MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing.

Germline pathogenic variants in the DNA mismatch repair genes (MMR): MLH1, MSH2, MSH6, and PMS2, are causative of Lynch syndrome (LS). However, many of the variants mapping outside the invariant splice site positions (IVS ± 1, IVS ± 2) are classified as variants of unknown significance (VUS). Three such variants (MLH1 c.588+5G>C, c.588+5G>T and c.677+5G>A) were identified in 8 unrelated LS families from Argentina, Brazil and Chile. Herein, we collected clinical information on these families and performed segregation analysis and RNA splicing studies to assess the implication of these VUS in LS etiology. Pedigrees showed a clear pattern of variant co-segregation with colorectal cancer and/or other LS-associated malignancies. Tumors presented deficient expression of MLH1-PMS2 proteins in 7/7 of the LS families, and MSI-high status in 3/3 cases. Moreover, RNA analyses revealed that c.588+5G>C and c.588+5G>T induce skipping of exon 7 whereas c.677+5G>A causes skipping of exon 8. In sum, we report that the combined clinical findings in the families and the molecular studies provided the evidences needed to demonstrate that the three MLH1 variants are causative of LS and to classify c.588+5G>C and c.677+5G>A as class 5 (pathogenic), and c.588+5G>T as class 4 (likely-pathogenic). Our findings underline the importance of performing clinical and family analyses, as well as RNA splicing assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives.

A snapshot of current genetic testing practice in Lynch syndrome: The results of a representative survey of 33 Latin American existing centres/registries.

We aimed to assess the current genetics practice to manage patients with Lynch syndrome (LS) across Latin America. A Latin American LS survey was sent out to 52 centres/registries, comprising a total of 12 countries from the region. Overall, 33 centres completed the survey, of which the oldest LS registry was established in 1992 in Sao Paulo (Brazil), and the youngest this year in San Jose (Costa Rica). In total, 87% (26/30) of the participating centres/registries belonging to the nine countries are performing genetic testing. Overall, 1352 suspected families were sequenced. Pathogenic variants were identified in 34% of the families, with slightly differing distribution of variants between females and males. Path_MLH1 variants were identified in 39% of females and 50% of males (p = 0.023), while path_MSH2 were identified in 37% of females and males, followed by path_PMS2 in 11% of females and 8% of males, path_MSH6 in 13% of females and 3% of males (p < 0.001) and path_EPCAM in 0.3% of females and 2% of males. In Latin America, 9 of 12 (75%) participating countries had implemented healthcare for LS. LS screening is inconsistently applied within Latin America healthcare systems because of structural differences in the healthcare systems between the countries.

From colorectal cancer pattern to the characterization of individuals at risk: Picture for genetic research in Latin America.

Colorectal cancer (CRC) is one of the most common cancers in Latin America and the Caribbean, with the highest rates reported for Uruguay, Brazil and Argentina. We provide a global snapshot of the CRC patterns, how screening is performed, and compared/contrasted to the genetic profile of Lynch syndrome (LS) in the region. From the literature, we find that only nine (20%) of the Latin America and the Caribbean countries have developed guidelines for early detection of CRC, and also with a low adherence. We describe a genetic profile of LS, including a total of 2,685 suspected families, where confirmed LS ranged from 8% in Uruguay and Argentina to 60% in Peru. Among confirmed LS, path_MLH1 variants were most commonly identified in Peru (82%), Mexico (80%), Chile (60%), and path_MSH2/EPCAM variants were most frequently identified in Colombia (80%) and Argentina (47%). Path_MSH6 and path_PMS2 variants were less common, but they showed important presence in Brazil (15%) and Chile (10%), respectively. Important differences exist at identifying LS families in Latin American countries, where the spectrum of path_MLH1 and path_MSH2 variants are those most frequently identified. Our findings have an impact on the evaluation of the patients and their relatives at risk for LS, derived from the gene affected. Although the awareness of hereditary cancer and genetic testing has improved in the last decade, it is remains deficient, with 39%-80% of the families not being identified for LS among those who actually met both the clinical criteria for LS and showed MMR deficiency.

Disparities in colorectal cancer mortality across Brazilian States. / Disparidades na mortalidade de câncer colorretal nos estados brasileiros.

OBJECTIVE: To analyze the trend of colorectal cancer mortality adjusted for selected indicators, according to sex, by Brazilian federative units and regions, and countrywide from 1996 to 2012. METHODS: This is a temporal time series on colorectal cancer mortality rates, using linear regression analysis, in which the independent variable was the centered year. Models were adjusted for selected indicators. RESULTS: There was an increase in standardized colorectal cancer mortality rates for males in all states and for females in 21 states. In the model adjusted for mortality rate from ill-defined causes, for gross domestic product, and for Gini coefficient, the upward trend remained statistically significant (p < 0.05) countrywide only for men, with 0.17 deaths per 100 thousand inhabitants per year (py). In the States of Piauí (0.09 and 0.20 py), Ceará (0.17 and 0.19 py) and Rio Grande do Sul (0.61 and 0.42 py), there was an increase for both men and women, respectively; only among men in the States of Paraíba (0.16 py), Espírito Santo (0.28 py), São Paulo (0.24 py) and Goiás (0.31 py); and among women in Roraima (0.41 py), Amapá (0.97 P/Y), Maranhão (0.10 py), Sergipe (0.46 P/Y), Mato Grosso do Sul (0.47 py), and the Federal District (0.69 py). CONCLUSION: The increase in colorectal cancer mortality remained significant when assessing Brazil as a whole only among men; in seven States among men, and in nine States among women, regardless of the studied indicators. These differences could be related to the possible increase in incidence and to late access to diagnosis and treatment.

OBJETIVO: Analisar a tendência da mortalidade por câncer colorretal, ajustado por indicadores selecionados, segundo sexo, para unidades federativas, regiões e Brasil, no período de 1996 a 2012. MÉTODOS: Estudo ecológico de série temporal das taxas de mortalidade por câncer colorretal, feita análise de regressão linear, sendo o ano centralizado a variável independente. Os modelos foram ajustados por indicadores selecionados. RESULTADOS: Houve aumento nas taxas de mortalidade padronizadas por câncer colorretal em todos os estados para o sexo masculino e em 21 estados para o sexo feminino. No modelo ajustado por taxa de mortalidade por causas mal definidas, produto interno bruto e coeficiente de Gini, a tendência de aumento foi significativa (p < 0,05) no Brasil, somente para os homens, com 0,17 óbitos por 100 mil habitantes ao ano (aa). Nos estados do Piauí (0,09 e 0,20 aa), Ceará (0,17 e 0,19 aa) e Rio Grande do Sul (0,61 e 0,42 aa) ocorreu aumento em homens e mulheres, respectivamente; somente em homens nos estados da Paraíba (0,16 aa), no Espírito Santo (0,28 aa), em São Paulo (0,24 aa) e Goiás (0,31 aa); e em mulheres nos estados de Roraima (0,41 aa), do Amapá (0,97 aa), Maranhão (0,10 aa), Sergipe (0,46 aa), Mato Grosso do Sul (0,47 aa) e Distrito Federal (0,69 aa). CONCLUSÃO: O aumento da taxa de mortalidade por câncer colorretal manteve-se significativo no Brasil somente entre os homens; em sete estados, entre homens; e em nove estados, entre mulheres, independentemente dos indicadores estudados. Essas diferenças podem estar relacionadas ao possível aumento da incidência e ao acesso tardio ao diagnóstico e tratamento.

Evaluation of MLH1 variants of unclear significance.

Inactivating mutations in the MLH1 gene cause the cancer predisposition Lynch syndrome, but for small coding genetic variants it is mostly unclear if they are inactivating or not. Nine such MLH1 variants have been identified in South American colorectal cancer (CRC) patients (p.Tyr97Asp, p.His112Gln, p.Pro141Ala, p.Arg265Pro, p.Asn338Ser, p.Ile501del, p.Arg575Lys, p.Lys618del, p.Leu676Pro), and evidence of pathogenicity or neutrality was not available for the majority of these variants. We therefore performed biochemical laboratory testing of the variant proteins and compared the results to protein in silico predictions on structure and conservation. Additionally, we collected all available clinical information of the families to come to a conclusion concerning their pathogenic potential and facilitate clinical diagnosis in the affected families. We provide evidence that four of the alterations are causative for Lynch syndrome, four are likely neutral and one shows compromised activity which can currently not be classified with respect to its pathogenic potential. The work demonstrates that biochemical testing, corroborated by congruent evolutionary and structural information, can serve to reliably classify uncertain variants when other data are insufficient.

Disparidades na mortalidade de câncer colorretal nos estados brasileiros / Disparities in colorectal cancer mortality across Brazilian States

RESUMO: Objetivo: Analisar a tendência da mortalidade por câncer colorretal, ajustado por indicadores selecionados, segundo sexo, para unidades federativas, regiões e Brasil, no período de 1996 a 2012. Métodos: Estudo ecológico de série temporal das taxas de mortalidade por câncer colorretal, feita análise de regressão linear, sendo o ano centralizado a variável independente. Os modelos foram ajustados por indicadores selecionados. Resultados: Houve aumento nas taxas de mortalidade padronizadas por câncer colorretal em todos os estados para o sexo masculino e em 21 estados para o sexo feminino. No modelo ajustado por taxa de mortalidade por causas mal definidas, produto interno bruto e coeficiente de Gini, a tendência de aumento foi significativa (p < 0,05) no Brasil, somente para os homens, com 0,17 óbitos por 100 mil habitantes ao ano (aa). Nos estados do Piauí (0,09 e 0,20 aa), Ceará (0,17 e 0,19 aa) e Rio Grande do Sul (0,61 e 0,42 aa) ocorreu aumento em homens e mulheres, respectivamente; somente em homens nos estados da Paraíba (0,16 aa), no Espírito Santo (0,28 aa), em São Paulo (0,24 aa) e Goiás (0,31 aa); e em mulheres nos estados de Roraima (0,41 aa), do Amapá (0,97 aa), Maranhão (0,10 aa), Sergipe (0,46 aa), Mato Grosso do Sul (0,47 aa) e Distrito Federal (0,69 aa). Conclusão: O aumento da taxa de mortalidade por câncer colorretal manteve-se significativo no Brasil somente entre os homens; em sete estados, entre homens; e em nove estados, entre mulheres, independentemente dos indicadores estudados. Essas diferenças podem estar relacionadas ao possível aumento da incidência e ao acesso tardio ao diagnóstico e tratamento.

ABSTRACT: Objective: To analyze the trend of colorectal cancer mortality adjusted for selected indicators, according to sex, by Brazilian federative units and regions, and countrywide from 1996 to 2012. Methods: This is a temporal time series on colorectal cancer mortality rates, using linear regression analysis, in which the independent variable was the centered year. Models were adjusted for selected indicators. Results: There was an increase in standardized colorectal cancer mortality rates for males in all states and for females in 21 states. In the model adjusted for mortality rate from ill-defined causes, for gross domestic product, and for Gini coefficient, the upward trend remained statistically significant (p < 0.05) countrywide only for men, with 0.17 deaths per 100 thousand inhabitants per year (py). In the States of Piauí (0.09 and 0.20 py), Ceará (0.17 and 0.19 py) and Rio Grande do Sul (0.61 and 0.42 py), there was an increase for both men and women, respectively; only among men in the States of Paraíba (0.16 py), Espírito Santo (0.28 py), São Paulo (0.24 py) and Goiás (0.31 py); and among women in Roraima (0.41 py), Amapá (0.97 P/Y), Maranhão (0.10 py), Sergipe (0.46 P/Y), Mato Grosso do Sul (0.47 py), and the Federal District (0.69 py). Conclusion: The increase in colorectal cancer mortality remained significant when assessing Brazil as a whole only among men; in seven States among men, and in nine States among women, regardless of the studied indicators. These differences could be related to the possible increase in incidence and to late access to diagnosis and treatment.

A survey of the clinicopathological and molecular characteristics of patients with suspected Lynch syndrome in Latin America.

BACKGROUND: Genetic counselling and testing for Lynch syndrome (LS) have recently been introduced in several Latin America countries. We aimed to characterize the clinical, molecular and mismatch repair (MMR) variants spectrum of patients with suspected LS in Latin America. METHODS: Eleven LS hereditary cancer registries and 34 published LS databases were used to identify unrelated families that fulfilled the Amsterdam II (AMSII) criteria and/or the Bethesda guidelines or suggestive of a dominant colorectal (CRC) inheritance syndrome. RESULTS: We performed a thorough investigation of 15 countries and identified 6 countries where germline genetic testing for LS is available and 3 countries where tumor testing is used in the LS diagnosis. The spectrum of pathogenic MMR variants included MLH1 up to 54%, MSH2 up to 43%, MSH6 up to 10%, PMS2 up to 3% and EPCAM up to 0.8%. The Latin America MMR spectrum is broad with a total of 220 different variants which 80% were private and 20% were recurrent. Frequent regions included exons 11 of MLH1 (15%), exon 3 and 7 of MSH2 (17 and 15%, respectively), exon 4 of MSH6 (65%), exons 11 and 13 of PMS2 (31% and 23%, respectively). Sixteen international founder variants in MLH1, MSH2 and MSH6 were identified and 41 (19%) variants have not previously been reported, thus representing novel genetic variants in the MMR genes. The AMSII criteria was the most used clinical criteria to identify pathogenic MMR carriers although microsatellite instability, immunohistochemistry and family history are still the primary methods in several countries where no genetic testing for LS is available yet. CONCLUSION: The Latin America LS pathogenic MMR variants spectrum included new variants, frequently altered genetic regions and potential founder effects, emphasizing the relevance implementing Lynch syndrome genetic testing and counseling in all of Latin America countries.

Clinical and Molecular Characterization of Brazilian Patients Suspected to Have Lynch Syndrome.

Lynch syndrome (LS) accounts for 3-5% of all colorectal cancers (CRC) and is inherited in an autosomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous/metachronous tumors and extra-colonic tumors. Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five mismatch repair genes (MMR), mostly in MLH1 and MSH2, but also in MSH6, PMS1 and PMS2. To comprehensively characterize 116 Brazilian patients suspected for LS, we assessed the frequency of germline mutations in the three minor genes MSH6, PMS1 and PMS2 in 82 patients negative for point mutations in MLH1 and MSH2. We also assessed large genomic rearrangements by MLPA for detecting copy number variations (CNVs) in MLH1, MSH2 and MSH6 generating a broad characterization of MMR genes. The complete analysis of the five MMR genes revealed 45 carriers of pathogenic mutations, including 25 in MSH2, 15 in MLH1, four in MSH6 and one in PMS2. Eleven novel pathogenic mutations (6 in MSH2, 4 in MSH6 and one in PMS2), and 11 variants of unknown significance (VUS) were found. Mutations in the MLH1 and MSH2 genes represented 89% of all mutations (40/45), whereas the three MMR genes (MSH6, PMS1 and PMS2) accounted for 11% (5/45). We also investigated the MLH1 p.Leu676Pro VUS located in the PMS2 interaction domain and our results revealed that this variant displayed no defective function in terms of cellular location and heterodimer interaction. Additionally, we assessed the tumor phenotype of a subset of patients and also the frequency of CRC and extra-colonic tumors in 2,365 individuals of the 116 families, generating the first comprehensive portrait of the genetic and clinical aspects of patients suspected of LS in a Brazilian cohort.

Angiogenesis-related protein expression in bevacizumab-treated metastatic colorectal cancer: NOTCH1 detrimental to overall survival.

BACKGROUND: The development of targeted therapies has undoubtedly broadened therapeutic options for patients with colorectal cancer (CRC). The use of bevacizumab to reduce angiogenesis has been associated with improved clinical outcomes. However, an urgent need for prognostic/predictive biomarkers for anti-angiogenic therapies still exists. METHODS: Clinical data of 105 CRC patients treated with bevacizumab in conjunction with chemotherapy were analyzed. The expression of vascular endothelial growth factor (VEGF) receptors, NOTCH1 receptor and its ligand DLL4 were determined by immunohistochemistry. Tumor samples were arranged on a tissue microarray. The association between protein expression and clinicopathological characteristics and outcomes was determined. RESULTS: Bevacizumab was administered as a first-line of treatment in 70.5 % of our cases. The median progression-free survival (PFS) was 10.2 months. The median overall survival (OS) of the total cohort was 24.4 months. Bevacizumab, as the first-line of treatment, and the presence of liver metastasis were independently associated with objective response rate. Membrane VEGFR1 and VEGFR3 expressions were associated with the presence of lung metastasis; interestingly, VEGFR3 was associated with less liver metastasis. NOTCH1 expression was associated with lymph node metastasis. There was a trend toward association between improved PFS and lower NOTCH1 expression (p = 0.06). Improved OS was significantly associated with lower NOTCH1 expression (p = 0.01). In a multivariate analysis, ECOG (Eastern Cooperative Oncology Group) performance status, liver metastasis, histological grade, and NOTCH1 expression were independently associated with OS. CONCLUSION: Our findings illustrated the expression profile of angiogenesis-related proteins and their association with clinicopathological characteristics and outcomes. NOTCH1 expression is a detrimental prognostic factor in metastatic CRC patients treated with chemotherapy plus bevacizumab.

Association of the p53 codon 72 polymorphism with clinicopathological characteristics of colorectal cancer through mRNA analysis.

TP53 represents a suitable candidate for a colorectal cancer susceptibility locus. The polymorphism in the p53 72nd codon involves a proline to arginine substitution, leading to changes in gene transcription activity, interaction with other proteins and modulation of apoptosis. Studies evaluating the association between this polymorphism and colorectal cancer (CRC) have shown inconsistent results, and none have evaluated the mRNA status of TP53. The aim of the present study was to evaluate the association between this SNP expression at the mRNA level in CRC samples and patient clinicopathological variables and prognosis, p53 protein expression and TP53 mutation. This is the first report to describe the mRNA expression of p53 codon 72 alleles in CRC. We evaluated 101 non-related patients with CRC treated at the A.C. Camargo Cancer Center in Brazil. RNA was isolated from frozen tumor tissues using a trizol-based protocol. The polymorphism was detected using RT-PCR followed by Sanger sequencing. Associations were analyzed using Pearson's Chi-square or Fisher's exact tests, logistic regression and Cox. This polymorphism was significantly associated with clinicopathological variables related to increased tumor aggressiveness. The expression of Arg72 (OR, 3.83; CI 1.02-14.35; P=0.046) and the TNM stage (OR, 7.15; CI 1.45-35.29; P=0.016) were found to be independent predictors for recurrence. These data suggest that the mRNA expression of the Pro72 allele is associated with less favorable tumor features. The allele frequency of the p53 Pro72 was 0.26. The analysis of mRNA is important to determine the specific contribution of the allele expressed. These results suggest that this polymorphism may play a role in CRC.

Mutational spectrum of the APC and MUTYH genes and genotype-phenotype correlations in Brazilian FAP, AFAP, and MAP patients.

BACKGROUND: Patients with multiple colorectal adenomas are currently screened for germline mutations in two genes, APC and MUTYH. APC-mutated patients present classic or attenuated familial adenomatous polyposis (FAP/AFAP), while patients carrying biallelic MUTYH mutations exhibit MUTYH-associated polyposis (MAP). The spectrum of mutations as well as the genotype-phenotype correlations in polyposis syndromes present clinical impact and can be population specific, making important to obtain genetic and clinical data from different populations. METHODS: DNA sequencing of the complete coding region of the APC and MUTYH genes was performed in 23 unrelated Brazilian polyposis patients. In addition, mutation-negative patients were screened for large genomic rearrangements by multiplex ligation-dependent probe amplification, array-comparative genomic hybridization, and duplex quantitative PCR. Biallelic MUTYH mutations were confirmed by allele-specific PCR. Clinical data of the index cases and their affected relatives were used to assess genotype-phenotype correlations. RESULTS: Pathogenic mutations were identified in 20 of the 23 probands (87%): 14 in the APC gene and six in the MUTYH gene; six of them (30%) were described for the first time in this series. Genotype-phenotype correlations revealed divergent results compared with those described in other studies, particularly regarding the extent of polyposis and the occurrence of desmoid tumors in families with mutations before codon 1444 (6/8 families with desmoid). CONCLUSIONS: This first comprehensive investigation of the APC and MUTYH mutation spectrum in Brazilian polyposis patients showed a high detection rate and identified novel pathogenic mutations. Notably, a significant number of APC-positive families were not consistent with the predicted genotype-phenotype correlations from other populations.

Evaluation of MLH1 I219V polymorphism in unrelated South American individuals suspected of having Lynch syndrome.

BACKGROUND: Some single-nucleotide polymorphisms are associated with higher risk of colorectal cancer development and are suggested to explain part of the genetic contribution to Lynch syndrome. AIM: To evaluate the mutL homolog 1 (MLH1) I219V polymorphism in 124 unrelated South American individuals suspected of having Lynch syndrome, based on frequency, association with pathogenic MLH1 and mutS homolog 2 (MSH2) mutation and clinical features. MATERIALS AND METHODS: DNA was obtained from peripheral blood and polymerase chain reaction (PCR) was performed, followed by direct sequencing. RESULTS: The Val allelic of the I219V polymorphism was found in 51.61% (64/124) of the individuals, with an allelic frequency of 0.3. MLH1 or MHS2 pathogenic mutations were found in 32.81% (21/64) and in 23.33% (14/60) of Val-carriers and non-carriers, respectively. CONCLUSION: The Val-carrying genotype was frequent in the studied population; however, it does not appear to exert any modifier effect on MLH1 or MSH2 pathogenic mutations and the development of colorectal cancer.

A novel SYBR-based duplex qPCR for the detection of gene dosage: detection of an APC large deletion in a familial adenomatous polyposis patient with an unusual phenotype.

BACKGROUND: Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome caused by a loss of function of the APC gene. Large deletions in APC are a common cause of FAP; despite the existence of a variety of gene dosage detection methodologies, most are labor intensive and time and resource consuming. METHODS: We describe a new duplex qPCR method for gene dosage analysis based on the coamplification of a target and a reference gene in a SYBR Green reaction, followed by a comparison of the ratio between the target and the reference peaks of the melting curve for the test (patient) and control samples. The reliability of the described duplex qPCR was validated for several genes (APC, HPRT1, ATM, PTEN and BRCA1). RESULTS: Using this novel gene dosage method, we have identified an APC gene deletion in a FAP patient undergoing genetic testing. Comparative genomic hybridization based on microarrays (aCGH) was used to confirm and map the extent of the deletion, revealing a 5.2 MB rearrangement (5q21.3-q22.3) encompassing the entire APC and 19 additional genes. CONCLUSION: The novel assay accurately detected losses and gains of one copy of the target sequences, representing a reliable and flexible alternative to other gene dosage techniques. In addition, we described a FAP patient harboring a gross deletion at 5q21.3-q22.3 with an unusual phenotype of the absence of mental impairment and dysmorphic features.