Claudia Hurtado

Spectrum of MLH1 and MSH2 mutations in Chilean families with suspected Lynch syndrome.

PURPOSE: Lynch syndrome is the most common inherited syndrome of colorectal cancer, caused principally by germline mutations in MLH1 and MSH2. We report our experience with genetic screening in the diagnosis of Lynch syndrome in Chile, a country previously underserved in the capacity to diagnose hereditary colorectal cancer. METHODS: Families from our Familial Colorectal Cancer Registry were selected for this study if they fulfilled either Amsterdam I/II or Bethesda criteria for classification of Lynch syndrome. Analysis of colorectal tumors from probands included a microsatellite instability study and immunohistochemical evaluation for MLH1 and MSH2. Screening of germline mutations was performed by single-strand conformation polymorphism analysis and DNA sequencing. RESULTS: A total of 21 families were evaluated, 14 meeting Amsterdam criteria and 7 meeting Bethesda criteria. Tumors in 20 families (95%) showed microsatellite instability (19 high and 1 low) and 9 of these 20 families (45%) harbored a germline mutation (7 of 13 Amsterdam and 2 of 7 Bethesda families). Of the 9 mutations identified, 6 were in MLH1 and 3 in MSH2. Two of the mutations were novel, 3 were previously found in 1 to 2 European populations, and 4 were previously found in various ethnic populations worldwide. Only 2 mutations were previously found in another Latin American population (Colombia). In our probands, colorectal cancer was located mainly (57%) in the right or transverse colon. Pedigree information from 104 family affected members of 21 studied families showed endometrial cancer to be the most frequent primary extracolonic tumor, accounting for 15.1% of total cases, followed by stomach (13.2%) and breast cancer (11.3%). Analysis of mitochondrial DNA haplotypes showed a strong Amerindian genetic component in 15 (71.4%) of the 21 families analyzed. CONCLUSION: The study of Lynch syndrome in families of different ethnic origins contributes to the definition of genetic and clinical differences among populations. Wide distribution in other ethnic populations strongly suggests varying origins of 4 the mutations found. Although cancer phenotype was consistent with those from other Latin American populations, only 2 of 9 mutations were shared with other South American populations and 2 novel mutations were found. The Chilean population is considered to be an admixture of Amerindian and European—mainly Spanish—populations, producing an ethnic group with significant genetic differences from populations previously studied.

Síndrome de Lynch: selección de pacientes para el estudio genético mediante análisis de inestabilidad microsatelital e inmunohistoquímica

BACKGROUND Selection of patients with Lynch Syndrome (LS) for a genetic study involves the application of clinical criteria. To increase the rate of identification of mutations, the use of molecular studies as Microsatellite Instability (MSI) and Immunohistochemistry (IHC) in the tumor has been proposed. AIM To demonstrate the usefulness of MSI and IHC in the detection of mutations in patients with LS. MATERIAL AND METHODS From our Familial Colorectal Cancer Registry, families suspected of LS were selected according to Amsterdam or Bethesda clinical criteria. Screening of germline mutations of MLH1, MSH2 and MSH6 genes was performed. In addition, analysis of MSI and IHC were performed in colorectal tumors. RESULTS A total of 35 families were studied (19 met Amsterdam and 16 met Bethesda criteria). Twenty one families harbored a germline alteration in MLH1, MSH2 or MSH6 (18 Amsterdam and 3 Bethesda). In these families, eighteen different alterations were found, 15 of which were mutations and 3 corresponded to variants of uncertain pathogenicity. On the other hand, 80% of the tumors showed positive microsatellite instability (27 MSI-high and 1 MSI-low), and immunohistochemical testing showed that 77% of tumors had the loss of a protein. Correlation between results of tumor molecular studies and the finding of germline nucleotide change showed that IHC and MSI predicted mutations in 81 and 100% of patients, respectively. CONCLUSIONS MSI and IHC can efficiently select patients with a high probability of carrying a mutation in DNA repair genes.

Caracterización de pacientes con cáncer colorrectal esporádico basado en la nueva subclasificación molecular de consenso

BACKGROUND Colorectal cancer (CRC) is an heterogeneous disease. Three carcinogenic pathways determine its molecular profile: microsatellite instability (MSI), chromosomal instability (CIN) and CpG island methylator phenotype (CIMP). Based on the new molecular classification, four consensus CRC molecular subtypes (CMS) are established, which are related to clinical, pathological and biological characteristics of the tumor. AIM To classify Chilean patients with sporadic CRC according to the new consensus molecular subtypes of carcinogenic pathways. MATERIAL AND METHODS Prospective analytical study of 53 patients with a mean age of 70 years (55% males) with CRC, operated at a private clinic, without neoadjuvant treatment. From normal and tumor tissue DNA of each patient, CIN, MSI and CIMP were analyzed. Combining these variables, tumors were classified as CMS1/MSI-immune, CMS2/canonical, CMS3/metabolic and CMS4/mesenchymal. RESULTS CMS1 tumors (19%) were located in the right colon, were in early stages, had MMR complex deficiencies and 67% had an activating mutation of the BRAF oncogene. CMS2 tumors (31%) were located in the left colon, had moderate differentiation, absence of vascular invasion, lymphatic and mucin. CMS3 tumors (29%) were also left-sided, with absence of vascular and lymphatic invasion, and 29% had an activating mutation of the KRAS oncogene. CMS4 tumors (21%) showed advanced stages and presence of metastases. CONCLUSIONS This new molecular classification contributes to understanding the heterogeneity of tumors. It is possible to differentiate molecular subgroups of a single pathological diagnosis of adenocarcinoma, opening the door to personalized medicine.

Prevalencia y características de mutaciones somáticas del gen KRAS en pacientes chilenos con cáncer colorrectal

BACKGROUND The molecular testing of KRAS mutation status in metastatic colorectal cancer patients is mandatory to identify patients eligible for anti-epidermal growth factor receptor monoclonal antibody therapy. AIM To report the frequency of KRAS gene mutations in Chilean patients with colorectal cancer (CRC). MATERIAL AND METHODS A cohort of 262 Chilean patients with CRC aged 26 to 90 years (53% males), was studied. KRAS mutation status was analyzed by real-time polymerase chain reaction and correlated with clinicopathological data. RESULTS Ninety-eight patients (37%) were positive for KRAS mutations. G12D was the most common mutation with a frequency of 36.7%, followed by G12V (25.5%), G13D (17.3%), G12A (7.1%), G12C (6.1%), G12S (5.1%) and G12R (2%). The frequency of the mutation in left, right colon and rectal tumors was 37.8, 32.6 and 44.9%, respectively. Among tumors with mutations, 86.7% were well or moderately differentiated tumors and the rest were poorly differentiated. No significant associations between KRAS gene mutations and other clinicopathological features of the tumor were observed. CONCLUSIONS The frequencies of KRAS mutations reported in this study are similar to frequencies reported for European and North-American populations, lower than in a Spanish study and higher than in a Peruvian study.Background: The molecular testing of KRAS mutation status in metastatic colorectal cancer patients is mandatory to identify patients eligible for anti- epidermal growth factor receptor monoclonal antibody therapy. Aim: To report the frequency of KRAS gene mutations in Chilean patients with colorectal cancer (CRC). Material and Methods: A cohort of 262 Chilean patients with CRC aged 26 to 90 years (53% males), was studied. KRAS mutation status was analyzed by real-time polymerase chain reaction and correlated with clinicopathological data. Results: Ninety-eight patients (37%) were positive for KRAS mutations. G12D was the most common mutation with a frequency of 36.7%, followed by G12V (25.5%), G13D (17.3%), G12A (7.1%), G12C (6.1%), G12S (5.1%) and G12R (2%). The frequency of the mutation in left, right colon and rectal tumors was 37.8, 32.6 and 44.9%, respectively. Among tumors with mutations, 86.7% were well or moderately differentiated tumors and the rest were poorly differentiated. No significant associations between KRAS gene mutations and other clinicopathological features of the tumor were observed. Conclusions: The frequencies of KRAS mutations reported in this study are similar to frequencies reported for European and North-American populations, lower than in a Spanish study and higher than in a Peruvian study. (Rev Med Chile 2014; 142: 1407-1414)

Abstract 2114: Deletions/Duplications detection by MLPA in Chilean families with hereditary colorectal cancer: Lynch syndrome and FAP

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The main variants of colorectal cancer hereditary are Familial Adenomatous Polyposis (FAP) and Lynch Syndrome, which account for approximately 5% of total cases of colorectal cancer. Point mutations in APC and MMR genes are responsible of about 85% and 50% of Chilean FAP (classic phenotype) and Lynch Syndrome (Amsterdam criteria) families, respectively. Different molecular strategies are available for the detection of mutations in these genes. Studies in other populations have identified deletions and duplications of one or more consecutive exons account for around 50% and 10-20% of the total alterations in MMR and APC genes, respectively. The aim of our work was the detection of genomic deletion/duplication through Multiplex Ligation-Dependent Probe Amplification (MLPA) in Lynch Syndrome and FAP patients in Chile. In this study, we analized alterations in MLH1 and MSH2 in 26 Lynch Syndrome families (5 Amsterdam and 21 Bethesda families), and in APC in 10 FAP families (6 classic and 4 attenuated), all of them non carriers of point mutations in these genes. We identified 3 different alterations in the MLH1 in four Amsterdam families, all of them are 0.5 times deletion, which are located: exon 1 in one family, exon 19 in two families, and exons 14 and 15 in one family. In the MSH2, in one Amsterdam family we detected a 0.5 time deletion of exon 2. In the APC, we found 2 different alterations in three classic FAP families: in one family we detected a 0.5 times deletion of the whole gene including promoter region, and in two families we found a 1.5 time amplification of exons 1, 2 and 3. These alterations were confirmed in other relatives of these families and in two independent MLPA analysis. The deletion of exon 19 in MLH1 and the amplification of exons 1, 2 and 3 in APC have not been previously described in other populations. In conclusion, genomic deletions/duplications were only detected in 5 Lynch Syndrome families that fulfilled Amsterdam criteria, and in 3 FAP families with classic phenotype. The detection of different mutations involved in these syndromes, allows the development of prevention strategies of these diseases. Financed by Cleveland Clinic Foundation and Las Condes Clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2114.

Abstract 5167: Expression levels of orexin receptor 1 in different stages of colorectal cancer

Introduction: Colorectal cancer (CRC) is the fourth leading cause of cancer death in Chile. Currently, treatment is surgical and adjuvance is based on chemotherapy mainly. One of the risk factors of CRC is obesity and metabolic syndrome. In search of factors that induce cell apoptosis, orexins were identified. Orexins are neuropeptides that regulate appetite, inhibit satiety and increase energy expenditure. The altered expression of orexin or its dysregulation has been associated with a wide range of human diseases such as narcolepsy, obesity, drug addiction and cancer. In rats and mice lacking orexin receptor, an increase in body mass index (BMI) was observed. Orexin Receptor 1 (OX1R) is overexpressed in CRC, but is not detected in normal colon tissue. Its activation by Orexin-A promotes apoptosis in cancer cell lines, and can be modulated by diet. Aim: To determine OX1R expression in the adenoma-carcinoma progression of CRC as possible therapeutic target, and its correlation with BMI. Material and Methods: Patients with neoplastic colorectal lesions undergoing surgical or endoscopic treatment between 2014 and 2015 were prospectively enrolled. Tissue samples of 29 patients were divided into 4 groups. Group A: control (n = 5), Group B: low-grade adenoma (n = 5), Group C: high-grade adenoma (n = 7), Group D: adenocarcinoma (n = 12). Formalin-fixed-paraffin-embedded samples were used to perform immunohistochemistry (IHC) of OX1R. Fresh tissues from Group D were used to assess protein and mRNA expression of OX1R by Western-blot and quantitative RT-PCR, respectively. Primary cultures were established from fresh tumor tissue of patients with adenocarcinoma. To compare OX1R expression levels we used the t-student test. Results: IHC expression of OX1R was detected both in the luminal membrane of colorectal epithelium and in the cytoplasm. We observed stained cells in 0-1% of the total area in Group A, B and C. In Group D, 7 samples showed staining 0-1%, 2 samples 1-10% and 3 samples 10-20%. High mRNA and protein expression was detected in stage III- IV adenocarcinoma samples compared to stage 0, I and II tumor samples. Normal adyacent tissue of CRC patients does not express OX1R. BMI is inversely associated with OX1R expression. Orexin A was able to induce apoptosis in primary cell cultures from advanced tumors in a dose dependent manner. Conclusions: OX1R is expressed scarcely in early stages of carcinogenesis. Its expression is induced significantly only in the most advanced adenocarcinomas, both at protein and mRNA levels, and is inversely associated with BMI. In primary cultures of patients with CRC, orexin A is able to induce apoptosis in a dose dependent manner. FONDECYT 1140012. Citation Format: Ana M. Wielandt, Cynthia Villarroel, Claudia Hurtado, Kento Inada, Hiroshi Kawachi, Daniela Simian, Maria T. Vial, Marcela Figueroa, Magdalena Castro, Udo Kronberg, Francisco Lopez-Kostner. Expression levels of orexin receptor 1 in different stages of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5167.

Análisis molecular del cáncer de colon esporádico

Background: In Chile, colorectal cancer (CRC) is often diagnosed in late stages. Thus, surgical treatment must be complemented with chemotherapy. KRAS mutations and microsatellite instability have been detected in these tumors. However, the response to treatment in patients without KRAS mutations varies and requires a better understanding. Aim: To determine the frequency and distribution of somatic point mutations in KRAS, BRAF and PIK3CA genes and microsatellite instability status (MSI) in patients with colon cancer (CC). Material and Methods: A prospective observational study of patients undergoing surgery for colon cancer. Tumor-derived DNA was analyzed by polymerase chain reaction (PCR) for the most frequent mutations of KRAS, BRAF and PIK3CA. PCR was also used to analyze MSI. Results: Fifty-eight patients with sporadic CC were analyzed, 16 showed KRAS mutations (G12R, G12D, G12V, G13D) and out of the 42 patients that did not show any mutation, 10 had mutations in BRAF (V600E) and PIK3CA (E542K, E545D, E545K, Q546E, H1047R). BRAF mutations alone or in combination with PIK3CA mutations were observed in 27% of high MSI tumors and in 2% of tumors without instability (p<0.049). A higher percentage of high MSI tumors were located in the right colon (p<0.001), and showed BRAF mutation (p <0.020). Conclusions: The highest percentage of high MSI and BRAF mutations was observed in the right colon. Therefore, this study suggests the presence of different molecular features between right and left colon tumors that should be considered when defining the therapeutic management.

Abstract 5551: Somatic mutations in KRAS, BRAF and PIK3CA genes in Chilean patients with sporadic colorectal cancer: Correlation with clinical and histological features

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: KRAS, BRAF and PIK3CA proto-oncogenes encode for proteins involved in epidermal growth factor receptor (EGFR) signaling pathways. Activating mutations in these genes may contribute to tumor development and induce resistance to biological therapies in patients with metastatic colorectal cancer (CRC). Furthermore, microsatellite instability (MSI) has been proposed as a prognostic and predictive marker in sporadic CRC. Patients and methods: In tumors of patients undergoing surgery for sporadic CRC at our institution, somatic mutations in KRAS, BRAF and PIK3CA genes were assessed by single-strand conformation polymorphism(SSCP) and direct sequencing. MSI analysis was performed using the 5 markers of the standard NIH panel. Clinical and histopathological data of the patients were obtained from our prospectively maintained database. Results: A total of 58 patients with a nearly equal gender distribution and a mean age of 62 years were included. A total of 31 mutations in 26 patients were identified. The frequency of mutations was 28% for KRAS, 9% for BRAF and 17% for PIK3CA. No difference was found in the mutation frecuency between the different tumor locations (42,1% in right colon vs. 46,2% in left colon/rectum, P= NS) or lymphnode status (37,5% in node positive vs. 50% in node negative tumors, P=0,346). Tumors with deeper invasion showed a higher frecuency of mutations, but this tendency did not reach statistical significance (32% in pT1-2 vs. 51% in pT3-4, P=0,157). In 26% of the patients, MSI-high was observed, more frecuently in tumors of the right colon (57,9% vs 10,3%, P<0,001). Patients with BRAF mutations, a higher proportion of MSI-high was found compared to BRAF wildtype (27% vs. 2,3%, P=0,013). There was no correlation between the lymphnode positivity and MSI status. Conclusion: Our results show that the frequency of mutations in patients with sporadic CRC in Chile is similar to that described in other countries. The presence of MSI-high is correlated with tumors in the right colon and with mutations in the BRAF gene. The tendency of finding more mutations in locally advanced and node-negative tumors needs to be confirmed in future studies with a larger number of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5551. doi:1538-7445.AM2012-5551

Globalización y salud pública

Conocimiento, actitudes y practicas sobre salud oral en ninos de 12 anos Frecuencia de malos habitos bucales en ninos de 4 a 11 anos de Rancagua Integracion del sistema de salud en Mexico: el caso de la atencion de emergencias obstetricas Modelo de pesquisa de cancer colorrectal. Resultados del proyecto Previcolon 2007-2009 Perfil del paciente dental adulto que acude a la HUAP (Posta Central) Rol de un registro en familiares asintomaticos de pacientes con poliposis adenomatosa familiar Salud de inmigrantes en Chile: observando mas alla del efecto del migrante sano