Identificación del fenotipo de inestabilidad microsatelital en carcinoma colorrectal mediante el análisis de la expresión de proteínas reparadoras del ADN: Revisión narrativa / Identification of microsatellite instability phenotype in colorectal carcinoma through expression analysis of DNA mismatch repair proteins: Narrative review

El carcinoma colorrectal (CCR) es de las primeras causas de mortalidad del mundo, presentando Guatemala una incidencia anual de 7.4/millón de habitantes. El síndrome de Lynch se caracteriza clínicamente por un inicio temprano del CCR con lesiones causadas por alteraciones en genes que codifican proteínas reparadoras.Los microsatélites son regiones del ADN con una unidad repetitiva de uno o más nucleótidos y son susceptibles a errores durante la replicación de ADN de los enterocitos. Existe un sistema de reparación que corrige estos errores. Cuando las proteínas reparadoras de este sistema están mutadas o ausentes, dichos errores del ADN persisten. Estas proteínas reparadoras se expresan en el núcleo de las células colónicas normales y son detecta-bles utilizando estudios de inmunohistoquímica (IHQ). Los genes MLH1 y MSH2 pueden encontrarse mutados en el 90% de los casos de cáncer colorrectal y el resto corresponde a MSH6 y PMS2. Esta vía oncogénica se caracteriza por alteración del sistema de reparación de errores durante la replicación del ADN, controlado por los genes MMR (mismatch repair), principalmente MLH1, MSH2, MSH6 y PMS2. Se realizó una revisión extensa de la literatura en PubMed, Springer y JAMA, usando las palabras clave: fenotipo de CCR, Síndrome de Lynch e inestabilidad microsatelital, detectándose 55 artículos. El objetivo de esta revisión es describir la importancia de la identificación del fenotipo del CCR por medios de IHQ y de pruebas moleculares para el eficaz tratamiento con inmunoterapia anti-PD1/PD-L1.

Colorectal cancer (CRC) is one of the leading causes of mortality in the world. In Guatemala it's an important cause of morbidity (7.4 per million inhabitants). Lynch syndrome is clinically characterized by an early onset of nonpolyposis colorectal carcinoma, with multiple lesions and neoplasms. The syndrome is caused by mutations in genes encoding DNA mismatch repair proteins. The microsatellites are regions of the DNA that repeat between one or more nucleotides and are susceptible to errors during replication, these are corrected by a repair system, when genes are mutated, the errors persist. The genes encoding repair proteins are expressed in the nuclei of normal colonic cells which can be observed using immunohistochemical studies. The MLH1, MSH2 genes are found to be mutated in 90% of the cases and the rest corresponds to the MSH6 and PMS2 genes. This oncogenic pathway characteristically consists of an alteration in the DNA repair system that is controlled by mismatch repair genes (MMR). An extensive research was conducted on PubMed, Springer and JAMA, using the keyword: CRC phenotype, Lynch syndrome and microsatellite instability. 55 articles were found. This review«s objective is to understand the mechanisms of nonpolyposis colorectal cancer and the importance of identifying patients with a mutant phenotype as a predictive factor for the efficacy of the anti-PD1/PDL1 immunotherapy and for prognosis.

Síndrome de Lynch: aspectos genéticos, clínicos y diagnósticos / Lynch syndrome: genetic, clinical and diagnostic aspects

Esta revisión tiene como objetivo dar a conocer los aspectos genéticos, clínicos y diagnósticos del síndrome de Lynch, además de brindar la información más relevante acerca de la asesoría genética en estos pacientes y las recomendaciones actuales para su seguimiento.

This review aims to present the genetic, clinical and diagnostic aspects of Lynch syndrome, as well as providing the most relevant information about genetic counseling in these patients and the current recommendations for their surveillance.

Mutación fundadora en síndrome de Lynch tipo II / Founder mutation in Lynch syndrome

El síndrome de Lynch es la más frecuente de las neoplasias colorrectales hereditarias. Se origina por mutaciones germinales deletéreas familia-específicas en los genes que codifican proteínas de reparación del ADN: MLH1 (homólogo humano de mutL), MSH2 y MSH6 (homólogo humano de mutS 2 y 6, respectivamente), PMS2 (homólogo humano de PMS1 2) y MUTYH (homólogo humano de la ADN-glycosilasa mutY). La mutación c.2252_2253delAA, p.Lys751Serfs*3 en el exón 19 del gen MLH1 segrega con un haplotipo descripto en la región norte de Italia y cuyo origen fue atribuido a un efecto fundador. Esta mutación co-segrega con características típicas del síndrome de Lynch, incluyendo afectación temprana y múltiples tumores primarios en el mismo individuo, una alta frecuencia de cáncer pancreático, elevada inestabilidad microsatelital y falta de expresión de PMS2. En el presente trabajo se comunica dicha mutación en una paciente argentina con adenocarcinoma endometroide de útero en cuya historia familiar existen antecedentes de cáncer de colon diagnosticado antes de los 50 años en familiares de primer grado, reuniendo los criterios de Ámsterdam I y síndrome de Lynch II. Los polimorfismos presentes en la paciente coinciden con el haplotipo descripto en una región del norte de Italia. El alto grado de patogenicidad asociada a esta mutación hace imprescindible el estudio de todos los integrantes de las familias con cáncer hereditario permitiendo el diagnóstico genético pre-sintomático, la instauración de tratamientos o conductas preventivas y su seguimiento.

Lynch syndrome is the most frequent syndrome in hereditary colorectal cancer, a family-specific deleterious mutations in genes encoding DNA reparation proteins: MLH1 (mutL homolog 1), MSH2, MSH6 (mutS homolog 2 y 6, respectively), PMS2 (PMS1 homolog 2, mismatch repair system component) y MUTYH (mutY DNA glycosylase).The c.2252_2253delAA, p.Lys751Serfs*3 mutation in MLH1 gene segregates with a haplotype reported in the northern region of Italy and whose origin was attributed to a founder effect. This mutation co-segregates with typical characteristics of Lynch syndrome, including early age at onset and multiple primary tumors in the same individual, a high frequency of pancreatic cancer, high microsatellite instability and lack of PMS2 expression. This report describes a mutation in an Argentinian patient with endometrioid adenocarcinoma of uterus. Her first-degree relatives had a history of colon cancer diagnosed before 50 years, fulfilling the Amsterdam Criteria I and Lynch syndrome II. The high pathogenicity associated to this mutation makes necessary the study of all members from families with hereditary cancer, allowing pre-symptomatic genetic diagnosis, early assessment and the instauration of preventive treatments.

Completeness of pedigree and family cancer history for ovarian cancer patients / 부인종양

OBJECTIVE: To investigate the completeness of pedigree and of number of pedigree analysis to know the acceptable familial history in Korean women with ovarian cancer. METHODS: Interview was conducted in 50 ovarian cancer patients for obtaining familial history three times over the 6 weeks. The completeness of pedigree is estimated in terms of familial history of disease (cancer), health status (health living, disease and death), and onset age of disease and death. RESULTS: The completion of pedigree was 79.3, 85.1, and 85.6% at the 1st, 2nd, and 3rd time of interview and the time for pedigree analysis was 34.3, 10.8, and 3.1 minutes, respectively. The factors limiting pedigree analysis were as follows: out of contact with their relatives (38%), no living ancestors who know the family history (34%), dispersed family member because of the Korean War (16%), unknown cause of death (12%), reluctance to ask medical history of relatives (10%), and concealing their ovarian cancer (10%). The percentage of cancers revealed in 1st (2%) and 2nd degree (8%) relatives were increasing through surveys, especially colorectal cancer related with Lynch syndrome (4%). CONCLUSION: Analysis of pedigree at least two times is acceptable in Korean woman with ovarian cancer from the first study. The completion of pedigree is increasing, while time to take family history is decreasing during three time survey.

Genómica en el diagnóstico del cáncer colorrectal hereditario / Genetics in diagnosis of hereditary colorectal cancer

Introducción: El cáncer colorrectal (CCR) se presenta en el 3 - 5% como formas hereditarias. Existen 4 síndromes en donde se han descubierto mutaciones responsables, el síndrome de Lynch (SL), la Poliposis Adenotamosa Familiar (PAF), la Poliposis Juvenil (PJ) y el síndrome de Peutz-Jeghers (SPJ). Cada uno de ellos presenta una patente genética distinta, vías de carcinogénesis y comportamientos distintos. Los conocimientos actuales sobre las mismas son limitados y los abordajes diagnósticos controvertidos. Material y Métodos: Se ha realizado la búsqueda bibliográfica sobre las técnicas de biología molecular que permiten detectar las mutaciones en los síndromes de CCR hereditario, así como las guías y estrategias diagnósticas. Se presenta una breve reseña sobre conceptos básicos de genómica y técnicas de biología molecular y luego sus usos en la práctica clínica en estas 4 enfermedades. Resultados: Hemos encontrado que con el avance de las técnicas de biología molecular cada día es mayor el conocimiento con respecto a la base genética de estas enfermedades. Esto provoca un impacto tanto en el diagnóstico como en la terapéutica y seguimiento. Las guías actuales de manejo muestran consenso en gran cantidad de puntos aunque todavía queda campo por explorar. Conclusiones: Hacen falta futuros ensayos que nos permitan arribar a estrategias de manejo en cada subgrupo de pacientes seguramente basados en las distintas patentes genotípicas. Esto permitirá un manejo más personalizado en el futuro del cáncer colorrectal hereditario.

Introduction: 3 - 5% of colorectal cancer (CRC) occurs as hereditary forms. There are 4 syndromes in which mutations have been found responsible, the Lynch syndrome (LS), the Family Adenotamosa polyposis (FAP), Juvenile Polyposis (JP) and Peutz-Jeghers syndrome (PJS). Each one has a distinct genetic patent, different process of carcinogenesis and different behaviors. Current knowledge about them is limited and the diagnostic approaches are controversial. Material and methods: We performed literature searches on molecular biology techniques to detect mutations in hereditary CRC syndromes and diagnostic guidelines and strategies. A review of basic concepts of genomics and molecular biology techniques are presented and then their uses in clinical practice in these 4 diseases. Results: We have found that with the progress of molecular biology techniques is growing the knowledge about the genetic basis of these diseases. This causes an impact on both diagnosis and therapy and monitoring. Current guidelines show consensus in handling large amount of points but there is still room to explore. Conclusions: Future trials are needed to enable us to arrive at management strategies in each subgroup of patients likely based in different genotypic patents. This will allow a more personalized management in the future of hereditary colorectal cáncer.

Síndrome de Lynch: selección de pacientes para el estudio genético mediante análisis de inestabilidad microsatelital e inmunohistoquímica / Lynch syndrome: selection of families by microsatellite instability and immunohistochemistry

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 Im-munohistochemistry (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.

Mismatch repair genes expression defects & association with clinicopathological characteristics in colorectal carcinoma.

Background & objectives: DNA mismatch repair gene (MMR) abnormalities are seen in 95 per cent of hereditary nonpolyposis colorectal cancer (HNPCC) and 10-15 per cent of sporadic colorectal cancers. There are no data on MMR abnormalities in Malaysian colorectal cancer patients. This study was aimed to determine the frequency of abnormal MMR gene protein expression in colorectal carcinoma in Northern Peninsular Malaysia using immunohistochemistry. Methods: Clinicopathological information was obtained from 148 patients’ records who underwent bowel resection for colorectal cancer (CRC) at the three hospitals in Malaysia. Immunohistochemistry for MLH1, MSH2, MSH6 and PMS2 proteins were performed on paraffin embedded tissue containing carcinoma. Results: A total of 148 subjects and 150 colorectal carcinomas of sporadic and hereditary types were assessed. Three patients had synchronous tumours. Twenty eight cancers (18.6%) from 26 subjects (17.6%) had absent immunohistochemical expression of any one of the MMR gene proteins. This comprised absent MLH1 only – 3 cancers, absent MSH2 only – 3, absent MSH6 only – 2, absent PMS2 only – 3, absent MLH1 and PMS2 – 14, absent MSH2 and MSH6 – 2 and absent MLH1, MSH6 and PMS2 – 1. There was significant association between abnormal MMR gene protein expression and proximal colon cancers, mucinous, signet ring and poorly differentiated morphology. Interpretation & conclusions: Cancers with abnormal MMR gene expression were associated with microsatellite instability-high (MSI-H) phenotype. About 15 per cent demonstrated absent MSH2, MSH6 and PMS2 protein expression in isolation or in combination with other MMR genes, which often predicts a germline mutation, synonymous with a diagnosis of HNPCC. This appears to be high frequency compared to reported data.

Cáncer hereditario de colon: Aportes del diagnóstico genético molecular / Molecular and genetic studies for hereditary colon cancer in two patients and their families

Background: About 30 percent of cases of colon cancer (CC) have a family history of CC, and only 5 percent are hereditary forms. Hereditary forms have an increased risk of CC and other tumors. Aim: To report the molecular and genetic study in two families with hereditary CC. Material and Methods: Molecular analysis of the adenomatous polyposis coli (APC) gene of familial adenomatous polyposis (FAP), was done in a patient with multiple benign polyps and his children. Molecular analysis was performed for MLH1 gene mutation of hereditary non-polyposis colon cancer (HNPCC) in an asymptomatic patient with family history of multiple cancers and his mother with a confrmed mutation in the MLH1 gene. Results: The patient with FAP had an insertion of 17 base pairs in exon 9 of the APC gene and two of his children had the same mutation. The patient with history of HNPCC did not have the family mutation on MLH1. Conclusions: In the case of FAP, molecular study was performed in his children since manifestations in carriers of the mutation may begin in childhood. If the second patient would have had the mutation, the study of his children could have been postponed until the age of 18, when the risk for CC is increased.

Mutación fundadora en una familia argentina con cáncer colorrectal hereditario / Detection of a founder mutation in an Argentine family with hereditary non polyposis colorectal cancer

El cáncer colorrectal hereditario no poliposo (CCHNP) se relaciona con mutaciones en los genes reparadores de ADN (MLH1, MSH2 y MSH6). La mayoría de estas alteraciones son familia-específicas y su detección suele requerir la secuenciación completa de los genes relacionados. Se detectó una mutación puntual (2269-2270insT) en el último codón del gen MLH1 en familias de un área del norte de Italia (Reggio Emilia) y su origen se considera debido a un efecto fundador. En este trabajo presentamos una familia mendocina con CCHNP portadora de la misma mutación, cuyos ancestros eran oriundos de Reggio Emilia. Para la detección de la mutación se diseñó una estrategia basada en PCR y posterior corte enzimático. La mutación fue hallada en tres integrantes de la familia estudiada, dos de los cuales no presentaban sintomatología clínica. Estos pacientes fueron seguidos preventivamente mediante colonoscopias. La metodología utilizada en nuestro laboratorio fue específica y sensible para la detección de una mutación previamente registrada y permitió realizar el diagnóstico genético molecular en el país, evitando el envío de muestras al extranjero. Es de importancia destacar que el diagnóstico genético pre-sintomático de cáncer hereditario, enfocado desde un grupo multidisciplinario de profesionales, permite un mejor seguimiento y apoyo a las familias afectadas.

Hereditary non polyposis colorectal cancer (HNPCC) has been related to mutations in the DNA mismatch repair genes (MLH1, MSH2 y MSH6). Mutation detection analysis requires the complete sequencing of these genes, given the high frequency of family-specific alterations. A point mutation (2269- 2270insT) in the last codon of the MLH1 gene has been detected in families from a northern region of Italy (Reggio Emilia).Given that this alteration was registered only in people from this region, it has been considered a founder mutation. In this work, we present an Argentine HNPCC family whose ancestors were natives from the Reggio Emilia, Italy, and who were carriers for this mutation. In order to detect the genetic alteration, a PCR was developed followed by a restriction enzyme incubation assay. The mutation was detected in 3 family members, two of them without clinical symptoms. The PCR/restriction enzyme methodology has been sensitive and specific for the detection of this mutation. It has allowed the performance of a pre-symptomatic genetic diagnosis in the Argentine HNPCC family, avoiding sending samples abroad. It is worth mentioning that pre-symptomatic diagnosis of hereditary cancers allows enhanced surveillance and support for the affected families when it is performed by a multidisciplinary group.

R659X mutation in the MLH1 gene in hereditary non-polyposis colorectal cancer (HNPCC) in an Indian extended family.

Background & objective: Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome), is a genetically heterogeneous disorder that is believed to account for 2–10 per cent of all the colorectal cancer cases. The disease follows autosomal dominant inheritance pattern with high penetrance (85%) and younger age of onset when compared to patients with sporadic tumours. HNPCC is associated with germ-line mutations in the DNA mismatch repair (MMR) genes namely MLH1, MSH2, MSH6, and PMS2. The present study was aimed at analyzing mismatch repair gene(s) in an extended Indian family satisfying the Amsterdam criteria, and extending the analysis to general population to estimate frequency of the mutations/polymorphisms observed. Methods: A total 12 members of the HNPCC family were studied for genetic investigation. Ethnically matched 250 normal individuals were also included as controls to study the observed mutations/ polymorphisms at population level. Results: The analysis resulted in identification of a 1975C>T mutation in exon 17, resulting in substitution of arginine residue with stop codon at codon 659. 655A>G substitution was also observed, resulting in replacement of isoleucine with valine at codon 219. Similar analysis on 250 ethnically matched control subjects revealed complete absence of R659X mutation, while I219V variant was found in 9.8 per cent of the controls. Interpretation & conclusion: R659X mutation correlates with disease phenotype, and 655A>G locus is highly polymorphic. Our study suggested that R659X substitution was prime cause for the disease phenotype in this family. I219V substitution is a polymorphism having no association with the disease onset or segregation. The family members harbouring this mutation were advised to be under regular medical surveillance.

Mismatch repair genes in Lynch syndrome: a review: [review] / Genes de reparo de DNA na síndrome de Lynch: uma revisão: [revisão]

Lynch syndrome represents 1-7 percent of all cases of colorectal cancer and is an autosomal-dominant inherited cancer predisposition syndrome caused by germline mutations in deoxyribonucleic acid (DNA) mismatch repair genes. Since the discovery of the major human genes with DNA mismatch repair function, mutations in five of them have been correlated with susceptibility to Lynch syndrome: mutS homolog 2 (MSH2); mutL homolog 1 (MLH1); mutS homolog 6 (MSH6); postmeiotic segregation increased 2 (PMS2); and postmeiotic segregation increased 1 (PMS1). It has been proposed that one additional mismatch repair gene, mutL homolog 3 (MLH3), also plays a role in Lynch syndrome predisposition, but the clinical significance of mutations in this gene is less clear. According to the InSiGHT database (International Society for Gastrointestinal Hereditary Tumors), approximately 500 different LS-associated mismatch repair gene mutations are known, primarily involving MLH1 (50 percent) and MSH2 (40 percent), while others account for 10 percent. Much progress has been made in understanding the molecular basis of Lynch Syndrome. Molecular characterization will be the most accurate way of defining Lynch syndrome and will provide predictive information of greater accuracy regarding the risks of colon and extracolonic cancer and enable optimal cancer surveillance regimens.

A síndrome de Lynch representa de 1-7 por cento de todos os casos de câncer colorretal. É uma síndrome de herança autossômica dominante que predispõe ao câncer e é causada por mutações nos genes de reparo de ácido desoxirribonucléico (DNA). Desde a descoberta dos principais genes com função de reparo de DNA, mutações nos genes MSH2, MLH1, MSH6, PMS2 e PMS1 estão relacionadas com a susceptibilidade à síndrome de Lynch. Outro gene, MLH3, tem sido proposto como tendo papel na predisposição à síndrome de Lynch, porém mutações de significância clínica nesse gene não são claras. De acordo com o banco de dados InSiGHT (International Society for Gastrointestinal Hereditary Tumors), aproximadamente 500 diferentes mutações associadas à síndrome de Lynch são conhecidas, envolvendo primeiramente MLH1 (50 por cento), MSH2 (40 por cento) e outros (10 por cento). Grandes progressos têm ocorrido para nosso entendimento das bases moleculares da síndrome de Lynch. A caracterização molecular será a forma mais precisa para definirmos a síndrome de Lynch e irá fornecer informações preditivas mais precisas sobre o risco de câncer colorretal e extra-colônico, além de permitir regimes otimizados de manejo.

Síndrome de Lynch: Caracterización genético clínica. Caso clínico / Hereditary non-polyposis colorectal cancer: Report of four siblings

Hereditary non-polyposis colorectal cancer (HNPCC) or Lynch Syndrome is an autosomic dominant syndrome involving 596-1096 of colorectal cancer patients. Mutations in MLH1 and MSH2 genes account for most cases. These two genes particípate in the DNA mismatch repair pathway. Therefore mutation carriers show microsatellite instability (MSI) in tumors. This syndrome is characterized by the early development of colorectal cancer (before 50 years) and an increased incidence of cancer in other organs. We report four siblings from a family diagnosed with HNPCC. All of them were subjected to colonic surgery for colorectal cancer Moreover, one patient developed an ampulloma after her colon surgery. The molecular-genetic analysis revealed three brothers with microsatellite instability in the tumor tissue, the absence of the MLH1 protein, and the presence of a germ Une mutation localized in introm 15 ofthe MLH1 gene.

Bases genéticas del cáncer colorrectal hereditario / Genetic basis of hereditary colorectal cancer

Las variantes hereditarias representan aproximadamente el 5 por ciento de los casos de cáncer colorrectal. Las formas más frecuentes son la Poliposis Adenomatosa Familiar (PAF) y el Cáncer Colorrectal Hereditario No Polipósico (HNPCC) o Síndrome de Lynch. Esta revisión analiza los genes relacionados, sus mecanismos moleculares y la importancia de los estudios genéticos en el manejo de los pacientes y las familias comprometidas. En la PAl; diferentes mutaciones del gen APC son responsables del desarrollo de cientos de pólipos en colon y recto, y sus manifestaciones extracolónicas como tumores desmoides, pólipos gástricos y duodenales. En el HNPCC, mutaciones principalmente en los genes MLH1, MSH2 Y MSH6 son la causa de tumores en el colon, endometrio, ovario, estómago, urotelio e intestino delgado.

Hereditary variants account for approximately 5 percent of colorectal cancers. Within them, Familiar Adenomatous Polyposis (FAP) and Hereditary Non Polyposis Colorectal Cancer (HNPCC) or Lynch's syndrome are the most frequent. The underlyinggenes and their molecular pathways are reviewed, and the importance of genetic testing for the clinical management of patients and their families is analyzed. In FARmutations on the APC gene are responsible for the development of hundreds of colorectal polyps and extracolonic manifestations such as desmoid tumors, gastric and duodenal polyps. In HNPCC, mutations on MLH1, MSH2 and MSH6 genes are the most frequent causes of colonic, endometrial, ovarian, gastric, urothelial and small bowel tumors.

Expresion inmunohistoquimica e inestabilidad microsatelital en el sindrome de lynch / Immunohistochemical expression and microsatellite instability in Lynch syndrome

Las mutaciones de los genes MLH1 y MSH2 son frecuentemente implicadas en el síndrome de Lynch. La expresión inmunohistoquímica (IHQ) es una forma simple de selección para pruebas moleculares. Se analizó la IHQ de MLH1 y MSH2 en pacientes con síndrome de Lynch (16 casos) y pacientes menores de 50 años sin antecedentes familiares (25 casos). Se estudiaron 41 tumores de un grupo de pacientes (64% mujeres) de edad promedio 40.7 años (rango: 16-75). Se obtuvieron resultados concluyentes en 40 casos (97.6%). Dieciocho casos (45%) presentaron falta de expresión (MLH1 negativa: 11 casos; MSH2 negativa: 6 casos; MLH1 negativa y MSH2 negativa: 1 caso), con una incidencia significativamente mayor en pacientes con síndrome de Lynch (68.7% vs. 28%, p=0.01). Entre los casos esporádicos, 5 casos (20%) mostraron falta de expresión MLH1 y 2 casos (8%) con falta de expresión MSH2. La falta de expresión IHQ presentó una fuerte asociación con inestabilidad microsatelital alta (IMS): expresión normal: 5.9%, expresión negativa: 92.3%, P<0.0001. Los índices de sensibilidad y especificidad de la IHQ para detectar IMS fueron de 92.3% y 94.1% respectivamente. Los patrones de IHQ y de IMS no se relacionaron a ninguna característica histopatológica. En conclusión, el análisis inmunohistoquímico de las proteínas MLH1 y MSH2 fue altamente sensible y específico para detectar IMS y permitió identificar en un 45% de los casos la proteína alterada. El índice de falta de expresión IHQ entre los casos esporádicos diagnosticados antes de los 50 años justifica su implementación sistemática en este grupo de pacientes.

Mutation of the mismatch repair genes MLH1 and MSH2 account for the majority of the genetic abnormalities in Lynch syndrome. Immunohistochemical detection of their protein products is becoming an increasingly common method to detect these mutations. The aim of this study was to compare the expression of MLH1 and MSH2 by immunohistochemistry and its relationship with a group of clinical and histological variables in patients with known Lynch syndrome (n=16) and in cohort of young patients (less than 50 years) who did not meet Amsterdam criteria (n=25). The mean age was 40.7 and 64% were women. Conclusive results were obtained in 40 cases (97.6%). Eighteen cases (45%) showed abnormal expression of either MLH1 (11 cases) or MSH2 (6 cases) and both stains (1 case). Alteration of the normal staining pattern was seen more commonly in patients with Lynch syndrome than in the sporadic group (68.7% vs 28%, p=0.01). A significant correlation was obtained between abnormal protein expression and microsatellite instability (MSI): normal expression: 5.9%, lack of expression: 92.3%, p<0.0001. The sensitivity and specificity of the immunohistochemical to predict MSI were 92.3% and 94.1% respectively. Immunohistochemistry and MSI results did not correlate with any histopathological parameter. In conclusion, in our experience abnormal staining of MLH and MSH correlates strongly with the presence of MSI. In addition it appears that in our population a significant proportion of young patients (< 50 years old) demonstrate alterations in the mismatch repair gene products suggesting an important role of these molecules in tumorigenesis.

Análisis genético en pacientes con cáncer colorrectal / Microsatellite instability among patients with colorectal cancer

Background: In patients with colorectal carcinoma, insertions or deletions of short sequences of DNA, a phenomenon called microsatellite instability, are observed. Aim: To look for microsatellite instability and mutations of MLH1 and MSH2 gene mutations in patients with colorectal carcinoma. Material and Methods: Ten patients with sporadic colorectal carcinoma and 31 patients fulfilling criteria for hereditary nonpolyposis colon cancer (HNPCC), aged 9 to 70 years, were studied. Microsatellite instability was studied in samples of tumor and peripheral blood mononuclear cell DNA. Six markers were amplified by polymerase chain reaction and capillary electrophoresis. In samples with microsatellite instability, mutations of MLH1 and MSH2 genes were studied by direct sequencing. Results: Thirty four percent of patients had microsatellite instability and among these, 76 percent had a high degree of instability. BAT40 marker had the higher frequency of instability. No mutations for MLH1 and MSH2 genes were observed. However a new polymorphism, C399T, was identified in exon 3 of MSH2 gene. This polymorphism was observed both in patients with sporadic colorectal carcinoma and patients with HNPCC. Conclusions: There is a high frequency of microsatellite instability among patients with colorectal cancer. A new polymorphism, not previously reported, was identified in MSH2 gene.

Cáncer colorrectal hereditario: análisis molecular de los genes APC y MLH1 / Hereditary colorectal cancer: Molecular analysis of APC and MLH1 genes

Background: Among colorectal cancer hereditary variants, two syndromes show a predisposition to the disease based on germline mutations: Familial Adenomatous Polyposis (FAP) and Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Aim: To screen mutations in FAP and HNPCC families in Chile. Materials and Methods: Two FAP and one HNPCC families were studied. The APC gene (for FAP patients) and the MLH1 gene (for HNPCC patients), were screened for mutations on genomic DNA. The molecular analysis was performed through polymerase chain reaction, Single Strand Conformer Polymorphism (SSCP) and DNA sequencing. Mutations were defined as changes in the DNA sequence leading into a stop codon and a truncated protein. Results: In the two FAP families the analysis revealed a mutation consisting in the deletion of five nucleotides named c.3927_3931delAAAGA. The genetic study of the HNPCC family demonstrated the insertion of one adenine in codon 168 of exon 6, named c.504insA. Discussion: Germ-line mutations were identified in the three families. The relevance of these studies in a better knowledge of cancer susceptibility, and the possibility of identifying in relatives in risk by molecular diagnosis.