RESUMEN
BACKGROUND: Lynch syndrome is an inherited tumour predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes. Mutation carriers have a high risk of developing colorectal cancer, but do not present with polyposis, a typical feature of other colorectal cancer syndromes such as familial adenomatous polyposis, in which polyposis reflects the high frequency of biallelic APC gene inactivation. We asked whether in Lynch syndrome biallelic inactivation of MMR genes occurred at a similar frequency to that of APC gene, and whether MMR inactivation resulted in detectable lesions within the intestinal mucosa. METHODS: Resections done for small and large bowel cancer between January, 2002, and January, 2011, were retrieved. We systematically analysed non-tumorous mucosa from carriers of a Lynch syndrome mutation (set 1: ten patients) and control patients without Lynch syndrome (set 1: nine patients) for MMR protein expression (MLH1, MSH2, and EPCAM) with immunohistochemistry. We validated the findings in an independent sample set (set 2: 30 Lynch syndrome patients, 79 controls). We did an analysis of microsatellite instability by PCR analysis to test lesions for mismatch repair deficiency. We applied a Poisson regression model to analyse the distribution of MMR-deficient crypt foci counts and a Fisher's exact test to compare the prevalence of these foci between mutation carriers and control patients. FINDINGS: 20 crypt foci with no MMR protein expression were detected in 20·1 cm(2) of non-tumorous mucosa from Lynch syndrome patients (set 1), an additional five were detected upon resectioning of two samples. In an independent validation set (set 2), two MMR-deficient crypt foci were noted in 2·2 cm(2) of mucosa. No MMR-deficient crypt foci were noted in non-tumorous mucosa from control patients without evidence for Lynch syndrome (set 1: 3·7 cm(2), set 2: 4·8 cm(2)). Microsatellite instability was detected in all seven MMR-deficient crypt foci analysed. A subset of these foci displayed unusual architectural and cytological abnormalities, although they had no polypous or adenomatous appearance. INTERPRETATION: We identified a novel type of lesion, the MMR-deficient crypt focus, as the manifestation of biallelic MMR gene inactivation in Lynch syndrome. The abundance of MMR-deficient crypt foci indicates a high frequency of biallelic MMR gene inactivation, which is in sharp contrast with the low number of clinically manifest cancers in Lynch syndrome. This discrepancy suggests that most MMR-deficient crypt foci do not progress to cancer. We propose Lynch syndrome as a unique model syndrome for studying initial steps of MMR deficiency, tumour initiation and, possibly, elimination. FUNDING: German Cancer Aid and German Research Foundation.
Asunto(s)
Focos de Criptas Aberrantes/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Reparación de la Incompatibilidad de ADN , Predisposición Genética a la Enfermedad/epidemiología , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Focos de Criptas Aberrantes/genética , Adulto , Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Molécula de Adhesión Celular Epitelial , Femenino , Mutación de Línea Germinal , Heterocigoto , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Intestino Delgado/patología , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Adhesión en Parafina , Distribución de Poisson , Prevalencia , Valores de ReferenciaRESUMEN
Germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene lead to silencing of MSH2 and cause Lynch syndrome. We have recently reported that lack of EPCAM expression occurs in many, but not all tumors from Lynch syndrome patients with EPCAM germline deletions. The differences in EPCAM expression were not related to the localization of EPCAM germline deletions. We therefore hypothesized that the type of the second somatic hit, which leads to MSH2 inactivation during tumor development, determines EPCAM expression in the tumor cells. To test this hypothesis and to evaluate whether lack of EPCAM expression can already be detected in Lynch syndrome-associated adenomas, we analyzed four carcinomas and two adenomas from EPCAM germline deletion carriers for EPCAM protein expression and allelic deletion status of the EPCAM gene region by multiplex ligation-dependent probe amplification. In four out of six tumors we observed lack of EPCAM expression accompanied by biallelic deletions affecting the EPCAM gene. In contrast, monoallelic retention of the EPCAM gene was observed in the remaining two tumors with retained EPCAM protein expression. These results demonstrate that EPCAM expression in tumors from EPCAM deletion carriers depends on the localization of the second somatic hit that inactivates MSH2. Moreover, we report lack of EPCAM protein expression in a colorectal adenoma, suggesting that EPCAM immunohistochemistry may detect EPCAM germline deletions already at a precancerous stage.
Asunto(s)
Adenoma/genética , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Carcinoma/genética , Moléculas de Adhesión Celular/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Mutación de Línea Germinal , Eliminación de Secuencia , Adenoma/química , Adenoma/patología , Adulto , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Carcinoma/química , Carcinoma/patología , Moléculas de Adhesión Celular/análisis , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/química , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Regulación hacia Abajo , Molécula de Adhesión Celular Epitelial , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Alemania , Humanos , Inmunohistoquímica , Pérdida de Heterocigocidad , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Proteína 2 Homóloga a MutS/genética , Linaje , FenotipoRESUMEN
PURPOSE: Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and dissemination. EMT occurs predominantly at the tumor edge where it is induced by cytokines, the extracellular matrix environment, or hypoxia. In the tumor cell, it is further mediated by several transcription factors and microRNAs. The aim of this study was to explore the expression of EMT-associated genes at the invasive front in colorectal cancer and to evaluate their prognostic significance. EXPERIMENTAL DESIGN: We evaluated the expression of 13 EMT-associated genes at the invasion front of 30 colorectal liver metastases by quantitative real-time PCR. Immunostaining against zinc finger E-box-binding homeobox 2 (ZEB2) was carried out on 175 primary colorectal cancer specimens and 30 colorectal liver metastases and correlated to clinical and histopathologic data. DLD-1 cells were transfected with siRNA and subjected to migration and invasion assays. RESULTS: Gene expression analysis and immunohistochemistry showed an upregulation of ZEB2 at the invasion front in primary colorectal cancer and liver metastases. Overexpression of ZEB2 at the invasion front correlated significantly with tumor stage in primary colorectal cancer. Moreover, univariate and multivariate analysis revealed overexpression of ZEB2 at the invasion front as an independent prognostic marker for cancer-specific survival. Downregulation of ZEB2 by siRNA decreased the migration and invasion capacity of DLD-1 cells in vitro. CONCLUSIONS: Overexpression of ZEB2 at the invasion front correlates with tumor progression and predicts cancer-specific survival in primary colorectal cancer. Therefore, ZEB2 may be interesting as biomarker and potential target for treatment of colorectal cancer.