Functional characterization of MLH1 missense variants unveils mechanisms of pathogenicity and clarifies role in cancer.

Lynch syndrome is a heritable condition caused by a heterozygous germline inactivating mutation of the DNA mismatch repair (MMR) genes, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants, for which the clinical significance is unclear in many cases. We have identified three MLH1 missense alterations (p.(Glu736Lys), p.(Pro640Thr) and p.(Leu73Pro)) in six individuals from large Tunisian families. For none of these alterations, a classification of pathogenicity was available, consequently diagnosis, predictive testing and targeted surveillance in affected families was impossible. We therefore performed functional laboratory testing using a system testing stability as well as catalytic activity that includes clinically validated reference variants. Both p.(Leu73Pro) and p.(Pro640Thr) were found to be non-functional due to severe defects in protein stability and catalytic activity. In contrast, p.(Glu736Lys) was comparable to the wildtype protein and therefore considered a neutral substitution. Analysis of residue conservation and of the structural roles of the substituted residues corroborated these findings. In conjunction with the available clinical data, two variants fulfil classification criteria for class 4 "likely pathogenic". The findings of this work clarify the mechanism of pathogenicity of two unclear MLH1 variants and enables predictive testing and targeted surveillance in members of carrier families worldwide.

Clinicopathological and prognostic significance of DNA mismatch-repair protein expression in gastric adenocarcinoma

Although the significance of DNA mismatch repair (MMR) protein expression in colorectal cancer is well-established, it remains contentious in extra-colorectal cancers and mainly in gastric adenocarcinoma. Data from Africa and Arab world remain limited. This study explored the MMR expression in gastric adenocarcinoma and evaluated its clinicopathological and prognostic signification among Tunisian patients. A retrospective study of 72 gastric adenocarcinomas was carried out. Clinicopathological particularities and patient outcomes were recorded. MMR expression was determined by immunohistochemistry on whole sections of archived material. Survival analysis was realized utilizing the Kaplan-Meier estimates and Log-Rank test. Expression of MMR proteins was observed in 84.7% of gastric adenocarcinoma samples. The 11 remaining samples (15.3%) exhibited an altered pattern of MMR protein. A significant association was identified between deficient MMR expression and advanced age (p = 0.03), intestinal type (p = 0.04) and lymph node metastases (p = 0.04). No other significant relationship was observed with the remaining selected tumor features. Patient survival was significantly associated with lymph node invasion (p = 0.002), distant metastases (p = 0.02) and tumor differentiation (p = 0.03), but not with MMR status (p = 0.83). MMR deficiency was related to advanced-age, intestinal type and nodal metastasis, but not to survival of Tunisian patients with gastric adenocarcinoma. Larger multicenter studies with additional molecular investigation are required to more explore these tumors.

Bien que l'importance de l'expression des protéines de réparation des mésappariements de l'ADN (MMR) dans le cancer colorectal soit bien établie, elle reste controversée dans les cancers extra-colorectaux et principalement dans l'adénocarcinome gastrique. Les données de l'Afrique et du monde arabe restent limitées. Cette étude a exploré l'expression des protéines MMR dans l'adénocarcinome gastrique et a évalué sa signification clinicopathologique et pronostique chez les patients tunisiens. Une étude rétrospective de 72 adénocarcinomes gastriques a été réalisée. Les particularités clinicopathologiques et pronostiques des patients ont été enregistrées. L'expression des protéines MMR a été déterminée par immunohistochimie. L'analyse de survie a été réalisée en utilisant la méthode de Kaplan-Meier et le test Log-Rank. L'expression des protéines MMR a été observée dans 84,7 % des échantillons d'adénocarcinome gastrique. Les 11 cas restants (15,3 %) présentaient un profil d'expression altérée des protéines MMR. Une association significative a été identifiée entre l'expression déficiente de MMR et l'âge avancé (p = 0,03), le type intestinal (p = 0,04) et les métastases ganglionnaires (p = 0,04). Aucune autre relation significative n'a été observée avec les autres caractéristiques tumorales sélectionnées. La survie des patients était significativement associée à l'envahissement des ganglions lymphatiques (Log Rank, p = 0,002), aux métastases à distance (Log Rank, p = 0,02) et à la différenciation tumorale (Log Rank, p = 0,03), mais pas à l'expression de MMR (Log Rank, p = 0,03). Rang, p = 0,83). Le déficit de l'expression des protéines MMR était lié à l'âge avancé, au type intestinal et aux métastases ganglionnaires, mais pas à la survie des patients tunisiens ayant un adénocarcinome gastrique. Des études multicentriques avec des investigations moléculaires supplémentaires sont nécessaires pour explorer davantage le cancer gastrique avec expression déficiente des protéines MMR.

Clinicopathological significance of cancer stem cell markers CD44 and ALDH1 expression in breast cancer.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) has been reputed to be cancer stem cell (CSC) markers in breast cancer. Yet, the clinicopathologic and prognostic significance of these markers remain unclear. In this study, we have investigated the expression of these markers and their relation with conventional clinicopathologic tumor characteristic including molecular subtype. METHODS: CD44 and ALDH1 expression were investigated by immunohistochemistry in a series of 157 formalin-fixed paraffin-embedded breast cancer tissues. RESULTS: Overall, CD44 and ALDH1 are, respectively, detected in 33% (52 of 157) and 7% (10 of 157) of breast cancer cases. We also observed that CD44 expression was associated with histological grade (p = 0.005). For ALDH1, we found that its expression is more frequent with elderly women (> 50 years, p = 0.03). The investigation of relationship between the stem cell phenotype and breast cancer molecular subtype, revealed that CD44 and ALDH1 expression was more frequent in basal-like tumors (p = 0.005). Among the two cancer stem cell markers tested, ALDH1 showed a strong association with the basal marker EGFR (p = 0.05). CONCLUSIONS: These findings suggest that CD44 and ALDH1 play a role in the clinical behavior in breast cancer and might be interesting biomarkers and therapeutic targets.

Stromal CD10 expression in breast cancer correlates with tumor invasion and cancer stem cell phenotype.

BACKGROUND: Previous investigations have indicated that CD10 is associated with biological aggressivity in human cancers, but the use of this marker for diagnosis and prognosis is more complex. The aim of this study was to evaluate the expression of CD10 in breast cancer and its association with the clinicopathological features. In addition, we investigated whether a relationship exists between CD10 expression and cancer stem cells. METHODS: CD10 expression was examined by the immunohistochemistry in a series of 133 invasive breast carcinoma cases. Results were correlated to several clinicopathological parameters. Cancer stem cell phenotype was assessed by the immunohistochemical analysis of CD44 and ALDH1. RESULTS: Significant CD10 expression was found in the fusiform stromal cells in 19.5% of the cases and in the neoplastic cells in 7% of the cases. The stromal CD10 positivity was more frequently found in tumors with lymph node metastasis (p = 0.01) and a high histological grade (p = 0.01). However, CD10 expression by the neoplastic cells correlates with a high histological grade (p = 0.03) and the absence of estrogen (p = 0.002) as well as progesterone (p = 0.001) receptor expression. We also found that CD10 expression by the stromal cells, but not by the neoplastic cells, correlates significantly with the expression of cancer stem cell markers (CD44+/ALDH1+) (p = 0.002). CONCLUSION: These findings support the role of the stromal CD10 expression in breast cancer progression and dissemination, and suggest a relationship with cancer stem cells.

Changing patterns in the Epstein-Barr virus (EBV)and Hodgkin lymphoma association in Tunisia.

We compared the features of the Epstein-Barr virus (EBV) and Hodgkin lymphoma (HL) association in Tunisia in two periods of time, 1991-2001 (111 cases) and 2002-2012 (122 cases). The investigation of the EBV status by EBER in situ hybridization showed a significant decrease in the prevalence of EBV-positive HL from 69.3 % for the period 1991-2001 to 40.1 % for the 2002-2012 period (p = 0.00001). EBV positivity has decreased in all age groups but was more pronounced among young patients, in the 15-24-year age group (46.1 vs 10.3 %, p = 0.003), in the 25-34-year age group (56.2 vs 25 %, p = 0.04), and among children (88.4 vs 59.2 %, p = 0.01). This decrease in EBV-positive HL over time contrasted with a remarkable increase in EBV-negative HL in young adults aged 15-34 years (51.2 vs 83 %; p = 0.001), especially among women (59.1 vs 91.2 %; p = 0.01). The decrease in EBV-positive HL over time concerns particularly the nodular sclerosis histological subtype (69.2 vs 31.6 %, p = 0.000001). These results indicate that the epidemiology of HL and its association with EBV are changing over time, with a trend toward a Western profile, and point toward the emergence of other environmental causative factors, especially among young women, which remain to be identified.