Mutations in the WTX - gene are found in some high-grade microsatellite instable (MSI-H) colorectal cancers

Background: Genetically, colorectal cancers (CRCs) can be subdivided into tumors with chromosomal instability (CIN) or microsatellite instability (MSI). In both types of CRCs genes that are involved in the degradation of beta-CATENIN are frequently mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli) is affected in most cases, high grade MSI (MSI-H) CRCs frequently display mutations in various genes, like the APC-, AXIN2- or CTNNBI (beta-CATENIN) gene itself. Recently in Wilms tumors, WTX (Wilms tumor gene on the X-chromosome) was discovered as another gene involved in the destruction of beta-CATENIN. As the WTX-gene harbors a short T(6)-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in the WTX gene, thus additionally contributing to the stabilization of beta-CATENIN in human CRCs. Methods: DNA was extracted from 632 formalin-fixed, paraffin-embedded metastatic CRCs (UICCIV) and analyzed for MSI-H by investigating the stability of the highly sensitive microsatellite markers BAT25 and BAT26 applying fluorescence capillary electrophoresis (FCE). Then, in the MSI-H cases, well described mutational hot spot regions from the APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations by didesoxy-sequencing while the WTX T(6)-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T(5)-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, the KRAS and the BRAF proto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression of WTX from MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT-) PCR and FCE. Results: In our cohort of 632 metastatic CRCs (UICCIV) we identified 41 MSI-H cases (6.5%). Two of the 41 MSI-H cases (4.8%) displayed a frameshift mutation in the T(6)-repeat resulting in a T(5) sequence. Only one case, a male patient, expressed the mutated WTX gene while being wild type for all other investigated genes. Conclusion: Mutations in the WTX-gene might compromise the function of the beta-CATENIN destruction complex in only a small fraction of MSI-H CRCs thus contributing to the process of carcinogenesis

The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis

Background:Tumour-infiltrating lymphocytes (TILs) are often found in tumours, presumably reflecting an immune response against the tumour. We carried out a systematic review and meta-analysis, aiming to establish pooled estimates for survival outcomes based on the presence of TILs in cancer.Methods:A Pubmed and Embase literature search was designed. Studies were included, in which the prognostic significance of intratumoural CD3+, CD4+, CD8+, and FoxP3+ lymphocytes, as well as ratios between these subsets, were determined in solid tumours.Results:In pooled analysis, CD3+ TILs had a positive effect on survival with a hazard ratio (HR) of 0.58 (95% confidence interval (CI) 0.43-0.78) for death, as did CD8+ TILs with a HR of 0.71 (95% CI 0.62-0.82). FoxP3+ regulatory TILs were not linked to overall survival, with a HR of 1.19 (95% CI 0.84-1.67). The CD8/FoxP3 ratio produced a more impressive HR (risk of death: HR 0.48, 95% CI 0.34-0.68), but was used in relatively few studies. Sample size and follow-up time seemed to influence study outcomes.Conclusion:Any future studies should be carefully designed, to prevent overestimating the effect of TILs on prognosis. In this context, ratios between TIL subsets may be more informative.British Journal of Cancer advance online publication, 31 May 2011; doi:10.1038/bjc.2011.189 www.bjcancer.com

STAT3 in epithelial cells regulates inflammation and tumor progression to malignant state in colon

AbstractChronic inflammation is an important risk factor for the development of colorectal cancer; however, the mechanism of tumorigenesis especially tumor progression to malignancy in the inflamed colon is still unclear. Our study shows that epithelial signal transducer and activator of transcription 3 (STAT3), persistently activated in inflamed colon, is not required for inflammation-induced epithelial overproliferation and the development of early-stage tumors; however, it is essential for tumor progression to advanced malignancy. We found that one of the mechanisms that epithelial STAT3 regulates in tumor progression might be to modify leukocytic infiltration in the large intestine. Activation of epithelial STAT3 promotes the infiltration of the CD8+ lymphocyte population but inhibits the recruitment of regulatory T (Treg) lymphocytes. The loss of Stat3 in epithelial cells promoted the expression of cytokines/chemokines including CCL19, CCL28, and RANTES, which are known to be able to recruit Treg lymphocytes. Linked to these changes was the pathway mediated by sphingosine 1-phosphate receptor 1 and sphingosine 1-phosphate kinases, which is activated in colonic epithelial cells in inflamed colon with functional STAT3 but not in epithelial cells deleted of STAT3. Our data suggest that epithelial STAT3 plays a critical role in inflammation-induced tumor progression through regulation of leukocytic recruitment especially the infiltration of Treg cells in the large intestine

Tumour-infiltrating CD8+ lymphocytes and colorectal cancer recurrence by tumour and nodal stage

Background Intratumoural T-cell infiltrate intensity cortes wrelaith clinical outcome in stage II/III colorectal cancer (CRC). We aimed to determine whether this association varies across this heterogeneous group. Methods We performed a pooled analysis of 1804 CRCs from the QUASAR2 and VICTOR trials. Intratumoural CD8+ and CD3+ densities were quantified by immunohistochemistry in tissue microarray (TMA) cores, and their association with clinical outcome analysed by Cox regression. We validated our results using publicly available gene expression data in a pooled analysis of 1375 CRCs from seven independent series. Results In QUASAR2, intratumoural CD8+ was a stronger predictor of CRC recurrence than CD3+ and showed similar discriminative ability to both markers in combination. Pooled multivariable analysis of both trials showed increasing CD8+ density was associated with reduced recurrence risk independent of confounders including DNA mismatch repair deficiency, POLE mutation and chromosomal instability (multivariable hazard ratio [HR] for each two-fold increase = 0.92, 95%CI = 0.87–0.97, P = 3.6 × 10−3). This association was not uniform across risk strata defined by tumour and nodal stage: absent in low-risk (pT3,N0) cases (HR = 1.03, 95%CI = 0.87–1.21, P = 0.75), modest in intermediate-risk (pT4,N0 or pT1-3,N1-2) cases (HR = 0.92, 95%CI = 0.86–1.0, P = 0.046) and strong in high-risk (pT4,N1-2) cases (HR = 0.87, 95%CI = 0.79–0.97, P = 9.4 × 10−3); PINTERACTION = 0.090. Analysis of tumour CD8A expression in the independent validation cohort revealed similar variation in prognostic value across risk strata (PINTERACTION = 0.048). Conclusions The prognostic value of intratumoural CD8+ cell infiltration in stage II/III CRC varies across tumour and nodal risk strata. </p

KRAS mutation detection and prognostic potential in sporadic colorectal cancer using high-resolution melting analysis

BACKGROUND: The development of targeted therapies has created a pressing clinical need for molecular characterisation of cancers. In this retrospective study, high-resolution melting analysis (HRMA) was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect KRAS hot-spot mutations and to evaluate its prognostic value. Direct sequencing was used to confirm and characterise HRMA results. METHODS: After establishing its sensitivity, HRMA was validated on seven cell lines and inter- and intra-variation were analysed. The prognostic value of KRAS mutations in CRC was evaluated using survival analysis. RESULTS: HRMA revealed abnormal melting patterns in 34.1% CRC samples. Kaplan–Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation. In the Cox regression analysis, only when colon and rectal cancer were analysed separately, KRAS mutation was a negative predictor for OS in patients with rectal cancer and DFS in those with stage II colon cancer. CONCLUSIONS: HRMA was found to be a valid screening method for KRAS mutation detection. The KRAS mutation came forward as a negative predictive factor for OS in patients with rectal cancer and for DFS in stage II colon cancer patients