Inti Zlobec

Prognostic significance of the wnt signalling pathway molecules APC, β‐catenin and E‐cadherin in colorectal cancer—a tissue microarray‐based analysis

Aims:u2002 To investigate dysregulation of the wnt signalling pathway by assessing β‐catenin expression/increasing expression and loss of cytoplasmic adenomatous polyposis coli (APC) and membranous E‐cadherin in colorectal cancer (CRC) and determining the prognostic significance of these variables.

Loss of Raf-1 Kinase Inhibitor Protein Expression Is Associated With Tumor Progression and Metastasis in Colorectal Cancer

Raf-1 kinase inhibitor protein (RKIP) is known as a critical down-regulator of the mitogen-activated protein kinase signaling pathway and a potential molecular determinant of malignant metastasis. The aim of this study was to determine the prognostic significance of RKIP expression in colorectal cancer (CRC). Immunohistochemical staining for RKIP was performed on a tissue microarray comprising 1,197 mismatch repair (MMR)-proficient and 141 MMRdeficient CRCs. The association of RKIP with clinicopathologic features was analyzed. Loss of cytoplasmic RKIP was associated with distant metastasis (P = .038), higher N stage (P = .032), vascular invasion (P = .01), and worse survival (P = .001) in the MMR-proficient group. In MMR-deficient CRCs, loss of cytoplasmic RKIP was associated with distant metastasis (P = .043) and independently predicted worse survival (P = .004). Methylation analysis of 28 cases showed that loss of RKIP expression is unlikely to be due to promoter methylation.Loss of RKIP expression is a marker of tumor progression and distant metastasis in MMR-proficient and MMR-deficient CRCs.

Role of APAF-1, E-cadherin and peritumoural lymphocytic infiltration in tumour budding in colorectal cancer†

Tumour budding or dedifferentiation at the invasive margin of colorectal cancer (CRC) is an important prognostic marker and linked mechanistically to dysregulation of Wnt pathway signalling. Since budding is observed in only 40% of CRCs, we hypothesized that Wnt pathway dysregulation may be a necessary but insufficient explanation for budding and that buds may be destroyed selectively by tumour immune mechanisms. Twenty potential markers of tumour budding were evaluated in tissue microarrays (TMAs) obtained from the main tumour body of 1164 DNA mismatch repair‐proficient CRCs and the findings were correlated with tumour budding, lymphocytic infiltration and survival. Loss of expression of E‐cadherin and APAF‐1 were independent predictors of budding (sensitivity 70.3% and specificity 48.2% when one or the other was lost). Peritumoural lymphocytes (PTLs) were observed more frequently in CRCs with loss of either E‐cadherin or APAF‐1 that were budding‐negative. PTLs and tumour‐infiltrating lymphocytes (TILs) were strongly correlated. The absence of TILs increased the adverse prognostic impact of E‐cadherin and APAF‐1 loss. Co‐occurrence of E‐cadherin loss, APAF‐1 loss and low TIL counts in CRCs was an independent prognostic factor. The findings were verified in whole tissue sections from 88 CRCs with known KRAS mutation status (which was not associated with budding). Loss of E‐cadherin and APAF‐1 within the main body of CRCs are independent predictors of tumour budding. The prognostic benefit of lymphocytic infiltration may be explained by the immune destruction of budding cells. Copyright

Scoring of p53, VEGF, Bcl-2 and APAF-1 immunohistochemistry and interobserver reliability in colorectal cancer.

Molecular tumor markers are often studied in colorectal cancer using immunohistochemistry to determine their prognostic or predictive value. Protein expression is typically assigned a ‘positive’ score based on a predetermined cutoff. A semiquantitative scoring method that evaluates the percentage of positive tumor cells (0–100%) may provide a better understanding of the prognostic or predictive significance of these markers. The aim of this study was to assess and compare the interobserver agreement of immunohistochemistry scores using a percentage scoring method and three categorical scoring systems. Immunohistochemistry for p53, Bcl-2, vascular endothelial growth factor (VEGF) and apoptotic protease activating factor-1 (APAF-1) was performed on 87 tumor biopsies from patients with rectal carcinoma and scored independently by four pathologists as the percentage of positive tumor cells. Interobserver agreement was assessed by the intraclass correlation coefficient. The intraclass correlation coefficients for p53 and VEGF (>0.6) indicate substantial agreement between observers. The distribution of Bcl-2 and APAF-1 scores in addition to weaker interobserver agreement by percentage scoring suggest that this approach may not be appropriate for these proteins. In conclusion, p53 and VEGF protein expression assessed by immunohistochemistry in colorectal cancer and scored as a percentage of positive tumor cells may be a viable alternative scoring method.

Prognostic significance of mammalian sterile20-like kinase 1 in colorectal cancer.

Mammalian Sterile20-like kinase 1 (Mst1) is a member of the yeast Ste20-related kinase family known to be involved in the process of apoptosis initiated by a variety of stimuli. The aim of this study was to determine the prognostic significance of Mst1 expression in colorectal cancer. A series of 1197 mismatch-repair-proficient colorectal cancers retrieved from a tissue microarray were randomized into two study groups. On the first group (n=599) receiver operating characteristic curves were used to determine the most clinically useful cutoffs to describe Mst1 expression with respect to T stage, N stage, tumor grade, vascular invasion and overall survival. The association of Mst1 expression and each outcome was immunohistochemically evaluated on the second study group (n=598) as well as on a third study group comprising 141 mismatch-repair-deficient colorectal cancers. A univariate analysis in the second study group showed that loss of cytoplasmic Mst1 was associated with higher T stage (P=0.001), higher N stage (P=0.029), vascular invasion (P=0.017) and overall survival (P=0.014). Nuclear Mst1 expression was not significantly associated with N stage, T stage or vascular invasion but was associated with tumor grade. In mismatch-repair-deficient colorectal cancers, loss of cytoplasmic Mst1 was associated with higher N stage (P=0.019) and shortened survival (P=0.0001). In a multivariate analysis, loss of cytoplasmic Mst1 was an independent adverse prognostic factor in this group of patients. Methylation analysis on 32 cases showed that the loss of cytoplasmic Mst1 expression is not likely due to promoter methylation. In summary, loss of cytoplasmic Mst1 expression is a marker of tumor progression in mismatch-repair-proficient as well as mismatch-repair-deficient colorectal cancers. These results are suggestive of a tumor suppressor role for Mst1 in human colorectal cancer.

VEGF as a predictive marker of rectal tumor response to preoperative radiotherapy.

Neoadjuvant radiotherapy for rectal cancer may result in tumor downstaging or complete tumor regression leading to greater sphincter preservation. The identification of molecular predictive markers of tumor response to preoperative radiotherapy would provide an additional tool for selecting patients most likely to benefit from treatment. The aim of this study was to determine whether VEGF expression in preirradiation tumor biopsies is a useful predictive marker of tumor response in patients with rectal cancer undergoing preoperative radiotherapy.

Overexpression of the receptor for hyaluronic acid mediated motility is an independent adverse prognostic factor in colorectal cancer.

RHAMM, a member of the microtubule-associated protein family that interacts with the mitogen-activated protein kinase pathway, is associated with tumor progression, aggressive disease and shortened survival in several tumor types. This study aimed to determine the prognostic value of RHAMM in colorectal cancer (CRC). A series of 1420 unselected, nonconsecutive CRC resections were subdivided into three groups: (1) DNA mismatch repair (MMR)-proficient, (2) MLH1 negative and (3) presumed Lynch syndrome. Immunohistochemical analysis of RHAMM expression (0 vs >0%), increasing expression (increasing percentage positivity) and complete expression (100 vs <100%) was performed using tissue microarray technique and the results were correlated with clinicopathological parameters. Fifty-seven tissue samples of normal colonic mucosa were included as a control group. In a univariate analysis increasing and complete expression of RHAMM were associated with higher N stage (P=0.023 and 0.021) and worse survival (P<0.0001) in MMR-proficient CRC. Complete expression of RHAMM was associated with worse survival in presumed Lynch syndrome (P=0.016). In MLH1-negative CRC there was no association between RHAMM expression and the clinicopathological features. In a multivariate analysis, increasing RHAMM expression was an independent adverse prognostic factor in MMR-proficient CRC (P<0.0001) and complete expression in MMR-proficient CRC and presumed Lynch syndrome (P<0.0001 and P=0.031, respectively). Nuclear pERK expression was associated with increasing RHAMM expression in MMR-proficient CRC (P=0.012) and with complete RHAMM expression in presumed HNPCC (P=0.03). Increasing and complete RHAMM expressions are independent adverse prognostic factors in MMR-proficient CRC and presumed Lynch syndrome.

A Predictive Model of Rectal Tumor Response to Preoperative Radiotherapy Using Classification and Regression Tree Methods

Purpose: The ability to predict rectal tumor response to preoperative radiotherapy before treatment would significantly impact patient selection. In this study, classification and regression tree (CART) methods were used to model tumor response to preoperative conformal high-dose rate brachytherapy by assessing the predictive value of vascular endothelial growth factor (VEGF), Bcl-2, p21, p53, and APAF-1. Experimental Design: Immunohistochemistry was used to detect VEGF, Bcl-2, p21, p53, and APAF-1 from 62 pretreatment rectal tumor biopsies. Scores were assigned as percentages of positive tumor cell staining and were used in CART analysis to identify the proteins that best predicted response to radiotherapy. Ten-fold cross-validation was used to prevent overfitting and multiple cross-validation experiments were run to estimate the prediction error. Results: Postoperative pathologic evaluation of the irradiated tumor bed revealed 43 responsive tumors [20 with complete response (T0) and 23 with partial response] and 19 nonresponsive tumors. The optimal tree resulting from CART analysis had five terminal nodes with a misclassification rate of 18%. Of the five proteins selected for their predictive value, VEGF and Bcl-2 contributed most to the classification of responsive and nonresponsive tumors. All 10 tumors with no VEGF were completely responsive (T0) to radiotherapy; 85% of those with VEGF and negative for Bcl-2 were responsive to therapy. Conclusions: VEGF and Bcl-2 status in pretreatment rectal tumor biopsies may be predictive of response to preoperative high-dose rate brachytherapy.

Napoleon Bonaparte's gastric cancer: a clinicopathologic approach to staging, pathogenesis, and etiology

Background Numerous hypotheses on the cause of Napoleon Bonapartes death have been proposed, including hereditary gastric cancer, arsenic poisoning, and inappropriate medical treatment. We aimed to determine the etiology and pathogenesis of Napoleons illness by a comparison of historical information with current clinicopathologic knowledge.Investigations Evaluation of Napoleons clinical history, original autopsy reports, and of historical documents. The clinicopathologic data from 135 gastric cancer patients were used for comparison with the data available on Napoleon.Diagnosis At least T3N1M0 (stage IIIA) gastric cancer. Napoleons tumor extended from the cardia to the pylorus (>10 cm) without infiltration of adjacent structures, which provides strong evidence for at least stage T3. The N1 stage was determined by the presence of several enlarged and hardened regional (perigastric) lymph nodes, and the M0 stage by the absence of distant metastasis. Analysis of the available historical documents indicates that Napoleons main risk factor might have been Helicobacter pylori infection rather than a familial predisposition.Conclusions Our analysis suggests that Napoleons illness was a sporadic gastric carcinoma of advanced stage. Patients with such tumors have a notoriously poor prognosis.

The predictive value of apoptosis protease-activating factor 1 in rectal tumors treated with preoperative, high-dose-rate brachytherapy.

The objective of this study was to assess the value of apoptosis protease‐activating factor 1 (APAF‐1) as a predictive marker of response in rectal tumors treated with preoperative, high‐dose‐rate endorectal brachytherapy.