How reliable is immunohistochemical staining for DNA mismatch repair proteins performed after neoadjuvant chemoradiation?

Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) is currently being used primarily in colorectal cancer resection specimens. We aimed to compare the results of IHC staining performed on biopsy specimens obtained at endoscopy with that performed on surgical specimens after neoadjuvant therapy. Thirty-two rectal cancer subjects had paired preneoadjuvant and postneoadjuvant tissue available for IHC staining (MLH1, MSH2, MSH6, and PMS2), whereas 39 rectosigmoid cancer patients who did not receive neoadjuvant treatment served as controls. Each slide received a qualitative (absent, focal, and strong) and quantitative score (immunoreactivity [0-3] × percent positivity [0-4]). The quantitative scores of MMRP from the operative material were significantly lower in the neoadjuvant group than in the control (P < .05 for all).The scores of all MMRP from endoscopic biopsies were not significantly different between the neoadjuvant and the control groups. Disagreement between the endoscopic biopsy and the operative material was evident in 23 of 128 stains (18.5%) in the neoadjuvant group and in 12 of 156 stains (7.7%) in the control group (P = .009). In the neoadjuvant group, a disagreement pattern of "endoscopic strong operative focal" was observed in 28.1% for PMS2, 12.5% for MSH6, 12.5% for MLH1, and 6.3% for MSH2, and in the control group, this same disagreement pattern was found in 12.8% for PMS2, 7.7% for MSH6, 7.7% for MLH1, and 0% for MSH2. Based on our findings, we suggest that for rectal cancer, the endoscopic material rather than the operative material should serve as the primary material for IHC staining.

The antibiotic monensin causes cell cycle disruption of Toxoplasma gondii mediated through the DNA repair enzyme TgMSH-1.

Monensin is a polyether ionophore antibiotic that is widely used in the control of coccidia in animals. Despite its significance in veterinary medicine, little is known about its mode of action and potential mechanisms of resistance in coccidian parasites. Here we show that monensin causes accumulation of the coccidian Toxoplasma gondii at an apparent late-S-phase cell cycle checkpoint. In addition, experiments utilizing a monensin-resistant T. gondii mutant show that this effect of monensin is dependent on the function of a mitochondrial homologue of the MutS DNA damage repair enzyme (TgMSH-1). Furthermore, the same TgMSH-1-dependent cell cycle disruption is observed with the antiparasitic ionophore salinomycin and the DNA alkylating agent methyl nitrosourea. Our results suggest a novel mechanism for the mode of action of monensin and salinomycin on coccidial parasites, in which the drug activates an MSH-1-dependent cell cycle checkpoint by an unknown mechanism, ultimately leading to the death of the parasite. This model would indicate that cell cycle disruption is an important mediator of drug susceptibility and resistance to ionophoric antibiotics in coccidian parasites.

Effects of calcium and vitamin D on MLH1 and MSH2 expression in rectal mucosa of sporadic colorectal adenoma patients.

To further clarify and develop calcium and vitamin D as chemopreventive agents against colorectal cancer in humans and develop modifiable biomarkers of risk for colorectal cancer, we conducted a pilot, randomized, double-blind, placebo-controlled, 2 x 2 factorial clinical trial to test the effects of calcium and vitamin D(3), alone and in combination, on key DNA mismatch repair proteins in the normal colorectal mucosa. Ninety-two men and women with at least one pathology-confirmed colorectal adenoma were treated with 2.0 g/d calcium or 800 IU/d vitamin D(3), alone or in combination, versus placebo over 6 months. Colorectal crypt overall expression and distribution of MSH2 and MLH1 proteins in biopsies of normal-appearing rectal mucosa were detected by automated immunohistochemistry and quantified by image analysis. After 6 months of treatment, MSH2 expression along the full lengths of crypts increased by 61% (P = 0.11) and 30% (P = 0.36) in the vitamin D and calcium groups, respectively, relative to the placebo group. The estimated calcium and vitamin D treatment effects were more pronounced in the upper 40% of crypts (differentiation zone) in which MSH2 expression increased by 169% (P = 0.04) and 107% (P = 0.13) in the vitamin D and calcium groups, respectively. These findings suggest that higher calcium and vitamin D intakes may result in increased DNA MMR system activity in the normal colorectal mucosa of sporadic adenoma patients and that the strongest effects may be vitamin D related and in the differentiation zone of the colorectal crypt.

Thymidylate synthase and microsatellite instability in colorectal cancer: implications for disease free survival, treatment response and survival with metastases.

BACKGROUND: Colorectal cancer (CRC) cell lines displaying microsatellite instability (MSI) are resistant to 5-fluorouracil (5-FU) in vitro, which can be overcome by restoring DNA mismatch repair (MMR) competence. Thymidylate synthase (TS) is inhibited by 5-FU, being another potential mediator of therapeutic resistance to 5-FU. The clinical relevance of these observations remains unclear. OBJECTIVE: We examined the expression of TS and two MMR proteins (hMLH1 and hMSH2) in advanced CRC patients, to determine a) their mutual relationship, b) association to therapeutic response and c) impact on disease outcome. MATERIAL AND METHODS: Tumour samples from 73 patients CRC who were treated in advanced stage with either irinotecan alone or in combination with 5-FU/leucovorin, were analysed for expression of TS, hMLH1 and hMSH2 using immunohistochemistry (IHC). RESULTS: TS expression was closely correlated with hMLH1 expression (negative-weak/moderate-strong) (p=0.0001). TS-MMR expression was significantly (p=0.029 for whole series; p=0.004 for the 5-FU treated cases) related to response to treatment; tumours with low levels of both TS and MMR responded better (n=14/27, 51.8%) than those with high TS and MMR (n=3/18, 16.6%). Patients with high TS-MMR expression had a significantly longer DFS (47 months vs. 9 months, n=26) than those with low TS-MMR index (p=0.015), while the reverse was true concerning survival with metastases (WMS) (p=0.018) in all the patients (n=73). CONCLUSIONS: The present data suggest that MSI patients with low TS and deficient MMR demonstrate a significantly shorter DFS and longer WMS than patients with high expression of both markers, and they are also more likely to obtain the greatest benefit from 5-FU based chemotherapy.