Mismatch Repair Proteins hMLH1 Research Articles

BRAFV600E immunohistochemistry can reliably substitute BRAF molecular testing in the Lynch syndrome screening algorithm in colorectal cancer.

The Lynch syndrome (LS) screening algorithm requires BRAF testing as a fundamental step to distinguish sporadic from LS-associated colorectal carcinomas (CRC). BRAF testing by immunohistochemistry (IHC) has shown variable results in the literature. Our aim was to analyse concordance between BRAFV600E IHC and BRAF molecular analysis in a large, mono-institutional CRC whole-slide, case series with laboratory validation. MisMatch repair (MMR) protein (hMLH1, hPMS2, hMSH2, and hMSH6) and BRAFV600E IHC were performed on all unselected cases of surgically resected CRCs (2018-2023). An in-house validation study for BRAFV600E IHC was performed in order to obtain optimal IHC stains. BRAFVV600E IHC was considered negative (score 0), positive (scores 2-3), and equivocal (score 1). Interobserver differences in BRAFV600E IHC scoring were noted in the first 150 cases prospectively collected. Nine-hundred and ninety CRCs cases (830 proficient (p)MMR/160 deficient (d)MMR) were included and all cases performed BRAFV600E IHC (BRAFV600E IHC-positive 13.5% of all series; 66.3% dMMR cases; 3.4% pMMR cases), while 333 also went to BRAF mutation analysis. Optimal agreement in IHC scoring between pathologists (P < 0.0001) was seen; concordance between BRAFV600E IHC and BRAF molecular analysis was extremely high (sensitivity 99.1%, specificity 99.5%; PPV 99.1%, and NPV 99.5%). Discordant cases were reevaluated; 1 score 3 + IHC/wildtype case was an interpretation error and one score 0 IHC/mutated case was related to heterogenous BRAFV600E IHC expression. Among the 12 IHC-equivocal score 1+ cases (which require BRAF molecular analysis), three were BRAF-mutated and nine BRAF-wildtype. BRAFV600E IHC can be used as a reliable surrogate of molecular testing after stringent in-house validation.

Tumor Budding and PDC Grade Are Stage Independent Predictors of Clinical Outcome in Mismatch Repair Deficient Colorectal Cancer.

Mismatch repair deficient (dMMR) colorectal cancer (CRC) despite its association with poor histologic grade often has improved prognosis compared with MMR proficient CRC. Tumor budding and poorly differentiated clusters (PDCs) may predict metastatic potential of colorectal adenocarcinoma (CRC). In addition, their assessment may be more reproducible than the evaluation of other histopathologic parameters. Therefore, we wished to determine their potential as prognostic indicators in a cohort of dMMR CRC patients relative to histologic grade. We investigated the predictive value of conventional WHO grade, budding, PDC grade and other histopathologic parameters on the presence of lymph node metastasis (LNM) and clinical outcome in 238 dMMR CRCs. MMR status was determined by immunohistochemistry for the mismatch repair proteins hMLH1, hMSH2, hMSH6, and hPMS2. Tumor budding and PDCs were highly correlated (r=0.701; P<0.000). Both budding and PDC grade were associated with WHO grade, perineural invasion, lympho-vascular invasion, and extramural vascular invasion, and the presence of LNM in dMMR CRC (P<0.009). Independent predictors of LNM were PDC grade (odds ratio, 4.12; 95% confidence interval [CI], 1.69-10.04; P=0.011) and EMVI (odds ratio, 3.81; 95% CI, 1.56-9.19; P<0.000). Only pTstage (hazard ratio [HR], 4.11; 95% CI, 1.48-11.36; P=0.007) and tumor budding (HR, 2.99; 95% CI, 1.72-5.19; P<0.000) were independently associated with worse disease-free survival (DFS). If tumor budding was excluded from the model, PDC grade became significant for DFS (HR, 2.34; 95% CI, 1.34-4.09; P=0.003). WHO Grade does not independently correlate with clinical outcome in dMMR CRC. PDC grade and extramural vascular invasion are independent predictors of LNM. Tumor budding and pTstage are the best predictors of DFS. If tumor budding cannot be assessed, PDC grade may be used as a prognostic surrogate.

Mismatch Repair Protein hMLH1, but not hMSH2, Enhances Estrogen-Induced Apoptosis of Colon Cancer Cells.

Background: Epidemiological studies suggest a protective role of estrogen against colon carcinogenesis; this effect appears to be dependent on mismatch repair (MMR) status. However, the underlying mechanism remains unclear. This study investigated the role of MMR proteins in apoptosis of colon cancer cells in the presence or absence of estrogen.Methods: Two major MMR proteins, human mutL homolog 1 (hMLH1) and mutS homolog 2 (hMSH2), as well as estrogen receptor-β (ERβ), were transiently expressed in either hMLH1-deficient HCT116 cells or hMSH2-deficient LoVo cells. Effects of estradiol on cell viability and apoptosis were assessed. Furthermore, we examined the apoptotic status of epithelial cells in colonic mucosa taken from previous healthy female subjects with menopausal syndrome before and after 6-month hormone replacement therapy (HRT).Results: In hMLH1-deficient HCT116 cells, re-expression of hMLH1 led to a significantly decreased cell viability and increased apoptosis, which were further enhanced by estradiol, including marked increase of activated caspase-3 and caspase-9, as well as Bax and P53. The effect of hMLH1 overexpression in LoVo cells resulted in a similar increase in apoptosis that was greatly stimulated by estradiol. The enhanced apoptosis by hMLH1 and estradiol was further validated by FACS analyses of Annexin V expression. Re-expression of hMSH2 or overexpression of ERβ in HCT116 cells also enhanced apoptosis; however, the effects were independent of estradiol. Furthermore, studies on healthy menopausal women before and after 6-month HRT demonstrated a significant HRT-mediated upregulation of the hMLH1 expression, with concomitant elevation of caspase-3 and caspase-9 activation in the colonic mucosa.Conclusion: We present the first evidence that hMLH1 and hMSH2 have similar but distinct roles in the apoptosis of colon cancer cells: an increased expression of either one can promote apoptosis, while only the effect of hMLH1 but not hMSH2 is estradiol-dependent. Our data suggest that MMR status should be assessed before hormone replacement therapy or future application of estrogen-based chemoprevention.

ERK/pERK expression and B-raf mutations in colon adenocarcinomas: correlation with clinicopathological characteristics

BackgroundColorectal (CRC) carcinogenesis through various morphological stages has been linked to several genetic and epigenetic changes. The Raf/MEK/ERK (MAPK) signal transduction cascade is an important mediator of a number of cellular fates.MethodsIn this study, we investigated the presence of B-raf and K-ras mutations in 94 consecutive cases of primary colon adenocarcinoma in correlation with the immunohistochemical expression of total and activated ERK and the expression of mismatch repair proteins (MMR) hMLH1 and hMSH2 as well as their correlations with standard clinicopathological parameters.ResultsThe immunostaining pattern for total and activated ERK was nuclear and cytoplasmic. hMLH1 and hMSH2 proteins were preserved in 45/63 (71.43%) cases and 35/53 (66.04%) cases respectively. Total ERK nuclear expression, was positively correlated with tumor stage (p = 0.049), whereas nuclear pERK expression was positively correlated with histological grade (p = 0.0113) and tumor stage (p = 0.0952), although the latter relationship was of marginal significance. DNA sequencing showed that 12 samples (12.7%) had a mutation in B-RAF Exon 15 and none in Exon 11, whereas 22 (23.4%) had a K-ras mutation. Disruption of the MAP kinase pathway-either through K-ras or B-raf mutation-was detected in 37% of all the examined cases, although the overexpression of total and activated ERK1/2 was not correlated with the mutational status of K-ras or B-raf genes. Finally, the preservation of hMLH1 or hMSH2 immunoexpression was not correlated with the presence of B-raf and/or K-ras mutations.ConclusionsIn this study, we present evidence that ERK activation occurs in a K-ras or B-raf -independent manner in the majority of primary colon cancer cases. Moreover, B-raf mutations are not associated with mismatch-repair deficiency through loss of hMLH1 or hMSH2 expression. Activated ERK could possibly be implicated in tumor invasiveness as well as in the acquisition of a more aggressive phenotype.

Abstract 850: Selenium compounds activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells

Abstract Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G2/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 850. doi:10.1158/1538-7445.AM2011-850

Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ( 788QIKLNELW 795). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390–490 and 787–846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.

Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.

Abstract 3927: Distinct roles of hMLH1-hMRE11 in homologous recombination and heteroduplex DNA repair at a single chromosomal locus

Abstract Increasing evidence indicates that the mismatch repair protein hMLH1 and DNA double-strand break (DSB) repair protein hMRE11 play multifunctional roles in various processes of DNA repair and damage response in human cells. Our previous studies have revealed a physical interaction between these two proteins and have suggested a role for hMRE11 in heteroduplex DNA repair and hMLH1-dependent DNA damage-induced G2 arrest. Here, we analyzed the effects of hMLH1-hMRE11 on homologous recombinational repair of an induced DSB and consequential heteroduplex repair at a single chromosomal locus by a newly developed reporter system. The results of these studies indicate that hMLH1 and hMRE11 display a synergistic anti-recombination effect and the hMLH1-mediated anti-recombination is at least partially dependent on its interaction with hMRE11, whereas the repair of heteroduplex DNA requires functional hMLH1 and hMRE11. Consistent with these results, chromatin immunoprecipitation (ChIP) analysis of the reporter locus demonstrates that DSB triggers the loading of hMLH1 and hMRE11 proteins to both the proximal region and the site containing heteroduplex DNA. However, in contrast to the proximal regions, loading of hMLH1-hMRE11 at the site of heteroduplex DNA also requires hMSH2, suggesting the involvement of DNA mismatch repair. In summary, our studies have provided evidence to suggest that, in addition to DNA damage response, hMLH1-hMRE11 are involved in at least two other DNA damage repair processes: anti-recombination and heteroduplex DNA repair. Our results implicate that mutations impairing the hMLH1-hMRE11 interaction might promote spontaneous chromosomal recombination between ectopic repetitive sequences and thereby increasing gene conversion and genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3927.

The mismatch repair protein hMLH1 regulates selenium‐induced DNA damage response in colorectal cancer cells

Epidemiological studies indicate that selenium supplementation in humans suppresses risk of colorectal and other cancers. The majority of colorectal cancer is characterized by a defective DNA mismatch repair (MMR) system. The hMLH1‐hPMS2 heterodimer plays a critical role at the early stage of MMR repair. Here, we employed HCH116 colorectal cancer cells with intrinsic hMLH1 deficiency and the cell complemented with hMLH1, and treated them with sodium selenite (2–10 μM), methane seleninic acid (2–10 μM) and methyl‐seleno‐L‐cysteine (50–300 μM). We showed that complementation of hMLH1 sensitizes HCT116 cells to cell death after treatment with the selenium compounds. Interestingly, cellular exposure to organic forms of selenium compounds resulted in increased hMLH1 protein expression and hMLH1‐hPMS2 association. Furthermore, hMLH1 is involved in proper S and G2/M phase checkpoint responses and DNA‐PKcs phosphorylation at Thr‐2647, an early event of DNA damage response, after cellular exposure to selenite. Our results suggest that DNA damage checkpoint response, a barrier of tumorigenesis, is deficient in HCT116 cells after selenium treatment. Consequently, a lack of functional hMLH1‐hPMS2 complex may predispose colorectal cancer cells to genomic instability.Grant Funding Source: University of Maryland

The role of expression of mismatch repair proteins hMSH2 and hMLH1 in gastric carcinogenesis and its clinical significance

Objective To investigate the expression of the mismatch repair proteins hMSH2 and hMLH1, and to examine the clinical significance of the intracellular expression site (ICES) in gastric carcinogenesis.

Hsp27, Hsp70 and mismatch repair proteins hMLH1 and hMSH2 expression in peripheral blood lymphocytes from healthy subjects and cancer patients

Mismatch repair (MMR) deficiency and higher expression levels of heat shock proteins (Hsps) have been implicated with drug resistance to topoisomerase II poisons (doxorubicin) and to platinum compounds (cisplatin). This study was designed to determine individual influences of doxorubicin and cisplatin treatment on the expression of Hsp27, Hsp70, hMLH1 and hMSH2 proteins and in the DNA damage status in peripheral blood lymphocytes (PBLs). In addition, we studied whether these proteins and the DNA damage correlated with the survival of cancer patients. PBLs from 10 healthy donors and 25 cancer patients (before and after three cycles of chemotherapy) were exposed to in vitro treatments: C (control), HS (heat shock at 42 °C), Do or Pt (doxorubicin or cisplatin alone), and HS + Do or HS + Pt (heat shock + doxorubicin or heat shock + cisplatin). PBLs were collected at time 0 (T0: immediately after drug treatment) and after 24 h of repair (T24). Hsp27, Hsp70, hMLH1 and hMSH2 were studied by immunocytochemistry and the DNA damage by alkaline comet assay. Immunofluorescence studies and confocal microscopy revealed that hMLH1 and hMSH2 colocalized with Hsp27 and Hsp72 (inducible form of Hsp70). hMLH1 and hMSH2 were significantly induced by Pt and HS + Pt at T24 in cancer patients, but only modestly influenced by Do. Cancer patients presented higher basal expression of total and nuclear Hsp27 and Hsp70 than controls, and these proteins were also increased by HS, Do and HS + Do. The Hsp70 induction by Pt and HS + Pt was noted in cancer patients, especially nuclear Hsp70. In cancer patients, basal DNA damage was slightly higher than in healthy persons; and after Pt and HS + Pt treatments, DNA migration and number of apoptotic cells were higher than controls. Hsps accomplished a cytoprotective function in pre-chemotherapy PBLs (HS before Do or Pt), but not in post-chemotherapy samples. In Pt-treated patients the ratio N/C (nuclear/cytoplasmic) of Hsp27 was related to disease free survival and overall survival, and hMSH2 correlated with overall survival. The results point to the utility of these molecules and of the comet assay as possible predictive markers.

Clinicopathological Characteristics of Colorectal Cancer with Family History: an Evaluation of Family History as a Predictive Factor for Microsatellite Instability

To determine whether family history of cancer may be a risk factor for the mutator phenotype in colorectal cancer, we recruited 143 consecutive colorectal cancer patients with a family history of accompanying cancers not meeting the Amsterdam criteria. Microsatellite instability (MSI) at 5 markers, hMLH1-promoter methylation, and expression of mismatch repair (MMR) proteins (hMLH1, hMSH2, hMSH6, hMPS1, and hPMS2) were determined. Among the relatives of familial colorectal cancer patients, colorectal cancer was the most common tumor type. Of the proband colorectal cancers, 26 (18.2%) showed high-level MSI (MSI-H); 47 additional tumors with mutator phenotype (32.9%) were identified by hMLH1-promoter methylation and/or loss of MMR protein expression. Mutator phenotype was associated with right-sided colon cancer and the type of accompanying cancer. Family history, which was differentially quantified according to the degree of relatives and the type of accompanying cancers, effectively discriminated MSI-H from microsatellite stable (MSS) and low-level microsatellite instability (MSI-L) and mutator phenotypes. Our findings indicate that familial colorectal cancer may be associated with multiple occurrences of colorectal or accompanying cancers and that family history could be correlated with microsatellite instability.

Genomic aberrations are rare in urothelial neoplasms of patients 19 years or younger

Urothelial neoplasms in patients 19 years of age or younger are rare, and the data regarding clinical outcome are conflicting. Molecular data are not available. Urothelial tumours from 14 patients aged 4 to 19 years were analysed, including FGFR3 and TP53 mutation screening, comparative genomic hybridization (CGH), UroVysion FISH analysis, polymerase chain reaction for human papillomavirus (HPV), microsatellite analysis using the NIH consensus panel for detection of microsatellite instability (MSI) and six markers for loss of heterozygosity on chromosome arms 9p, 9q, and 17p and immunohistochemistry for TP53, Ki-67, CK20 and the mismatch repair proteins (MRPs) hMSH2, hMLH1, and hMSH6. Based on the 2004 WHO classification, one urothelial papilloma, seven papillary urothelial neoplasms of low malignant potential (PUNLMPs), five low-grade, and one high-grade papillary urothelial carcinoma were included. No multifocal tumours were found and recurrence was seen in only one patient with a urothelial papilloma. All patients were alive with no evidence of disease at a median follow-up of 3.0 years. We found no mutations in FGFR3, deletions of chromosome arms 9p, 9q or 17p, MSI or MRP loss, or HPV positivity in any of the patients. Three cases showed chromosome alterations in CGH analyses, urothelial dedifferentiation with CK20 overexpression, or aneuploidy, and one TP53 mutation with TP53 overexpression was found. Urothelial neoplasms in people younger than 20 years are predominantly low grade and are associated with a favourable clinical outcome. Genetic alterations frequently seen in older adults are extremely rare in young patients. Urothelial neoplasms in children and young adults appear to be biologically distinct and lack genetic instability in most cases.

Frequency of Immunohistochemical Loss of Mismatch Repair Protein in Double Primary Cancers of the Colorectum and Stomach in Japan

Colorectal cancer and gastric cancer are the two most commonly associated malignancies in Japan. We examined mismatch repair deficiency in the tumors of patients with primary colorectal and gastric cancers retrospectively. In 103 cases and 102 healthy control subjects, surgical specimens of colorectal and gastric cancer underwent immunohistochemical analysis of mismatch repair proteins (hMLH1 and hMSH2) and microsatellite instability testing. Immunohistochemical and microsatellite instability testing produced similar results. High microsatellite instability in colorectal cancer was found in 23 of 103 cases (23 percent) with colorectal and gastric cancers, and in 8 of 102 healthy control subjects (8 percent). Twelve (12 percent) had mismatch repair deficiency in both colorectal and gastric cancers, and both tumors had loss of the same mismatch repair protein (hMLH1, n = 5; hMSH2, n = 7). They had the first cancer at a younger age, with a higher frequency of familial colorectal cancer than the others. Seventeen had mismatch repair deficiency in either tumor, which showed loss of expression of hMLH1. Multiple cancers and right-sided colon cancers developed more frequently in patients with mismatch repair deficiency. Patients with both colorectal and gastric cancers are more likely to have phenotypic evidence of hereditary nonpolyposis colorectal cancer than patients with colorectal cancer only. Among patients with double tumors, 12 percent showed a common deficiency in the same mismatch repair protein in both tumors by immunohistochemistry, and they should undergo genetic counseling for germline mutational analysis. Immunohistochemistry was effective in detecting mismatch repair deficiency of colorectal and gastric cancer as well as microsatellite instability testing, and may be more practical to perform phenotypic analysis of tumors because of its cost-effectiveness.

Elevated Microsatellite Instability at Selected Tetranucleotide Repeats does not Correlate with Clinicopathologic Features of Bladder Cancer

IntroductionThe aim of the present study was to evaluate whether elevated microsatellite instability (MSI) at selected tetranucleotide repeats (EMAST) is associated with clinicopathologic and molecular parameters in urinary bladder cancer (BC). MethodsA consecutive series of 117 nonselected urothelial BCs was studied. Microsatellite analysis and detection of loss of heterozygosity (LOH) was performed after microdissection by using 12 markers selected for sensitive detection of EMAST. Furthermore, five markers (BAT25, BAT26, D2S123, APC, and D17S250) of the Bethesda consensus panel for detection of colorectal cancer within the hereditary non-polyposis colon cancer syndrome (HNPCC) were analyzed. Expression of TP53 and mismatch repair (MMR) proteins hMSH2, hMLH1, and hMSH6 was investigated by immunohistochemistry. ResultsEMAST was detected in 10 of 117 patients (8.5%), whereas MSI in the HNPCC panel was found in one of 114 cases (0.9%). LOH in EMAST markers was seen in six of 114 (5.3%) cases. All tumours showed normal expression of hMSH2 and hMLH1. Overexpression of TP53 was observed in nine of 117 evaluated cases (7.7%). No association of EMAST and TP53 expression could be demonstrated. None of the clinicopathologic or molecular parameters were associated with EMAST. ConclusionsEMAST is another distinct form of MSI in urothelial BC. However, EMAST seems to be a rare event in non-selected urothelial BC and does not seem to be associated with commonly used clinicopathologic and clinical features in patients with BC.

Mismatch repair protein expression and microsatellite instability: a comparison of clear cell sarcoma of soft parts and metastatic melanoma

Clear cell sarcoma of soft parts is a rare soft tissue malignancy that shows phenotypic overlap with cutaneous melanoma but can be distinguished by the presence of a t(12;22) translocation. Microsatellite instability (MSI), a variation in the lengths of short repeat DNA segments in the genome, has been implicated in melanoma tumorigenesis, but is rare or absent in clear cell sarcoma. Defects in the mismatch repair (MMR) enzyme complex correlate with MSI in some tumor types, allowing the use of immunohistochemistry for the MMR proteins hMLH1 and hMSH2 to predict the presence of MSI. To determine if the association between MMR defects and MSI extends to clear cell sarcoma, we compared a group of nine clear cell sarcomas to 11 metastatic melanomas on the basis of MSI and the expression of MMR proteins. MSI was studied using fluorescence-based multiplexed PCR of five loci. Immunohistochemistry was evaluated on formalin-fixed paraffin-embedded tissue for hMLH1, hMHS2 and hMSH6. MSI was present in only 1/9 (11%) clear cell sarcoma case and in 8/11 (73%) melanoma cases. Immunostaining for hMLH1 and hMSH2 was preserved in all the clear cell sarcomas but loss of immunostaining for one or both proteins was seen in 6/11 melanomas (55%). hMSH6 was detected in 7/9 (78%) clear cell sarcomas and 10/11 (91%) of melanomas. Clear cell sarcoma and metastatic melanoma differed significantly with respect to the presence of MSI (P=0.010) and staining for hMLH1 and/or hMSH2 (P=0.014) but not hMSH6 (P=0.57). Mismatch repair, and consequently genomic instability may contribute to tumorigenesis in melanoma but not clear cell sarcoma. Immunostaining for hMLH1 and hMSH2 and MSI analysis may be helpful in the differential diagnosis of large soft tissue or visceral malignancies with melanocytic differentiation.

Expression of the MutL Homologue hMLH3 in Human Cells and its Role in DNA Mismatch Repair

The human mismatch repair (MMR) proteins hMLH1 and hPMS2 function in MMR as a heterodimer. Cells lacking either protein have a strong mutator phenotype and display microsatellite instability, yet mutations in the hMLH1 gene account for approximately 50% of hereditary nonpolyposis colon cancer families, whereas hPMS2 mutations are substantially less frequent and less penetrant. Similarly, in the mouse model, Mlh1-/- animals are highly cancer prone and present with gastrointestinal tumors at an early age, whereas Pms2-/- mice succumb to cancer much later in life and do not present with gastrointestinal tumors. This evidence suggested that MLH1 might functionally interact with another MutL homologue, which compensates, at least in part, for a deficiency in PMS2. Sterility of Mlh1-/-, Pms2-/-, and Mlh3-/- mice implicated the Mlh1/Pms2 and Mlh1/Mlh3 heterodimers in meiotic recombination. We now show that the hMLH1/hMLH3 heterodimer, hMutLgamma, can also assist in the repair of base-base mismatches and single extrahelical nucleotides in vitro. Analysis of hMLH3 expression in colon cancer cell lines indicated that the protein levels vary substantially and independently of hMLH1. If hMLH3 participates in MMR in vivo, its partial redundancy with hPMS2, coupled with the fluctuating expression levels of hMLH3, may help explain the low penetrance of hPMS2 mutations in hereditary nonpolyposis colon cancer families.

Slipped (CTG)•(CAG) repeats can be correctly repaired, escape repair or undergo error-prone repair

Expansion of (CTG)*(CAG) repeats, the cause of 14 or more diseases, is presumed to arise through escaped repair of slipped DNAs. We report the fidelity of slipped-DNA repair using human cell extracts and DNAs with slip-outs of (CAG)(20) or (CTG)(20). Three outcomes occurred: correct repair, escaped repair and error-prone repair. The choice of repair path depended on nick location and slip-out composition (CAG or CTG). A new form of error-prone repair was detected whereby excess repeats were incompletely excised, constituting a previously unknown path to generate expansions but not deletions. Neuron-like cell extracts yielded each of the three repair outcomes, supporting a role for these processes in (CTG)*(CAG) instability in patient post-mitotic brain cells. Mismatch repair (MMR) and nucleotide excision repair (NER) proteins hMSH2, hMSH3, hMLH1, XPF, XPG or polymerase beta were not required-indicating that their role in instability may precede that of slip-out processing. Differential processing of slipped repeats may explain the differences in mutation patterns between various disease loci or tissues.

Fruits, Vegetables, and hMLH1 Protein–Deficient and –Proficient Colon Cancer: The Netherlands Cohort Study

Clinical and pathologic differences exist between colon carcinomas deficient and proficient in the mismatch repair protein hMLH1. Animal and in vitro studies suggest that fruits, vegetables, folate, and antioxidants are associated with colonic expression of mismatch repair genes. Associations between consumption of fruits and vegetables and hMLH1 protein-deficient and -proficient colon cancer were evaluated in the Netherlands Cohort Study on diet and cancer using a case-cohort approach. A self-administered food frequency questionnaire was completed, in 1986, by 120,852 individuals ages 55 to 69 years. Using immunohistochemistry, hMLH1 protein expression was assessed in colon cancer tissue obtained from 441 patients who were identified over 7.3 years of follow-up excluding the initial 2.3 years. Incidence rate ratios (RR) were estimated for hMLH1 protein-deficient and -proficient colon cancer. hMLH1 protein expression was absent in 54 tumors (12.2%) and present in 387 tumors. Fruit consumption was associated with hMLH1 protein-deficient colon cancer [highest versus lowest tertile, RR, 0.46; 95% confidence interval (95% CI), 0.23-0.90; P(trend) = 0.029] but not with hMLH1 protein-proficient tumors (highest versus lowest tertile, RR, 1.03; 95% CI, 0.78-1.35; P(trend) = 0.81). Total consumption of vegetables was not associated with either type of tumor (hMLH1 protein deficient: RR, 0.86; 95% CI, 0.45-1.65; P(trend) = 0.67; hMLH1 protein proficient: RR, 0.94; 95% CI, 0.72-1.23; P(trend) = 0.72). No associations were observed for folate, fiber, antioxidants, or subgroups of vegetables. These analyses indicate that an inverse association between consumption of fruits and colon cancer may be confined to the subgroup of tumors with a deficient mismatch repair system.

Mutations of the BRAF gene in ulcerative colitis‐related colorectal carcinoma

The Raf/MEK/ERK (MAPK) signal transduction cascade is an important mediator of a number of cellular fates including growth, proliferation and survival. The BRAF gene is activated by oncogenic Ras, leading to cooperative effects in cells responding to growth factor signals. Our study was performed to elucidate a possible function of BRAF in ulcerative colitis (UC)-related colorectal carcinogenesis. Mutations of BRAF and KRAS were determined in 33 UC-related colorectal cancers by direct DNA sequencing analyses after microdissection. Mismatch-repair deficiency was assessed by immunohistochemistry for major mismatch-repair proteins hMLH1, hMSH2 and hMSH6 and microsatellite analyses of the BAT25 and BAT26 loci. Hypermethylation of the hMLH1 promoter was also tested. The results obtained were correlated with histopathologic variables. Activating BRAF missense mutations were identified in 3/33 UC-related cancers (9%), 2 of which exhibited a loss of hMLH1-protein expression and hypermethylation of the hMLH1 promoter. Corresponding nondysplastic UC-mucosa of these patients did not show BRAF mutations. KRAS mutations were found in 6/33 (18%) UC cancers. All 6 UC cancers with KRAS mutations had an intact BRAF gene as the 3 cancers with BRAF mutations had an intact KRAS gene. There was no significant correlation between BRAF or KRAS status and clinicopathologic variables. Our data indicate that BRAF mutations are not an initiating event in UC-related carcinogenesis and are associated with mismatch-repair deficiency through hMLH1-promoter hypermethylation. Disruption of the Raf/MEK/ERK (MAPK) kinase pathway-either through RAS or BRAF mutation-was detected in 27% of all UC-related cancers and thus plays an important role in UC-related carcinogenesis.