Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal?

Background

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by germline mutations in MisMatch Repair (MMR) genes, particularly in MLH1, MSH2 and MSH6. Patients with LS seem to have a more favourable prognosis than those with sporadic CRC, although the prognostic impact of different mutation types is unknown.Aim of our study is to compare survival outcomes of different types of MMR mutations in patients with LS-related CRC.

Methods

302 CRC patients were prospectively selected on the basis of Amsterdam or Revised Bethesda criteria to undergo genetic testing: direct sequencing of DNA and MLPA were used to examine the entire MLH1, MSH2 and MSH6 coding sequence.Patients were classified as mutation-positive or negative according to the genetic testing result.

Results

A deleterious MMR mutation was found in 38/302 patients. Median overall survival (OS) was significantly higher in mutation-positive vs mutation-negative patients (102.6 vs 77.7 months, HR:0.63, 95%CI:0.46–0.89, p = 0.0083). Different types of mutation were significantly related with OS: missense or splicing-site mutations were associated with better OS compared with rearrangement, frameshift or non-sense mutations (132.5 vs 82.5 months, HR:0.46, 95%CI:0.16–0.82, p = 0.0153).

Conclusions

Our study confirms improved OS for LS-patients compared with mutation-negative CRC patients. In addition, not all mutations could be considered equal: the better prognosis in CRC patients with MMR pathogenic missense or splicing site mutation could be due to different functional activity of the encoded MMR protein. This matter should be investigated by use of functional assays in the future.     

Cancer risk in MLH1, MSH2 and MSH6 mutation carriers; different risk profiles may influence clinical management

Background

Lynch syndrome (LS) is associated with a high risk for colorectal cancer (CRC) and extracolonic malignancies, such as endometrial carcinoma (EC). The risk is dependent of the affected mismatch repair gene. The aim of the present study was to calculate the cumulative risk of LS related cancers in proven MLH1, MSH2 and MSH6 mutation carriers.

Methods

The studypopulation consisted out of 67 proven LS families. Clinical information including mutation status and tumour diagnosis was collected. Cumulative risks were calculated and compared using Kaplan Meier survival analysis.

Results

MSH6 mutation carriers, both males and females had the lowest risk for developing CRC at age 70 years, 54% and 30% respectively and the age of onset was delayed by 3-5 years in males. With respect to endometrial carcinoma, female MSH6 mutation carriers had the highest risk at age 70 years (61%) compared to MLH1 (25%) and MSH2 (49%). Also, the age of EC onset was delayed by 5-10 years in comparison with MLH1 and MSH2.

Conclusions

Although the cumulative lifetime risk of LS related cancer is similar, MLH1, MSH2 and MSH6 mutations seem to cause distinguishable cancer risk profiles. Female MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. As a consequence, surveillance colonoscopy starting at age 30 years instead of 20-25 years is more suitable. Also, prophylactic hysterectomy may be more indicated in female MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers.     

DNA mismatch repair-related protein loss as a prognostic factor in endometrial cancers

Objective

Recent investigations have revealed DNA mismatch repair (MMR) gene mutations are closely related with carcinogenesis of endometrial cancer; however the impact of MMR protein expression on prognosis is not determined. Correlations between MMR-related protein expression and clinicopathological factors of endometrial cancers are analyzed in the present study.

Methods

A total of 191 endometrial cancer tissues treated between 1990 and 2007 in our hospital were enrolled. Immunoreactions for MSH2, MLH1, MSH6, and PMS2 on tissue microarray specimens and clinicopathological features were analyzed retrospectively.

Results

Seventy-six cases (40%) had at least one immunohistochemical alteration in MMR proteins (MMR-deficient group). There were statistically significant differences of histology, International Federation of Gynecology and Obstetrics (FIGO) stage, and histological grade between MMR-deficient group and the other cases (MMR-retained group). Response rate of first-line chemotherapy in evaluable cases was slightly higher in MMR-deficient cases (67% vs. 44%, p=0.34). MMR-deficient cases had significantly better progression-free and overall survival (OS) compared with MMR-retained cases. Multivariate analysis revealed MMR status was an independent prognostic factor for OS in endometrial cancers.

Conclusion

MMR-related proteins expression was identified as an independent prognostic factor for OS, suggesting that MMR was a key biomarker for further investigations of endometrial cancers.     

Tumours with loss of MSH6 expression are MSI-H when screened with a pentaplex of five mononucleotide repeats

Background:

Microsatellite instability (MSI) is commonly screened using a panel of two mononucleotide and three dinucleotide repeats as recommended by a consensus meeting on MSI tumours held at the National Cancer Institute (Bethesda, MD, USA). According to these recommendations, tumours are classified as MSI-H when at least two of the five microsatellite markers show instability, MSI-L when only one marker shows instability and MSS when none of the markers show instability. Almost all MSI-H tumours are characterised by alterations in one of the four major proteins of the mismatch repair (MMR) system (MLH1, MSH2, MSH6 or PMS2) that renders them MMR deficient, whereas MSI-L and MSS tumours are generally MMR proficient. However, tumours from patients with a pathogenic germline mutation in MSH6 can sometimes present an MSI-L phenotype with the NCI panel. The MSH6 protein is not involved in the repair of mismatches of two nucleotides in length and consequently the three dinucleotide repeats of the NCI panel often show stability in MSH6-deficient tumours.

Methods:

A pentaplex panel comprising five mononucleotide repeats has been recommended as an alternative to the NCI panel to determine tumour MSI status. Several studies have confirmed the sensitivity, specificity and ease of use of the pentaplex panel; however, its sensitivity for the detection of MSH6-deficient tumours is so far unknown. Here, we used the pentaplex panel to evaluate MSI status in 29 tumours known to harbour an MSH6 defect.

Results:

MSI-H status was confirmed in 15 out of 15 (100%) cases where matching normal DNA was available and in 28 out of 29 (97%) cases where matching DNA was not available or was not analysed.

Conclusion:

These results show that the pentaplex assay efficiently discriminates the MSI status of tumours with an MSH6 defect.     

Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress

Background:

DNA mismatch repair deficiency is present in a significant proportion of a number of solid tumours and is associated with distinct clinical behaviour.

Methods:

To identify the therapeutic agents that might show selectivity for mismatch repair-deficient tumour cells, we screened a pair of isogenic MLH1-deficient and MLH1-proficient tumour cell lines with a library of clinically used drugs. To test the generality of hits in the screen, selective agents were retested in cells deficient in the MSH2 mismatch repair gene.

Results:

We identified cytarabine and other related cytosine-based nucleoside analogues as being selectively toxic to MLH1 and MSH2-deficient tumour cells. The selective cytotoxicity we observed was likely caused by increased levels of cellular oxidative stress, as it could be abrogated by antioxidants.

Conclusion:

We propose that cytarabine-based chemotherapy regimens may represent a tumour-selective treatment strategy for mismatch repair-deficient cancers.     

Proficiency of DNA repair genes and microsatellite instability in operated colorectal cancer patients with clinical suspicion of lynch syndrome

Background

Lynch syndrome (LS) diagnosis is underestimated, and most of the patients remain undetected after colorectal resections. The study aims to assess the frequency of LS in patients undergoing surgical treatment for colorectal cancer (CRC).

Methods

A total of 458 CRC patients were operated from January 2005 to December 2008. Positive CRC family history (FH) was present in 118 (25.8%) patients. Histologic sections were reviewed for microsatellite instability (MSI) criteria (Bethesda guidelines), immunohistochemical (IHC) analysis for MLH1, MSH2, MSH6, PMS2 proteins, through the avidin-biotin-peroxidase complex, MSI (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and BRAF somatic mutation.

Results

Of the 118 patients with FH, 61 (51.69%) met at least one of the revised Bethesda criteria. IHC was abnormal in 8 (13.1%) and MSI in 12 patients (20%). BRAF was negative in all cases. MSI histopathological included: intratumoral lymphocytes (47.5%), expansive tumors (29.5%) mucinous component (27.8%) and Crohn’s like reaction in (14.7%). There was an association between the revised Bethesda criteria with: sex, mucinous histology and Crohn’s like reaction; MSI and IHC with PMS2 and MLH1. Revised Bethesda criteria 4 had 10.6 increased chances to display positive MSI. We have proposed a score to contribute as a practical tool in the diagnosis of LS.

Conclusions

The frequence of LS in resected CRC patients was 2.6%. The criterion 4 Revised Bethesda was associated more strongly with the presence of MSI.     

Synchronous gynecologic malignancy and preliminary results of Lynch syndrome

Objective

Lynch syndrome is a hereditary cancer syndrome that increases the risks of colorectal and gynecologic malignancies such as endometrial and ovarian cancer. Studies have shown that mutations in mismatch repair genes (MSH2, MSH6, and MLH1) are associated with Lynch syndrome. The aim of our study was to estimate the value of MSH2, MSH6, and MLH1 immunohistochemistry based on family history in a Korean sample.

Methods

Thirty six women with synchronous gynecologic tumors of endometrial and ovarian cancer were identified among patients being treated at our institution. Among them, 32 patients had tumor blocks (total 62 slides) available for analysis. According to a diagnostic algorithm, we performed immunohistochemistry analyses. Staining was scored based on intensity and proportion (negative or 0: intensity undetectable or minimal, proportion <5%; weak or 1+: intensity mild, proportion 5-30%; strong or 2+: intensity moderate to marked, proportion 30-99%).

Results

Among 32 eligible patients, 9 (28%) had a family history of cancer. Six patients (19%) were negative for MLH1; among them, four (4/6) were negative at both sites. Nine patients (28%) were negative for MSH2 or MSH6 at both sites or negative for both MSH2 and MSH6. Among these three patients showed negative staining for both sites. The three patients showing negative staining for MLH1, MSH2, and MSH6 at both sites with family history were considered to be the screening positive groups of Lynch syndrome.

Conclusion

In this study, the frequency of Lynch syndrome associated immunohistochemical staining (MLH1, MSH2, and MSH6) group was estimated as 9% (3/32) among Korean women with synchronous gynecologic tumors.     

Genotyping of BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes in a male patient with secondary breast cancer

Background

Some tumour suppressor genes (BRCA2) and mismatch repair genes (MSH2, MLH1) are correlated with an increased risk for male breast cancer.

Case report

Our patient developed secondary breast cancer after the treatment for Hodgkin’s disease in childhood. DNA was isolated from the patients’ blood and screened for mutations, polymorphisms and variants in BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes. We found no mutations but common polymorphisms, and three variants in mismatch repair genes.

Conclusions

Nucleotide variants c.2006-6T>C and p.G322D in MSH2 might be correlated with male breast cancer.     

Mismatch repair deficiency: a temozolomide resistance factor in medulloblastoma cell lines that is uncommon in primary medulloblastoma tumours

Background:

Tumours are responsive to temozolomide (TMZ) if they are deficient in O⁶-methylguanine-DNA methyltransferase (MGMT), and mismatch repair (MMR) proficient.

Methods:

The effect of TMZ on medulloblastoma (MB) cell killing was analysed with clonogenic survival assays. Expression of DNA repair genes and enzymes was investigated using microarrays, western blot, and immunohistochemistry. DNA sequencing and promoter methylation analysis were employed to investigate the cause of loss of the expression of MMR gene MLH1.

Results:

Temozolomide exhibited potent cytotoxic activity in D425Med (MGMT deficient, MLH1 proficient; IC₅₀=1.7 μℳ), moderate activity against D341Med (MGMT proficient, MLH1 deficient), and DAOY MB cells (MGMT proficient, MLH1 proficient). MGMT inhibitor O⁶-benzylguanine sensitised DAOY, but not D341Med cells to TMZ. Of 12 MB cell lines, D341Med, D283Med, and 1580WÜ cells exhibited MMR deficiency due to MLH1 promoter hypermethylation. DNA sequencing of these cells provided no evidence for somatic genetic alterations in MLH1. Expression analyses of MMR and MGMT in MB revealed that all patient specimens (n=74; expression array, n=61; immunostaining, n=13) are most likely MMR proficient, whereas some tumours had low MGMT expression levels (according to expression array) or were totally MGMT deficient (3 out of 13 according to immunohistochemistry).

Conclusion:

A subset of MB may respond to TMZ as some patient specimens are MGMT deficient, and tumours appear to be MMR proficient.     

Mismatch repair gene defects in sporadic colorectal cancer enhance immune surveillance

Background

There is evidence that colorectal cancers (CRC) with DNA mismatch repair deficiency (MMR-D) are associated with a better prognosis than the generality of large bowel malignancies. Since an active immune surveillance process has been demonstrated to influence CRC outcome, we investigated whether MMR-D can enhance the immune response in CRC.

Patients and Methods

A group of 113 consecutive patients operated for CRC (42 stage I or II and 71 with stage III or IV) was retrospectively analyzed. The expression of MMR genes (MSH2, MLH1, MSH6 and PSM2) and co-stimulatory molecule CD80 was assessed by tissue microarray immunohistochemistry. In addition, tumor infiltrating mononuclear cells (TIMC) and T cell subpopulations (CD4, CD8, T-bet and FoxP-3) were quantified. The effect of specific siRNA (siMSH2, siMLH1, siMSH6 and siPSM2) transfection in HT29 on CD80 expression was quantified by flow cytometry. Non parametric statistics and survival analysis were used.

Results

Patients with MMR-D showed a higher T-bet/CD4 ratio (p = 0.02), a higher rate of CD80 expression and CD8 lymphocyte infiltration compared to those with no MMR-D. Moreover, in the MMR-D group, the Treg marker FoxP-3 was not expressed (p = 0.05). MMR-D patients with stage I or II and T-bet expression had a significant better survival (p = 0.009). Silencing of MSH2, MLH1 and MSH6, but not PSM2, significantly increased the rate of CD80+ HT29 cells (p = 0.007, p = 0.023 and p = 0.015, respectively).

Conclusions

CRC with MMR-D showed a higher CD80 expression, and CD8+ and Th1 T-cell infiltration. In vitro silencing of MSH2, MLH1 and MSH6 significantly increased CD80+ cell rate. These results suggest an enhanced immune surveillance mechanism in presence of MMR-D.     

Body mass index in early adulthood and colorectal cancer risk for carriers and non-carriers of germline mutations in DNA mismatch repair genes

Background:

Carriers of germline mutations in DNA mismatch repair (MMR) genes have a high risk of colorectal cancer (CRC), but the modifiers of this risk are not well established. We estimated an association between body mass index (BMI) in early adulthood and subsequent risk of CRC for carriers and, as a comparison, estimated the association for non-carriers.

Methods:

A weighted Cox regression was used to analyse height and weight at 20 years reported by 1324 carriers of MMR gene mutations (500 MLH1, 648 MSH2, 117 MSH6 and 59 PMS2) and 1219 non-carriers from the Colon Cancer Family Registry.

Results:

During 122 304 person-years of observation, we observed diagnoses of CRC for 659 carriers (50%) and 36 non-carriers (3%). For carriers, the risk of CRC increased by 30% for each 5 kg m^–2 increment in BMI in early adulthood (hazard ratio, HR: 1.30; 95% confidence interval, CI: 1.08–1.58; P=0.01), and increased by 64% for non-carriers (HR: 1.64; 95% CI: 1.02–2.64; P=0.04) after adjusting for sex, country, cigarette smoking and alcohol drinking (and the MMR gene that was mutated in carriers). The difference in HRs for carriers and non-carriers was not statistically significant (P=0.50). For MLH1 and PMS2 (MutLα heterodimer) mutation carriers combined, the corresponding increase was 36% (HR: 1.36; 95% CI: 1.05–1.76; P=0.02). For MSH2 and MSH6 (MutSα heterodimer) mutation carriers combined, the HR was 1.26 (95% CI: 0.96–1.65; P=0.09). There was no significant difference between the HRs for MutLα and MutSα heterodimer carriers (P=0.56).

Conclusion:

Body mass index in early adulthood is positively associated with risk of CRC for MMR gene mutation carriers and non-carriers.     

DNA mismatch repair gene polymorphisms affect survival in pancreatic cancer

Purpose.

DNA mismatch repair (MMR) maintains genomic stability and mediates cellular response to DNA damage. We aim to demonstrate whether MMR genetic variants affect overall survival (OS) in pancreatic cancer.

Materials and Methods.

Using the Sequenom method in genomic DNA, we retrospectively genotyped 102 single-nucleotide polymorphisms (SNPs) of 13 MMR genes from 706 patients with pancreatic adenocarcinoma seen at The University of Texas MD Anderson Cancer Center. Association between genotype and OS was evaluated using multivariable Cox proportional hazard regression models.

Results.

At a false discovery rate of 1% (p ≤ .0015), 15 SNPs of EXO1, MLH1, MSH2, MSH3, MSH6, PMS2, PMS2L3, TP73, and TREX1 in patients with localized disease (n = 333) and 6 SNPs of MSH3, MSH6, and TP73 in patients with locally advanced or metastatic disease (n = 373) were significantly associated with OS. In multivariable Cox proportional hazard regression models, SNPs of EXO1, MSH2, MSH3, PMS2L3, and TP73 in patients with localized disease, MSH2, MSH3, MSH6, and TP73 in patients with locally advanced or metastatic disease, and EXO1, MGMT, MSH2, MSH3, MSH6, PMS2L3, and TP73 in all patients remained significant predictors for OS (p ≤ .0015) after adjusting for all clinical predictors and all SNPs with p ≤ .0015 in single-locus analysis. Sixteen haplotypes of EXO1, MLH1, MSH2, MSH3, MSH6, PMS2, PMS2L3, RECQL, TP73, and TREX1 significantly correlated with OS in all patients (p ≤ .001).

Conclusion.

MMR gene variants may have potential value as prognostic markers for OS in pancreatic cancer patients.     

Target gene mutational pattern in Lynch syndrome colorectal carcinomas according to tumour location and germline mutation

Background:

We previously reported that the target genes in sporadic mismatch repair (MMR)-deficient colorectal carcinomas (CRCs) in the distal colon differ from those occurring elsewhere in the colon. This study aimed to compare the target gene mutational pattern in microsatellite instability (MSI) CRC from Lynch syndrome patients stratified by tumour location and germline mutation, as well as with that of sporadic disease.

Methods:

A series of CRC from Lynch syndrome patients was analysed for MSI in genes predicted to be selective MSI targets and known to be involved in several pathways of colorectal carcinogenesis.

Results:

The most frequently mutated genes belong to the TGF-β superfamily pathway, namely ACVR2A and TGFBR2. A significantly higher frequency of target gene mutations was observed in CRC from patients with germline mutations in MLH1 or MSH2 when compared with MSH6. Mutations in microsatellite sequences (A)7 of BMPR2 and (A)8 of MSH3 were significantly more frequent in the distal CRC. Additionally, we observed differences in MSH3 and TGFBR2 mutational frequency between Lynch syndrome and sporadic MSI CRC regarding tumour location.

Conclusions:

Our results indicate that the pattern of genetic changes differs in CRC depending on tumour location and between Lynch syndrome and sporadic MSI CRC, suggesting that carcinogenesis can occur by different pathways even if driven by generalised MSI.     

MutS�� exceeds MutS�� in dinucleotide loop repair

Backround:

The target substrates of DNA mismatch recognising factors MutSα (MSH2+MSH6) and MutSβ (MSH2+MSH3) have already been widely researched. However, the extent of their functional redundancy and clinical substance remains unclear. Mismatch repair (MMR)-deficient tumours are strongly associated with microsatellite instability (MSI) and the degree and type of MSI seem to be dependent on the MMR gene affected, and is linked to its substrate specificities. Deficiency in MSH2 and MSH6 is associated with both mononucleotide and dinucleotide repeat instability. Although no pathogenic MSH3 mutations have been reported, its deficiency is also suggested to cause low dinucleotide repeat instability.

Methods:

To assess the substrate specificities and functionality of MutSα and MutSβ we performed an in vitro MMR assay using three substrate constructs, GT mismatch, 1 and 2 nucleotide insertion/deletion loops (IDLs) in three different cell lines.

Results:

Our results show that though MutSα alone seems to be responsible for GT and IDL1 repair, MutSα and MutSβ indeed have functional redundancy in IDL2 repair and in contrast with earlier studies, MutSβ seems to exceed MutSα.

Conclusion:

The finding is clinically relevant because the strong role of MutSβ in IDL2 repair indicates MSH3 deficiency in tumours with low dinucleotide and no mononucleotide repeat instability.     

Frequency of mutations in mismatch repair genes in a population-based study of women with ovarian cancer

Background:

Mutations in genes for hereditary non-polyposis colorectal cancer (HNPCC) in ovarian cancer patients remains poorly defined. We sought to estimate the frequency and characteristics of HNPCC gene mutations in a population-based sample of women with epithelial ovarian cancer.

Methods:

The analysis included 1893 women with epithelial ovarian cancer ascertained from three population-based studies. Full-germline DNA sequencing of the coding regions was performed on three HNPCC genes, MLH1, MSH2 and MSH6. Collection of demographic, clinical and family history information was attempted in all women.

Results:

Nine clearly pathogenic mutations were identified, including five in MSH6, two each in MLH1 and MSH2. In addition, 28 unique predicted pathogenic missense variants were identified in 55 patients. Pathogenic mutation carriers had an earlier mean age at diagnosis of ovarian cancer, overrepresentation of cancers with non-serous histologies and a higher number of relatives with HNPCC-related cancers.

Conclusions:

Our findings suggest that fewer than 1% of women with ovarian cancer harbour a germline mutation in the HNPCC genes, with overrepresentation of MSH6 mutations. This represents a lower-range estimate due to the large number of predicted pathogenic variants in which pathogenicity could not definitively be determined. Identification of mismatch repair gene mutations has the potential to impact screening and treatment decisions in these women.     

Muir–Torre syndrome or phenocopy? The value of the immunohistochemical expression of mismatch repair proteins in sebaceous tumors of immunocompromised patients

Primary and secondary immunodepressive conditions are associated with an increased incidence of sebaceous tumors. Microsatellite instability (MSI) and lack of expression of mismatch repair (MMR) proteins, typical markers of Muir–Torre/Lynch heredo-familial settings, can be recognized also in immunocompromised patients. We aimed to carry on a systematic examination of clinical, immunohistochemical, biomolecular features of sebaceous tumors arising in immunocompromised and immunocompetent patients between 1986 and 2012. Microsatellite screening, immunohistochemical analysis and genetic testing were performed for hMLH1, hMSH2 and hMSH6. Methylation status of MMR genes was checked in cases with immunohistochemistry (IHC) loss of MMR proteins expression and no germline mutations. Fifteen patients had a personal history of visceral carcinomas fulfilling diagnostic criteria for Muir–Torre syndrome. In this cohort, IHC analysis, MSI status and genetic testing were in agreement, showing eight MSH2 and two MLH1 germline mutations. Five patients were immunosuppressed and their sebaceous tumors showed a lack of MSH2/MSH6 expression, although just one case with positive family history for visceral cancer harbored a germline mutation. In immunosuppressed patients, loss of IHC for MMR proteins is not necessarily secondary to MMR germline mutations. IHC false positives are probably due to epigenetic alterations. MSI and lack of expression of MMR proteins can be recognized also in immunocompromised patients without MMR germline mutations.     

Early-onset breast cancer in a Lebanese family with Lynch syndrome due to MSH2 gene mutation

Background

There are still controversies about the integration of breast cancer as a part of the disease spectrum in Lynch syndrome.

Methods

A regular follow-up of a Lebanese pedigree with Lynch syndrome due to a point mutation of MSH2 gene at the splice donor site of intron 3 started in 1996.

Results

A 26-year-old pregnant woman, mutation carrier, developed an aggressive breast cancer, refractory to standard chemotherapy regimens. The microsatellite analysis of the tumor showed an unstable pattern for markers BAT25 and BAT26. The immunohistochemical staining was negative for MSH2 and MSH6 and normal for MLH1 and PMS6 enzymes.

Conclusion

The segregation of the mutation with the disease phenotype and these results suggest that MSH2 inactivation may be involved in the accelerated breast carcinogenesis and might be considered in the cancer screening program.     

Evaluation of a new panel of six mononucleotide repeat markers for the detection of DNA mismatch repair-deficient tumours

Background:

Microsatellite instability (MSI) is a molecular phenotype due to defective DNA mismatch repair (MMR) system. It is used to predict outcome of colorectal tumours and to screen tumours for Lynch syndrome (LS). A pentaplex panel composed of five mononucleotide markers has been largely recommended for determination of the MSI status. However, its sensitivity may be taken in default in occasional situations. The aim of the study was to optimise this panel for the detection of MSI.

Methods:

We developed an assay allowing co-amplification of six mononucleotide repeat markers (BAT25, BAT26, BAT40, NR21, NR22, NR27) and one polymorphic dinucleotide marker (D3S1260) in a single reaction. Performances of the new panel were evaluated on a cohort of patients suspected of LS.

Results:

We demonstrate that our assay is technically as easy to use as the pentaplex assay. The hexaplex panel shows similar performances for the identification of colorectal and non-MSH6-deficient tumours. On the other hand, the hexaplex panel has higher sensitivity for the identification of MSH6-deficient tumours (94.7% vs 84.2%) and MMR-deficient tumours other than colorectal cancer (92.9% vs 85.7%).

Conclusion:

The hexaplex panel could thus be an attractive alternative to the pentaplex panel for the identification of patients with LS.     

Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours

Background

Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT.

Methods

The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined.

Results

MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity.

Conclusion

This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis.

     

Loss of MSH2 and MSH6 due to heterozygous germline defects in <Emphasis Type="Italic">MSH3</Emphasis> and <Emphasis Type="Italic">MSH6</Emphasis>

Lynch Syndrome (LS) is the most common dominantly inherited colorectal cancer (CRC) predisposition and is caused by a heterozygous germline defect in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. High microsatellite instability (MSI-H) and loss of MMR protein expression in tumours reflecting a defective MMR are indicators for LS, as well as a positive family history of early onset CRC. MSH2 and MSH6 form a major functional heterodimer, and MSH3 is an alternative binding partner for MSH2. So far, the role of germline MSH3 variants remains unclear, as to our knowledge heterozygous truncating variants are not regarded causative for LS, but were detected in patients with CRC, and recently biallelic MSH3 defects have been identified in two patients with adenomatous polyposis. By gene screening we investigated the role of MSH3 in 11 LS patients with truncating MSH6 germline variants and an unexplained MSH2 protein loss in their corresponding MSI-H tumours. We report the first two LS patients harbouring heterozygous germline variants c.1035del and c.2732T>G in MSH3 coincidentally with truncating variants in MSH6. In the patient with truncating germline variants in MSH3 and MSH6, two additional somatic second hits in both genes abrogate all binding partners for the MSH2 protein which might subsequently be degraded. The clinical relevance of MSH3 germline variants is currently under re-evaluation, and heterozygous MSH3 defects alone do not seem to induce a LS phenotype, but might aggravate the MSH6 phenotype in affected family members.