MSI-H Colorectal Carcinomas Research Articles

121P Spectrum of druggable gene fusions in microsatellite-unstable colorectal tumors

Colorectal carcinomas (CRCs) with high-level microsatellite instability (MSI-H) often contain gene fusions, which involve receptor tyrosine kinases. This study included 105 MSI-H CRCs. NTRK1/2/3, ALK, ROS1 and RET gene fusions were analyzed by PCR test for 5’/3’-end unbalanced expression, which is capable of detecting all translocation variants. The type of rearrangements was subsequently determined by variant-specific PCR for common fusions, and, wherever necessary, by targeted RNA next-generation sequencing (NGS). NTRK1/2/3 translocations were the most common, being detected in 8/105 (8%) CRCs (TPM3-NTRK1 (T8;N10): n = 2; ETV6-NTRK2 (E5;N15): n = 2; ETV6-NTRK3 (E5;N15): n = 4). There were 2 tumors with RET rearrangements (CCDC6-RET (C1;R12) and NCOA4-RET (N9del501;R12), respectively) and 1 instance of SPTBN1-ALK (S7;A20) chimera. 76/105 (72%) CRCs were negative for KRAS, NRAS or BRAF mutations; gene rearrangements were detected in 10 (13%) of these tumors. 29/105 (28%) carcinomas carried activating lesions in RAS/RAF oncogenes; one of these CRCs had both ETV6-NTRK3 (E5;N15) translocation and BRAF V600E substitution. Microsatellite-unstable CRCs have high frequency of druggable gene rearrangements, with NTRK1/2/3 deserving particular attention.

B2M and JAK1/2-mutated MSI-H Colorectal Carcinomas Can Benefit From Anti-PD-1 Therapy.

β2-microglobulin (B2M) and Janus kinases 1 and 2 (JAK1/2) mutations have been suggested as genetic mechanisms of immune evasion for anti–programmed cell death protein 1 (PD-1) therapy. Whether B2M and JAK1/2 lose-of-function mutation can cause primary resistance to anti-PD-1 therapy in colorectal carcinoma (CRC) patients remains controversial. Here, we sought to compare the efficacy of anti-PD-1 therapy in DNA mismatch repair deficient/microsatellite instability–high CRC patients with or without B2M or JAK1/2 mutations. Thirty-Five CRC patients who received anti-PD-1 therapy were enrolled in this study. All tumor samples underwent next-generation sequencing. The clinical and molecular data from 110 CRC patients sequenced with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay and accessed through cBioportal were also analyzed in this study. Of the 35 CRC patients from our center, 10 (28.6%) had a B2M loss-of-function mutation, and 8 (22.9%) had a JAK1/2 loss-of-function mutation. Compared with B2M wild-type CRCs, B2M-mutated CRCs did not show a higher frequency of resistance to anti-PD-1 therapy (P=0.71). There was even better response to anti-PD-1 therapy in patients with JAK1/2 mutation than in those without (P=0.015). Of the 110 CRC patients in the MSK-IMPACT datasets, 13 (11.8%) had a B2M mutation, and 15 (13.6%) had a JAK1/2 mutation. After analyzing the response to anti-PD-1 therapy in these 110 patients, we found similar results (P=0.438 and 0.071, respectively). Moreover, patients with B2M or JAK1/2 mutation had a lower tumor mutational burden score compared with those without. B2M and JAK1/2 loss-of-function mutations occur frequently in microsatellite instability–high CRC. Our study demonstrated that patients with CRC harboring B2M or JAK1/2 mutations should not be excluded from anti-PD-1 therapy.

Value of histopathology in predicting microsatellite instability in colorectal cancer

Background: Identifying colorectal cancers (CRCs) with high levels of microsatellite instability (MSI-H) is clinically important. Few studies reported the role of histology in identifying MSI-H colorectal carcinomas. Hence the present study was undertaken to assess the value of histopathology in predicting MSI-H and its correlation with histopathologic prognostic features in CRC. Methods: Total 100 histo-pathologically confirmed cases of CRC were enrolled in the study. Tumour’s histopathological typing, staging, and sizing was conducted based on WHO criteria. Tumour grade, extent of mucin production, tumour growth pattern and presence of a Crohn’s like inflammatory infiltrate were determined. For immunohistochemistry (IHC) we used monoclonal antibodies (ES05 or 25D12). Results: Among 100 tumour samples, loss of MLH1 or MSH2 expression was detected in 13(13%) cases (MSI tumours) while 87(87%) tumours showed normal expression for MLH1 and MSH2 immuno-reactivity (MSS tumours). In terms of MSI/MSS status of tumour samples, statistical analysis showed that abnormal MMR protein expression was associated with tumour site, side of colon with cancer, size of tumour, tumour infiltrating lymphocytes and crohn’s like inflammatory infiltrate. Some histopathological and clinical features that appeared highly specific but much less sensitive in predicting MSI include stage, and some features were more sensitive and less specific which include tumour differentiation, CLR, TIL, dirty necrosis and pushing margin. Conclusion: MSI is an important prognostic factor in CRC and an important predictive factor of CRC chemotherapeutic treatment and outcome efficacy. Also result of the study is not consistent in describing various histopathological and clinical features in predicting MSI, CRC.

Mapping of colorectal carcinoma diseases with activation of Wnt/beta-catenin signalling pathway using hierarchical clustering approach

AimsTo map the colorectal carcinoma (CRC) diseases with significant Wnt signalling pathway activation for delineating their clinicopathological and molecular profiles.MethodsMapping is based on hierarchical clustering analyses of a series of...

Molecular and clinicopathological features of colorectal adenocarcinoma with enteroblastic differentiation.

Colorectal adenocarcinoma with enteroblastic differentiation (CAED) is a rare subtype of colorectal malignancy with expression of enteroblastic markers (glypican 3, SALL4, AFP); however, the clinicopathological and epidemiological features are not fully elucidated. The aims of this study were to elucidate and establish the molecular and clinicopathological characteristics of CAED. In addition to three cases recently diagnosed as CAED, colorectal carcinoma (CRC) with expression of enteroblastic markers were selected by using immunohistochemistry (IHC) on tissue microarrays of 988 advanced CRC. We employed next-generation sequencing (NGS) and Sanger sequencing for the detection of genetic alterations. IHC for p53 and HER2, HER2-FISH and MSI status was also investigated. Survival analyses for clinicopathological parameters were performed using Kaplan-Meier methods. Thirty-nine cases (4.0%) were positive for at least one enteroblastic marker. Histological evaluation of the total of 42 cases revealed that 10 contained tumour cells with clear cytoplasm. Enteroblastic marker-positive cases had aggressive behaviour and poor prognosis. NGS revealed TP53 as the most frequently mutated gene. The rate of HER2-positive cases and MSI-H cases was 9.5% (four of 42) and 12.2% (five of 41), respectively. Among these 42 cases, there were no molecular and clinicopathological differences according to the presence of tumour cells with clear cytoplasm. Enteroblastic marker-positive CRC could be grouped together as CAED regardless of clear cell cytoplasm. Using this definition, the frequency of CAED is 4.0% and has a poorer prognosis than that for conventional CRCs. HER2 targeted therapy would be a meaningful treatment for CAED, and CAEDs contain both MSI-H and MSI-stable CRCs, although the MSS phenotype is dominant.

Immunohistochemical Expression of MMR Proteins with Clinicopathological Correlation in Colorectal Cancer in Egypt.

BACKGROUND:Microsatellite instability (MSI) is the genetic pathway underlying 15% of sporadic colorectal carcinoma (CRC) and hereditary non-polyposis CRC. MSI-H CRC has a distinct clinicopathological characteristic including excess mucin and signet ring component, proximal colon, Crohn’s like reaction, lymphocytic infiltration, and better survival.AIM:This research aims to screen Egyptian CRC patients for MSI status by IHC testing of expression of the MMR proteins in correlation to its clinicopathological features.MATERIAL AND METHODS:Immunohistochemistry study for mismatch repair proteins (MMR) was done on 115 cases of CRC. Their expressions were assessed and correlated to clinicopathological parameters in an attempt to obtain the most significant predictors of MSI.RESULTS:MSI (low and high) represents 67% of the study cases. The most frequent expression pattern was combined loss of MLH, and PMS2 (38% of MSI) followed by a combined loss of MSH2, and MSH6 (29% of MSI). There was significant correlation of expression pattern of MMR proteins with the laterality, lymphovascular emboli, perineural invasion, grade, T stage, N stage, signet ring component, tumor infiltrating lymphocyte, and peritumoral lesion (0.014, 0.035, 0.012, 0.033, 0.013, 0.000, 0.041, 0.012, and 0.009 respectively). Proximal location (right sided) and lower grade, higher nodal stage, and marked TIL were selected as predictors of MS-H CRC (0.005, 0.031, 0.025, and 0.000 respectively).CONCLUSION:All clinicopathological and histological parameters should be assessed in CRC for the sake of predicting MSI. The optimal approach to MSI evaluation is (IHC) assessment of MMR proteins.

High microsatellite instability (MSI-H) colorectal carcinoma: a brief review of predictive biomarkers in the era of personalized medicine.

Approximately 15 % of colorectal carcinomas (CRC) display high level microsatellite instability (MSI-H) due to either a germline mutation in one of the genes responsible for DNA mismatch repair (Lynch syndrome, 3 %) or somatic inactivation of the same pathway, most commonly through hypermethylation of the MLH1 gene (sporadic MSI-H, 12 %). Although heterogeneous, MSI-H colorectal carcinomas as a group show some distinct biologic characteristics when compared to CRC with stable or low level microsatellite instability. In the present review we will highlight therapeutically relevant characteristics of MSI-H tumors which could lead to specific responses to some conventional chemotherapy or novel targeted therapy agents.

570P - Thymidylate Synthase Over-Expression Underlies the Observed Lack of 5-Fu Therapy Benefit for Msi-H Colorectal Cancers

ABSTRACT Aim: 5-FU based treatment remains the standard of care for the adjuvant treatment of colorectal carcinoma (CRC), in combination with oxaliplatin and irinotecan. CRC is molecularly heterogenous disease and patients with microsatellite stable (MSS) CRC have been previously shown to benefit from 5-FU based therapies while patients with high microsatellite instability (MSI-H) CRC did not. We investigated the expression thymidylate synthase, the targeted enzyme for 5-FU and topoisomerase I (Topo1), the targeted enzyme for irinotecan, in molecularly defined cohort of CRC. Methods: 106 patients with CRC (86 MSS and 20 MSI-H) were molecularly profiled using multiplatform approach [Caris Life Sciences] including immunohsitochemistry, PCR microsatellite analysis and gene sequencing (NGS) . Results: TS over-expression (over the ≥1+ and ≥10% threshold, and frequently >2+ and >50%) was observed in all 20 MSI-H cases (100%). In contrast, MSS cases overexpressed TS in only 31/86 (36%) of the cases (p Conclusions: Two most common molecular subtypes of colorectal cancer exhibit different expression patterns of TS and Topo1; overexpression of TS may explain the observed lack of benefit of 5-FU based therapy in MSI-H CRC patients. Additional genetic and molecular biomarkers could provide guidance to alternative chemotherapy modalities. Disclosure: Z. Gatalica: Stock options Caris Life Sciences; D. Bryant: Stock options Caris Life Sciences All other authors have declared no conflicts of interest.

BRAF-mutated microsatellite stable colorectal carcinoma: an aggressive adenocarcinoma with reduced CDX2 and increased cytokeratin 7 immunohistochemical expression.

Reduced CDX2 and cytokeratin 20 (CK20) expression in colorectal carcinoma with BRAF mutation and high-level microsatellite instability (MSI-H) has been well documented. The immunophenotype of BRAF-mutated microsatellite stable (MSS) colorectal carcinoma has not been reported. We analyzed 205 colorectal carcinomas including 28 BRAF-mutated MSS, 53 BRAF-mutated MSI-H, and 124 BRAF wild-type MSS tumors for CDX2, cytokeratin 7 (CK7), and CK20 immunohistochemical expression. CDX2 was scored semiquantitatively for both staining intensity and percent of tumor cells staining and a modified CDX2 H-score was calculated. Patients with BRAF-mutated MSS colorectal carcinomas were more frequently stage IV at presentation compared to patients with BRAF-mutated MSI-H colorectal carcinomas and BRAF wild-type MSS colorectal carcinomas (32% versus 8% versus 15%, P < .001). BRAF-mutated MSS colorectal carcinoma displayed reduced CDX2 expression compared to BRAF wild-type MSS colorectal carcinoma (75% versus 94%; mean CDX2 H-score 98 versus 150, P < .001). CK7 expression was more often identified in BRAF-mutated MSS colorectal carcinoma compared to both BRAF-mutated MSI-H colorectal carcinoma and BRAF wild-type MSS colorectal carcinoma (39% versus 6% versus 6%, P = .0001). BRAF-mutated MSI-H colorectal carcinomas were less often CK20 positive compared to BRAF-mutated MSS and BRAF wild-type MSS tumors (70% versus 93% versus 90%, P = 0.001). In summary, BRAF-mutated MSS colorectal carcinoma often displays reduced CDX2 and increased CK7 expression. Knowledge of this altered immunophenotype is important as patients with BRAF-mutated MSS colorectal carcinoma often present with metastatic disease and the altered tumor immunophenotype may lead to the erroneous assumption that origin from the colon/rectum is unlikely.

The analysis of microsatellite instability in extracolonic gastrointestinal malignancy

Microsatellite instability (MSI) is a genetic feature of sporadic and familial cancers of multiple sites and is related to defective mismatch repair (MMR) protein function. Lynch syndrome (LS) is a familial form of MMR deficiency that may present with a spectrum of MSI positive cancers including gastrointestinal (GI) malignancies. The incidence of high level MSI (MSI-H) in colorectal carcinoma is well defined in both familial and sporadic cases and these tumours portend a better overall prognosis in colorectal carcinoma (CRC). There are certain morphological features that suggest MSI-H CRC and international guidelines have been established for the evaluation of MSI in CRC. The prevalence and morphological features of extracolonic GI MSI-H tumours are less well documented. Furthermore, it is unclear whether the guidelines for the assessment of MSI in CRC are appropriate for application to extracolonic GI malignancies. This review aims to summarise the recent literature on MSI in extracolonic LS-related GI tract malignancies with special attention to the assessment of the MMR system by evaluation of specific microsatellite markers and/or immunohistochemical evaluation of MMR protein expression. The reported prevalence of sporadic and LS-related MSI-H tumours along with their associated unique morphological patterns and related prognostic or therapeutic implications will be discussed.

Lynch syndrome–associated colorectal carcinoma: frequent involvement of the left colon and rectum and late-onset presentation supports a universal screening approach

The optimal strategy for screening patients with colorectal carcinoma for Lynch syndrome (LS) is a subject of continued debate in the literature with some advocating universal screening while others arguing for selective screening. We evaluated 1292 colorectal carcinomas for DNA mismatch repair protein abnormalities and identified 150 (11.6%) tumors demonstrating high-levels of microsatellite instability (MSI-H). MSI-H colorectal carcinomas were divided into sporadic (112/1292, 8.7%) and LS/probable LS-associated (38/1292, 2.9%) groups based on BRAF V600E mutation, MLH1 promoter hypermethylation, cancer history, and germline mismatch repair gene mutation. All MSI-H colorectal carcinomas were analyzed for grade, location, and tumor histology. The utility of the revised Bethesda guidelines and published predictive pathology models for MSI-H colorectal carcinomas (PREDICT and MSPath) were evaluated. Left-sided MSI-H colorectal carcinomas were more frequently associated with LS compared with right-sided MSI-H colorectal carcinomas (12/21, 57% versus 26/129, 20%, P = .0008). There was no significant difference in histology between sporadic MSI-H and LS/probable LS-associated colorectal carcinomas except for a slightly higher proportion of sporadic MSI-H tumors demonstrating tumor-infiltrating lymphocytes (81% versus 61%, P = .015). Neither pathology predictive model identified all LS-associated colorectal carcinomas (PREDICT: 33/38, 87%; MSPath: 35/38, 92%). 12/117 (10%) MSI-H colorectal carcinomas identified in patients >60 years were LS/probable LS-associated. Our results demonstrate that models of predicting MSI-H fail to identify LS-associated colorectal carcinoma given their reliance on right-sided location. A significant proportion (32%) of LS-associated colorectal carcinoma is identified in patients >60 years. Finally, our results demonstrate similar morphologic features between LS-associated and sporadic MSI-H colorectal carcinomas.

Clinical and pathological tools for identifying microsatellite instability in colorectal cancer

AimTo assess practical accuracy of revised Bethesda criteria (BGrev), pathological predictive model (MsPath), and histopathological parameters for detection of high-frequency of microsatellite instability (MSI-H) phenotype in patients with colorectal carcinoma (CRC).MethodTumors from 150 patients with CRC were analyzed for MSI using a fluorescence-based pentaplex polymerase chain reaction technique. For all patients, we evaluated age, sex, family history of cancer, localization, tumor differentiation, mucin production, lymphocytic infiltration (TIL), and Union for International Cancer Control stage. Patients were classified according to the BGrev, and the groups were compared. The utility of the BGrev, MsPath, and clinical and histopathological parameters for predicting microsatellite tumor status were assessed by univariate logistic regression analysis and by calculating the sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values.ResultsFifteen out of 45 patients who met and 4 of 105 patients who did not meet the BGrev criteria had MSI-H CRC. Sensitivity, specificity, PPV, and NPV for BGrev were 78.9%, 77%, 30%, and 70%, respectively. MSI histology (the third BGrev criterion without age limit) was as sensitive as BGrev, but more specific. MsPath model was more sensitive than BGrev (86%), with similar specificity. Any BGrev criterion fulfillment, mucinous differentiation, and right-sided CRC were singled out as independent factors to identify MSI-H colorectal cancer.ConclusionThe BGrev, MsPath model, and MSI histology are useful tools for selecting patients for MSI testing.

Implication of the β2-microglobulin gene in the generation of tumor escape phenotypes.

Classical MHC molecules present processed peptides from endogenous protein antigens on the cell surface, which allows CD8(+) cytotoxic T lymphocytes (CTLs) to recognize and respond to the abnormal antigen repertoire of hazardous cells, including tumor cells. The light chain, β2-microglobulin (β2m), is an essential constant component of all trimeric MHC class I molecules. There is convincing evidence that β2m deficiency generates immune escape phenotypes in different tumor entities, with an exceptionally high frequency in colorectal carcinoma (CRC) and melanoma. Damage of a single β2m gene by LOH on chromosome 15 may be sufficient to generate a tumor cell precommitted to escape. In addition, this genetic lesion is followed in some tumors by a mutation of the second gene (point mutation or insertion/deletion), which produces a tumor cell unable to express any HLA class I molecule. The pattern of mutations found in microsatellite unstable colorectal carcinoma (MSI-H CRC) and melanoma showed a striking similarity, namely the predominance of frameshift mutations in repetitive CT elements. This review emphasizes common but also distinct molecular mechanisms of β2m loss in both tumor types. It also summarizes recent studies that point to an acquired β2m deficiency in response to cancer immunotherapy, a barrier to successful vaccination or adoptive cellular therapy.

T cell responses against microsatellite instability-induced frameshift peptides and influence of regulatory T cells in colorectal cancer

High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.

Identification of an MSI-H Tumor-Specific Cytotoxic T Cell Epitope Generated by the (−1) Frame ofU79260(FTO)

Microsatellite instability (MSI-H) induced by defects of the DNA mismatch repair system results in insertion or deletion of single nucleotides at short repetitive DNA sequences. About 15% of sporadic and approximately 90% of hereditary nonpolyposis colorectal cancers display MSI-H. When affecting coding regions, MSI-H results in frameshift mutations and expression of corresponding frameshift peptides (FSPs). Functional tumor promoting relevance has been demonstrated for a growing number of genes frequently hit by MSI-H. Contrary, immune reactions against FSPs are involved in the immune surveillance of MSI-H cancers. Here, we provide conclusive data that the (−1) frame of U79260(FTO) encodes an HLA-A0201-restricted cytotoxic T cell epitope (FSP11; TLSPGWSAV). T cells specific for FSP11 efficiently recognized HLA-A0201(pos) tumor cells harboring the mutated reading frame. Considering the exceptionally high mutation rate of U79260(FTO) in MSI-H colorectal carcinoma (81.8%), this recommends that FSP11 be a component of future vaccines.

Clinicopathologic Characteristics of Left-Sided Colon Cancers with High Microsatellite Instability

Background : High microsatellite instability (MSI-H) colorectal carcinomas (CRCs) with numerous mutations in the microsatellite sequence are characterized by a right-sided preponderance, frequent peritumoral and intratumoral lymphocytic infiltration, and frequent mucin production. However, no study has correlated anatomic site and type of genetic changes with clinicopathologic changes. Methods : We analyzed the histopathologic features of 135 MSI-H CRCs and compared them to 140 microsatellite stable (MSS) CRCs. Histopathologic changes in MSI-H were further analyzed according to anatomic sites and genetic changes. Results : MSI-H CRCs showed previously reported clinicopathologic findings; a right-sided preponderance, an increased number of mucinous carcinomas, and peritumoral lymphoid reactions (p<0.001 for each variable). Increased serum CEA levels showed an MSS CRC preponderance (p=0.013). We further analyzed the histologic differences between right- and left-sided MSI-H tumors. We found that MSI-H CRCs on both sides had similar clinicopathologic findings, except for higher tumor stage (p=0.048) and less frequent abnormal CEA levels in left-sided MSI-H tumors (p=0.027). We found that not all clinicopathologic features were different between hereditary nonpolyposis colorectal cancers (HNPCCs) and sporadic MSI-H CRCs. Conclusions : These findings indicate that MSI-H CRCs of the left colon have similar clinicopathologic characteristics as right-sided MSI-H CRCs. We did not find any significant clinicopathological difference between HNPCCs and sporadic MSI-H CRCs.

High Frequency of LMAN1 Abnormalities in Colorectal Tumors with Microsatellite Instability

Glycosyl epitopes have been identified as tumor-specific markers in colorectal tumors and various lines of evidence indicate the significance of altered synthesis, transport, and secretion of glycoproteins in tumorigenesis. However, aberrant glycosylation has been largely ignored in microsatellite unstable (MSI-H) colorectal tumors. Therefore, we analyzed mutation frequencies of genes of the cellular glycosylation machinery in MSI-H tumors, focusing on frameshift mutations in coding MNRs (cMNRs). Among 28 candidate genes, LMAN1/ERGIC53, a mannose-specific lectin mediating endoplasmatic reticulum (ER)-to-Golgi transit of glycosylated proteins, showed high mutation frequency in MSI-H colorectal cancer cell lines (52%; 12 of 23), carcinomas (45%; 72 of 161), and adenomas (40%; 8 of 20). Biallelic mutations were observed in 17% (4 of 23) of MSI-H colorectal cancer cell lines. LMAN1 was found to be transcribed but truncated protein remained undetectable in these LMAN1-mutant cell lines. Immunohistochemical and molecular analysis of LMAN1-mutated carcinomas and adenomas revealed regional loss of LMAN1 expression due to biallelic LMAN1 cMNR frameshift mutations. In LMAN1-deficient colorectal cancer cell lines, secretion of the LMAN1 client protein alpha-1-antitrypsin (A1AT), an inhibitor of angiogenesis and tumor growth, was significantly impaired but could be restored upon LMAN1 re-expression. These results suggest that LMAN1 mutational inactivation is a frequent and early event potentially contributing to MSI-H tumorigenesis.

Restoring TGFβ function in microsatellite unstable (MSI-H) colorectal cancer reduces tumourigenicity but increases metastasis formation

TGFbeta is an important cell growth regulator which may have a role in metastasis formation. Microsatellite unstable (MSI-H) colon cancer serves as a unique model to demonstrate this as most MSI-H colon cancers have a mutation in the transforming growth factor beta receptor II (TGFbetaRII) gene and a low metastatic rate. To demonstrate an increase in invasion and metastasis in a MSI-H colorectal cancer cell line with a known mutation in TGFbetaRII. By restoring the wild-type TGFbetaRII gene in the KM12C MSI-H colorectal carcinoma cell line with a known mutation in TGFbetaRII, we have demonstrated that both invasion and metastasis in this cell line was significantly increased. A mouse metastatic model have shown that liver metastases were increased in mice inoculated with cells containing a wild-type TGFbetaRII gene (42% for the transfected group compared with 15% for the control group; p = 0.0379), despite a reduction in the size of primary tumours. This study highlights an important mechanism which may contribute to the low metastatic rate of MSI-H colon cancers and demonstrates the importance of TGFbeta signalling in metastasis formation. Previous studies involving breast cancer cell lines have shown that blocking TGFbeta signalling results in a reduction in metastasis formation. This study is the first study to use a cell line with a low metastatic rate and TGFbetaRII mutations to demonstrate that restoring TGFbeta signalling increases the metastatic rate.

Beta2‐microglobulin mutations in microsatellite unstable colorectal tumors

Defects of DNA mismatch repair (MMR) cause the high level microsatellite instability (MSI-H) phenotype. MSI-H cancers may develop either sporadically or in the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome that is caused by germline mutations of MMR genes. In colorectal cancer (CRC), MSI-H is characterized by a dense lymphocytic infiltration, reflecting a high immunogenicity of these cancers. As a consequence of immunoselection, MSI-H CRCs frequently display a loss of human leukocyte antigen (HLA) class I antigen presentation caused by mutations of the beta2-microglobulin (beta2m) gene. To examine the implications of beta2m mutations during MSI-H colorectal tumor development, we analyzed the prevalence of beta2m mutations in MSI-H colorectal adenomas (n=38) and carcinomas (n=104) of different stages. Mutations were observed in 6/38 (15.8%) MSI-H adenomas and 29/104 (27.9%) MSI-H CRCs. A higher frequency of beta2m mutations was observed in MSI-H CRC patients with germline mutations of MMR genes MLH1 or MSH2 (36.4%) compared with patients without germline mutations (15.4%). The high frequency of beta2m mutations in HNPCC-associated MSI-H CRCs is in line with the hypothesis that immunoselection may be particularly pronounced in HNPCC patients with inherited predisposition to develop MSI-H cancers. beta2m mutations were positively related to stage in tumors without distant metastases (UICC I-III), suggesting that loss of beta2m expression may promote local progression of colorectal MSI-H tumors. However, no beta2m mutations were observed in metastasized CRCs (UICC stage IV, p=0.04). These results suggest that functional beta2m may be necessary for distant metastasis formation in CRC patients.

Microsatellite instability in colorectal cancer is associated with local lymphocyte infiltration and low frequency of distant metastases

Colorectal carcinomas (CRCs) with high microsatellite instability (MSI-H) share clinicopathological features distinctly different from their microsatellite stable (MSS) counterparts. Unlike MSS cancers, MSI-H CRCs occur predominantly in the right-sided colon and are often characterised by a strong lymphocyte infiltration. A poor differentiation pattern is found in most MSI-H CRCs, even though patients with MSI-H carcinomas seem to have a significantly longer survival after surgical resection. To clarify which factors contribute to the obvious paradoxon of a more favourable prognosis of MSI tumours, several clinical and histopathological features as well as the microsatellite status were evaluated in 120 colorectal cancer cases fulfilling clinical criteria (Bethesda) indicative for familial colorectal cancer. Microsatellite instablity status and lymphocyte infiltration were related to tumour stage and patients' follow-up. Statistical analysis confirmed well-known relations, such as enhanced lymphocyte infiltration accompanied by Crohn's like reaction (CLR) in MSI-H cancers (CLR+ in 27 out of 47 MSI-H vs 14 out of 71 MSS CRCs, P<0.001). However, after stratification for depth of local invasion and penetration of the primary tumour, T3 tumours displaying MSI had a significantly lower rate of distant metastases (M1 in four out of 35 MSI-H vs 20 out of 41 MSS CRCs, P<0.001). A similar tendency was observed for CLR-positive CRCs (M1 in six out of 29 CLR+ vs 17 out of 45 CLR− CRCs, P=0.13). In a logistic regression model, the MSI-H phenotype and the presence of CLR were independent predictors of a low UICC stage (P=0.006 and 0.04, respectively). These data, together with the recent definition of highly immunogenic neo-antigens expressed in MSI-H tumour cells, suggest that MSI-H CRCs elicit a protective host response that may prevent metastasis formation.