Microsatellite Unstable Colorectal Cancer Research Articles

Abstract 2422: Microsatellite stable colorectal cancers stratified by the BRAF V600E mutation show distinct patterns of chromosomal instability

Abstract The BRAF (V600E) mutation in colorectal cancers that are microsatellite stable (MSS) confers a very poor patient prognosis, whereas BRAF mutant microsatellite-unstable (MSI) colorectal cancers have an excellent prognosis. BRAF wild type cancers are typically MSS and display chromosomal instability (CIN). CIN is associated with a poor prognosis but has not been extensively studied on a genome-wide basis in relation to BRAF mutational status in colorectal cancer. BRAF mutant/MSS (BRAFmut/MSS) cancers (n=33) and BRAF mutant/MSI (BRAFmut/MSI) cancers (n=30) were compared for presence of copy number aberrations (CNAs) indicative of CIN, with BRAF wild type/MSS (BRAFwt/MSS) cancers (n=18) using Illumina CytoSNP-12 arrays. BRAFmut/MSS and BRAFwt/MSS cancers showed comparable numbers of CNAs per cancer at 32.8 and 29.8 respectively. However there were distinct differences in patterns of CNA length between MSS cohorts, with BRAFmut/MSS cancers having a significantly greater proportion of focal CNAs compared to BRAFwt/MSS cancers (p<0.0001); whereas whole chromosomal arm CNAs were more common in BRAFwt/MSS cancers (p<0.0001). This related to a reduced average CNA length in BRAFmut/MSS compared to BRAFwt/MSS cancers (20.7Mb vs 33.4Mb; p<0.0001); and a smaller average percentage of CIN affected genomes in BRAFmut/MSS compared to BRAFwt/MSS cancers (23.9% vs 34.9% respectively). BRAFmut/MSI cancers were confirmed to have low CNA rates (5.4 per cancer) and minimal CIN-affected genomes (average of 4.5%) compared to MSS cohorts (p<0.0001). BRAFmut/MSS cancers had more frequent deletion CNAs compared to BRAFwt/MSS cancers on 17q and 6p at loci not typically correlated with colorectal cancer, and more frequent amplification CNAs on 8q and 18q compared to BRAFwt/MSS cancers. These results indicate that although comparable rates of CIN occur between MSS subgroups, significant differences in their patterns of instability exist as BRAFmut/MSS cancers display a ‘focal pattern’ and BRAFwt/MSS cancers have more a ‘whole arm pattern' of CIN. This and the genomic loci more frequently affected in BRAFmut/MSS cancers may warrant extended studies to ascertain correlations with clinical data. Overall this investigation has identified that differing patterns of genomic instability exist between the two MSS colorectal subgroups and provides further evidence of the biological distinctions of the aggressive BRAFmut/MSS cancers. Citation Format: Catherine E. Bond, Derek J. Nancarrow, Leesa F. Wockner, Leanne Wallace, Grant W. Montgomery, Barbara A. Leggett, Vicki LJ Whitehall. Microsatellite stable colorectal cancers stratified by the BRAF V600E mutation show distinct patterns of chromosomal instability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2422. doi:10.1158/1538-7445.AM2014-2422

High incidence of LRAT promoter hypermethylation in colorectal cancer correlates with tumor stage.

Lecithin:retinol acyltransferase (LRAT) is a major enzyme involved in vitamin A/retinol metabolism, which regulates various physiological processes like cell proliferation and differentiation. LRAT expression is reduced in numerous cancers, yet the underlying mechanisms have remained undefined. We hypothesized that methylation silencing may contribute to decreased LRAT gene expression in colorectal cancer (CRC). LRAT hypermethylation status was analyzed in five CRC cell lines, 167 colorectal tumors, and 69 adjacent normal colonic mucosae, using a quantitative bisulfite/PCR/LDR/Universal Array assay. LRAT transcription levels were determined by real-time RT-PCR in a subset of tumors and matched normal tissues and in CRC cell lines that were treated with a demethylating agent, 5-aza-2'-deoxycytidine. The incidence of LRAT hypermethylation was significantly higher in colorectal tumors than in adjacent normal mucosae (p = 0.0025). Aberrant methylation occurred in 51 % of microsatellite-stable CRCs, in 84 % of microsatellite-unstable CRCs, and in 12 out of 13 colonic polyps. The number of hypermethylated LRAT events was inversely correlated with CRC stage (p < 0.0001). Importantly, LRAT hypermethylation was associated with decreased mRNA level in CRC clinical specimens, and demethylation treatment resulted in LRAT transcriptional reactivation. Our data support the idea that LRAT promoter hypermethylation associates with LRAT gene expression in CRC. The higher frequency of LRAT hypermethylation in colonic polyps and early-stage CRCs indicates that it may occur early in malignant progression.

Microsatellite Stable Colorectal Cancers Stratified by the BRAF V600E Mutation Show Distinct Patterns of Chromosomal Instability

The BRAF (V600E) mutation in colorectal cancers that are microsatellite stable (MSS) confers a poor patient prognosis, whereas BRAF mutant microsatellite-unstable (MSI) colorectal cancers have an excellent prognosis. BRAF wild type cancers are typically MSS and display chromosomal instability (CIN). CIN has not been extensively studied on a genome-wide basis in relation to BRAF mutational status in colorectal cancer. BRAF mutant/MSS (BRAFmut/MSS) cancers (n = 33) and BRAF mutant/MSI (BRAFmut/MSI) cancers (n = 30) were compared for presence of copy number aberrations (CNAs) indicative of CIN, with BRAF wild type/MSS (BRAFwt/MSS) cancers (n = 18) using Illumina CytoSNP-12 arrays. BRAFmut/MSS and BRAFwt/MSS cancers showed comparable numbers of CNAs/cancer at 32.8 and 29.8 respectively. However, there were differences in patterns of CNA length between MSS cohorts, with BRAFmut/MSS cancers having significantly greater proportions of focal CNAs compared to BRAFwt/MSS cancers (p<0.0001); whereas whole chromosomal arm CNAs were more common in BRAFwt/MSS cancers (p<0.0001). This related to a reduced average CNA length in BRAFmut/MSS compared to BRAFwt/MSS cancers (20.7 Mb vs 33.4 Mb;p<0.0001); and a smaller average percent of CIN affected genomes in BRAFmut/MSS compared to BRAFwt/MSS cancers (23.9% vs 34.9% respectively). BRAFmut/MSI cancers were confirmed to have low CNA rates (5.4/cancer) and minimal CIN-affected genomes (average of 4.5%) compared to MSS cohorts (p<0.0001). BRAFmut/MSS cancers had more frequent deletion CNAs compared to BRAFwt/MSS cancers on 6p and 17q at loci not typically correlated with colorectal cancer, and greater amplification CNAs on 8q and 18q compared to BRAFwt/MSS cancers. These results indicate that comparable rates of CIN occur between MSS subgroups, however significant differences in their patterns of instability exist, with BRAFmut/MSS cancers showing a ‘focal pattern’ and BRAFwt/MSS cancers having a ‘whole arm pattern’ of CIN. This and the genomic loci more frequently affected in BRAFmut/MSS cancers provides further evidence of the biological distinctions of this important cancer subgroup.

Expression status of wild-type HSP110 correlates with HSP110 T17 deletion size and patient prognosis in microsatellite-unstable colorectal cancer

It has been recently suggested that the expression levels of mutant HSP110 could be a prognostic marker in colorectal cancer with a high level of microsatellite instability (MSI-H). The aim of our study was to validate the prognostic significance of HSP110 mutation using immunohistochemistry and DNA testing in MSI-H colorectal cancer. Wild-type HSP110 (HSP110wt)-specific immunohistochemistry was performed in 168 MSI-H colorectal cancer tissues, and their expression levels were evaluated using a four-tier scoring system (0/1+/2+/3+). Of these tissues, 167 cases were analyzed for HSP110 T17 deletion. Associations with clinicopathological, molecular and survival parameters were statistically analyzed. The low-level expression of HSP110wt (0/1+) was observed in 40 MSI-H colorectal cancers (24%) and was significantly related to large HSP110 T17 deletions (≥ 4 bp, P<0.001). In survival analysis, patients with low HSP110wt expression (0/1+) showed better disease-free survival compared with those with high expression (2+/3+; P=0.005). This significance in survival difference was maintained in patients with 5-fluorouracil-based chemotherapy-treated tumors (P=0.024) and in those with stage III/IV tumors (P=0.032). Multivariate analysis confirmed the role of HSP110wt expression as an independent prognostic factor (P=0.016, hazard ratio=4.32). In MSI-H colorectal cancer, a low expression of HSP110wt is associated with large HSP110 T17 deletions and better clinical outcome. Immunohistochemistry of HSP110wt can be a simple and valuable tool for the prognostic and therapeutic stratification of patients with MSI-H colorectal cancer.

Exome sequencing reveals frequent inactivating mutations inARID1A, ARID1B, ARID2andARID4Ain microsatellite unstable colorectal cancer

ARID1A has been identified as a novel tumor suppressor gene in ovarian cancer and subsequently in various other tumor types. ARID1A belongs to the ARID domain containing gene family, which comprises of 15 genes involved, for example, in transcriptional regulation, proliferation and chromatin remodeling. In this study, we used exome sequencing data to analyze the mutation frequency of all the ARID domain containing genes in 25 microsatellite unstable (MSI) colorectal cancers (CRCs) as a first systematic effort to characterize the mutation pattern of the whole ARID gene family. Genes which fulfilled the selection criteria in this discovery set (mutations in at least 4/25 [16%] samples, including at least one nonsense or splice site mutation) were chosen for further analysis in an independent validation set of 21 MSI CRCs. We found that in addition to ARID1A, which was mutated in 39% of the tumors (18/46), also ARID1B (13%, 6/46), ARID2 (13%, 6/46) and ARID4A (20%, 9/46) were frequently mutated. In all these genes, the mutations were distributed along the entire length of the gene, thus distinguishing them from typical MSI target genes previously described. Our results indicate that in addition to ARID1A, other members of the ARID gene family may play a role in MSI CRC.

Molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer.

Colorectal cancers (CRCs) with a high level of microsatellite instability (MSI-H) are clinicopathologically distinct tumors characterized by predominance in females, proximal colonic localization, poor differentiation, mucinous histology, tumor-infiltrating lymphocytes, a Crohn's-like lymphoid reaction and a favorable prognosis. In terms of their molecular features, MSI-H CRCs are heterogeneous tumors associated with various genetic and epigenetic alterations, including DNA mismatch repair deficiency, target microsatellite mutations, BRAF mutations, a CpG island methylator phenotype-high (CIMP-H) status, and a low level of genomic hypomethylation. The molecular heterogeneity of MSI-H CRCs also depends on ethnic differences; for example, in Eastern Asian countries, relatively low frequencies of CIMP-H and BRAF mutations have been observed in MSI-H CRCs compared to Western countries. Although the prognostic features of MSI-H CRCs include a favorable survival of patients and low benefit of adjuvant chemotherapy, there may be prognostic differences based on the molecular heterogeneity of MSI-H CRCs. Here, we have reviewed and discussed the molecular and prognostic features of MSI-H CRCs, as well as several putative prognostic or predictive molecular markers, including HSP110 expression, beta2-microglobulin mutations, myosin 1a expression, CDX2/CK20 expression, SMAD4 expression, CIMP status and LINE-1 methylation levels.

Identification of driver genes in microsatellite-unstable colorectal cancers

SUMMARY Somatic mutations in key driver genes are necessary for colorectal tumor formation. Whereas the majority of sporadic colorectal cancer (CRC) cases have chromosomal instability, a sixth of cases generate somatic mutations by a deficiency in the mismatch repair system. This choice of mutational mechanism has a strong impact on which genes in CRC pathways are the most likely to be mutated in an individual tumor. A specific challenge in understanding CRC with microsatellite instability is to discriminate driver from passenger genes given the large number of nucleotide repeats that are frequently found mutated in patient tumors. This review, therefore, discusses strategies for the identification and validation of cancer genes in microsatellite-unstable colorectal cancers.

Role of topoisomerase I and thymidylate synthase expression in sporadic colorectal cancer: Associations with clinicopathological and molecular features

Topoisomerase I (Topo I) and thymidylate synthase (TS) are essential enzymes for the replication, transcription and repair of DNA, and are potential biomarkers in colorectal cancer (CRC). The aim of the study was to correlate the tissue expression of Topo I and TS in sporadic CRCs with relevant pathological and molecular features and patients’ outcome.Topo I and TS expression was assessed by immunostaining in 112 consecutive primary CRCs. Increased expression of Topo I was found in 36% of tumors, preferentially rectal (50%) and with not otherwise specified (NOS) histology (44%). Topo I expression was associated with 18q allelic loss (LOH), (p=0.013), microsatellite stable phenotype (p=0.002) and normal expression of mismatch proteins hMLH1 and hMSH2 (p=0.0012 and p=0.02, respectively). High TS expression was found in 60% of tumors, more frequently in distal sites (62%) and with NOS histology (66%); no association with microsatellite instability was observed.Topo I seems to be involved in the chromosomal instability pathway of sporadic CRCs. Conversely, high TS expression is unlikely to affect the clinical behavior of microsatellite unstable CRCs.

Loss of CDX2/CK20 Expression Is Associated With Poorly Differentiated Carcinoma, the CpG Island Methylator Phenotype, and Adverse Prognosis in Microsatellite-unstable Colorectal Cancer

Several previous studies have demonstrated that the CDX2-negative (CDX2) and/or CK20-negative (CK20) phenotypes of colorectal cancers (CRCs) might be associated with high levels of microsatellite instability (MSI-H). The aim of this study was to investigate the clinicopathologic and molecular features of MSI-H CRCs with different CDX2/CK20 expression statuses. The CDX2 and CK20 expression statuses were immunohistochemically evaluated in 109 MSI-H CRC tissue samples, and the correlations of these statuses with clinicopathologic, molecular, and survival data were statistically analyzed. Of the 109 MSI-H CRCs, 15 were CDX2 (13.8%), and 19 were CK20 (17.4%). The simultaneous loss of CDX2 and CK20 expression (CDX2/CK20) was observed in 9 cases (8.3%). CDX2 loss was correlated with lymph node metastasis, poor differentiation, MLH1 loss, the mutation of BRAF, and CpG island methylator phenotype-high (CIMP-H) status. Right-sided tumor location, nodal metastasis, poor differentiation, and CIMP-H status were significant characteristics of CK20 tumors. The CDX2/CK20 phenotype was associated with older age (above 56 y), higher stage (stage III or IV), deep invasion (pT3 or pT4), lymph node metastasis (pN1 or pN2), poor differentiation (nonmedullary/non-signet ring cell type), the mutation of BRAF, and CIMP-H status among MSI-H CRCs. Patients with CDX2/CK20 tumors exhibited worse overall and disease-free survival compared with the patients with CDX2 and/or CK20 tumors (P<0.001). In the multivariate analysis for disease-free survival, the CDX2/CK20 phenotype was an independent prognostic factor for MSI-H CRC (P=0.030, hazard ratio=3.288). The CDX2/CK20 phenotype defines a distinct subgroup of MSI-H CRCs with poor differentiation, CIMP-H status, and unfavorable prognosis.

Microsatellite Instability Affecting the T17 Repeats in Intron 8 of HSP110 , as Well as Five Mononucleotide Repeats in Patients with Colorectal Carcinoma

To investigate mononucleotide markers: BAT-25, BAT-26, NR-21, NR-22 and NR-24 in patients with colorectal cancer (CRC), and the status of HSP110T17, KRAS, BRAF and the MLH1 promoter mutations in microsatellite unstable CRC. Genetic assessments were performed on samples obtained following resection of CRC in 200 patients. Allelic variations of HSP110T17 were found in all 18 patients with microsatellite instabilities (MSIs) in at least three markers (high-frequency MSI). By contrast, mutations of HSP110T17 were absent in all 20 patients with no MSI frequency. Eight out of 182 patients with low (instability in one marker) or no frequency MSI had allelic shifts due to polymorphisms of BAT-25 (1.5%), NR-21 (1.75%) and NR-24 (1.5%). BRAF mutations were associated with >5 bp shortening of HSP110T17. Patients with high-frequency MSI CRC had allelic variations of HSP110T17. BRAF mutations occur along with greater shortening in HSP110T17 during oncogenesis via the MSI pathway.

Subsets of microsatellite-unstable colorectal cancers exhibit discordance between the CpG island methylator phenotype and MLH1 methylation status

Although the presence of MLH1 methylation in microsatellite-unstable colorectal cancer generally indicates involvement of the CpG island methylator phenotype (CIMP) in the development of the tumor, these two conditions do not always correlate. A minority of microsatellite-unstable colorectal cancers exhibit discordance between CIMP and MLH1 methylation statuses. However, the clinicopathological features of such microsatellite-unstable colorectal cancers with discrepant MLH1 methylation and CIMP statuses remain poorly studied. Microsatellite-unstable colorectal cancers (n=220) were analyzed for CIMP and MLH1 methylation statuses using the MethyLight assay. Based on the combinatorial CIMP and MLH1 methylation statuses, the microsatellite-unstable colorectal cancers were grouped into four subtypes (CIMP-high (CIMP-H) MLH1 methylation-positive (MLH1m+), CIMP-H MLH1 methylation-negative, CIMP-low/0 (CIMP-L/0) MLH1m+, and CIMP-L/0 MLH1 methylation-negative), which were compared in terms of their associations with clinicopathological and molecular features. The CIMP-L/0 MLH1 methylation-negative and CIMP-H MLH1m+ subtypes were predominant, comprising 63.6 and 24.1% of total microsatellite-unstable colorectal cancers, respectively. The discordant subtypes, CIMP-H MLH1 methylation-negative and CIMP-L/0 MLH1m+, were found in 5 and 7% of microsatellite-unstable colorectal cancers, respectively. The CIMP-H MLH1 methylation-negative subtype exhibited elevated incidence rates in male patients and was associated with larger tumor size, more frequent loss of MSH2 expression, increased frequency of KRAS mutation, and advanced cancer stage. The CIMP-L/0 MLH1m+ subtype was associated with onset at an earlier age, a predominance of MLH1 loss, and earlier cancer stage. None of the CIMP-L/0 MLH1m+ subtype patients succumbed to death during the follow-up. Our findings suggest that the discordant subtypes of colorectal cancers exhibit distinct clinicopathological and molecular features, although the proportion of discordant subtypes is low. The microsatellite-unstable colorectal cancers of the same CIMP status tended to exhibit different clinicopathological features depending on MLH1 methylation status.

Su1087 Clinicopathological Characterization and Nutritional Assessment of Hispanics With Sporadic Microsatellite Unstable Colorectal Cancers

Su1087 Clinicopathological Characterization and Nutritional Assessment of Hispanics With Sporadic Microsatellite Unstable Colorectal Cancers

BRAF V600E‐specific immunohistochemistry for the exclusion of Lynch syndrome in MSI‐H colorectal cancer

The differentiation between hereditary and sporadic microsatellite-unstable (MSI-H) colorectal cancer is a crucial step in Lynch syndrome diagnostics. Within MSI-H colorectal cancers, the BRAF V600E mutation is strongly associated with sporadic origin. Here, we asked whether BRAF V600E-specific immunohistochemistry (clone VE1) is helpful in separating sporadic from Lynch syndrome-associated MSI-H colorectal cancers. To that end, we performed VE1 immunohistochemistry and BRAF sequencing in a series of 91 MSI-H colorectal cancer specimens from patients tested for Lynch syndrome. Concordance of VE1 immunohistochemistry and molecular BRAF mutation status was observed in 90 of 91 (98.9%) MSI-H samples. All 11 tumors classified as BRAF V600E mutation-positive by Sanger sequencing were immunopositive, and 79 (98.8%) of 80 tumors classified as BRAF wild type showed negative staining. All VE1-positive tumors were MLH1- and PMS2-negative by immunohistochemistry. None of the tumors from mismatch repair (MMR) gene germline mutation carriers (n = 28) displayed positive VE1 staining, indicating that BRAF V600E mutation-specific immunostaining has a low risk of excluding Lynch syndrome patients from germline mutation analysis. In conclusion, implementation of VE1 immunohistochemistry was able to detect BRAF-mutated MSI-H colorectal cancers with a sensitivity of 100% and a specificity of 98.8%. Among MLH1-negative colorectal cancers, the rate of VE1-positive lesions was 21%, offering the exclusion of these patients from MMR germline testing. Therefore, we suggest the integration of VE1 immunohistochemistry into the diagnostic panel of Lynch syndrome.

Inactivation of TGF-β signaling and loss of PTEN cooperate to induce colon cancer in vivo.

The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-β) signaling pathway, which is often inactivated by mutations of TGF-β type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in ∼30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite-unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperates to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2(IEKO)) alone is not sufficient for intestinal tumor formation and lack of Pten (Pten(IEKO)) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (Pten(IEKO);Tgfbr2(IEKO)) led to malignant tumors in both the small intestine and colon in 86% of the mice and to metastases in 8% of the tumor-bearing mice. Moreover, these tumors arose via a β-catenin-independent mechanism. Inactivation of TGF-β signaling and loss of Pten in the tumors led to increased cell proliferation, decreased apoptosis and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-β signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.

Abstract 3156: New candidate oncogenes discovered in microsatellite unstable colorectal cancer.

Abstract Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI). A characteristic high passenger mutation load has discouraged systematic mutation screens in MSI CRCs. To systematically search for novel MSI CRC oncogenes, the exomes of 25 MSI CRC-normal pairs were sequenced as the discovery set. Observed hot spots were confirmed by Sanger sequencing and further validated in a set of 86 MSI CRCs. Mutational hot spots were confirmed in 15 genes and three genes showed additional hot spot mutations in the validation set. These three sites were highly conserved across species. The hot spot sites of these three genes were screened in 75 microsatellite stable CRCs and 12 MSI CRC cell lines with negative results. Next, we analyzed the subcellular localization of wild-type and mutant proteins. The findings of this study support the idea that cancer genomes are heterogeneous and characterized by few, frequently mutated “mountains” (e.g. BRAF and KRAS) and numerous, less frequently mutated “hills”. The identified mutational hot spots may prove important in developing personalized tumor profiling and therapy. Citation Format: Alexandra E. Gylfe, Johanna Kondelin, Mikko Turunen, Heikki Ristolainen, Riku Katainen, Esa Pitkänen, Eevi Kaasinen, Ville Rantanen, Tomas Tanskanen, Heli J. Lehtonen, Kimmo Palin, Minna Taipale, Jussi Taipale, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Ari Ristimäki, Sari Tuupanen, Auli Karhu, Outi Kilpivaara, Pia Vahteristo, Lauri A. Aaltonen. New candidate oncogenes discovered in microsatellite unstable colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3156. doi:10.1158/1538-7445.AM2013-3156

Regulatory T Lymphocytes Are Associated with Less Aggressive Histologic Features in Microsatellite-Unstable Colorectal Cancers

BackgroundColorectal cancers (CRCs) with microsatellite instability (MSI) are associated with a good prognosis and a high density of tumor-infiltrating lymphocytes (TILs). We have undertaken to determine the link between TIL densities and MSI CRC histologic features.Patients and MethodsUsing tissue microarrays, T-cell sub-population infiltration, including T cells (CD3), cytotoxic T cells (CD8) and regulatory T cells (FoxP3) were studied in 86 MSI CRCs. We separately analyzed TILs of the stromal and epithelial compartments in the tumor center, the tumoral invasion margin and associated normal tissue.ResultsFor FoxP3+ TIL density in the tumor center stromal compartment, we found a strong negative correlation with T4 stage (p = 0.01), node invasion (p<0.001) and VELIPI (vascular emboli, lymphatic invasion and perinervous invasion) criteria (p = 0.002).ConclusionThe strong correlation between regulatory T cell density and the absence of VELIPI criteria suggests that this sub-group of T cells is preferentially associated with less invasive tumors.

Abstract 654: Do epimutations affect MLH1 alone or a broad spectrum of genes to increase the severity of the associated cancer phenotype.

Abstract Germline mutations of the DNA mismatch repair genes, MLH1 and MSH2, are the major cause of Lynch syndrome, characterised by a predisposition to the development of microsatellite unstable colorectal, endometrial and additional cancers below 50 years of age. However, approximately one third of cases with a clinical suspicion have no identifiable mismatch repair sequence mutation. In recent years, constitutive epigenetic dysregulation of MLH1, so-called “constitutional epimutation," has been identified in a small proportion of these mutation-negative cases. The mechanism of onset and stochastic intergenerational transmission of this defect remains unknown, but may involve the co-inheritance of a trans-acting factor that causes aberrant epigenetic programming of MLH1, and possibly additional loci, in the germline or early embryo. The aim of this work is to determine if constitutional MLH1 epimutations occur in a focal manner, or in the context of a more generalised epigenomic mechanism that may exacerbate the cancer phenotype. Using the Illumina HumanMethylation450k BeadChip we studied 6 confirmed carriers of an MLH1 epimutation and 6 healthy controls matched for age and gender to determine if MLH1 epimutations are accompanied by additional epigenetic defects. Preliminary results have shown that other genes are indeed concomitantly and aberrantly methylated or hypomethylated alongside MLH1 and the methylation status of these genes is currently under validation. The finding of concomitant methylation or hypomethylation of additional loci would provide strong evidence for a defective trans-acting factor as the underlying cause of epimutations and may also contribute to the phenotypic expression of this aetiological mechanism. Citation Format: Amanda G. Silva, Konsta Duesing, Robyn Ward, Megan Hitchins. Do epimutations affect MLH1 alone or a broad spectrum of genes to increase the severity of the associated cancer phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 654. doi:10.1158/1538-7445.AM2013-654

Abstract 109: Clinicopathological characterization and nutritional assessment of Hispanics with sporadic microsatellite unstable colorectal cancers.

Abstract Introduction: Approximately 10% to 15% of all sporadic colorectal cancers have microsatellite instability (MSI). MSI has not been well studied in Hispanics, an ethnic minority at high risk for colorectal cancer (CRC). Patients with MSI tumors present with better prognosis, higher 5-year survival and limited response to 5-FU based chemotherapy, compared to microsatellite stable (MSS) tumors. The aim of this study was to define the clinic-pathologic phenotype of Hispanics with sporadic MSI CRC and to evaluate the association of dietary factors and tumor MSI-status. Methods: The case-case study evaluated the MSI status of sporadic CRC diagnosed during the period January 1, 2002 to October 31, 2012. MSI analysis was performed using six markers (BAT26, BAT25, NR21, NR22, NR24 and NR27). Tumors were classified as MSI-high if two or more of the six microsatellite repeats were mutated and MSS if none of the replicates was mutated. Frequency analysis, chi-square, Fisher's exact test and Wilcoxon rank-sum test were used for statistical analysis methods. Unconditional logistic regression was employed to estimate the odds ratio (OR) between exposures and MSI using STATA 10.0. Results: A total of 85 participants (mean age 57.5 ± 12.5 yrs, 51.8% males) were examined. 80 patients had MSS tumors, while 5 (6%) tumors had MSI. The colorectal neoplasia specimens analyzed were mostly adenocarcinomas (96.5%) located in the distal colon (72.0%) with TNM stage III (34.4%). Tumors with MSI did not significantly differ from MSS tumors with regards to gender (p=0.36), age (p=0.66), education (p&amp;gt;0.99), family history of CRC (p=0.58) or obesity (p&amp;gt;0.99). MSI tumors were more likely located in the proximal colon compared to MSS tumors (OR=12.21; 95% CI 1.4-306.6). There were no statistical significant associations between MSI and reported consumption of dark green leaf vegetables (2-4 times/week) (OR=0.77; 95% CI 0.11-6.88), fruits (≥ 7 times/week) (OR=3.54; 95% CI 0.24-108.52) and red meats (once per day) (OR=1.08; 95% CI: 0.04-9.55). Conclusions: We report a lower prevalence of MSI in Hispanics with sporadic CRC than other Western populations. As previously reported in other non-Hispanic populations, MSI tumors were mostly located in the proximal colon. There were no statistical significant associations between sociodemographic, clinical and nutritional characteristics and MSI. Ongoing efforts to continue to examine additional Hispanic patients are underway to determine if dietary patterns in Hispanics are associated with CRC MSI status. Citation Format: Yaritza Diaz-Algorri, Sofia Margarita Lopez-Diaz, Maria del Mar Gonzalez-Pons, Katerina Freyre, Maritere Olascoaga, Sylvia B. Saldaña-Villafañe, Mercedes Y. Lacourt-Ventura, Sharon C. Fonseca-Williams, Raul D. Bernabe-Dones, Xavier LLord, Rosa Xicola, Katherine Tucker, Marcia R. Cruz-Correa. Clinicopathological characterization and nutritional assessment of Hispanics with sporadic microsatellite unstable colorectal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 109. doi:10.1158/1538-7445.AM2013-109

Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

BackgroundElevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1’s promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.AimTo examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.MethodsWe measured −4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1−/−, hMSH6−/−, and hMSH3−/− CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.Results D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1−/− and hMSH3−/− cells, respectively, than in hMSH6−/− or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10−4 mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10−4 mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H2O2 to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H2O2 treatment.ConclusionEMAST is dependent upon the MMR background, with hMSH3−/− more prone to frameshift mutations than hMSH6−/−, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3−/− mimics complete MMR failure (hMLH1−/−) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3’s subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.

MSH3 Protein Expression and Nodal Status in MLH1-Deficient Colorectal Cancers

Patients with colorectal cancers (CRC) and high microsatellite instability (MSI) have a better outcome than their chromosome-unstable counterpart. Given the heterogeneity of microsatellite-unstable CRCs, we wanted to see whether any MSI-associated molecular features are specifically associated with prognosis. One hundred and nine MSI-high CRCs were typed for primary mismatch repair (MMR) defect and for secondary loss of MMR proteins. Frameshifts at seven target genes, mutations in the RAS pathway, and methylation at MLH1/CDKN2A promoters were also searched. The interplay of molecular findings with clinicopathologic features and patient survival was analyzed. Of 84 MLH1-deficient CRCs, 31 (36.9%) had MSH3 and 11 (13.1%) had MSH6 loss (P < 0.001), biallelic frameshift mutations at mononucleotide repeats accounting for most (78%) MSH3 losses. As compared with MSH3-retaining cancers, MLH1-deficient tumors with MSH3 loss showed a higher number of mutated target genes (3.94 ± 1.56 vs. 2.79 ± 1.75; P = 0.001), absence of nodal involvement at pathology [N0; OR, 0.11; 95% confidence interval (CI), 0.04-0.43, P < 0.001], and better disease-free survival (P = 0.06). No prognostic value was observed for KRAS status and for MLH1/CDKN2A promoter methylation. The association between MSH3 loss and N0 was confirmed in an independent cohort of 71 MLH1-deficient CRCs (OR, 0.23; 95% CI, 0.06-0.83, P = 0.02). MLH1-deficient CRCs not expressing MSH3 have a more severe MSI, a lower rate of nodal involvement, and a better postsurgical outcome.