Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways

BACKGROUND: 10% of sporadic colorectal cancers are characterised by a low level of microsatellite instability (MSI-L). These are not thought to differ substantially from microsatelite-stable (MSS) cancers, but MSI-L and MSS cancers are distinguished clinicopathologically and in their spectrum of genetic alterations from cancers showing high level microsatellite instability (MSI-H). AIMS: To study the distribution of molecular alterations in a series of colorectal cancers stratified by DNA microsatellite instability. METHODS: A subset of an unselected series of colorectal cancers was grouped by the finding of DNA MSI at 0 loci (MSS) (n = 51), 1-2 loci (MSI-L) (n = 38) and 3-6 loci (MSI-H) (n = 25). The frequency of K-ras mutation, loss of heterozygosity (LOH) at 5q, 17p and 18q, and patterns of p53 and beta catenin immunohistochemistry was determined in the three groups. RESULTS: MSI-H cancers had a low frequency of K-ras mutation (7%), LOH on chromosomes 5q (0%), 17p (0%) and 18q (12.5%), and a normal pattern of immunostaining for p53 and beta catenin. MSI-L cancers differed from MSS cancers in terms of a higher frequency of K-ras mutation (54% v 27%) (p = 0.01) and lower frequency of 5q LOH (23% v 48%) (p = 0.047). Whereas aberrant beta catenin expression and 5q LOH were concordant (both present or both absent) in 57% of MSS cancers, concordance was observed in only 20% of MSI-L cancers (p = 0.01). CONCLUSIONS: MSI-L colorectal cancers are distinct from both MSI-H and MSS cancers. This subset combines features of the suppressor and mutator pathways, may be more dependent on K-ras than on the APC gene in the early stages of neoplastic evolution, and a proportion may be related histogenetically to the serrated (hyperplastic) polyp.     

Frequent activation of the beta-catenin-Tcf signaling pathway in nonfamilial colorectal carcinomas with microsatellite instability

It has been reported that wild-type APC protein forms a complex with beta-Catenin and GSK3beta, inducing degradation of beta-Catenin in normal cells. Both beta-Catenin and APC gene mutations have recently been shown to activate the same signaling pathway. Frequent mutations of beta-Catenin in hereditary nonpolyposis colorectal carcinomas have also been reported. It was, however, controversial whether the mutation of the beta-Catenin gene was frequent in nonfamilial colorectal carcinomas with high-frequency microsatellite instability (MSI-H). We analyzed the mutations of the APC and beta-Catenin genes in 56 nonfamilial colorectal carcinomas stratified according to the presence or absence of microsatellite instability (MSI). APC mutations were identified in 11 of 22 (50%) cases of MSI-H and 14 of 34 (41%) cases of microsatellite-stable (MSS)/low-frequency microsatellite instability (MSI-L). In contrast, the frequency of beta-Catenin mutations was significantly higher in MSI-H (6/22; 27%) than in MSS/MSI-L (1/34; 3%) (P = 0.01). beta-Catenin mutations were not detected in carcinomas with APC mutation. APC mutation occurred irrespective of MSI status. beta-Catenin mutation, however, occurred frequently in MSI-H carcinomas. Our data suggest that activation of the beta-Catenin-Tcf signaling pathway, through either beta-Catenin or APC mutation, frequently contributes to MSI-H nonfamilial colorectal carcinomas (17/22; 77%).     

Colorectal carcinogenesis: MSI-H versus MSI-L

Microsatellite instability (MSI) is a well-recognized phenomenon that is classically a feature of tumors in the hereditary non-polyposis colorectal syndrome. Ten to 15% of sporadic colorectal cancers, however, will have MSI. Microsatellite unstable tumors can be divided into two distinct MSI phenotypes: MSI-high (MSI-H) and MSI-low (MSI-L). MSI sporadic colorectal cancers with a high level of MSI (MSI-H) form a well defined group with distinct clinicopathologic features characterized by an overall better long-term prognosis. These sporadic MSI-H colorectal tumors most often arise from the epigenetic silencing of the mismatch repair gene MLH1. In contrast, MSI-L colorectal tumors have not been shown to differ in their clinicopathologic features or in most molecular features from microsatellite stable (MSS) tumors. Unlike MSI-H tumors, MSI-L tumors appear to arise through the chromosomal instability carcinogenesis pathway, similar to MSS tumors. Some groups have reported more frequent mutations in K-ras and in the methylation of methylguanine transferase in MSI-L tumors, but others have questioned these findings. Therefore, although the use of the MSI-L category is widespread, there continues to be some debate as to whether a discrete MSI-L group truly exists. Rather, it has been suggested that MSI-L tumors differ quantitatively from MSS tumors but do not differ qualitatively. Future studies will need to evaluate the specific mutations in non-MSI-H tumors in an attempt to sub-classify MSI-L tumors with regard to MSS tumors so that subtle differences between these two sub-groups can be identified.     

Infrequent loss of heterozygosity of APC/MCC and DCC genes in gastric cancer showing DNA microsatellite instability

AIM: To investigate the role of DNA microsatellite instability (MSI) in gastric carcinogenesis by studying associations between MSI status, clinicopathological features, and loss of genetic loci. METHODS: Six microsatellite loci and loss of heterozygosity at APC, DCC, and MCC were analysed by polymerase chain reaction based methods in 53 cases of advanced gastric cancer. RESULTS: MSI was observed in 32.1% of gastric carcinomas (17/53) and 20% of foci of intestinal metaplasia (3/15). Seven gastric carcinomas (13.7%) were MSI-high (MSI-H) (three loci or more) and 10 (18.9%) were MSI-low (MSI-L) (one or two loci). The frequency of MSI-H was higher in intestinal (25.0%) than in diffuse carcinomas (3.7%) (p < 0.05). None of the MSI-H tumours showed loss of heterozygosity at APC, MCC, or DCC loci. CONCLUSIONS: MSI may have an important and early role in a subset of gastric cancers, particularly the intestinal type. The MSI-H subset of gastric cancer has features in common with its colorectal counterpart, whereas MSI-L and microsatellite stable cancers appear to develop through the loss of heterozygosity pathway.     

Angiogenic factor VEGF is decreased in human colorectal neoplasms showing DNA microsatellite instability.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two important determinants of angiogenesis in human cancers. Expression of VEGF and bFGF was examined by immunohistochemistry in 120 colorectal cancers. Neoplasms were classified according to the presence or absence of microsatellite instability determined at six microsatellite loci and labelled as a high microsatellite instability (MSI-H), low microsatellite instability (MSI-L) or microsatellite stable (MSS). Only 4/30 MSI-H cancers expressed VEGF (13 per cent), compared with 24/64 MSS cancers (38 per cent; p< 0.01). Fewer MSI-H cancers showed bFGF expression (38 per cent) than MSS cancers (53 per cent; p< 0.09). MSI-L cancers showed the same pattern as MSS cancers. Western blotting and immunohistochemistry showed that the tumour suppressor gene p53 was mutated infrequently in MSI-H cancers (8 per cent; p< 0. 001). Microvessel density counts using CD31 and UEA-1 demonstrated no difference in the number of blood vessels in MSI-H and MSS cancers. Although these results are consistent with the known role of wild-type p53 in down-regulating VEGF, no association was found between a mutation in p53 and VEGF or bFGF levels in all colonic neoplasms. This is the first evidence that MSI-H cancers may follow a different pathway to angiogenesis. The low frequency of VEGF expression amongst MSI-H cancers may partially explain why these cancers are less aggressive, with a better overall prognosis.     

Morphology and microsatellite instability in sporadic serrated and non-serrated colorectal cancer

Colorectal serrated adenocarcinoma originates from serrated adenoma, but definite histological criteria have not yet been established. It presents with frequent DNA microsatellite instability (MSI), but the frequency of low-level (MSI-L) and high-level MSI (MSI-H) and the expression of mismatch-repair (MMR) enzymes in serrated adenocarcinoma are not known. To address these questions, morphological criteria for serrated cancers were established, their validity was tested, and MSI analysis was performed with NIH consensus markers and MMR enzyme immunohistochemistry for hMLH1, hMSH2, and hMSH6 in 35 serrated and 75 non-serrated colorectal carcinomas. Serrated carcinomas frequently showed a serrated, mucinous or trabecular growth pattern; abundant eosinophilic cytoplasm; chromatin condensation; preserved polarity; and the absence of necrosis. With these features, it was possible to distinguish them from non-serrated cancers, with the mean kappa score for five observers being 0.509. MSI analysis was successful in 31 serrated and 73 non-serrated carcinomas. 54.8% of serrated carcinomas were microsatellite-stable (MSS), 29.0% presented with MSI-L, and 16.1% presented with MSI-H, whereas 78.1% of non-serrated carcinomas were MSS, 13.7% were MSI-L, and 8.2% were MSI-H. MSI-L was more common in serrated cancers (p=0.035) and it was associated with patchy immunohistochemical staining (33.3%) of MLH1. MSI-H did not differ between serrated and non-serrated cancers (p=0.14). These results suggest that the biological background of serrated carcinomas differs from sporadic non-serrated colorectal cancer, but is not directly related to MSI.     

Characterization of mutator pathway in younger-age-onset colorectal adenocarcinomas

The high-frequency microsatellite instability (MSI-H) phenotype, frequently identified in hereditary nonpolyposis colorectal cancer (HNPCC), also accounts for approximately 15% of sporadic colorectal cancers. Microsatellite instability (MSI) occurs from the mutational inactivation of the DNA mismatch repair genes, i.e. hMSH2 and hMLH1 in HNPCC, as well as from epigenetic inactivation of hMLH1 in sporadic colorectal tumors. The mutator pathway including microsatellite instability, hMLH1 promoter methylation, and hMSH2 and hMLH1 mutation patterns were identified in 21 sporadic colorectal adenocarcinoma patients younger than 30 yr excluding HNPCC. More than half of tumors showed MSI, with five MSI-H and six MSI-L (low-frequency microsatellite instability). Three of six MSI-H tumors showed the hMLH1 promoter methylation and did not express the hMLH1 protein. On the other hand, all MSI-L and all MSS (microsatellite stable) tumors expressed both hMSH2 and hMLH1 proteins. Two novel mutations, i.e. a missense mutation in hMLH1 and a splice-site alteration in hMSH2, were identified in two patients respectively. Although mutator pathway was implicated in younger-age-onset colorectal carcinogenesis, many tumors appeared to evolve from different genetic events other than hMSH2 and hMLH1 mutations frequently identified in HNPCC.     

Distinct clinicopathologic and genetic profiles in sporadic gastric cancer with different mutator phenotypes

A subset of sporadic gastric cancers (GC) exhibits microsatellite instability (MSI). To define the precise role of MSI in GC, a total of 100 patients with sporadic GC were classified into three groups, i.e., high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), based on 10 microsatellite markers. Mutational analyses of TGFbetaRII, IGFIIR, BAX, MSH3, MSH6, E2F4, MSH2, MLH1, and TP53 genes, and methylation and protein expression of MLH1 and MSH2 were performed and correlated. Twenty-seven percent of GC showed MSI at least in one locus and could be further graded as MSI-H (14%) and MSI-L (13%). No clinicopathologic difference was noted between GC with MSI-L and MSS. Compared with GC with MSI-L or MSS, GC with MSI-H had a significantly higher frequency of antral location, intestinal subtype, H. pylori seropositivity, but a lower incidence of lymph node metastasis, and displayed a higher frequency of frameshift mutations of TGFbetaRII, IGFIIR, BAX, MSH3, and E2F4 genes but a lower incidence of TP53 mutations. Furthermore, hypermethylation of the MLH1 promoter was responsible for the loss of protein function in 13 of 14 MSI-H tumors. It was concluded that a specific phenotype and a distinct profile of genetic alterations exist in MSI-H GC. We speculate that epigenetic inactivation of MLH1 by methylation plays a crucial role in initiating such a pathway of carcinogenesis. In contrast, GCs with MSS and MSI-L exhibit clinicopathologic features that are distinct from MSI-H tumors and have a higher frequency of TP53 mutations, suggesting that they may evolve through an entirely different pathway.     

Histologic features distinguish microsatellite-high from microsatellite-low and microsatellite-stable colorectal carcinomas, but do not differentiate germline mutations from methylation of the MLH1 promoter

The detection of microsatellite-unstable (microsatellite instability [MSI]) colorectal carcinomas (CRCs) has prognostic value and can help screen for Lynch syndrome. We determined which histologic features are associated with MSI status and presence of germline mutation and/or methylation of MLH1 promoter. Patients diagnosed with CRC were offered participation in the Columbus-area hereditary nonpolyposis colorectal cancer syndrome study regardless of age or family history. Tumors were evaluated for MSI using a modified Bethesda panel of microsatellite markers. Methylation status of the MLH1 promoter was evaluated by methylation-specific polymerase chain reaction and bisulfite PCR followed by restriction digestion of tumor DNA. All patients with microsatellite-unstable tumors underwent mutation analysis of the MLH1, MSH2, and MSH6 genes by full sequencing of genomic DNA and by multiplex ligation probe assay of MLH1 and MSH2. Histologic end points were tumor type, grade, percentage of mucin, border, and lymphoid host response. Of the 482 CRCs, 87 were MSI with 69 MSI high (MSI-H), 18 MSI low (MSI-L), and 395 microsatellite stable (MSS). Of 87 MSI tumors, 12 had germline mutations and 34 had methylation of the MLH1 promoter. Younger age, but not histologic features, was significantly associated with a germline mutation. Percentage of mucin, histologic type, grade, and lymphoid host response differed significantly between MSI-H when compared with MSI-L or MSS. No difference was found between MSI-L versus MSS. Histologic features are associated with MSI-H CRC and are helpful to differentiate MSI-H from MSI-L and MSS. These features are not useful to distinguish MSI-L from MSS carcinomas, and those with a deleterious germline hereditary nonpolyposis colorectal cancer syndrome mutation from those with methylation of the MLH1 promoter region.     

Microsatellite instability and alteration of the expression of hMLH1 and hMSH2 in ovarian clear cell carcinoma

Microsatellite instability (MSI) is commonly seen in tumors associated with the hereditary nonpolyposis colorectal cancer syndrome and is caused by defects in the DNA mismatch repair genes. MSI has also been observed in various sporadic cancers, including colorectal, gastric, and endometrial. The role and incidence of MSI in ovarian clear cell carcinoma remain unknown. This study was conducted to evaluate the frequency of MSI in ovarian clear cell carcinomas and to evaluate the sensitivity and specificity of immunohistochemistry in predicting mismatch-repair gene deficiency. A total of 42 ovarian clear cell carcinomas were analyzed for MSI using a panel of 5 microsatellite markers (BAT25, BAT26, D5S346, D2S123, and D17S250). Alterations in the expression of hMLH1 and hMSH2 proteins in these tumors were examined. Of the 42 ovarian clear cell tumors analyzed, 6 demonstrated a high level of MSI (MSI-H), 3 demonstrated a low level of MSI (MSI-L), and the remaining 33 exhibited microsatellite stability (MSS). No correlation was found between MSI level and patient age or tumor stage or size (P >0.05). Loss of expression of either hMLH1 or hMSH2 was observed in 4 of the 6 (67.7%) MSI-H tumors, whereas 34 of the 36 (94.4%) MSI-L or MSS tumors expressed both the hMLH1 and hMSH2 gene products. Our results indicate that MSI-H is involved in the development of a subset of ovarian clear cell carcinomas. A strong correlation exists between alterations in the expression of hMLH1 and hMSH2 and the presence of MSI-H in these tumors. However, immunohistochemical testing alone may miss a small fraction of cases with MSI-H.     

RAS突变和微卫星不稳定与Ⅲ期结直肠癌患者临床病理特征及预后的相关性分析

目的:检测Ⅲ期结直肠癌患者中RAS和BRAF基因突变以及微卫星不稳定(microsatellite instability,MSI)状态,并分析其临床病理关系及预后。方法:收集2010~2015年广东省人民医院共281例经病理学证实的Ⅲ期结直肠癌组织标本,采用PCR-Sanger测序法和免疫组织化学法对石蜡切片进行分析,检测RAS/BRAF基因突变和MSI状态,并探讨其与结直肠癌临床病理特征和预后的关系。结果:281例患者中,RAS/BRAF突变率为48.4%(136/281),其中KRAS突变率最高(116/281,41.3%)。RAS/BRAF基因突变与癌胚抗原水平密切相关(P0.05)。免疫组织化学法检测到高度MSI(MSI-H)患者18例(6.4%),MSI-H状态在淋巴结转移N2b期患者中更为常见(P0.05)。BRAF基因在MSI-H肿瘤中的突变率较高(P0.01)。RAS/BRAF野生型或者MSI-H患者的总生存期和无进展生存期均明显高于突变型或低度MSI/微卫星稳定患者。结论:RAS/BRAF突变和MSI检测有助于结直肠癌生物学行为分析和患者预后判断。     

Classification of colorectal cancer based on correlation of clinical, morphological and molecular features

Over the last 20 years it has become clear that colorectal cancer (CRC) evolves through multiple pathways. These pathways may be defined on the basis of two molecular features: (i) DNA microsatellite instability (MSI) status stratified as MSI-high (MSI-H), MSI-low (MSI-L) and MS stable (MSS), and (ii) CpG island methylator phenotype (CIMP) stratified as CIMP-high, CIMP-low and CIMP-negative (CIMP-neg). In this review the morphological correlates of five molecular subtypes are outlined: Type 1 (CIMP-high/MSI-H/BRAF mutation), Type 2 (CIMP-high/MSI-L or MSS/BRAF mutation), Type 3 (CIMP-low/MSS or MSI-L/KRAS mutation), Type 4 (CIMP-neg/MSS) and Type 5 or Lynch syndrome (CIMP-neg/MSI-H). The molecular pathways are determined at an early evolutionary stage and are fully established within precancerous lesions. Serrated polyps are the precursors of Types 1 and 2 CRC, whereas Types 4 and 5 evolve through the adenoma-carcinoma sequence. Type 3 CRC may arise within either type of polyp. Types 1 and 4 are conceived as having few, if any, molecular overlaps with each other, whereas Types 2, 3 and 5 combine the molecular features of Types 1 and 4 in different ways. This approach to the classification of CRC should accelerate understanding of causation and will impact on clinical management in the areas of both prevention and treatment.     

Differential involvement of the hypermethylator phenotype in hereditary and sporadic colorectal cancers with high-frequency microsatellite instability.

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair occurs in the majority of hereditary nonpolyposis colorectal cancers (HNPCCs) and in a subset of sporadic malignant tumors. Clinicopathologic and genotypic features of MSI-H colorectal tumors in HNPCC patients and those in sporadic cases are very similar but not identical. Correlation between the MSI phenotype and aberrant DNA methylation has been highlighted recently. A strong association between MSI and CpG island methylation has been well characterized in sporadic colorectal cancers with MSI-H but not in those of hereditary origin. To address the issue, we analyzed hereditary and sporadic colorectal cancers for aberrant DNA methylation of target genes using methylation-specific polymerase chain reaction. DNA methylation of the MLH1, CDKN2A, MGMT, THBS1, RARB, APC, and p14ARF genes was found in 0%, 23%, 10%, 3%, 73%, 53%, and 33% of 30 MSI-H cancers in HNPCC patients and in 80%, 55%, 23%, 23%, 58%, 35%, and 50% of 40 sporadic colorectal cancers with MSI-H, respectively. Cases showing methylation at three or more loci of six genes other than MLH1 were defined as CpG island methylator phenotype-positive (CIMP +), and 23% of HNPCC tumors and 53% of sporadic cancers with MSI-H were CIMP+ (P = 0.018). Differences in the extent of CpG island methylation, coupled with the differential involvement of several genes by methylation, in HNPCC tumors and sporadic MSI-H colorectal cancers may be associated with diverging developmental pathways in hereditary and sporadic cancers despite similar MSI-H phenotypes.     

Somatic frameshift mutations of bone morphogenic protein receptor 2 gene in gastric and colorectal cancers with microsatellite instability

Mounting evidence exists that perturbation of bone morphogenic protein (BMP) signaling is involved in cancer development, especially in gastrointestinal cancers. However, somatic mutations of the genes encoding BMP and BMP receptors have not yet been discovered in human cancer tissues. By analyzing a public database, we found that BMP receptor 2 (BMPR2) and BMP1 genes had mononucleotide repeats in their coding sequences that could be mutation targets in cancers with microsatellite instability (MSI). In this study, we analyzed the mutation of BMPR2 and BMP1 genes in gastric (GC) and colorectal cancers (CRC) with MSI [31 GC with high MSI (MSI-H), 13 GC with low MSI (MSI-L), 38 CRC with MSI-H and 15 CRC with MSI-L] by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found seven frameshift mutations in the BMPR2 gene, but not in the BMP1 gene. The mutations were an identical deletion mutation of one base in the repeats (c.1748delA) that would result in premature stops of the amino acid synthesis (p.Asn583ThrfsX44). The BMPR2 mutations were detected in 6.5% of GC and 13.2% of CRC with MSI-H. All the cancers with the BMPR2 mutation showed loss of BMPR2 expression. Our data indicate that frameshift mutation of BMPR2 gene occurs in GC and CRC with MSI-H, and suggest that the BMPR2 mutation might contribute to cancer pathogenesis by inactivating BMPR2-mediated BMP signaling.     

Clinical Implications of Microsatellite Instability in T1 Colorectal Cancer

Purpose

The estimation of regional lymph node metastasis (LNM) risk in T1 colorectal cancer is based on histologic examination and imaging of the primary tumor. High-frequency microsatellite instability (MSI-H) is likely to decrease the possibility of metastasis to either regional lymph nodes or distant organs in colorectal cancers. This study evaluated the clinical implications of MSI in T1 colorectal cancer with emphasis on the usefulness of MSI as a predictive factor for regional LNM.

Materials and Methods

A total of 133 patients who underwent radical resection for T1 colorectal cancer were included. Genomic DNA was extracted from normal and tumor tissues and amplified by polymerase chain reaction (PCR). Five microsatellite markers, BAT-25, BAT-26, D2S123, D5S346, and D17S250, were used. MSI and clinicopathological parameters were evaluated as potential predictors of LNM using univariate and multivariate analyses.

Results

Among 133 T1 colorectal cancer patients, MSI-H, low-frequency microsatellite instability (MSI-L), and microsatellite stable (MSS) colorectal cancers accounted for 7.5%, 6%, and 86.5%, respectively. MSI-H tumors showed a female predominance, a proximal location and more retrieved lymph nodes. Twenty-two patients (16.5%) had regional LNM. Lymphovascular invasion and depth of invasion were significantly associated with LNM. There was no LNM in 10 MSI-H patients; however, MSI status was not significantly correlated with LNM. Disease-free survival did not differ between patients with MSI-H and those with MSI-L/MSS.

Conclusion

MSI status could serve as a negative predictive factor in estimating LNM in T1 colorectal cancer, given that LNM was not detected in MSI-H patients. However, validation of our result in a different cohort is necessary.     

Characterization of tumour-infiltrating lymphocytes and apoptosis in colitis-associated neoplasia: comparison with sporadic colorectal cancer

The development of colorectal cancer is a major complication for patients with chronic idiopathic colitis. Colitis-associated tumours tend to occur at a younger age and be more aggressive than sporadic colorectal cancers. While we have previously associated the presence of tumour-infiltrating lymphocytes (TILs) and increased apoptosis in sporadic colorectal cancer with high-level microsatellite instability and improved prognosis, little is known of the relationship between these variables in colitis-associated colorectal cancer. The aim of this study was to correlate TILs and tumour cell apoptosis in colitis-associated neoplasms stratified according to microsatellite instability. Twenty tumour and 11 dysplastic samples resected from 21 patients with long-standing colitis were analysed for microsatellite instability at 10 microsatellite markers. TIL distribution (CD3, CD8) and function (granzyme B) were quantified by immunohistochemistry. Neoplastic cell apoptosis was assessed using the M30 CytoDEATH antibody. These findings were compared with 40 microsatellite stable (MSS) sporadic colorectal cancers previously evaluated for TILs and neoplastic apoptosis. Low-level microsatellite instability was found in 1/20 colitis-associated tumours. All other colitis-associated lesions were designated MSS. CD3(+) and CD8(+) TIL counts were significantly higher in colitis-associated lesions compared with MSS sporadic colorectal cancer (p < 0.0001, p = 0.001 respectively). Despite their higher TIL density, colitis-associated tumours were more likely to present late (Dukes' stage C or D) (p = 0.02). Functionally, colitis-associated TILs demonstrated significantly less granzyme B expression compared to sporadic cancers (p = 0.002). The level of tumour cell apoptosis was similar between the two groups (sporadic, 1.53%; colitis cancers, 1.45%). In conclusion, MSS colitis-associated tumours have a higher prevalence of CD3(+)/CD8(+) TILs but no associated increase in tumour cell killing by apoptosis. Unlike cytotoxic T cells in sporadic colorectal cancer, TILs do not appear to enhance the prognosis of colitis-associated colorectal cancer. This may be related to an impairment of granzyme B expression within these lesions.     

Robust microsatellite instability (MSI) analysis by denaturing high-performance liquid chromatography (DHPLC)

Microsatellite instability (MSI) plays an important biological role in various types of cancers, and especially in colorectal cancers. This study aimed to develop a simple, efficient, new method for robust MSI analysis. DNA was extracted from 175 (105 proximal colon and 70 distal colorectal) cancer samples and matched normal tissues, and five Bethesda microsatellite markers (BAT-25, BAT-26, D5S346, D2S123, and D17S250) were examined for MSI by denaturing high-performance liquid chromatography (DHPLC) analysis at a temperature of 50°C and a flow rate of 0.9 ml/min. It took just 9 min per PCR product to determine MSI or microsatellite stability (MSS) using the new protocol. The DHPLC results were confirmed with conventional gel-based electrophoresis and capillary-based sequencing method. Of 175 samples, 45 (26%) showed high microsatellite instability (MSI-H), 12 (7%) showed low microsatellite instability (MSI-L), and 118 (67%) showed MSS. All MSI samples were deletion mutants and all 12 MSI-L cases had MSI in dinucleotide markers (D5S346, D2S123, and D17S250). MSI was significantly associated with proximal colon cancers (p<0.0001), as previously reported. The MSI-H tumors were also associated with tumor node metastasis (TNM) I/II stages (p=0.05) and high-grade tumors (p<0.01). Here, we propose a DHPLC-based method as an alternative for MSI analysis.This work was presented in part at the meeting of AACR Oncogenomics 2003, Phoenix, AZ, USA, January 2003.     

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ≥ 4/5 methylated genes), MSI (MSI-H: ≥ 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.     

The "Fas counterattack" is not an active mode of tumor immune evasion in colorectal cancer with high-level microsatellite instability

Microsatellite instability (MSI) is an alternative pathway of colorectal carcinogenesis. It is found in 10% to 15% of sporadic colorectal neoplasms and is characterized by failure of the DNA mismatch-repair system. High-level MSI (MSI-H) is associated with tumor-infiltrating lymphocytes (TILs) and a favorable prognosis. Expression of Fas ligand (FasL/CD95L) by cancer cells may mediate tumor immune privilege by inducing apoptosis of antitumor immune cells. The aim of this study was to investigate the relationship between FasL expression and MSI status in primary colon tumors. Using immunohistochemistry, we detected FasL expression in 91 colorectal carcinoma specimens, previously classified according to the level of MSI as MSI-H (n = 26), MSI-low (MSI-L) (n = 29), and microsatellite stable (n = 36). Tumor-infiltrating lymphocyte density was quantified by immunohistochemical staining for CD3. MSI-H tumors were significantly associated with reduced frequency (P = .04) and intensity (P = .066) of FasL expression relative to non-MSI-H (ie, microsatellite stable and MSI-L) tumors. Higher FasL staining intensity correlated with reduced TIL density (P = .059). Together, these findings suggest that the abundance of TILs found in MSI-H tumors may be due to the failure of these tumor cells to up-regulate FasL and may explain, in part, the improved prognosis associated with these tumors.     

Elevated nuclear maspin expression is associated with microsatellite instability and high tumour grade in colorectal cancer

Maspin, a member of the serpin family, has been reported to suppress metastasis and angiogenesis in breast and prostate cancers. Overexpression of maspin was associated with adverse prognostic features in several other tumours. In this study, expression of maspin was analysed in 41 colorectal carcinomas with high-frequency microsatellite instability (MSI-H) and 159 microsatellite stable colorectal cancers (MSS/MSI-L) by immunohistochemistry (IHC) and partly by relative quantitative real-time RT-PCR and western blot analyses. Significant upregulation of maspin expression was found in MSI-H tumours compared to both MSS/MSI-L tumours and matched benign colonic mucosa. Increased maspin expression was also found in three MSI-H colon cancer cell lines, but not in three MSS colon cancer cell lines by RT-PCR and western blot analyses. Regulation of maspin expression depended on promoter methylation as tissue specimens and cell lines expressing maspin showed unmethylated maspin promoters, whereas promoter hypermethylation was found in specimens with loss of maspin expression. Intense nuclear maspin immunostaining was seen specifically in MSI-H tumours (p = 0.013), de-differentiated tumours (p = 0.006), and at the invasion front. These findings provide new insights into the role of maspin in colorectal cancer progression and may be useful for diagnosis and treatment strategies.