Chromosomal abnormalities and microsatellite instability in sporadic endometrial cancer

Defective DNA mismatch repair and nonfunctional mechanisms controlling the proper progression of the cell cycle have been proposed as being responsible for the genomic instability and accumulation of karyotypic alterations in endometrial cancer (EC). To assess whether numerical chromosomal anomalies (aneuploidy) and microsatellite instability (MSI) might be representative of distinctive tumour behaviour, paraffin-embedded tissue samples from 86 patients with sporadic EC were evaluated by both fluorescence in situ hybridisation (FISH) and microsatellite analysis, using free nuclei and genomic DNAs (respectively). Approximately one-third of the tumours analysed (24/74; 32%) exhibited MSI, whereas 38/86 (44%) of the EC samples displayed aneuploidy. The majority of the unstable cases (15/24; 63%) were from advanced-stage patients. Conversely, 23 (61%) out of the 38 tumours with aneuploidy were from early-stage patients. No apparent correlation was found between MSI and aneuploidy, whereas the immunohistochemical (IHC) analysis revealed that inactivation of the MLH1 mismatch repair gene may be involved in the majority of the MSI+ sporadic ECs. No genetic or cytogenetic alteration analysed here seems to add any significant predictive value to the stage of disease.     

MutL homolog 1 methylation and microsatellite instability in sporadic colorectal tumors among Filipinos

BACKGROUNDColorectal cancer (CRC) ranks third in terms of incidence and second in mortality worldwide. In CRC, the silencing of mismatch repair genes, including the mutL homolog 1 (hMLH1) has been linked to microsatellite instability (MSI), the lengthening or shortening of microsatellite repeats. Very limited data have been presented so far on the link of hMLH1 methylation and MSI in Southeast Asia populations with sporadic CRC, and on its clinical significance.AIMTo investigate the significance of the MSI status and hMLH1 methylation in CRC Filipino patients.METHODSFifty-four sporadic CRC patients with complete clinical data were included in this study. Genomic DNA from CRC tumor biopsies and their normal tissue counterparts were profiled for MSI by high resolution melting (HRM) analysis using the Bethesda Panel of Markers (BAT25, BAT26, D2S123, D5S346, and D17S250). hMLH1 methylation screening was performed using bisulfite conversion and methylation specific polymerase chain reaction. Statistical analysis was conducted to calculate their associations to clinicopathological characteristics and survival relevance (Kaplan-Meier curves and the log-rank test).RESULTS hMLH1 methylation was observed in 9% and 35% of CRC and normal samples, respectively. Higher incidence of consistently methylated hMLH1 found in both normal and CRC was noticed for relation to location of tumor (P < 0.05). As for MSI status, D2S123 the most common unstable microsatellite and MSI-high (MSI-H) was the most common MSI profile, counted for 46% and 50% of normal and CRC tissues, respectively. The presence of MSI-low (MSI-L) and microsatellite stable (MSS) was 43% and 11% for normal, and 31% and 19% for CRC samples. The mean month of patients’ survival was shorter in patients whose normal and tumor tissues had methylated compared to those with unmethylated hMLH1 and with MSI-H compared to those with MSI-L/MSS (P < 0.05). This was supported by significant difference in Kaplan-Meier with log-rank analysis. This data indicated that hMLH1 methylation and high MSI status have prognostic value.CONCLUSIONThis study showed the clinical significance of hMLH1 methylation and MSI status in sporadic CRC Filipino patients, especially in the normal part of the tumor.     

Low frequency of microsatellite instability in sporadic breast cancer

In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. The purpose of this study was to determine the presence of MSI in breast cancer and to correlate its occurrence with clinicopathological parameters, including expression of estrogen and progesterone receptors. We have analyzed the status of 10 different microsatellite loci (mono and dinucleotide repeats). Mmicrosatellite size patterns and LOH were compared in 88 paired breast-cancer/peripheral-blood DNA samples. Fluorescent polymerase chain reaction (PCR) for typing microsatellites coupled with DNA fragment analysis in an automated DNA sequencer was applied. Microsatellite instability in at least two microsatellite markers was observed in 6 out of 88 (7%) of the cases, all beloging to stage II or III. LOH was found in 48/88 (55%) of the cases. Five of the six cases with MSI also had LOH in other markers different from those of MSI. These MI and LOH data were analysed using a range of clinicopathological parameters, no correlation between MSI and histopathological characteristics were found. A significant correlation was observed between MSI and negative expression of both estrogen and progesterone receptors (p<0.02), indicating a possible relatioship between specific genetic changes at these microsatellite regions and hormonal deregulation in the progresion of breast cancer.     

The clinical importance and prognostic implications of microsatellite instability in sporadic cancer.

AIMS: The genetic abnormality known as microsatellite instability (MSI), first identified in colorectal cancer in 1993, has subsequently been recognised in other malignancies. These cancers are caused by a defect in the nuclear mismatch repair system, allowing mutations to accumulate with every cellular division. Hereditary Non Polyposis Colon Cancers (HNPCC) and associated malignancies demonstrating MSI have a unique histological appearance, improved prognosis and altered response to chemotherapy and radiotherapy. This review examines the incidence of MSI and its clinical significance in commonly occurring solid malignancies. METHOD: A medline based literature search was performed using the key words 'Microsatellite Instability' and the name of the specific malignancy being investigated. Additional original papers were obtained from citations in those articles identified in the original medline search. RESULTS: MSI has been detected in many solid malignancies although the definition of instability applied has been variable. It is most commonly found in sporadic malignancies that also occur in the HNPCC syndrome such as colorectal, stomach, endometrial and ovarian cancer. MSI may impart a favorable prognosis in colorectal, gastric, pancreatic and probably oesophageal cancers but a poor prognosis in non small cell lung cancer. In clinical studies colorectal cancers demonstrating MSI respond better to chemotherapy while in vitro studies using MSI positive cell lines show resistance to radiotherapy and chemotherapy. CONCLUSION: MSI may be a useful genetic marker in prognosis and could be an influential factor in deciding treatment options. However, in many cancers its significance remains unclear and more evaluation is required.     

Infrequent occurrence of microsatellite instability in sporadic and familial breast cancer

Microsatellite instability was analysed in 93 primary breast tumours at 13 chromosomal loci frequently altered in breast cancer. RER (replication errors) were observed at a low (5%) frequency in sporadic, familial and hereditary breast tumours, as well as in breast tumours from patients with multiple primary cancers. Our study suggests that the RER+ phenotype is rare in breast tumours, and that breast cancer is not included in the hereditary non-polyposis colon cancer (HNPCC) syndrome. Moreover, the RER+ tumours revealed an atypical pattern of microsatellite alteration as compared with those usually seen in HNPCC tumours. In agreement with the findings in HNPCC tumours, all RER+ breast tumours were diploid, although having a similar frequency of allelic imbalance as RER— tumours. Thus, mismatch repair deficiency is rare in breast cancer, is most likely caused by somatic mutations, and possibly in a set of DNA repair genes different from that involved in the HNPCC syndrome.     

Proliferation, apoptosis, and survival in high-level microsatellite instability sporadic colorectal cancer.

Sporadic colorectal cancer (CRC) characterized by high-level DNA microsatellite instability (MSI-H) has a favorable prognosis. The reason for this MSI-H survival advantage is not known. The aim of this study was to correlate proliferation, apoptosis, and prognosis in CRC stratified by MSI status. The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody in a selected series of 100 sporadic colorectal cancers classified according to the level of MSI as 31 MSI-H, 29 MSI-Low (MSI-L), and 40 microsatellite stable (MSS). The Ki-67 index was significantly higher in MSI-H cancers (P < 0.0001) in which the PI was 90.1 +/- 1.2% (mean +/- SE) compared with 69.5 +/- 3.1% and 69.5 +/- 2.3% in MSI-L and MSS subgroups, respectively. There was a positive linear correlation between the apoptotic index (AI) and PI (r = 0.51; P < 0.001), with MSI-H cancers demonstrating an increased AI:PI ratio indicative of a lower index of cell production. A high PI showed a trend toward predicting improved survival within MSI-H cancers (P = 0.09) but did not predict survival in MSI-L or MSS cancers. The AI was not associated with survival in any MSI subgroup. In conclusion, this is the first study to show that sporadic MSI-H cancers are characterized by a higher AI:PI ratio and increased proliferative activity compared with MSI-L and MSS cancers, and that an elevated PI may confer a survival advantage within the MSI-H subset.     

Oncogenic mutations in gastric cancer with microsatellite instability

Aim

Mitogen-activated protein kinase (MAPK) cascade and phosphatidylinositol 3-kinase (PI3K) survival pathways are frequently activated in the progression of gastrointestinal malignancies. In this study, we aimed to determine the frequency of gene mutations in members of these pathways - Epithelial Growth Factor Receptor (EGFR), KRAS, BRAF, PIK3CA and MLK3 in a series of 63 gastric carcinomas with high levels of microsatellite instability (MSI).

Methods

Gene mutation analysis was performed by PCR amplification followed by direct sequencing. In selected tumour cases, EGFR expression was evaluated by immunohistochemistry. Association studies between molecular data and clinicopathologic characteristics were performed.

Results

Mutations in EGFR (3′-untranslated region [UTR] polyA repeat), KRAS, PIK3CA and MLK3 genes occurred in 30 (47.6%), 11 (17.5%), 9 (14.3%) and 2 (3.2%) of the MSI gastric cancer (GC) cases, respectively. No BRAF or EGFR hotspot mutations were identified. Overall, mutations in at least one of these genes were found in 55.6% (35/63) of gastric carcinomas. From those mutant cases 40.0% (14/35) of them had concomitant gene mutations, always involving EGFR polyA deletions. Interestingly, we observed significant associations between oncogenic mutations and female gender (p = 0.046) old age of diagnosis (p = 0.001) and intestinal subtype (p = 0.043).

Conclusion

Our results show that MSI gastric carcinoma frequently shows activation of EGFR-MAPK and PI3K pathways. Within all alterations found, deletions of the A13 repeats of EGFR were common, suggesting this molecular event as an important biomarker for stratification of GC patients for treatment with EGFR inhibitors.     

Loss of p27 expression and microsatellite instability in sporadic colorectal cancer

BACKGROUND: The role of the loss of p27 protein expression in the oncogenesis of colorectal cancer is still in debate. In this study, we prospectively examined the immunohistochemical expression of p27 in 108 consecutive colorectal cancers, and we analysed the relationship with the results, the clinicopathological data, microsatellite instability (MSI) and other genetic alterations of tumours. METHODS: Unselected patients (108) who underwent curative colorectal resection for sporadic colorectal cancer in a three-year period were evaluated for MSI using 6 microsatellite markers, and for the presence of p27, p53, Fhit, Mlh1 and Msh2 proteins by means of immunostaining. The relationships between these markers were analysed. p27 protein expression was examined for association with disease recurrences and survival. RESULTS: Lack of p27 expression was noted in 33 out of 108 (30.5%) colorectal cancer cases (P<0.05). This altered expression was significantly higher in proximal cancers (P<0.05), mucinous tumours (P<0.001), poorly differentiated histology (P<0.01), cancers with MSI (P<0.05), tumours with altered expression of Mlh1 (P<0.01), of Msh2 (P<0.05), and of Fhit (P<0.01). Overall survival was better in the patient group with altered level of phenotypic p27 expression, although the difference does not reach statistical significance (P=0.069). The analysis performed only for patients with tumour at stage II showed significantly better survival when the tumour exhibited altered p27 expression (P<0.02). CONCLUSIONS: The results of the present study support the hypothesis that altered expression of p27 may be part of the genetic pathway involving MSI, which is responsible for the development of some colorectal cancers.     

散发性结直肠癌中微卫星不稳定研究及其临床病理意义

目的检测散发性结直肠癌(SCRC)中的微卫星不稳定状态,并探讨其与临床病理特征的关系。方法应用荧光标记多重PCR法扩增微卫星位点BAT-25和BAT-26,将PCR扩增产物在荧光毛细管中进行电泳,以GenScan3.1软件扫描分析两个微卫星位点状况。结果112例SCRC患者中,BAT-25和BAT-26两个位点均阳性者14例,MSI-H发生率为12.5(14/112);BAT-25单位点阳性者4例,BAT-26单位点阳性者6例;两个位点均阴性者88例,MSS发生率为78.6。SCRC中MSI-H和MSS两组的比较显示:MSI-H者发病部位以近端结肠癌多见(P=0.034),组织学类型以黏液腺癌、印戒细胞癌相对多见(P=0.023),分化程度以低分化癌多见(P=0.039),而MSI-H与MSS两者在性别、年龄、肿瘤浸润深度、淋巴结转移、远处转移等方面差异无统计学意义。结论SCRC中微卫星不稳定是一个常见的分子事件,微卫星不稳定的SCRC好发于右半结肠,具有低分化的倾向。     

Frequent and multiple mutations at minisatellite loci in sporadic human colorectal and gastric cancers--possible mechanistic differences from microsatellite instability in cancer cells.

Minisatellites (MNs), composed of 5 to 100 nucleotide repeat units, range from 0.5 to 30 kb in length, and have been reported to be mutated in various human malignancies. In this study, frequencies of MN mutations in sporadic human colorectal (34 cases) and gastric cancers (24 cases) at various clinicopathological stages were assessed by multilocus DNA fingerprint analysis with three MN probes, Pc-1, 33.6 and 33.15. MN mutations were observed in both colorectal and gastric cancers, but at a significantly higher frequency in the former (56%) than in the latter (25%). Multiplicities of MN mutations were 1.50 +/- 1.81 and 0.46 +/- 1.10 in colorectal and gastric cancers, respectively, and the difference was also significant. Neither the presence nor multiplicity of MN mutations in either colorectal or gastric cancer cases had any correlation with the pathological stage, histological grading or the presence of microsatellite instability (MSI). Although the biological relevance of MN mutations still remains to be clarified, a subset of colorectal and gastric cancers could feature a new type of genomic instability, distinct from MSI.     

Adverse prognostic effect of methylation in colorectal cancer is reversed by microsatellite instability.

PURPOSE: DNA methylation is an important biologic event in colorectal cancer and in some cases is associated with the development of microsatellite instability (MSI). In this study, we sought to determine the prognostic significance of DNA methylation, both in univariate analysis and in concert with other clinicopathologic factors known to influence outcome. PATIENTS AND METHODS: Fresh tissue (625 cancers) was obtained from 605 individuals (age range, 29 to 99 years) undergoing curative surgery for colorectal cancer at one institution during a period of 8 years. Clinicopathologic details were recorded for all tumors, including stage, grade, type, vascular space invasion, and clinical follow-up to 5 years. Microsatellite status was assessed using standard markers. Methylation of p16 and hMLH1 promoters was determined by methylation-specific polymerase chain reaction (PCR), whereas methylation at methylated-in-tumor loci (MINT)1, MINT2, MINT12, and MINT31 loci were assessed by bisulfite-PCR. RESULTS: Patients with microsatellite unstable tumors (12%) had better disease-specific survival than those with microsatellite stable (MSS) tumors (univariate analysis: hazard ratio [HR], 0.53; 95% CI, 0.27 to 1.0). Overall survival of individuals with MSS tumors was influenced by three independently significant factors: tumor stage (HR, 7.3; 95% CI, 5.1 to 10.4), heavy tumor methylation (HR, 2.1; 95% CI, 1.1 to 4.0), and vascular space invasion (HR, 1.9; 95% CI, 1.3 to 2.9). In MSS tumors, methylation at any single site was not independently predictive of survival. Neither methylation nor microsatellite status predicted a favorable response to chemotherapy. CONCLUSION: DNA methylation is associated with a worse outcome in colorectal cancer, but this adverse prognostic influence is lost in those methylated tumors showing MSI. The mechanisms of these events warrant additional investigation.     

The role of MYH and microsatellite instability in the development of sporadic colorectal cancer

Biallelic germline mutations in MYH are associated with colorectal neoplasms, which develop through a pathway involving somatic inactivation of APC. In this study, we investigated the incidence of the common MYH mutations in an Australian cohort of sporadic colorectal cancers, the clinicopathological features of MYH cancers, and determined whether inactivation of mismatch repair and base excision repair (BER) were mutually exclusive. The MYH gene was sequenced from lymphocyte DNA of 872 colorectal cancer patients and 478 controls. Two compound heterozygotes were identified in the cancer population and all three cancers from these individuals displayed a prominent infiltration of intraepithelial lymphocytes. In total, 11 heterozygotes were found in the cancer group and five in the control group. One tumour from an individual with biallelic germline mutation of MYH also demonstrated microsatellite instability (MSI) as a result of biallelic hypermethylation of the MLH1 promoter. Although MYH-associated cancers are rare in a sporadic colorectal population, this study shows that these tumours can develop through either a chromosomal or MSI pathway. Tumours arising in the setting of BER or mismatch repair deficiency may share a biological characteristic, which promotes lymphocytic infiltration.     

Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer.     

Abnormal Fhit protein expression and high frequency of microsatellite instability in sporadic colorectal cancer

The role of Fhit protein in the oncogenesis of colorectal cancer is still in debate. Recent studies have revealed that reduced Fhit protein expression is associated with a deficiency of the mismatch repair protein. One hundred and twenty unselected patients who underwent curative resection for sporadic colorectal cancer in a three-year period were evaluated for microsatellite instability (MSI) using six microsatellite markers, and for the presence of Fhit and mismatch repair (MMR) proteins (Mlh1 and Msh2) by means of immunostaining. The relations between these markers were analysed. Reduced or absent Fhit expression was noted in 18 out of 118 patients. This altered expression was significantly higher in right-sided cancer (P = 0.005), mucinous tumours (P = 0.005) and in poorly differentiated histological types (P = 0.0001). MSI was found in 22 out of 109 patients, more so in right-sided cancer (P = 0.0001), poorly differentiated histology (P = 0.0001), and mucinous tumours (P = 0.0001). No association was found with TNM stage. MSI was present in 66.7% of tumours with altered Fhit expression and in only 10% of tumours with preserved or intermediate Fhit expression (P = 0.0001). Of the tumours with reduced or absent Fhit expression, 72.2% had loss of nuclear Mlh1 or Msh2 expression compared with only 14% of the preserved or intermediate Fhit expression tumours (P = 0.0001). These results support the hypothesis that deficiency in a MMR gene could be a cause of the high frequency of alterations in Fhit expression, and they permit the suggestion that FHIT gene alteration may be part of the genetic pathway involving MSI through which some colorectal cancers arise.     

Differences between familial and sporadic forms of colorectal cancer with DNA microsatellite instability

Microsatellite instability (MSI) is observed in approximately 13% of colorectal cancers. Genes containing a mononucleotide microsatellite in the coding sequence are particularly prone to inactivation in MSI tumourigenesis, and much work has been conducted to identify genes with high repetitive tract mutation rates in these tumours. MSI caused by deficient DNA mismatch-repair functions is a hallmark of cancers associated with the hereditary non-polyposis colorectal cancer syndrome but is also found in about 15% of all sporadic tumours.     

Frameshift mutation of the STK11 gene in a sporadic gastrointestinal cancer with microsatellite instability.

Germline mutations of the STK11 gene lead to emergence of hamartomas in the gastrointestinal tract of patients with Peutz-Jeghers syndrome, who bear an increased risk of malignancies of the gastrointestinal tract, genital tract, and other organs. We analyzed 80 sporadic colorectal cancers, six small-intestinal cancers, and 40 gastric cancers for somatic mutations of STK11 by SSCP methods. Among them only one colorectal cancer, which showed a phenotype of microsatellite instability, was found to possess a deleterious mutation in this gene, a frameshift involving deletion of one base at codons 279-281. This region of the gene contains a mononucleotide-repeat sequence, CCCCCC. The other allele of STK11 had been lost in this tumor. If the STK11 gene is one of the mutational targets of microsatellite instability, its inactivation may be associated with tumor development in a small proportion of colorectal cancers.     

Somatic mutation in mitochondrial DNA and nuclear microsatellite instability in gastric cancer

It was reported that somatic mutations in the mitochondrial DNA (mtDNA) are associated with high-frequency microsatellite instability (MSI-H) of the nuclear in gastric cancers. However, no correlation between mtDNA mutations and nuclear MSI-H was found in colorectal, breast, and renal cancers. Therefore, the association between mtDNA mutations and nuclear MSI-H in gastric cancers is controversial. We examined mtDNA mutations and nuclear MSI in a large panel of gastric cancers. One-hundred and five gastric cancers were selected. Mutations in the mononucleotide repeat (D310) of mtDNA and nuclear MSI at 5 microsatellite loci were examined by microsatellite assay. Somatic mutations in the mtDNA and nuclear MSI-H were detected in 16 (15%) and 14 (13%) of the gastric cancers, respectively. mtDNA mutations were detected in 2 of the 14 (14%) and 14 of the 91 (15%) tumors with and without nuclear MSI-H, respectively. There was no significant difference between them. These results suggest that somatic mutations in the mtDNA and nuclear MSI-H play important roles in gastric carcinogenesis, and that mtDNA mutations may not be associated with nuclear MSI-H in gastric cancers.