NORMAL GENOMIC

• Velho S, Fernandes MS, Leite M, Figueiredo C, Seruca R
• Causes and consequences of microsatellite instability in gastric carcinogenesis.
• World J Gastroenterol. 2014; 20: 16433-42
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• Loss of DNA mismatch repair (MMR) function, due to somatic or germline epi/genetic alterations of MMR genes leads to the accumulation of numerous mutations across the genome, creating a molecular phenotype known as microsatellite instability (MSI). In gastric cancer (GC), MSI occurs in about 15% to 30% of the cases. This review summarizes the current knowledge on the molecular mechanisms underlying the acquisition of MSI in GC as well as on the clinic, pathologic and molecular consequences of the MSI phenotype. Additionally, current therapeutic strategies for GC and their applicability in the MSI subset are also discussed.

• Rajan Kd A, Burris C, Iliff N, Grant M, Eshleman JR, Eberhart CG
• DNA mismatch repair defects and microsatellite instability status in periocular sebaceous carcinoma.
• Am J Ophthalmol. 2014; 157: 640-7
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• PURPOSE: To characterize mismatch repair protein expression and the role of DNA repair abnormalities in sebaceous carcinomas of the ocular adnexa. DESIGN: Retrospective case-series study. METHODS: We reviewed 10 cases of sporadic sebaceous carcinoma and 1 case involving a patient with a family history consistent with Muir-Torre syndrome. Immunohistochemistry was used to analyze the presence of 4 mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) in these tumors. DNA was extracted from 7 of the larger tumors as well as from adjacent normal control tissue and microsatellite instability (MSI) analysis using 5 highly sensitive mononucleotides and 2 pentanucleotides was performed. RESULTS: All 10 sporadic periocular sebaceous carcinomas maintained strong staining of the 4 mismatch repair genes, while tumor from the patient with Muir-Torre syndrome showed loss of staining for the mismatch repair genes MSH2 and MSH6. MSI testing of 7 tumors identified no changes in sporadic cases and yielded results supporting presence of repeat sequence instability in the Muir-Torre-associated case. CONCLUSIONS: Sporadic sebaceous carcinoma of the ocular adnexa is not commonly associated with a loss of mismatch repair genes or microsatellite instability.

• Vilar E et al.
• Role of microsatellite instability-low as a diagnostic biomarker of Lynch syndrome in colorectal cancer.
• Cancer Genet. 2014; 207: 495-502
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• Lynch syndrome is the most common Mendelian disorder predisposing persons to hereditary colorectal cancer. Carriers of MSH6 mutations constitute less than 10% of the total of cases with Lynch syndrome and present with a weaker clinical phenotype, including low levels of microsatellite instability (MSI-L) in colorectal tumors. The frequency of MSH6 mutation carriers among patients presenting with MSI-L colorectal cancer has yet to be determined, as has the appropriate genetic workup in this context. We have reviewed here the clinicopathologic characteristics, immunohistochemistry, and genetic testing results for 71 patients at a single institution diagnosed with MSI-L colorectal cancers. Of 71 patients with MSI-L tumors, 21 underwent genetic testing for MSH6 mutations, three of whom presented with loss of staining of MSH6 and only one of whom carried a pathogenic germline MSH6 mutation in exon 4 (c.2677_2678delCT; p.Leu893Alafs*6). This latter patient had a significant family history of cancer and had a rectal primary tumor that showed instability only in mononucleotide markers. In this cohort of MSI-L patients, we detected no notable clinicopathologic or molecular characteristic that would help to distinguish a group most likely to harbor germline MSH6 mutations. Therefore, we conclude that the prevalence of MSH6 mutations among patients with MSI-L tumors is very low. Microsatellite instability analysis combined with immunohistochemistry of mismatch repair proteins adequately detects potential MSH6 mutation carriers among MSI-L colorectal cancers.

• Pai RK et al.
• BRAF-mutated, microsatellite-stable adenocarcinoma of the proximal colon: an aggressive adenocarcinoma with poor survival, mucinous differentiation, and adverse morphologic features.
• Am J Surg Pathol. 2012; 36: 744-52
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• The association of BRAF V600E mutation and the presence of the CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) often confound analysis of BRAF mutation status and survival in colorectal carcinoma. We evaluated a consecutive series of proximal colonic adenocarcinomas for mismatch repair protein abnormalities/MSI, BRAF V600E mutation, and KRAS mutations in an attempt to determine the prognostic significance of these abnormalities and to correlate histopathologic features with molecular alterations. Of the 259 proximal colon adenocarcinomas analyzed for mismatch repair protein abnormalities and/or MSI, 181 proximal colonic adenocarcinomas demonstrated proficient DNA mismatch repair using either MSI PCR (n=78), mismatch repair protein immunohistochemistry (n=91), or both MSI PCR and mismatch repair immunohistochemistry (n=12); these were tested for the BRAF V600E mutation and KRAS mutations. Compared with BRAF wild-type adenocarcinomas, BRAF-mutated adenocarcinomas more frequently demonstrated adverse histologic features such as lymphatic invasion (16/20, 80% vs. 75/161, 47%; P=0.008), mean number of lymph node metastases (4.5 vs. 2.2; P=0.01), perineural invasion (8/20, 40% vs. 13/161, 8%; P=0.0004), and high tumor budding (16/20, 80% vs. 83/161, 52%; P=0.02). BRAF-mutated adenocarcinomas frequently contained areas with mucinous histology (P=0.0002) and signet ring histology (P=0.03), compared with KRAS-mutated and KRAS/BRAF wild-type adenocarcinomas. Clinical follow-up data were available for 173 proximal colonic adenocarcinomas with proficient DNA mismatch repair. Patients with BRAF-mutated adenocarcinomas had a median survival of 12.3 months with a 1-year probability of survival of 54% and a 1-year disease-free survival of 56%. Patients with KRAS-mutated and KRAS/BRAF wild-type adenocarcinomas had significantly improved overall survival (unadjusted log-rank P=0.03 and unadjusted log-rank P=0.0002, respectively) and disease-free survival (unadjusted log-rank P=0.02 and unadjusted log-rank P=0.02, respectively) compared with patients with BRAF-mutated adenocarcinomas. When adjusting for tumor stage, survival analysis demonstrated that patients with BRAF-mutated adenocarcinoma had a significantly poor overall survival and disease-free survival (hazard ratios 6.63, 95% CI, 2.60-16.94; and 6.08, 95% CI, 2.11-17.56, respectively) compared with patients with KRAS/BRAF wild-type adenocarcinomas. No significant difference in overall or disease-free survival was identified between patients with KRAS-mutated and KRAS/BRAF wild-type adenocarcinomas. Our results demonstrate that BRAF-mutated proximal colon adenocarcinomas with proficient DNA mismatch repair have a dismal prognosis with an aggressive clinical course and often display mucinous differentiation, focal signet ring histology, and other adverse histologic features such as lymphatic and perineural invasion and high tumor budding.

• Shokal U, Sharma PC
• Implication of microsatellite instability in human gastric cancers.
• Indian J Med Res. 2012; 135: 599-613
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• Microsatellite instability, one of the phenomena implicated in gastric cancer, is mainly associated with the expansion or contraction of microsatellite sequences due to replication errors caused most frequently by mutations in the mismatch repair (MMR) and tumour suppressor genes. Tumours exhibiting microsatellite instability are proven to have truncated products resulting from frequent mutations in mononucleotide or dinucleotide runs in coding and non-coding regions of the targeted genes. Epigenetic changes like hypermethylation of the promoter region of MMR genes as well as gene silencing are also responsible for the microsatellite instability phenotypes. Assessing microsatellite instability in tumours has proved to be an efficient tool for the prognosis of various cancers including colorectal and gastric cancers. Such tumours are characterized by distinct clinicopathological profiles. Biotic agents like Epstein Barr Virus and H. pylori along with other factors like family history, diet and geographical location also play an important role in the onset of gastric carcinogenesis. Instability of mitochondrial DNA has also been investigated and claimed to be involved in the occurrence of gastric cancers in humans. Development of simplified but robust and reproducible microsatellite instability based molecular tools promises efficient prognostic assessment of gastric tumours.

• Arnason T et al.
• Loss of expression of DNA mismatch repair proteins is rare in pancreatic and small intestinal neuroendocrine tumors.
• Arch Pathol Lab Med. 2011; 135: 1539-44
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• CONTEXT: Two recent studies have identified a high rate of microsatellite instability (MSI) in pancreatic neuroendocrine tumors (pNETs). Microsatellite instability is rare in small intestinal neuroendocrine tumors (NETs). It is unclear why there is discordance in the frequency of MSI in the 2 studies of pNETs and why this mechanism is comparatively rare in small intestinal tumors. Loss of expression of DNA mismatch repair (MMR) proteins, which is known to correlate strongly with MSI, is not well studied in pancreatic or small intestinal NETs. OBJECTIVE: To determine if there is loss of expression of MMR protein expression in pancreatic or small intestinal NETs. DESIGN: Sixty-nine patients (31 male, 38 female; mean age, 59.2 years) were identified who had a resection for a primary pancreatic (n = 35) or primary small intestinal (n = 34) NET during an 18-year period. Immunohistochemical stains for MLH1, MSH2, MSH6, and PMS2 were applied to archived tissue from all cases. All pNETs with adequate tissue (n = 32) were also assessed by MSI analysis. RESULTS: There was preserved expression of MLH1, MSH2, MSH6, and PMS2 in all 35 pNETs. Of 32 pNETs tested by polymerase chain reaction, 28 were microsatellite stable and DNA did not amplify in 4. In 34 small intestinal NETs, 2 cases had indeterminate MLH1 and 1 case had indeterminate PMS2 expression. The remainder had intact MMR protein expression. CONCLUSION: Defects in DNA MMR proteins are rare in pancreatic and small intestinal NETs, raising doubt that MSI plays a significant role in the pathogenesis of these tumors.

• Esemuede I et al.
• Improved testing for microsatellite instability in colorectal cancer using a simplified 3-marker assay.
• Ann Surg Oncol. 2010; 17: 3370-8
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• BACKGROUND: In colorectal cancer (CRC), microsatellite instability (MSI) is a valuable marker of defective DNA mismatch repair that identifies cancers with distinct phenotypic properties, including favorable survival. However, the optimal assay for MSI status is unknown. We have evaluated a simplified 3-marker assay for MSI and compared it with the 5-marker (NCI) assay to see if technical variations in MSI testing are important. MATERIALS AND METHODS: DNA samples from 357 CRCs were evaluated for MSI using the 5 microsatellite markers recommended for the NCI assay (BAT 25, BAT26, D2S123, D5S346, and D17S250). Results were compared with a simplified 3-marker assay (BAT25, BAT26, and D2S123). CRCs identified as MSI were evaluated for their clinical, pathological, and genetic characteristics. RESULTS: The 5-marker assay identified 96 cancers as MSI. Only 56 of these were MSI by the 3-marker assay (3-marker+ group), leaving 40 cases identified as MSI only by NCI criteria (3-marker- group). The remaining 261 cancers were microsatellite stable (MSS). The 3-marker+ MSI tumors had features characteristic of MSI tumors: more proximal, poorly differentiated, associated with hereditary nonpolyposis colorectal cancer (HNPCC), more BRAF mutations, fewer KRAS mutations, better 5-year disease-specific survival, more frequent mismatch repair (MMR) protein loss, and less likely to be metastatic on presentation (P < .05). Chromosomal arm loss was observed only in 3-marker- MSI and MSS cancers (P < .05). CONCLUSION: The 3-marker MSI assay outperforms the traditional 5-marker assay for identifying patients with favorable prognosis and homogeneous clinical and genetic features. More accurate MSI testing should improve prognostic and predictive scoring systems for colorectal cancer.

• Arai T et al.
• Microsatellite-unstable mucinous colorectal carcinoma occurring in the elderly: comparison with medullary type poorly differentiated adenocarcinoma.
• Pathol Int. 2007; 57: 205-12
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• Mucinous carcinoma and poorly differentiated adenocarcinoma of the large intestine have a high frequency of microsatellite instability, and their occurrence increases gradually with age. To elucidate the clinicopathological and immunohistochemical features of microsatellite-unstable mucinous carcinoma and compare the tumor with medullary type poorly differentiated adenocarcinoma, the clinicopathological status and expression of mucin core and hMLH1 proteins were studied in 15 microsatellite-unstable and 20 microsatellite-stable mucinous colorectal carcinomas occurring in elderly patients, and compared with 23 cases of medullary type poorly differentiated adenocarcinoma in which 21 cases were microsatellite-unstable. Thirteen (87%) of 15 microsatellite-unstable carcinomas exhibited absent hMLH1 expression compared with three (15%) of 20 microsatellite-stable carcinomas (P < 0.01). The proportion (87%) of positive MUC5AC expression in microsatellite-unstable mucinous carcinoma was significantly higher than that (45%) in microsatellite-stable mucinous carcinoma (P = 0.01). Compared with microsatellite-stable mucinous carcinoma, microsatellite-unstable mucinous carcinomas were significantly associated with a proximal location, intra- and peritumoral inflammatory cell infiltration, frequent MUC5AC expression, a low incidence of lymph node metastasis and absent hMLH1 protein expression, which is not different to medullary type poorly differentiated adenocarcinoma except for MUC2 expression and age-related occurrence. These results suggest that microsatellite-unstable mucinous carcinoma occurring in the elderly shares clinicopathological and molecular features with medullary type poorly differentiated adenocarcinoma and that microsatellite instability with absent hMLH1 expression plays an important role in the development of these two carcinomas.

• Cesinaro AM, Ubiali A, Sighinolfi P, Trentini GP, Gentili F, Facchetti F
• Mismatch repair proteins expression and microsatellite instability in skin lesions with sebaceous differentiation: a study in different clinical subgroups with and without extracutaneous cancer.
• Am J Dermatopathol. 2007; 29: 351-8
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• Muir-Torre syndrome (MTS) is defined as the association of a sebaceous tumor or keratoacanthoma and an extracutaneous neoplasm, mainly from the gastrointestinal or genitourinary tracts. MTS is related to hereditary non-polyposis colorectal cancer (HNPCC), a syndrome with germline mutations in the mismatch repair (MMR) gene(s), leading to microsatellite instability (MSI). In this study, using immunohistochemistry and a microsatellite instability assay, we analyzed the incidence of MMR gene abnormalities in 79 sebaceous lesions from 70 patients, 26 of whom also had an extracutaneous visceral neoplasm. We were unable to investigate the family histories of our patients regarding other tumors in order to assess which of our cases met the Amsterdam criteria. Defective MMR protein expression (MMR-) was found in 18/70 (25.7%) patients, with an identical distribution between those having an isolated skin tumor (11/44, 25.0%) and those with an extracutaneous cancer (7/26, 25.4%). In the sporadic group, MMR negative lesions were significantly more frequent in extrafacial areas (P = 0.03). High concordance was found between MMR expression in sebaceous lesions and the extracutaneous neoplasm in the same patient (20/23, 86.9%), as well as between MMR expression and microsatellite status (18/20, 90%). In conclusion, this study confirms the value of immunohistochemistry to identify MMR defective tumors. However, since only a minority of sebaceous neoplasms in patients who also have an extracutaneous cancer display MMR defects, these techniques are of limited value for the identification of "clinically defined" MTS.

• Masuda H, Abe Y, Takayama T
• Microsatellite instability in poorly differentiated colorectal adenocarcinoma, particularly in relation to two subtypes.
• Hepatogastroenterology. 2005; 52: 82-5
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• BACKGROUND/AIMS: There have been a few reports indicating the characteristics of poorly differentiated colorectal adenocarcinoma by dividing it into subtypes. However, we have not found any reports describing microsatellite instability (MSI) of each subtype. In this study, we elucidated the clinicopathological features of subtypes in the poorly differentiated adenocarcinoma, especially the relationship between MSI and each subtype. METHODOLOGY: The present study included 28 cases with poorly differentiated adenocarcinoma. The 28 cases were classified into two groups; the solid group and the non-solid group. For each group, the clinicopathological aspects and MSI were examined. RESULTS: No significant differences were noted between the solid group and the non-solid group in terms of clinicopathological findings excluding male/female ratio. The 5-year survival rate of the solid group (38.5%) was significantly higher than that of the non-solid group (0.00%) (p=0.0013). The ratio of cases with MSI-H in the solid group (80.0%=12/15) was significantly higher than that of the non-solid group (30.8%=4/13) (p=0.0087). CONCLUSIONS: The incidence of MSI as well as the prognosis was different between solid and non-solid type with poorly differentiated colorectal adenocarcinoma. Therefore, we think that poorly differentiated colorectal adenocarcinoma should be classified into two subtypes: solid type and non-solid type when analysis for poorly differentiated adenocarcinoma is performed.

• Tomaszewska R, Okon K, Stachura J
• Expression of the DNA mismatch repair proteins (hMLH1 and hMSH2) in infiltrating pancreatic cancer and its relation to some phenotypic features.
• Pol J Pathol. 2003; 54: 31-7
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• DNA mismatch repair system defects cause microsatellite instability (MSI) and form an alternative pathway in cancer development. Germline mutations of DNA mismatch repair genes account for hereditary nonpolyposis colorectal cancer, which has a different morphology and biology than sporadic cancers. MSI has also been found in sporadic neoplasms and some inflammatory conditions (chronic pancreatitis, ulcerative colitis). The purpose of the present study was to evaluate the expression of hMLH1 and hMLH2 proteins in infiltrating pancreatic cancer and to find out whether there is a relationship between some phenotypic manifestations and expression of MMR genes. We studied 30 cases of infiltrating pancreatic cancer and apart from hMLH1 and hMLH2 expression cytokeratin 7 and chromogranin were measured as markers of ductal and endocrine differentiation, respectively. All ductal pancreatic cancers expressed cytokeratin 7. In most cases the expression was strong, present in 50-100% of cells in moderately differentiated cancers and in 80-100% of cells in poorly differentiated cancers. Chromogranin expression was seen in 5 moderately differentiated cancers and in 6 poorly differentiated cancers (up to 20% of positive cells). In all cases DNA mismatch repair genes expression was present. CONCLUSION: Ductal pancreatic carcinomas express hMLH1 and hMLH2 proteins irrespective of their differentiation. The expression of cytokeratin 7 is typical of ductal pancreatic carcinoma and its level is related to cancer differentiation. Some ductal pancreatic carcinomas irrespective of their differentiation show the expression of chromogranin, which is associated with the expression of hMSH2 gene.

• Szybka M, Bartkowiak J, Zakrzewski K, Polis L, Liberski P, Kordek R
• Microsatellite instability and expression of DNA mismatch repair genes in malignant astrocytic tumors from adult and pediatric patients.
• Clin Neuropathol. 2003; 22: 180-6
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• Microsatellite instability (MSI) is used as a molecular marker for defective DNA mismatch repair (MMR) genes. We report here alterations of MSI in 15 malignant astrocytomas (WHO grade III) and glioblastomas (GBM; WHO grade IV) of pediatric patients (2 - 21 years) and 12 GBM from adults (44 - 68 years) by comparative analysis of BAT25/BAT26 loci and 10 other microsatellite markers. High-level microsatellite instability (MSI-H) occurred in 4 of the 15 pediatric cases (26.7%) and in 1 of the 12 adult GBM cases (8.3%). Low-level microsatellite instability (MSI-L) was observed in 6 pediatric cases (40%) and 8 adult GBM (66.7%). Unstable BAT-25 locus was found in 1 of the MSI-H pediatric cases. Thus, 2 unstable cases showed no instability of this marker. For BAT-26, such a discordance was even more profound: in 1 of MSI-H cases, we obtained no PCR product and the remaining 3 showed no alterations of this marker. MSH2 (Human MutS, Homologue2) protein was detected in all but 3 pediatric cases (1 highly unstable and 2 low-level unstable) and in all adult cases. MLH1 (Human MutL, Homologue 1) protein was detected in all but 2 pediatric cases (1 highly unstable and 1 low-level unstable). Thus, 2 highly unstable pediatric cases showed no detectable MLH1/MSH2 proteins. Our data support earlier observations that MSI occurs predominantly in malignant astrocytic tumors of young patients, which lends support to the hypothesis of different molecular mechanisms of pediatric brain tumors. Surprisingly, we found no significant correlation between the status of 10 microsatellite markers and that of either BAT25 or BAT26 loci or with the expression of MMR genes.

• Oda S, Maehara Y, Sumiyoshi Y, Sugimachi K
• Microsatellite instability in cancer: what problems remain unanswered?
• Surgery. 2002; 131: 5562-5562
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• Analyses of microsatellite instability have been prevalent, particularly in the field of oncology. However, the literature on this subject is diverse. The discrepancies may derive from methodological problems in the conventional techniques used for analysis. Problems include low quantitativity in the detection systems, inaccurate migration in electrophoresis, and Taq polymerase-mediated modifications of polymerase chain reaction products. Indeed, use of a new fluorescent technique where these problems have been overcome has elucidated various intriguing and previously unrecognized aspects of microsatellite instability in human cancers. Patterns of microsatellite changes observed in various human cancers can be classified into 2 subtypes, those showing relatively small changes within 6 base pairs (type A) and those exhibiting drastic changes over 8 base pairs (type B). Although type A microsatellite instability has been connected to defective mismatch repair phenotype, the relationship between type B microsatellite instability and defective mismatch repair phenotype remains unclear. Nevertheless, as symbolized in cases of hereditary nonpolyposis colorectal cancer, connections between type B microsatellite instability and familial predisposition have been suggested in some cancers. The molecular background of type B microsatellite changes warrants particular attention.

• Guo RJ et al.
• Microsatellite instability of papillary subtype of human gastric adenocarcinoma and hMLH1 promoter hypermethylation in the surrounding mucosa.
• Pathol Int. 2001; 51: 240-7
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• Gastric cancer has striking heterogeneity in histological pattern, cellular phenotype, genotype, biomarkers, and biological behavior. We focused on the specific morphological papillary phenotype of gastric adenocarcinoma and attempted to identify its distinct molecular characteristics. In our comparative study, early stage papillary (papillary-dominant) gastric cancer showed a significantly higher and more widespread high-frequency microsatellite instability (MSI-H) than other morphological types. Analysis of mutations in a panel of five putative microsatellite instability (MSI)-associated genes in the MSI-H cases revealed that papillary or papillary-dominant cancer displays a unique profile of mutations compared to profiles previously reported in gastric cancer. Immunohistochemical staining and methylation analysis revealed that silencing of hMLH1 by methylation in its promoter region was responsible for the failure of mismatch repair in papillary-type gastric cancer, whereas aberrant promoter methylation of hMLH1 was not found in any cases without the unique mutator phenotype. Promoter hypermethylation of the hMLH1 genes was found to a lesser degree in the adjacent non-tumor mucosa in four of the 10 cases with tumor having the mutator phenotype. Microsatellite instability itself could not be detected in the adjacent non-tumor mucosa. Inactivation of hMLH1 expression by promoter hypermethylation may be an early event in carcinogenesis of this type of gastric cancer, preceding the development of the clear MSI phenotype of papillary carcinoma.

• Tamura G et al.
• Molecular characterization of undifferentiated-type gastric carcinoma.
• Lab Invest. 2001; 81: 593-8
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• As the great majority of gastric cancers develop histologically differentiated, and a significant proportion of differentiated-type carcinomas progress to become undifferentiated, both histological types are likely to share several common genetic abnormalities, such as p53 mutations at advanced stages. However, a subset of gastric cancers develop as undifferentiated carcinomas, including signet-ring cell carcinoma and poorly differentiated adenocarcinoma, and the molecular pathogenesis of this tumor type remains largely unknown. To characterize the molecular features of undifferentiated-type gastric carcinomas that developed as undifferentiated-type, we examined for p53, APC, and epithelial (E)-cadherin gene mutations, microsatellite alterations including loss of heterozygosity (LOH) and microsatellite instability (MSI), and hypermethylation of the E-cadherin gene promoter in 26 early undifferentiated gastric carcinomas, consisting of 14 signet-ring cell carcinomas and 12 poorly differentiated adenocarcinomas. E-cadherin expression was evaluated immunohistochemically. p53 mutations were detected in only one poorly differentiated adenocarcinoma sample (3.8%; 1/26), whereas no APC or E-cadherin mutations were found. LOH was present only at D8S261 on the short arm of chromosome 8 in 2 of 14 (14%) informative tumors, both of which were poorly differentiated adenocarcinomas, and MSI was not observed in any of the tumors. No signet-ring cell carcinomas have been found to carry gene mutations or microsatellite alterations. In contrast, hypermethylation of the E-cadherin promoter occurred in 69% (18/26) of the tumors; 57% (8/14) of signet-ring cell carcinomas, and 83% (10/12) of poorly differentiated adenocarcinomas, and was significantly associated with loss or reduced expression of E-cadherin. Thus, whereas tumor suppressor gene mutation, LOH, and MSI were less common in undifferentiated-type early gastric carcinomas, epigenetic inactivation of E-cadherin via promoter hypermethylation may be an early critical event in the development of undifferentiated tumors.

• Hinoi T et al.
• Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon.
• Am J Pathol. 2001; 159: 2239-48
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• Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and beta-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs.

• Gurin CC, Federici MG, Kang L, Boyd J
• Causes and consequences of microsatellite instability in endometrial carcinoma.
• Cancer Res. 1999; 59: 462-6
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• Genetic instability of microsatellite repeat sequences [microsatellite instability (MI)] is commonly seen in tumors associated with the hereditary nonpolyposis colorectal cancer syndrome and is a result of inactivating mutations in any of several genes involved in a particular pathway of DNA mismatch repair. Sporadic (i.e., nonhereditary) manifestations of several tumor types, including colorectal, gastric, and endometrial carcinomas, also exhibit MI in a significant fraction of cases. Many MI+ sporadic colorectal carcinomas are associated with somatic mutations of mismatch repair genes, and several genes with coding region microsatellites are frequently mutated as a result in these cancers. The molecular causes and consequences of MI in sporadic endometrial carcinomas remain obscure, however. The aims of this study were: (a) to identify a series of sporadic endometrial carcinomas with clear evidence of MI; (b) to determine the extent to which somatic alterations in mismatch repair genes are associated with this MI; and (c) to establish whether the genes containing coding region microsatellite repeats that are known to be disrupted in MI+ gastrointestinal cancers are also disrupted in MI+ endometrial carcinomas. Matched pairs of normal and tumor DNA from 57 consecutive cases of endometrial carcinoma were examined for evidence of MI using a consensus panel of microsatellite markers. Fourteen cases (25%) displayed unequivocal evidence of MI, consistent with previously published estimates of the incidence of MI+ sporadic endometrial carcinoma. These cases were subjected to a mutation screen of the coding regions and exon-intron boundaries of the mismatch repair genes MSH2 and MLH1. Although several polymorphisms were detected, no clearly deleterious mutations were found in either of these genes. Notably, however, hypermethylation of the MLH1 promoter region was identified in 10 of 14 (71%) MI+ cases. Somatic mutations in coding region microsatellite repeats in the TGFbetaIIR, IGFIIR, BAX, E2F4, MSH3, MSH6, BRCA1, and BRCA2 genes were generally rare. Four MI+ tumors (29%) contained somatic mutations in the PTEN gene, only one of which was likely the result of MI. These data indicate that somatic mutational inactivation of known mismatch repair genes does not account for the great majority of sporadic endometrial carcinomas with MI and that a significant fraction of these cases may instead be causally associated with hypermethylation of the MLH1 promoter. Furthermore, genes with coding region microsatellites that are frequently mutated in MI+ gastrointestinal cancers are rarely mutated in MI+ endometrial cancers, implying the existence of alternative molecular targets for the tumorigenic effects of MI in this tumor type.

• Fleisher AS et al.
• Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.
• Cancer Res. 1999; 59: 1090-5
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• Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers.

• Halling KC et al.
• Origin of microsatellite instability in gastric cancer.
• Am J Pathol. 1999; 155: 205-11
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• Microsatellite instability (MSI) is observed in 13-44% of gastric carcinoma. The etiology of MSI in gastric carcinoma has not been clearly defined. To assess the role of mismatch repair in the development of MSI in gastric cancer, expression of hMSH2 and hMLH1 was explored. We examined 117 gastric carcinomas for MSI and observed instability at one or more loci in 19 (16%) of these tumors. Of the 19 tumors with MSI, nine exhibited low-rate MSI (MSI-L) with instability at <17% of loci, whereas the remaining 10 exhibited high-rate MSI (MSI-H) with instability at >33% of loci examined. Immunohistochemical staining for hMLH1 and hMSH2 was performed on eight of the tumors with MSI-H, five with MSI-L, and 15 tumors without MSI. All eight tumors with MSI-H showed loss of staining for either hMLH1 (n = 5) or hMSH2 (n = 3). In contrast, tumors with MSI-L or without MSI all showed normal hMSH2 and hMLH1 protein expression patterns. Moreover, all eight of the tumors with MSI-H also showed instability at BAT-26, whereas none of the MSI-L tumors or tumors without instability showed instability at BAT-26. These findings suggest that the majority of high-level MSI in gastric cancer is associated with defects of the mismatch repair pathway. Although larger studies are needed, BAT-26 appears to be a sensitive and specific marker for the MSI-H phenotype in gastric carcinoma.

• Thompson MJ, Slack R, Double JA, Bibby MC
• Microsatellite instability, chemosensitivity and mutant frequency in a series of 1,2-dimethylhydrazine induced murine colon adenocarcinoma models.
• Int J Oncol. 1998; 13: 531-6
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• Loss of DNA mismatch repair has been described in a number of tumour types such as colorectal adenocarcinoma and leads to microsatellite instability. This may have clinical relevance due to mismatch repair defects altering chemosensitivity towards certain classes of anti-tumour agent. This study has examined microsatellite instability of eight murine colon adenocarcinoma tumour models induced by 1,2-dimethylhydrazine. Four microsatellite regions were examined suggesting that four of the tumour models exhibit a low level of microsatellite instability. Loss of heterozygosity was found in 5/8 tumours, suggesting that allelic loss may be a relatively common step in the carcinogenesis of these tumour models. Three of the allelic losses involved the D11MIT4 locus which is situated very close to the p53 tumour suppressor locus. Four tumour models are routinely cultured in vitro and these were used to examine whether there was any association between microsatellite instability, mutant frequency and chemo-sensitivity of these tumour models, comparing them with four human adenocarcinoma cell lines of known mismatch repair status. Two cell lines (MAC26 and MAC16) were found to be more chemoresistant towards cisplatin but not 6-thioguanine. No association was found between microsatellite instability and chemosensitivity for either the human or mouse cell lines.

• Macdonald GA, Greenson JK, Saito K, Cherian SP, Appelman HD, Boland CR
• Microsatellite instability and loss of heterozygosity at DNA mismatch repair gene loci occurs during hepatic carcinogenesis.
• Hepatology. 1998; 28: 90-7
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• DNA mismatch repair is an important mechanism involved in maintaining the fidelity of genomic DNA. Defective DNA mismatch repair is implicated in a variety of gastrointestinal and other tumors; however, its role in hepatocellular carcinoma (HCC) has not been assessed. Formalin-fixed, paraffin-embedded archival pathology tissues from 46 primary liver tumors were studied by microdissection and microsatellite analysis of extracted DNA to assess the degree of microsatellite instability, a marker of defective mismatch repair, and to determine the extent and timing of allelic loss of two DNA mismatch repair genes, human Mut S homologue-2 (hMSH2) and human Mut L homologue-1 (hMLH1), and the tumor suppressor genes adenomatous polyposis coli gene (APC), p53, and DPC4. Microsatellite instability was detected in 16 of the tumors (34.8%). Loss of heterozygosity at microsatellites linked to the DNA mismatch repair genes, hMSH2 and/or hMLH1, was found in 9 cases (19.6%), usually in association with microsatellite instability. Importantly, the pattern of allelic loss was uniform in 8 of these 9 tumors, suggesting that clonal loss had occurred. Moreover, loss at these loci also occurred in nonmalignant tissue adjacent to 4 of these tumors, where it was associated with marked allelic heterogeneity. There was relatively infrequent loss of APC, p53, or DPC4 loci that appeared unrelated to loss of hMSH2 or hMLH1 gene loci. Loss of heterozygosity at hMSH2 and/or hMLH1 gene loci, and the associated microsatellite instability in premalignant hepatic tissues suggests a possible causal role in hepatic carcinogenesis in a subset of hepatomas.

• Zhou X, Fang D, Luo Y
• [Microsatellite instability in gastric carcinoma and intestinal metaplasia].
• Zhonghua Bing Li Xue Za Zhi. 1998; 27: 356-8
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• OBJECTIVE: To investigate the role of microsatellite instability (MSI) in the carcinogenesis and development of gastric cancer (GC). METHODS: MSI was examined in 50 surgically resected gastric cancer specimens and 15 intestinal metaplasia specimens using PCR based methods. RESULTS: MSI was detected in 27/50 of GC and 3/15 of intestinal metaplasia at one or more loci. MSI was positive in all three cases of early GC. The incidence of MSI in well differentiated GC (86.6%) was significantly higher than that in poorly differentiated GC (P < 0.05). The frequency of MSI in intestinal type of GC (77.8%) was significantly higher than that in the diffused type (40%, P < 0.05). No significant correlation was found between MSI and age, sex, size, location, lymph node metastasis or clinical stage of GC. CONCLUSION: MSI is an early molecular marker in the carcinogenesis of GC and may play an important role in the development of GC.

• Hayden JD et al.
• Assessment of microsatellite alterations in young patients with gastric adenocarcinoma.
• Cancer. 1997; 79: 684-7
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• BACKGROUND: Genetic factors are probably important in the development of gastric carcinoma in young patients (younger than 40 years). The authors investigated early onset primary gastric adenocarcinomas for the presence of microsatellite instability, which is a phenotypic marker for the hereditary nonpolyposis colon carcinoma syndrome. METHODS: DNA was extracted from archival microdissected carcinoma and corresponding normal tissue from 10 British gastric carcinoma patients age 19 to 39 years at the time of diagnosis. A panel of 12 microsatellite loci were amplified by fluorescent polymerase chain reaction and analyzed using an automated DNA sequencer. RESULTS: There was no evidence of microsatellite instability. In contrast, allelic imbalance was recorded at D3S966, D3S1076, D10S197, D11S904, P53, NM23, and DCC microsatellite loci. CONCLUSIONS: The authors reported ten cases of early onset gastric carcinoma that demonstrated allelic imbalance but no evidence of instability at microsatellite loci. It is unlikely that defective DNA mismatch repair is important in this group of young patients.

• Dietmaier W, Wallinger S, Bocker T, Kullmann F, Fishel R, Ruschoff J
• Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression.
• Cancer Res. 1997; 57: 4749-56
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• Alterations of the length of simple repetitive genomic sequences (microsatellite instability, MSI) characterize a distinct mechanism of colorectal carcinogenesis. Such MSI has been found to be associated with hereditary nonpolyposis colorectal cancer (HNPCC) that involves mutation of the human mismatch repair genes hMSH2 and hMLH1 as well as many sporadic cancers of most tissue types. Although the study of MSI status is a useful tool for HNPCC screening and for the determination of tumor prognosis in sporadic cases of colorectal cancer, the reliability of MSI diagnosis is still a subject of debate. Here we have examined 58 primary colorectal tumors (selected from a cohort of 200) using 31 microsatellite markers that comprised the most frequent simple repeat types. The expression of the hMSH2 and hMLH1 mismatch repair proteins was studied by immunohistochemistry, and most patients were surveyed for at least 2 years. Reproducibility of gel interpretation, as well as diagnostic sensitivity and specificity of the MSI status, were determined. We found that unambiguous determination of band shifts as well as MSI diagnosis were closely related to the type of the marker repeat and that MSI could be subdivided into "high" MSI (>20% unstable loci), "low" MSI (<10% unstable loci), and microsatellite stable (0% unstable loci). One-half of the patients with high MSI tumors (n = 8) fulfilled either the Amsterdam criteria (n = 4), had at least one relative with HNPCC-related carcinoma (n = 2), or were diagnosed with colorectal cancer at an age below 45 years (n = 2). Fourteen of the 15 high MSI tumors had lost either hMSH2 (n = 8) or hMLH1 (n = 6) protein expression. In contrast, all of the low MSI tumors and the MSI-negative tumors displayed normal expression of hMSH2 and hMLH1. These studies provide a clear recommendation for the uniform use of a panel of 10 microsatellites and a definition of at least 40% instability (using these defined marker loci) in the diagnostic analysis of MSI.

• Lin JT, Wu MS, Shun CT, Lee WJ, Sheu JC, Wang TH
• Occurrence of microsatellite instability in gastric carcinoma is associated with enhanced expression of erbB-2 oncoprotein.
• Cancer Res. 1995; 55: 1428-30
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• To investigate the molecular mechanism of gastric carcinogenesis, we examined simultaneously the frequency of microsatellite instability and the immunoreactivities to ras, erbB-2, and p53 in 42 gastric adenocarcinoma tissues. Microsatellite instability, measured by DNA replication error, was detected in 33.3% (14/42) of patients with gastric carcinoma while positive immunostaining was demonstrated in 3.1% (1/32) for ras, 40.5% (17/42) for erbB-2, and 28.6% (12/42) for p53. There was no statistical difference between the intestinal type and the diffuse type of carcinoma with respect to microsatellite instability, ras, or erbB-2 expression. The expression of p53 occurred more frequently in the intestinal type of carcinoma (41.7%, 10/24) than in the diffuse type of carcinoma (11.1%, 2/18; P < 0.01). There was no association between microsatellite instability and ras or p53 expression, while enhanced expression of erbB-2 occurred more frequently in carcinomas with microsatellite instability (64.3%, 9/14) than in those without microsatellite instability (28.6%, 8/28; P < 0.05). Such a strong association between microsatellite instability and erbB-2 oncogene may be responsible for the increase of other oncogenic mutations and tumor progression in gastric carcinogenesis.

• Risinger JI et al.
• Microsatellite instability in gynecological sarcomas and in hMSH2 mutant uterine sarcoma cell lines defective in mismatch repair activity.
• Cancer Res. 1995; 55: 5664-9
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• We have examined a panel of gynecological sarcomas for microsatellite instability. The genomic DNA from 11 of 44 sarcomas contained somatic alterations in the lengths of one or more di-, tri-, tetra-, or pentanucleotide microsatellite sequence markers, and 6 of these cases had alterations in two or more markers. In addition, di-, tri-, and tetranucleotide microsatellites were found to be highly unstable in single cell clones of two cell lines derived from a uterine mixed mesodermal tumor. Since such instability is characteristic of cells defective in postreplication mismatch repair, we examined mismatch repair activity in extracts made from these lines. Both extracts were repair deficient, while an extract of another gynecological sarcoma cell line not exhibiting microsatellite instability was repair proficient. The repair deficiency was complemented by a colon tumor cell extract that was defective in the hMLH1 protein but not by an extract defective in hMSH2 protein. This suggested that the defect in the uterine sarcoma line could be in hMSH2. Subsequent analysis of the gene revealed a 2-bp deletion in exon 14, leading to premature truncation of the hMSH2 protein at codon 796 and no detectable wild-type gene present. These data suggest that the microsatellite instability observed in these cell lines, and possibly in a significant number of gynecological sarcomas, is due to defective postreplication mismatch repair. There was no apparent correlation with microsatellite instability and clinical outcome.