Overexpression of cyclooxygenase-2 protein is less frequent in gastric cancers with microsatellite instability.

Overexpression of cyclooxygenase-2 (COX-2) has been reported in gastric cancers. However, the relationship between expression of COX-2 and clinico-pathological or genotypic features has not been elucidated. To address the issue, expression of COX-2 protein was analyzed in 100 gastric cancers as well as 7 gastric cancer cell lines by using immunoblot analysis. Overexpression of COX-2 in cancer tissues compared with matched non-cancerous tissues was found in 70% of cases and was significantly associated with lymphatic involvement, lymph node metastasis and advanced tumor stage. Interestingly, overexpression of COX-2 was less frequent in gastric cancers with microsatellite instability (MSI) than in those without MSI (8/20 vs. 62/80, p < 0.01). Expression of COX-2 protein was detected in some gastric cancer cell lines without MSI at various levels, but not in those with MSI. Our results suggest that overexpression of COX-2 may play an important role in tumor progression of gastric cancer and also support the notion that gastric cancers with and without MSI represent distinctive pathways of carcinogenesis. We also observed a reduction of MSI phenotype after aspirin or sulindac treatment in a hMLH1-defective gastric cancer cell line SNU-1, which lacks COX-2 expression. Int. J. Cancer (Pred. Oncol.) 84:400-403, 1999.     

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.     

胃癌组织中环氧合酶-2和hMLH1及hMSH2 蛋白表达与微卫星不稳定的关系

目的 观察环氧合酶-2（COX-2）、hMLH1和hMSH2蛋白在胃癌组织中的表达,探讨其与微卫星不稳定（MSI）的关系。方法 采用Western blot和PCR技术,检测30例胃癌及相应癌旁正常组织COX-2、hMLH1和hMSH2蛋白表达及5个位点的MSI状态。结果 胃癌组织COX-2高表达率为66．7％,hMLH1、hMSH2低表达率分别为40．0％和33．3％,与癌旁正常组织差异均有统计学意义（P均〈0．05）。30例胃癌组织中微卫譬稳定（MSS）17例,MS1l3例,其中高频率MS1（MSI-H）9例,低频率MSI（MSI-L）4例。9例MS1-H中COX-2、hMLH1和hMSH2低表达者分别为6,8和5例,与MSS组差异均有统汁学意义（P均〈0．05）。结论 散发性胃癌发生过程中可能存在MSI途径。MSI途径常伴有hMI．Hl、hMSH2蛋白表达缺陷,并可能伴有COX-2表达降低。     

Distinct methylation pattern and microsatellite instability in sporadic gastric cancer.

Aberrant 5' CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor-suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5' CpG island hypermethylation of hMLH1, E-cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation-specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E-cadherin and p16 methylation respectively, and 8 (13.1%) presented high-frequency MSI (MSI-H). Of the 8 MSI-H patients, 5 presented hMLH1 methylation, whereas no low-frequency MSI (MSI-L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E-cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these 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.     

MRE11 expression is impaired in gastric cancer with microsatellite instability

Gastric carcinomas (GCs) with high-level microsatellite instability (MSI-H) are characterized by widespread mutations at coding and non-coding mononucleotide repeats. Deletions at coding mononucleotide tracts are predicted to cause frameshift mutations and alter normal protein functions. Mutations affecting non-coding mononucleotide repeats may lead to functional consequences if they occur in gene regulatory regions. To investigate whether mutations in non-coding polypyrimidine tracts within cancer-related genes may contribute to the phenotype of MSI-H GCs, we analysed the poly(T)11 tract constituting an accessory splicing signal within the intron 4 of the MRE11 gene. Mutations at the intronic MRE11 poly(T)11 were evaluated by PCR-based assay in 27 MSI-H, 22 MSI-low and 29 MSI-negative GCs derived from a well-characterized series of GCs identified in a high-risk area in Tuscany, Central Italy. Deletion of 2 and 1 bp at the MRE11poly(T)11 were identified in 33 and 48% MSI-H GCs, respectively. Biallelic mutations were frequently observed (77%) in GCs harbouring 2 bp deletions. The presence of MRE11poly(T)11 2 bp deletion was associated with a totally absent or strongly reduced MRE11 immunostaining (P < 0.001) and with a positive GC family history (P = 0.046). Immunoblotting assays confirmed the absence of MRE11 expression in GCs with a 2 bp deletion. The relatively high frequency of the MRE11poly(T)11 mutations, the occurrence of biallelic mutations and the evidence of loss of protein expression indicate MRE11 as novel mutational target in MSI-H GC. Overall, our results indicate that MSI-associated mutations occurring in non-coding repeats may affect protein expression in MSI-H GC.     

Evidence of tumor microsatellite instability in gastric cancer with familial aggregation

About 90% of gastric cancer (GC) cases appear in a sporadic setting. Nonetheless, in high incidence areas high familial aggregation rates have been recently described. Microsatellite instability (MSI) is thought to be an important molecular phenotype both in sporadic GC and in tumors of the HNPCC spectrum. The aim of this study was to assess the frequency of MSI in GC with familial aggregation. Five quasimonomorphic mononucleotide repeats (BAT-26, BAT-25, NR-24, NR-21 and NR-27) were analyzed in 250 GC patients. Seventy-five patients (30%) had at least one-first-degree family member affected by GC and 63 patients (25.2%) showed MSI. The frequency of MSI was significantly higher in patients with a positive family history of GC (38.7%) compared to patients with other tumor types within the family (15.7%) or with a negative oncological familial history (21.9%, P = 0.004). Within cases with a positive familial oncological history, the MSI frequency in families with GC only was similar to the one observed in families with GC and colon cancer (P = 0.96). Nonetheless, in families with GC and lung cancer, the frequency of MSI was significantly lower (5.6%, P = 0.007). MSI occurs in GCs with familial aggregation. Similar MSI rates have been observed in GC patients with other family members affected by GC or colon cancer. The same does not occur in families with other members affected by lung cancer. Our data seem to suggest that familial aggregation for either GC alone or gastric and colon cancer share common etiological factors in contrast to families with gastric and lung cancers. C. Pedrazzani and G. Corso are contributed equally.     

Prognostic significance of cyclooxygenase-2 overexpression in glottic cancer.

PURPOSE: Cyclooxygenase-2 (COX-2) overexpression has been associated with a poor prognosis in many cancers. However, the role of COX-2 overexpression in head and neck cancers remains undetermined. The objective of this study was to evaluate whether COX-2 is a prognostic factor in glottic cancer. EXPERIMENTAL DESIGN: This study was part of a phase III placebo-controlled randomized trial evaluating the efficacy of alpha-tocopherol in reducing second primary cancers (SPC) in head and neck cancer patients. Immunohistochemical analyses were conducted on pretreatment biopsies of 301 patients with early-stage glottic cancer treated by radiotherapy. The median value of 50% of positive tumor cells was the cutoff point used to define COX-2 overexpression. Outcomes considered in the statistical analysis were recurrence, SPC, and death. The Cox proportional hazards model was used to estimate the hazard ratios (HR) and their 95% confidence intervals (95% CI). RESULTS: The HR associated with COX-2 overexpression was 0.94 (95% CI, 0.55-1.62) for recurrence. The HR associated with SPC was 2.63 (95% CI, 1.32-5.23) for the first 3.5 years of follow-up and 0.55 (95% CI, 0.22-1.32) for the following 3.5 years. The HR associated with COX-2 overexpression was 1.57 (95% CI, 1.01-2.45) for overall mortality. CONCLUSIONS: COX-2 overexpression in glottic cancer was associated with increased overall mortality and an increased risk of SPC during the early follow-up period. Future studies are needed to explain observed effects on SPC. COX-2 expression may prove helpful in defining an individual patient's prognosis.     

微卫星不稳定性在胃癌预后及疗效预测中的意义

微卫星不稳定性(MSI)是由DNA错配修复(MMR)蛋白功能缺陷导致,与多种肿瘤发生发展、预后及疗效预测密切相关。微卫星高度不稳定(MSI-H)与错配修复蛋白缺乏(dMMR)可能是胃癌患者预后良好的预测因素,也可能是可切除胃癌化疗疗效的负性预测因素;同时MSI-H/dMMR是晚期胃癌免疫检查点抑制剂有效治疗的预测标志物,但对于晚期胃癌姑息化疗的预测作用尚未明确。文章就MSI/MMR状态与胃癌预后及疗效预测方面的相关性研究进展进行阐述。     

肺癌XPD基因表达与微卫星不稳定关系的探讨

目的:探讨肺癌中XPD基因表达及其与微卫星不稳定(MSI)的关联情况.方法:采用微卫星分析和RT-PCR方法检测了5个微卫星标记在肺癌中的状况及XPD转录水平的表达.结果:27.16%(22/81)的肺癌出现至少1个微卫星的不稳定,但是XPD没有明显的mRNA表达下调.MSI与肺癌分期、分级、淋巴结转移和患者性别等参数之间无相关性,P>0.05.结论:XPD基因微卫星的不稳定是肺癌的频发事件,与XPD基因表达可能不存在必然联系.     

Frequent microsatellite instability in oral cancer

In oder to confirm whether microsatellite instability (MI) contributes to oral carcinogenesis, we studied 30 unrelated patients with oral cancer by PCR-MI assay with 14 microsatellite markers. MI was detected in 60% of 30 primary tumors. Tn particular, 12 of these cases presented at least two loci with MI, which were considered as patients with replication error (RER). Moreover, an additional MI, which was not observed in the primary tumor and normal tissue, was observed in one lymph node metastasis. We found significant correlation between RER and lymph node metastasis. These results suggest that RER is a common event in the oncogenesis of oral cancer and may be correlated with the progression of this disease.     

胃癌组织中COX-2蛋白的表达研究

目的:探讨胃癌组织中环氧化酶-2(cyclooxygenase-2,COX-2)蛋白的表达及其与胃癌临床病理参数间的关系.方法:应用免疫组化Envision法检测手术切除的85例胃癌标本中COX-2蛋白的表达.结果:COX-2蛋白在85例胃癌标本中阳性表达率为81.2%(69/85).COX-2蛋白表达与患者的性别、年龄、肿瘤生长部位、分化程度、淋巴结转移及神经是否侵犯无明显相关性(P＞0.05).COX-2蛋白表达与肿瘤大小、Lauren分型、侵犯深度、脉管受侵及肿瘤病理分期显著相关(P＜0.05).结论:COX-2蛋白的表达与胃癌发生、发展、侵袭、转移等相关,可作为反应胃癌生物学行为的指标之一.     

胃癌微卫星不稳定性的临床研究

目的：检测微卫星不稳定性在胃癌中的发生情况,探讨其与临床病理参数的关系。方法：选取D2S123和D3S1298两个微卫星位点,采用PCR-银染法,对36例胃癌标本进行检测;胃癌微卫星不稳定性与临床病理参数的关系采用Χ^2检验或费歇尔精确检验。结果：两个位点共检出MSI阳性11例,发生率为30．6％（11／36）;与胃癌微卫星不稳定性有关的临床病理因素为肿瘤的组织学类型（P=0．048）及有无淋巴结转移（P=0．034）。结论：微卫星不稳定性在胃癌的发生及发展中可能起重要作用,是正常细胞癌变的可能途径之一;中-高分化腺癌或有淋巴结转移的胃癌患者微卫星不稳定的发生率高。     

The relationship between microsatellite instability and PTEN gene mutations in endometrial cancer

Microsatellite instability (MSI) and mutations in the PTEN gene are among the molecular alterations involved in endometrial carcinogenesis. There is conflicting information regarding to their role in this type of tumor. For this reason, we have studied both molecular lesions in a large population-based series of 205 patients with sporadic endometrial cancer. MSI was found in 41 (20.0%) of the tumors and PTEN mutations were found in 74 (36.1%). There were differences in genotype between tumors with and without MSI. Tumors with MSI showed both a higher frequency of PTEN mutations (58.5% vs. 30.4%) (p=0.002, Fisher's exact test) and a higher number of insertions or deletions (I/D) of one nucleotide within the mononucleotide tracts of the PTEN gene (45.8% vs. 11.4% out of all I/D, p=0.005). Conversely, G:C to A:T transitions in CpG dinucleotides were found mostly in microsatellite stable tumors (57.7% vs. 18.2% out of all single-base substitutions, p=0.037). Overall, 67.6% of tumors with mutated PTEN exhibited multiple mutations or allelic imbalance (AI). Multiple PTEN mutations in the same tumor were more frequent in tumors with MSI (60% vs. 25.7%); by contrast the presence of AI accompanying PTEN mutation was higher in microsatellite stable tumors (74.3% vs. 40%) (p=0.028). In addition, patients with both genetic alterations were diagnosed at more advanced stage of progression (54.2% for MSI vs. 20.0% for MSS, p=0.006), and exhibited a worse prognosis (hazard ratio [95% confidence interval]: 3.0 [1.1-13.1], p=0.034, log-rank test) than patients with only the PTEN gene mutated. Our data suggest that the DNA mismatch repair system status influences: (i) both the frequency and the mutational spectrum of PTEN; (ii) the nature of one of the hits that inactivate this tumor-suppressor gene; and (iii) the clinical condition and behavior of the patients.     

Methylation of the hMLH1 promoter in familial gastric cancer with microsatellite instability

Microsatellite instability (MSI), which is recognized as an important mechanism in tumorigenesis, has been reported in familial gastric cancers (FGC). However, genetic defects responsible for this phenotype, that is, mutations in mismatch-repair genes such as hMLH1 and hMSH2, have not been detected in most FGC cases. Earlier studies have shown that the promoter region of the hMLH1 gene was methylated in some sporadic colorectal and endometrial cancers. To determine how FGC acquire MSI, we examined the MSI status, hMLH1-protein expression and methylation status of the hMLH1-promoter region in FGC cases. Out of 9 cancers, 6 from 8 FGC kindreds showed MSI at one or more loci; no germline mutations in the hMLH1 or hMSH2 genes were detected; 4 cancers exhibiting MSI displayed aberrant hMLH1 expression: complete loss in one, decreased level in another, and partially staining pattern in the remaining 2. Methylation in the hMLH1-promoter region was found in these 4 cases. In contrast, the cancers displaying hMLH1-protein expression were not methylated in the hMLH1-promoter region. Our data show a significant association between the absence of hMLH1 expression and methylation of its promoter in FGC cases with MSI. This suggests that the mechanism of inactivation of hMLH1 is epigenetic and that there are other genes responsible for FGC.     

乳腺癌系统化疗与微卫星不稳定性的关系

目的探讨乳腺癌全身化疗与周围血微卫星不稳定的关系。方法应用PCR和免疫组化测定30例乳腺癌患者化疗前、中、后周围血单核细胞,并与正常组进行对照比较。结果90%(27/30)患者化疗后周围血中发现微卫星不稳定(microsatellitein-stability,MSI),其中6例出现杂合子丢失(lossofheterozygosity,LOH)。MSI与化疗明显相关;MSI与hMSH2呈负相关;hMSH2与治疗方法明显相关;MSI与p53、hMLH1、hPMS1和hPMS2不相关。结论化疗可导致患者周围血单核细胞MSI和LOH发生,且可能与hMSH2表达有关,从而导致部分患者发生继发血液系统异常,以及导致肿瘤基因的不稳定和增加化疗的耐药。     

幽门螺杆菌感染与胃癌与癌前病变的环氧合酶—2及p21表达的研究

目的 研究HP感染与胃癌及癌前病变中环氧合酶－2（cox-2)、p21表达的关系，以探讨HP致癌的可能机制。方法 经胃镜和病理诊断明确的病例共120例，包括慢性浅表性胃炎（CSG）、肠上皮化生、不典型增生、胃癌各30例。以ABC免疫组化检测上述标本的cox-2、p21，以改良Giemsa法检测HP。结果 胃癌组肠型胃癌HP阳性率为83．3％（20／24），弥漫型6例中HP阳性1例。胃癌HP阳性率与CSG比较差异有高度显著性（P＜0．01），肠型胃癌HP阳性率与胃癌阳性率比较有显著性差异（P＜0．05）。各组HP阳性的cox-2及p21与其HP阴性者作相应比较均有显著或高度显著性差异（P＜0．05－0．01）。结论 提示胃癌可能与HP感染有关，而HP感染与肠型胃癌的关系更为密切，HP感染可使胃黏膜中cox-2过度表达，导致细胞凋亡受抑制，并通过使癌基因激活从而可能引起胃癌的发生。     

Differences in genomic instability between intestinal- and diffuse-type gastric cancer

Background Microsatellite instability (MSI) and loss of heterozygosity (LOH) are lesions in the genome found with different frequencies in gastric carcinomas (GCAs). Despite a great body of studies, no systematic approach to the detailed classification of MSI and LOH in the two major types of GCA has been published. Methods Thirty-seven advanced GCAs, 25 intestinal-type (IGCAs) and 12 diffuse-type (DGCAs), were assayed with 15 autosomal tetranucleotide markers on 14 chromosomal arms. The observed frequencies and types of microsatellite alterations allowed stratification into subgroups, i.e., high- and low-grade MSI (MSI-H, MSI-L) or microsatellite-stable (MSS), and high- or low-grade, or non-detectable LOH (LOH-H, LOH-L, LOH-N). Results Collectively, the markers detected MSI-H tumors with sensitivity equal to that of BAT-26 (a single marker highly specific for MSI-H). Likewise, the markers detected LOH at chromosomal arms 5q, 18q, and 21q with a sensitivity equal to markers used previously. Seven (19%) MSI-H and six (16%) LOH-H tumors were found, with a significant association (P = 0.027) with IGCA: 92% of MSI-H and LOH-H occurred in IGCA patients only. Conversely, in DGCA, a significantly higher prevalence of a stable (LOH-N/MSS) phenotype was found as compared with IGCA (75.1% vs 28.0%; P = 0.035). The MSI-L phenotype was found in 57.9% of non-MSI-H IGCA tumors and was associated significantly (P = 0.015) with LOH-H. Conclusion A clear difference in genomic instability between IGCA and DGCA was found. In IGCA, the MSI and LOH pathways were more commonly involved, whereas in DGCA, a stable phenotype was predominant. As a novel finding, MSI-L as a true phenomenon and its association with LOH was observed in IGCA.