Targeted next generation sequencing reveals a common genetic pathway for colorectal cancers with chromosomal instability and those with microsatellite and chromosome stability

IntroductionMicrosatellite stable sporadic colorectal cancers (CRCs) can be classified as either tumours with chromosomal instability (CIN+) or tumours that are ‘Microsatellite and Chromosomal Stable’ (MACS). The CIN + tumours are aneuploid whilst MACS are near-diploid; little else is known about their differences. We compared the mutation profiles of CIN + and MACS CRCs.MethodTargeted Next Generation Sequencing for mutation in 26 driver genes (TruSight-26 kit) was undertaken in 46 CIN + and 35 MACSCRCs. Tumours were compared for mutation frequency, allelic imbalance and clonal heterogeneity.ResultsMutations were detected in 58% genes and, overall, mutation in driver genes was at expected frequencies. Comparison of classes revealed similar mutation frequencies in most genes and allelic imbalance atAPC and TP53. Differences were seen in mutation frequency in KRAS (41% CIN+ vs 68% MACS, p = 0.015) and GNAS (0% CIN+ vs 12% MACS, p = 0.032). Twenty percent CIN + CRCs harboured mutations only in TP53 - a profile not seen in the MACS tumours (p = 0.009). None of the differences were significant after multiple testing corrections.ConclusionsThe mutation profiles of CIN and MACS CRCs are similar. The events allowing aneuploidy (or forcing retention of diploidy) remain unknown.     

Contribution of microdissection for the detection of microsatellite instability in colorectal cancer

The determination ofmicrosatellite instability (MSI) is an important step in the identification of familial colorectal cancer such as hereditary nonpolyposis colon cancer. It could also be of interest in the therapeutic management of sporadic cancer. International criteria for the determination of MSI have been published, recommending the use of microdissection. The aim of this work was to evaluate the impact of contaminant normal DNA in tumor samples for MSI assessment in colorectal cancer using a microdissection technique. We performed a comparative analysis of the microsatellite status between total DNA (DNA extracted from whole tumor samples) and microdissected DNA in 3 different regions from 23 cases of colorectal cancer. Six microsatellites were amplified using fluorescent polymerase chain reaction. We analyzed 9 cases with MSI and 14 cases without instability, with similar results between total DNA and microdissected DNA. Moreover, within a same tumor, the MSI phenotype was observed regardless of the region analyzed. Thus, this work shows the reproducibility of the MSI phenotype throughout a tumor. However, we observed a regional heterogeneity of the MSI profile, consisting of variations in the number and the size of unstable alleles within different regions. This result reflects the genetic heterogeneity of colorectal cancer with MSI. In the 14 cases without instability, we observed an increase of more than 60% in the loss of heterozygosity detection rate after microdissection. Thus, this work confirms the contribution of microdissection for loss of heterozygosity assessment.     

Clinicopathologic characteristics of colorectal cancer with microsatellite instability

Colorectal cancer (CRC) can be classified according to the level of microsatellite instability (MSI) exhibited by the tumor. The aim of this study was to determine MSI status in CRC from Tunisia and to identify clinical and pathological characteristics of MSI-H tumors.     

Genetic progression in sporadic endometrial and gastrointestinal cancers with high microsatellite instability

This study selected a series of 136 MSI-H (microsatellite instability at high frequency) gastric, colorectal, and endometrial carcinomas combining immunohistochemical analysis for hMLH1 or hMSH2 gene products and microsatellite study. The clinico-pathological profile of all tumours was correlated with the overall instability rates at coding and non-coding repeats, in order to clarify the role and the mutation timing of seven target genes (TGFbetaRII, IGFIIR, BAX, hMSH3, hMSH6, CHK1, and BRCA2) in the progression of an MSI-H neoplasm. Regardless of the primary site, the results confirm a model of oncogenesis in which inactivation of hMLH1, or less frequently hMSH2, may initiate a pathway culminating in a progressive accumulation of frameshifts in coding region (CDR) microsatellites. Comparing gastrointestinal and endometrial tumours, significantly lower levels of microsatellite instability at both coding and non-coding repeats were observed. Among gastric and colorectal tumours, the detection of small shortening within Bat-26 and Bat-25 markers defines a subgroup of MSI-H gastrointestinal tumours invariably characterized by early stage at diagnosis. In these tumours, mutations of TGFbetaRII or BAX genes precede frameshifts in the other tested genes. The analysis of correlations between the mutational and clinico-pathological profiles of advanced gastrointestinal tumours revealed that the higher levels of microsatellite instability at both coding and non-coding repeats were not associated with a more advanced clinico-pathological stage or a less favourable outcome. A significant association was observed between a low number of CDR frameshifts and the presence of lymph-node metastasis in advanced gastrointestinal tumours. The existence of advanced MSI-H tumours with more aggressive behaviour and a 'mild mutator phenotype' could be explained by hypothesizing an overlapping of different mechanisms of tumourigenesis, including both the mutator and the suppressor pathways; this should be tested by further studies.     

Clinical significance of microsatellite instability in sporadic epithelial ovarian tumors

PURPOSE: We evaluated the expression of microsatellite instability (MSI) in sporadic ovarian tumors using 5 standard and 9 new MSI markers to determine the clinical significance of MSI in sporadic epithelial ovarian tumors. MATERIALS AND METHODS: MSI was examined in 21 borderline and 25 malignant ovarian tumors. Polymerase chain reaction (PCR) was performed using the 5 markers recommended by the National Cancer Institute (NCI) for colon cancer and 9 additional markers. MSI was determined using fractional analysis by mixing the PCR products and size markers. RESULTS: Using the 5 conventional MSI markers, MSI was found in 4 of 46 (8.6%) ovarian tumors, including 2 of 21 (9.5%) borderline ovarian tumors and 2 of 25 (8%) malignant ovarian tumors. Using the 9 additional MSI markers, MSI was observed in 7 of 46 (15.2%) ovarian tumors, including 3 of 21 (14.3%) borderline ovarian tumors and 4 of 25 (16%) malignant ovarian tumors. There was no statistically significant difference between MSI and clinicopathological factors, including histology and stage, although there was a trend toward an increased incidence of MSI in the serous type. CONCLUSION: MSI was infrequent in ovarian tumors, including both borderline and malignant tumors. MSI was found to be uncommon in sporadic ovarian tumors, even by using additional MSI markers. The clinical significance of MSI is not strong in patients with sporadic ovarian tumors.     

伴有微卫星不稳定性的散发性结直肠癌转化生长因子-betaⅡ型受体基因突变的研究

目的:为了探讨在散发性结直肠癌的发生过程中转化生长因子βⅡ型受体基因(RII)的突变与微卫星不稳定性(RER)间的关系。方法: 我们应用PCR-SSCP-银染方法检测了50例散发性结直肠癌中的RER状态及RII基因突变情况(近端结肠19例,远端31例)。结果: RER+的共有13例(8例在近端,5例在远端),RII基因突变的共有5例。所有5例RII基因突变者均同时伴有RER+,而所有RER-病例均无RII基因突变。其中4例RII基因突变者位于回盲部肿瘤中。结论: 这些数据提示在散发性结直肠癌中,尤其是在回盲部肿瘤中,TGF-β RII基因的A₁₀重复序列是微卫星不稳定性的靶点,在肿瘤形成过程中可能起重要作用。     

喉癌p16区域的杂合性丢失和微卫星序列不稳定性分析

目的 缩小喉癌抑癌基因的寻找范围,探讨Ｐ１６基因在喉癌发生中的作用。方法 选择Ｐ１６基因附近５个微卫星多态标记对６０例喉癌进行杂合性丢失和微卫星序列不稳定性分析。结果 ５个标记在喉癌中杂合性丢失频率均不高,最高仅达２３．１％,但２个标记的微卫星序列不稳定性的频率较高,其中示记微卫星序列恶性循环频率高达４６．１％。结论 提示Ｐ１６基因在喉癌的发生中不以缺失为主,在Ｄ９Ｓ１７５２附近可能存在参与喉癌演     

Mutational and expressional analysis of SMC2 gene in gastric and colorectal cancers with microsatellite instability

Structural maintenance of chromosomes 2 (SMC2) gene encodes condensin complexes that are required for proper chromosome segregation and maintenance of chromosomal stability. Although cells with defective chromosome segregation become aneuploid and are prone to harbor chromosome instability, pathologic implications of SMC2 gene alterations are largely unknown. In a public database, we found that SMC2 gene had mononucleotide repeats that could be mutated in cancers with microsatellite instability (MSI). In this study, we analyzed these repeats in 32 gastric cancers (GC) with high MSI (MSI‐H), 59 GC with low MSI (MSI‐L)/stable MSI (MSS), 43 colorectal cancers (CRC) with MSI‐H and 60 CRC with MSI‐L/MSS by single‐strand conformation polymorphism (SSCP) and DNA sequencing. We also analyzed SMC2 protein expression in GC and CRC tissues using immunohistochemistry. We found SMC2 frameshift mutations in two GC and two CRC that would result in truncation of SMC2. The mutations were detected exclusively in MSI‐H cancers, but not in MSI‐L/MSS cancers. Loss of SMC2 expression was observed in 22% of GC and 25% of CRC. Of note, all of the cancers with SMC2 frameshift mutations displayed loss of SMC2 expression. Also, both GC and CRC with MSI‐H had significantly higher incidences in SMC2 frameshift mutations and loss of SMC2 expression than those with MSI‐L/MSS. Our data indicate that SMC2 gene is altered by both frameshift mutation and loss of expression in GC and CRC with MSI‐H, and suggest that SMC2 gene alterations might be involved in pathogenesis of these cancers.     

No evidence of microsatellite instability but frequent loss of heterozygosity in primary resected lung cancer

We examined microsatellite instability and loss of heterozygosity (LOH) in primary lung tumors from 93 cancer patients, using 16 microsatellite markers. The cases studied included 87 non-small-cell lung cancers (NSCLC) and six small-cell lung cancers (SCLC). All the patients except two were current or former smokers. The microsatellite markers were all dinucleotide repeat sequences from chromosomal locations 1p, 3p, 5q, 8p, 9p, 10p, 11p, 13q, and 17q. None of the tumors showed microsatellite instability (0/93). In NSCLC, 28% (24/87) of the cases showed LOH in at least one locus, whereas, in SCLC, 67% (4/6) had allelic losses. The frequency of LOH differed between the various cell types of NSCLC. The highest frequency was seen in large cell carcinoma (3/6, 50%) followed by squamous cell carcinoma (16/43, 37%) and adenocarcinoma (5/35, 14%). The most common site of LOH was 3p, where markers D3S1284, D3S659, D3S1289, D3S966, D3S647, and D3S1038 were studied. LOH, studied with 9p markers (D9S126, D9S171, D9S162), was less common. The present results, together with earlier reports, suggest that smoking-related primary lung cancers seldom show microsatellite instability but are characterized by frequent LOH. Environ. Mol. Mutagen. 30:217–223, 1997. © 1997 Wiley-Liss, Inc.     

Frameshift mutations of Wnt pathway genes AXIN2 and TCF7L2 in gastric carcinomas with high microsatellite instability

Frameshift mutations of genes with mononucleotide repeats are features of colorectal and gastric cancers with microsatellite instability (MSI). Deregulation of Wnt pathway is involved in the mechanisms of cancer development, and mutations of the Wnt-pathway genes have frequently been detected in cancers, indicating somatic mutations are important deregulation mechanisms of the Wnt signaling in cancer development. Both AXIN2 and TCF7L2 genes in the Wnt pathway possess mononucleotide repeats in their coding sequences and are considered as candidate tumor suppressor genes. The aim of this study was to see whether AXIN2 and TCF7L2 are altered by frameshift mutations in gastric carcinomas with MSI. For this, we analyzed human AXIN2 exon 8 and TCF7L2 exon 14 in 32 gastric carcinomas with high MSI, 13 gastric carcinomas with low MSI, and 47 gastric carcinomas without MSI by a single-strand conformation polymorphism analysis. Overall, we detected 9 AXIN2 and 6 TCF7L2 frameshift mutations in the mononucleotide repeats in the cancers with MSH-H, and all of them were found in MSH-H cancers (AXIN2, 28.1%; TCF7L2, 18.8%). Of the 32 high MSI cancers, 13 cancers (40.6%) harbored at least one of AXIN2 and TCF7L2 mutation, whereas 19 cancers (59.4%) harbored neither. The present data indicate that frameshift mutations in both AXIN2 and TCF7L2 genes are common in gastric carcinomas with high MSI and suggest that these mutations may contribute to development of gastric cancers with high MSI by deregulating the Wnt signaling in the affected cancer cells.     

Epstein-Barr virus and microsatellite instability in gastric carcinogenesis

Little information is available concerning the relationship between transforming viruses and microsatellite instability (MSI). We evaluated Epstein-Barr virus (EBV) using in situ hybridization for EBV-encoded small RNAs and MSI using the polymerase chain reaction in surgically resected gastric cancer. The study subjects included 298 consecutive cases of solitary gastric carcinoma, 63 gastric carcinomas in young patients (相似文献   

中国人胃癌骨形态发生蛋白3基因遗传的不稳定和甲基化

目的 探讨骨形态发生蛋白3(BMP3)基因遗传不稳定性及异常甲基化与胃癌进展的关系,为揭示BMP3基因作用机制和胃癌发生发展机制提供实验依据. 方法 采用聚合酶链反应-单链构象多态(PCR-SSCP)技术分析中国人胃癌BMP3基因D4S2922、 D4S2964位点微卫星不稳定性(MSI)和杂合性缺失(LOH);使用甲基化特异PCR法检测BMP3基因启动子的甲基化状况. 结果 在本实验中,50例胃癌标本BMP3基因微卫星位点MSI、 LOH的阳性率分别为16.00%和20.00%,并且LOH检出率在胃癌的TNM Ⅲ+Ⅳ期高于Ⅰ+Ⅱ期(P<0.01),并随淋巴转移的发生而增高(P<0.01).45例胃癌标本中BMP3基因启动子区域甲基化阳性率为64.44%,其检出率与胃癌分化程度、浆膜浸润、淋巴转移及TNM分期均无关.此外,MSI阴性组的甲基化阳性率为71.05%, 显著高于MSI阴性组的28.58%(P<0.05). 结论 BMP3基因遗传不稳定和启动子甲基化存在于胃癌的发生发展中.LOH多发生于胃癌的晚期阶段并赋予胃癌高侵袭、预后差的表型.BMP3基因启动子高甲基化可能是胃癌发生因素之一.