Allelic imbalance in chromosome band 18q21 and SMAD4 mutations in ovarian cancers.

Recently, three candidate tumor suppressor genes, SMAD2 (MADR2/JV18-1), SMAD4 (DPC4), and DCC, were identified in chromosome band 18q21. We examined allelic imbalance (AI) in 18q21 using six polymorphic microsatellite markers in 38 primary ovarian cancers and four ovarian borderline tumors. AI at one or more loci was detected in 15 of 37 (41%) informative ovarian cancers and in none of the four borderline tumors. Frequent AI was detected at the D18S46 (31%) and D18S474 (36%) loci, which were adjacent to the SMAD4 gene, and at the D18S69 (33%) locus, which was telomeric to the DCC gene. Therefore, we searched for mutations of the SMAD4 gene in 42 primary tumors and eight cell lines by PCR-SSCP and sequencing analyses. Missense mutations were detected in two ovarian tumors and three ovarian cancer cell lines, whereas silent mutation was detected in a primary ovarian cancer. These results suggest that there are at least two tumor suppressor genes on chromosome arm 18q and that SMAD4 is of importance in ovarian tumorigenesis.     

Two somatic biallelic lesions within and near SMAD4 in a human breast cancer cell line

Loss of chromosome arm 18q is a common event in human pancreatic, colon, and breast cancers and is often interpreted as representing loss of one or more tumor-suppressor genes. In this article, we describe two novel biallelic deletions at chromosome band 18q21.1 in a recently characterized human breast cancer cell line, HCC-1428. One lesion deletes a fragment of approximately 300 kb between SMAD4 and DCC that encodes no known genes. The second lesion is an in-frame SMAD4 deletion (amino acids 49-51) that affects the level of SMAD4 protein but not the SMAD4 message. This change accelerates 26S proteasome-mediated degradation of both endogenous and exogenous mutant SMAD4. Examination of normal DNA from the same patient demonstrated that both lesions are somatic and associated with loss of both normal alleles. These data support the concept that two independent tumor-suppressor loci exist at chromosome segment 18q21.1, one at SMAD4 and the other potentially at an enhancer of DCC or an unrelated novel gene.     

High resolution analysis of chromosome 18 alterations in ulcerative colitis-related colorectal cancer

We previously have demonstrated by comparative genomic hybridization that 80% of ulcerative colitis-related cancers show loss of all or part of chromosome 18, the site of at least three candidate tumor suppressor genes: DCC, SMAD2, and SMAD4. To determine whether these genes are targeted in colitis-related carcinogenesis, we performed a high-resolution analysis of chromosome 18 alteractions in 32 colitis-related colorectal cancers by assessing allelic imbalance at 11 microsatellite markers distributed along the chromosome, and by the quantitative polymerase chain reaction (PCR) method (TaqMan). TaqMan analysis was used to determine the relative copy number of five test genes on chromosome 18 (PACAP on 18p and DCC, SMAD2, SMAD4, and GALNR on 18q). We found allelic imbalance, as assessed by loss of heterozygosity, in at least one marker on chromosome 18 in 25 of the 29 tumors (86%) successfully tested. In 14 tumors, allelic imbalance was detected at all informative markers on 18q, while the other 11 tumors showed only partial loss. Allelic imbalance was most commonly detected at D18S363 (78% of informative cases). This marker is in closest proximity to SMAD4. By quantitative PCR analysis, a relative loss of copy number of SMAD2, SMAD4, and DCC were detected in 40%, 57%, and 53%, respectively, of the colitis-related cancers. SMAD2 was retained in four tumors having loss of SMAD4 and DCC. Loss of SMAD4 alone was seen in one tumor. The present data indicate that the loss of SMAD4 and DCC occurs in the majority of colitis-related cancers.     

Identification of a novel 9 cM deletion unit on chromosome 6q23-24 in papillary serous carcinoma of the peritoneum

To define regions of deletion on chromosome 6q in papillary serous carcinoma of the peritoneum (PSCP), we analyzed 103 tumor tissues from 53 patients by using 11 polymorphic microsatellite markers spanning loci from 6q23 to 6q27. Allelic losses on 6q were observed in 42 of 53 (79.2%) cases. We identified 3 distinct regions with a high percentage (>40%) of loss of heterozygosity. The first region is located at 6q23-24 and defined by D6S311 (15 of 35 informative cases, 42.9%). Detailed deletion mapping of chromosome 6q23-24 in these tumor samples identified a novel 9 cM minimal deletion region flanked by D6S250 and ESR. The second one is located at 6q25.1-25.2 and defined by D6S448 (17 of 36 informative cases, 47.2%). A second minimal deletion region of 4 cM was flanked by D6S420 and D6S442. The third region is located at 6q27 and defined by D6S297 (9 of 19 informative cases, 47.4%). Comparing these results with our cases of advanced staged invasive serous epithelial ovarian carcinoma (SEOC), we observed that allelic losses at D6S311 (6q23) and D6S149 (6q27) were significantly higher for PSCP than for SEOC. The pattern of allelic loss at each tumor site within an individual patient was also studied. A total of 36 cases displayed allelic loss for at least 1 of multiple tumor sites, and 35 of these patients exhibited nonidentical patterns of allelic loss at various tumor sites of the same patient. Furthermore, an alternating pattern of allelic loss in the same patient was identified in 3 of 53 patients studied. These results show that allelic losses on 6q are very frequent in PSCP, and we show 2 discrete minimal deletion regions on 6q, suggesting the existence of at least 2 tumor suppressor genes within 6q that may be involved in the pathogenesis of PSCP. In addition, the finding of different patterns of allelic loss at different tumor sites within the same patient indicate a mutifocal origin in some PSCP cases. These results provide strong evidence to support our previous reports that PSCP is a multifocal disease entity.     

Substantial reduction of the gastric carcinoma critical region at 6q16.3-q23.1.

Deletions of the long arm of chromosome 6 are a common event in gastric carcinomas. In a previous study, deletion mapping of 6q identified two smallest regions of overlap (SROs) of heterozygous deletions: one interstitial, spanning 12-16 cM, bordered by D6S268 (6q16.3-q21) and ARG1 (6q22.3-q23.1), and one distal to IFNGR1 (6q23-q24), spanning more than 30 cM. Loss of heterozygosity (LOH) of the interstitial SRO was detected in 50% of informative tumors. We analyzed 60 primary gastric tumors with 19 highly polymorphic markers from 6q16.3-q23.3 to delimit the interstitial SRO further. Of the 50 tumors that were informative for at least one locus, 18 (36%) showed allelic imbalance (AI). The overlap of these cases allowed us to define an SRO of approximately 3 Mb flanked by D6S278 and D6S404. AI or LOH of this region occurs in all histologic types of gastric carcinoma and in early stages of development, indicating that loss of a gene from this region of 6q is a crucial step in a main route of gastric carcinogenesis. For cases with retention of 6q, alternative routes of gastric carcinogenesis may exist. Genes Chromosomes Cancer 26:29-34, 1999.     

Loss of cables, a novel gene on chromosome 18q, in ovarian cancer.

Cables, a cyclin-dependent kinase (cdk) interacting protein, has recently been identified and mapped to human chromosome 18q11. Cables appears to be primarily involved in cell cycle regulation and cell proliferation. Overexpression of Cables in Hela and other cell lines inhibits cell proliferation and tumor formation. We hypothesize that loss of Cables expression is associated with ovarian cancer. To test our hypothesis, we examined Cables expression in the four most common subtypes of ovarian carcinomas: serous, endometrioid, mucinous, and clear cell. In addition, mucinous and serous borderline tumors were also included. Loss of Cables expression was observed at high frequency in ovarian serous (11 of 14 cases, 79%) and endometrioid (5 of 10 cases, 50%) carcinomas. In contrast, strong Cables staining was detected in all clear cell carcinomas (10 cases) and mucinous tumors (5 carcinomas and 5 borderline tumors). The majority of serous borderline tumors (11 of 14 cases, 79%) showed positive Cables staining, with the rest showing focal loss of Cables expression. Furthermore, RT-PCR revealed the lack of Cables mRNA in a human ovarian cancer xenograft. No correlation was noted between loss of Cables and histologic grade, tumor stage, and survival. In conclusion, our results indicate that loss of Cables is common in ovarian serous and endometrioid carcinomas and imply that Cables may be involved in the pathogenesis of these two types of ovarian carcinomas.     

LOH at the sites of the DCC, APC, and TP53 tumor suppressor genes occurs in Barrett's metaplasia and dysplasia adjacent to adenocarcinoma of the esophagus

Barrett's esophagus carries a 30- to 100-fold increased risk of adenocarcinoma, which is thought to develop via a metaplasia-dysplasia-carcinoma progression. A common genetic abnormality detected in Barrett's adenocarcinoma is loss of heterozygosity (LOH) at the sites of known or putative tumor suppressor genes, of which there are at least 9 associated with esophageal adenocarcinoma. The aim of this study was to identify at which histological stage of carcinogenesis LOH at these sites occur. Microdissection of multiple paraffin-embedded tissue blocks from 17 esophagogastrectomy specimens of adenocarcinoma arising in Barrett's esophagus yielded areas of metaplasia, low-, intermediate- and high-grade dysplasia, and carcinoma. LOH analysis of microdissected tissues was performed using a double polymerase chain reaction technique with 11 microsatellite primers shown previously to have LOH in at least 30% of esophageal adenocarcinomas. Identical LOH was detected in premalignant and malignant tissues in 4 of 17 patients, and was located at 5q21-q22 (D5S346 primer), 17p11.1-p12 (TCF2 primer), 17p13.1 (TP53 primer), 18q21.1 (detected in colon cancer tumor suppressor gene [DCC] primer), and 18q23-qter (D18S70 primer). These results suggest that LOH at the sites of the DCC, adenomatous polyposis coli (APC), and TP53 tumor suppressor genes occur before the development of adenocarcinoma in Barrett's esophagus, and so merit further study as potential biomarkers of neoplastic progression in patients with Barrett's esophagus undergoing endoscopic and histological surveillance.     

Molecular and cytogenetic analysis of chromosome 7 in uterine leiomyomas

Uterine leiomyomas are benign tumors that arise clonally from smooth muscle cells of the myometrium. Cytogenetic studies of uterine leiomyomas have shown that about 40% have chromosome abnormalities and that deletion of 7q is a common finding. The observations suggest the possible location of a growth-suppressor gene within the 7q21-q22 region. Molecular genetic analysis of cytogenetically normal tumors has frequently shown somatic loss of specific tumor suppressor genes detected by loss of heterozygosity in the critical region. To test the hypothesis that chromosome region 7q21-q22 contains a growth-suppressor gene involved in the development of leiomyomas, we examined 92 leiomyomas for allelic loss of 7q markers spanning the cytogenetically defined critical region. Forty tumors with cytogenetically defined 7q deletion, 45 tumors without cytogenetically visible 7q deletion, and seven tumors with no cytogenetic information were examined for allelic loss of loci D7S489, D7S440, D7S492, D7S518, D7S471, D7S466, and D7S530. Loss of heterozygosity for one or more of these loci was observed in 23 of 40 (57.5%) of the tumors with deletion of 7q and in 2 of 45 cases without a cytogenetically visible deletion. The tumors with cytogenetic deletion of 7q, but no loss of 7q21-q22 markers, were mosaics, with only a minority of cells containing the cytogenetic deletion. The critical region of loss is defined by the markers D7S518 and D7S471, each showing loss in approximately 50% of informative cases. These markers define a 10 cM region of 7q21-q22 that is consistent with the cytogenetically defined smallest region of overlap and exclude loss of the MET oncogene locus and WNT1, the murine mammary tumor-virus integration site, from the critical region. Our results further define a region that is consistently lost in leiomyomas with cytogenetic deletion of chromosome arm 7q. This region may contain a tumor suppressor gene involved in the development of a subset of leiomyomas.     

Combined copy status of 18q21 genes in colorectal cancer shows frequent retention of SMAD7

Deletions of chromosome band 18q21 appear with very high frequency in a variety of carcinomas, especially in colorectal cancer. Potent tumor suppressor genes located in this region encode transforming growth factor beta (TGF-beta) signal transducers SMAD2 and SMAD4, and inactivation of either one leads to impaired TGF-beta-mediated cell growth/apoptosis. Following the assignment of SMAD7 to 18q21, we first refined the SMAD7 gene position within this region by genetically mapping SMAD7 between SMAD2 and SMAD4. Further, to compare the respective frequencies of genetic alterations of these three SMAD genes in colorectal cancer, we undertook a large-scale evaluation of the copy status of each of these genes on DNA samples from colorectal tumor biopsy material. Among a subset of 233 DNA samples for which data were available for all four genes, SMAD4, SMAD2, and the nearby gene DCC showed high deletion rates (66%, 64%, and 59%, respectively), whereas SMAD7 was deleted in only 48% of the tumors. Unexpectedly, we found some gene duplications; SMAD7 appears to be more frequently amplified (10%) than the three other genes (4-7%). Compiled data for SMAD genes in each tumor show that the most common combination (26% of all the tumors) consists of the simultaneous deletions of SMAD2 and SMAD4 associated with normal diploidy or even duplication of SMAD7. Since SMAD7 normally counteracts SMAD2 and SMAD4 in TGF-beta signaling, we hypothesize that the tumor might not benefit from simultaneous SMAD7 inactivation, thereby exerting selective pressure to retain or even to duplicate the SMAD7 gene.     

Diverse tumorigenic pathways in ovarian serous carcinoma

This study was undertaken to analyze genetic alterations in 108 sporadic serous ovarian neoplasms to elucidate ovarian serous carcinogenesis. Our results demonstrate that K-ras mutations occur in approximately 50% of serous borderline tumors (SBTs), non-invasive micropapillary serous carcinomas (MPSCs), and invasive micropapillary serous carcinomas, which represent a morphological continuum of tumor progression. Moreover, progressive increase in the degree of allelic imbalance of chromosomes 1p, 5q, 8p, 18q, 22q, and Xp was observed comparing serous borderline tumors to noninvasive and invasive micropapillary serous carcinomas. In contrast, high-grade (conventional serous carcinoma) tumors contained wild-type K-ras in all 23 cases studied and a high frequency of allelic imbalance even in small (early) primary tumors similar to that found in advanced stage tumors. Based on these findings, we propose a dualistic model for ovarian serous carcinogenesis. One pathway involves a stepwise progression from SBT to noninvasive and then invasive MPSC. The other pathway is characterized by rapid progression from the ovarian surface epithelium or inclusion cysts to a conventional (high-grade) serous carcinoma.     

Systematic screening of chromosome 18 for loss of heterozygosity in esophageal squamous cell carcinoma.

Esophageal cancer ranks among the 10 most common cancers in the world, and is almost uniformly fatal. The genetic events leading to the development of esophageal carcinoma are not well established. To identify genomic regions involved in esophageal carcinogenesis, we performed a systematic screening for loss of heterozygosity (LOH) in 24 samples of squamous cell carcinomas, initially focusing the analysis on chromosome 18. Thirteen short tandem repeat markers spanning 18p and 18q were used. We found a broad peak of LOH spanning 18p11.2 and 18q21.1 with the most frequent LOH (72%) at D18S978 on 18q12.2, which coincides with a known fragile site FRA18A. This region is 4 cM proximal to known tumor suppressor genes and therefore suggests the possible existence of a yet undiscovered tumor suppressor gene.     

上皮性卵巢癌组织脆性组氨酸三联基因甲基化和3p14的等位基因丢失

目的 探讨肿瘤抑制基因脆性组氨酸三联(FHIT)基因在上皮性卵巢癌(简称卵巢癌)组织中的甲基化状态以及该基因定位的3p14位点的等位基因丢失及其在卵巢癌的发生发展中的作用。方法采用甲基化特异性聚合酶链反应(MSP)方法检测61例卵巢癌组织及10例交界性上皮性卵巢肿瘤(简称交界性卵巢肿瘤)组织FHIT基因启动子CpG岛的甲基化频率,并采用微卫星多态性标志D3S1287检测45例卵巢癌组织3p14位点杂合子丢失(LOH)和纯合子丢失(HD)状态。结果卵巢癌组织中FHIT基因甲基化频率为39．3％(24／61),其中浆液性囊腺癌为45．2％(19／42),黏液性囊腺癌为14．3％(1／7),子宫内膜样癌为33．3％(4／12);交界性卵巢肿瘤组织中FHIT基因甲基化为6／10,其中交界性浆液性囊腺瘤1／3,交界性黏液性囊腺瘤5／7。卵巢癌组织甲基化频率与临床分期、细胞分化程度相关性无统计学意义。交界性卵巢肿瘤与卵巢癌之甲基化频率差异无统计学意义。43．5％(10／23)卵巢癌显示LOH;有信息的浆液性囊腺癌中33．3％(6／18)检测到HD。FHIT基因甲基化与基因丢失之间无明显相关性。结论首次证实卵巢癌FHIT基因启动子有较高的甲基化频率,这可能是FHIT基因沉默的重要原因,在卵巢癌的发生与发展过程中起着重要的作用;同时3p14位点等位基因的丢失可使基因完全失去功能,促进肿瘤的发生。     

Deletion mapping of 18q in conventional renal cell carcinoma

Loss of heterozygosity (LOH) is frequently associated with the inactivation of tumor suppressor genes. 18q LOH has been frequently reported in colorectal cancer and lung cancer; however, allelic loss on 18q has not been investigated in renal cell carcinoma (RCC). We evaluated LOH on 18q using nine microsatellite markers in 126 with conventional RCC (cRCC). LOH was observed in more than one 18q microsatellite locus in 24 cRCC (19%). We found the highest frequency of LOH (13.5%) at 18q21.3, where the DCC gene is located. We also assessed the relationship between LOH frequency and patient clinical parameters. Patients with a family history of cancer had a significantly higher frequency of 18q LOH than those without such a history (P=0.0017). No associations were found with other parameters, including gender, tumor grade, tumor stage, smoking status, and body mass index. The results suggest that inactivation of tumor suppressor genes at 18q21.3, including DCC and SMAD4 as candidates, may be involved in the tumorigenesis of some conventional RCCs.     

Frequent allelic loss of 21q11.1 approximately q21.1 region in advanced stage oral squamous cell carcinoma

A fine mapping of loss of heterozygosity (LOH) was performed in oral squamous cell carcinoma (OSCC), using 12 markers on 21q11.1 approximately q21.1. We studied 43 resected primary invasive tumors and their paired normal tissues, concurrent dysplasia or carcinoma in situ in separate areas from 8 of the specimens, and 6 local recurrent carcinomas. LOH status was compared between lesions of different phases of progression within the same patient. A high frequency of LOH was observed for D21S1410, D21S120, and D21S1433 (60% each) in the primary lesions, constituting two interstitial deleted regions encompassing eight known genes. Cases showing LOH of D21S120 were significantly associated with advanced clinical stages (III and IV; P=0.02). Consistent allelic loss was observed in 64.2% of the informative cases between the precursor lesions and their corresponding invasive tumors, and in 59.5% of those between the primary lesions and their recurrent counterparts. Fewer than half of the different lesions within a given patient showed discordant allelic loss for tested markers. Our results suggest that 21q11.1 approximately q21.1 harbors tumor suppressor genes in OSCC. Genetic divergence may develop during tumor clone evolution.     

Definition of a region of loss of heterozygosity at chromosome 11q in cervical carcinoma.

Chromosomal deletions at segment 11q23-q24 have been identified in a variety of human epithelial tumors, including cervical carcinoma (CC), indicating the presence in this region of at least a tumor suppressor gene (TSG) involved in the development of these neoplasms. To localize the 11q deletion target more precisely, 54 primary cervical carcinomas were examined for loss of heterozygosity (LOH) using a panel of microsatellite DNA markers mapping to 11p.15 and spanning region 11q23-qter. Nineteen tumors were found to have LOH at chromosome 11q. The highest frequency of LOH was observed at locus APOC-3, located in 11q23.1-q23.2, which was deleted in 42% of the informative cases. In contrast, LOH was infrequent at distal 11q in current series of CC. The smallest common region of loss included APOC-3 and was defined distally by marker D11S925 in region 11q23. The present data strongly suggest that the 11q suppressor gene(s) involved in cervical tumorigenesis is likely to be located at chromosome region 11q22-q23.     

Mapping and genome sequence analysis of chromosome 5 regions involved in bladder cancer progression.

We studied the evolution of allelic losses on chromosome 5 by whole-organ histologic and genetic mapping in 234 mucosal DNA samples of 5 cystectomy specimens with invasive bladder cancer and preneoplastic changes in adjacent urothelium. The frequency of alterations in individual loci was verified on 32 tumors and 29 voided urine samples from patients with bladder cancer. Finally, deleted regions on chromosome 5 were integrated with the human genome contigs and sequence-based databases. Deleted regions on chromosome 5 involved in intraurothelial phases of bladder neoplasia defined by their nearest flanking markers and predicted size were identified as follows: q13.3-q22 (D5S424-D5S656; 38.8 centimorgan [cM]); q22-q31.1 (D5S656-D5S808; 19.2 cM), q31.1-q32 (D5S816-SPARC; 11.5 cM), and q34 (GABRA1-D5S415; 6.4 cM). The two most frequently deleted neighbor markers (D5S2055 and D5S818) mapping to q22-q31.1 defined a 9 cM region, which may contain genes that play an important role in early phases of urinary bladder carcinogenesis. Human genome database analysis provided an accurate map of deleted regions with positions of 138 known genes and revealed several smaller gene-rich areas representing putative targets for further mapping. The strategic approach presented here, which combines whole-organ histologic and genetic mapping with analysis of the rapidly emerging human genome sequence database, facilitates identification of genes potentially involved in early phases of bladder carcinogenesis.     

Novel homozygous deletions of chromosomal band 18q22 in pancreatic adenocarcinoma identified by STS marker scanning.

The identification of homozygous deletions in sporadic neoplasms has been pivotal in the positional cloning of several tumor suppressor genes. Chromosomal arm 18q harbors the DPC4, SMAD2, and DCC genes and is suspected on the basis of high frequencies of allelic loss to harbor additional tumor suppressor genes. We applied high-resolution sequence-tagged site (STS) marker scanning to a panel of 106 pancreatic adenocarcinomas to identify novel regions of homozygous deletions on 18q. Three homozygous deletions were identified. Physical mapping of these deletions showed them to be nonoverlapping, but clustered in an approximately 7- to 10-Mb region of chromosome band 18q22. Each deletion spanned physical distances of nearly 1.3 to 3 Mb. A number of transcribed genes map within these deletions. The identification of these homozygous deletions might aid in the identification of novel tumor suppressor genes on chromosomal arm 18q. Genes Chromosomes Cancer 25:370-375, 1999.