Allelic imbalance on the long arm of chromosome 5 in oral squamous cell carcinoma

To obtain a more detailed estimate of chromosome 5 loci involvement in oral squamous cell carcinoma (SCC), thirty-two oral SCCs were examined for loss of heterozygosity (LOH) on the long arm of chromosome 5 (5q) by PCR-LOH assay using thirteen microsatellite markers. LOH was observed in 16 (51.6%) of 31 informative cases. The incidence or the number of regions showing LOH was significantly higher in moderately and poorly differentiated tumors than in the well differentiated ones. Among the loci tested, D5S178 exhibited LOH in 11 (39.3%) of 28 cases, suggesting this to be the site, at 5q, of a novel candidate tumor suppressor gene. These data indicate that the incidence of LOH at chromosome 5q is high and is associated with oral tumor differentiation.     

Allelic imbalance at rs6983267 suggests selection of the risk allele in somatic colorectal tumor evolution

A common single nucleotide polymorphism (SNP), rs6983267, at 8q24.21 has recently been shown to associate with colorectal cancer (CRC). Three independent SNP association studies showed that rs6983267 contributes to CRC with odds ratios (OR) of 1.17 to 1.22. Here, we genotyped a population-based series of 1,042 patients with CRC and 1,012 healthy controls for rs6983267 and determined the contribution of SNP to CRC in Finland, using germ line DNA, as well as the respective cancer DNA in heterozygous patients. The comprehensive clinical data available from the 1,042 patients and their first-degree relatives enabled us to thoroughly examine the possible association of this variant with different clinical features. As expected, a significant association between the G allele of rs6983267 and CRC [OR, 1.22; 95% confidence interval (CI), 1.08-1.38; P = 0.0018] was found, confirming the previous observations. A trend towards association of the G allele with microsatellite-stable cancer (OR, 1.37; 95% CI, 1.02-1.85; P = 0.04) and family history of cancers other than CRC was seen (OR, 1.20; 95% CI, 1-1.43; P = 0.05). Four hundred and sixty-six GT heterozygotes identified in this study were analyzed for allelic imbalance at rs6983267 in the respective cancer DNA. One hundred and one tumors showed allelic imbalance (22%). The risk allele G was favored in 67 versus 34 tumors (P = 0.0007). This finding implicates that the underlying germ line genetic defect in 8q24.21 is a target in the somatic evolution of CRC.     

Allelic imbalance on chromosome 18 in neuroblastoma

We previously demonstrated that chromosome 18 is frequently deleted in neuroblastoma. To further elucidate the role of chromosome 18 deletions in the development of neuroblastomas we examined 82 cases of neuroblastomas for allelic imbalance (AI) at 17 loci on chromosome 18 to define the common region of AI in neuroblastoma. AI at one or more loci on chromosome 18 was detected in 18/82 (22%) cases. AI on 18q was detected in 17/82 (21%) cases, whereas AI on 18p was detected in 4/82 (5%) cases. There was a distinct common region of AI at 18q21.1 between the D18S363 and D18S858 loci. In addition, cases 16 and 53, which did not show AI at 18q21.1, showed AI at 18pter-q12.3 between the D18S52 and D18S36 loci, indicating that another common region of AI may exist on chromosome 18. AI on chromosome 18 did not significantly correlate with any clinicopathological findings of patients with neuroblastoma. The common region of AI at 18q21.1 includes the DCC gene but not the Smad2 and Smad4 genes. However, our previous studies together with the present study indicated that the incidence of DCC mutation is much less than that of AI at 18q21.1 in neuroblastoma. These results indicate that novel tumour suppressor genes involved in the development of neuroblastoma are present at 18q21.1, and possibly at 18pter-q12.3.     

Allelic imbalance at NBS1 is frequent in both proximal and distal colorectal carcinoma

Nijmegen breakage syndrome (NBS) is a hereditary disorder involving chromosomal instability, cancer risk and radiosensitivity. NBS carriers have an increased risk of cancer, though the significance of mutations in the NBS1 gene in sporadic cancer has not yet been investigated. Because the loss of NBS1 is associated with increased chromosomal re-arrangements, and tumors of the colon are particularly prone to chromosomal anomalies, we have begun to study the NBS1 locus in colorectal cancer (CRC). DNA was isolated from 99 microdissected colorectal tumors, and microsatellite markers flanking the NBS1 locus at 8q21.3 as well as elsewhere on 8q were analyzed. Normal lymphocyte DNA from each patient served to normalize the amplification of each allele, and a reduction of at least 35% in the intensity of one allele was taken as evidence of allelic imbalance (AI). In proximal and distal CRCs we found 25.9 and 36.2% with AI at 8q21.3, respectively. AI in proximal CRC tended not to extend to marker D8S555 at 8q24.1, whereas in distal CRC the region of AI frequently included all the informative markers. AI of 8q21.3 was not associated with any clinical variable. These results suggest that 8q21.3 contains a tumor suppressor gene involved in proximal CRC, possibly NBS1. The large regions of AI make it difficult to determine the importance of AI at the NBS1 locus in distal CRC.     

前列腺癌可能存在多克隆起源

目的 :探寻人类前列腺癌发生和多发的细胞克隆来源。方法 :采用病理切片上病灶显微切割、P5 3基因突变的PCR SSCP分析及DNA测序测定 3例患者前列腺癌根治切除术标本中癌灶和高分级PIN灶组织 ;同时还采用免疫组织化学方法检测了p2 1WAF1/CIP1蛋白的表达。结果 :在人前列腺癌灶和高分级PIN灶组织中均发现有p5 3基因的点突变 ,同 1例患者标本中癌灶和高分级PIN灶所含点突变均在不同位点上。含p5 3基因的点突变的癌灶和高分级PIN灶组织中均未见有p2 1WAF1/CIP1蛋白的表达。结论 :人前列腺癌发生和多发可能有多细胞克隆来源 ,其p2 1WAF1/CIP1蛋白的表达系P5 3基因依赖性途径     

Allelic imbalance in the region of the BRCA1 gene in ductal carcinoma in situ of the breast.

Thirty-four cases of ductal carcinoma in situ (DCIS) of the breast, with or without associated benign or invasive disease, were analysed for allelic imbalance (AI) in the region of the BRCA1 gene. AI on 17q12-23 in DCIS was demonstrated in 74% of cases, and in the majority of cases the region of AI included the BRCA1 gene. However, two cases showed AI distal to BRCA1, supporting the presence of a second tumour-suppressor gene on 17q.     

Allelic imbalance of the mutant and wild-type RET allele in MEN 2A-associated medullary thyroid carcinoma.

Germline mutations of the RET proto-oncogene are responsible for the familial tumor syndrome called multiple endocrine neoplasia type 2 (MEN 2) that includes medullary thyroid carcinoma (MTC). Although inherited mutations of RET lead to tumor formation in patients with MEN 2, it is not understood why only selected cells develop into tumors. We have recently shown that duplication of the mutated RET allele or loss of the wild-type allele might represent mechanisms of tumorigenesis in patients with MEN 2A-related pheochromocytoma. We now analysed 19 DNA samples of MTC (15 of which were non-microdissected, four of which were microdissected) from patients with MEN 2A. Using polymorphic marker and phosphorimage densitometry analyses, we found allelic imbalance of the mutated and wild-type RET allele in six of 19 DNA MTC samples. Of note, two of the four microdissected tumor DNA samples showed allelic imbalance of RET, whereas only four of the 15 non-microdissected MTC samples did. These results underscore the significance of microdissection in the analysis of tumor DNA. In our study, some of the non-microdissected tumor DNA samples may have failed to display allelic imbalance of RET, because of contamination of tumor DNA with nonneoplastic DNA or noninformative microsatellite marker analysis. Taken together, our results suggest allelic imbalance between mutated and wild-type RET as a possible mechanism for tumor formation in some patients with MEN 2A-related MTC.     

Allelic imbalance and mutations of the PTEN gene in ovarian cancer

The PTEN/MMAC1/TEP1 tumor-suppressor gene, which maps to chromosome 10q23.3, is mutated and homozygously deleted in a variety of human tumors, including endometrioid-type ovarian tumors. We examined 33 primary ovarian cancers and 3 ovarian borderline tumors for allelic imbalance (AI) of the 10q23.3 region using 5 polymorphic markers, including an insertion/deletion-type polymorphic marker identified in intron 4 of the PTEN gene. AI at one or more loci was detected in 12 of 31 (39%) informative ovarian cancers and none of 3 ovarian borderline tumors. The commonly deleted region was mapped between the D10S215 and D10S541 loci, including the PTEN locus. Moreover, the incidence of AI at the PTEN locus (38%) was the highest among the 5 loci examined. Therefore, we searched for mutations in the entire coding region of the PTEN gene by PCR-SSCP and sequencing analyses in these tumors and 7 ovarian cancer cell lines. Mutations were detected in 3 of the 33 (9%) ovarian cancers: 2 cases with double mutations and 1 case with a mutation on 1 allele accompanied by deletions on both alleles in the poly T tract preceding the splice acceptor site in intron 7. An intragenic deletion was detected in 1 of the 7 (14%) ovarian cancer cell lines. PTEN mutations were detected not only in the endometrioid type but also in the serous and mucinous types of ovarian cancer. However, PTEN was not mutated in the 12 tumors that showed AI of the PTEN locus. Our results suggest that the PTEN gene plays an important role in the development of a subset but diverse histological types of ovarian tumors. However, it is possible that another tumor-suppressor gene in the close vicinity of the PTEN gene is also inactivated by AI of the 10q23.3 region.     

Allelic imbalance of chromosome 6q in ovarian tumours.

Previous work has implicated putative tumour-suppressor (ts) genes at 6q27 and a broad region at 6p12-q23. Here we report the results of a coded, randomised study of allelic imbalance at 12 loci on 6q on 40 pairs of coded tumour-blood pairs from patients with ovarian tumours. Our results provide clear evidence for the involvement of different regions of 6q in tumours of different histological subtypes. The involvement in serous tumours of a ts gene at the distal site is confirmed. However, proximal 6q presents a complex picture, with possibly three further ts genes: one at 6q21-23.3 involved at high frequency in benign and endometrioid tumours, another at 6q14-q15, also involved in endometrioid tumours, and a third suggested by a smallest region of deletion at 6q16.3-q21, between D6S275 and D6S300, that appears to be involved in early stage tumours. These observations point the way to a statistical study of the involvement of 6q in tumours of different histological type and staging performed on larger cohorts of samples.     

Allelic imbalance at chromosome region 11q23 in cervical carcinomas

The long arm of chromosome 11 has received much scrutiny as a high frequency of deletions of various sites has been observed in different tumour types, indicating the presence of putative tumour suppressor genes. In the present study, 81 primary cervical carcinomas were examined for allelic imbalance (AI) using nine microsatellite markers, mapping to the chromosomal region 11q23.1 where the ATM gene is located. AI at any locus in the region was found in 34 of 81 (42%) tumours. AI frequencies varied from 12 to 31% for the different markers used, with the highest frequency at marker D11S1294. Based on the findings of 17 cases with restricted areas of deletions, four chromosomal regions of possible importance in cervical carcinomas could be distinguished. The first region is located between the markers D11S1325 and D11S1819, the second region between D11S2179 and D11S1294, the third region between D11S1778 and D11S1818 and the fourth region between D11S1818 and D11S1347. The second region may thus contain part of the ATM gene. No association between AI of any marker and histopathological or clinical parameters was seen. When comparing the AI findings of the different loci with TP53 protein overexpression, the only significant association found was with D11S2179 located within the ATM gene. The results indicate that a tumour suppressor gene (or genes) on chromosome 11q.23.1 may be involved in carcinogenesis of the cervix and the involvement of the ATM gene remains a possibility.     

Chromosomal study demonstrating the clonal evolution and metastatic origin of a metachronous colorectal carcinoma

Detailed cytogenetic studies were performed on 3 samples of a metachronous rectal carcinoma. Two samples were obtained from the primary tumor, and one from a secondary growth which arose 10 months after surgical removal of the primary. All 149 R-banded metaphases analysed were abnormal and most likely derived from the same modified karyotype: 45, XY, -1, -18, +20 der(6) t(1;6) (q21.100;q22.3), i(17q). These data are consistent with our previous findings that the loss of chromosome 18 and of the short arm of chromosome 17 may be the primary chromosomal changes associated with carcinoma of the large bowel. From all the karyotypes observed, a typical clonal evolution could be reconstructed, indicating that the second tumor was indeed metastatic from the first one, based on similar chromosome markers. An identical secondary chromosomal anomaly, i.e. gain of chromosome 8 or X, occurred independently twice in two different cell populations during the evolution of this tumor.     

Allelic imbalance on chromosome 5q predicts long-term survival in neuroblastoma.

Neuroblastoma is the most common extracranial solid tumour of childhood. Amplification of the proto-oncogene, N-myc, confers a poor prognosis in neuroblastoma, while hyperdiploidy is associated with a favourable outcome. Little is known about the contribution of tumour-suppressor genes to the development or progression of neuroblastoma. We examined allelic imbalance at the locus of the tumour-suppressor gene, APC (adenomatous polyposis coli), on chromosome 5q using a polymerase chain reaction (PCR)-based assay. Nine of 24 (37.5%) informative neuroblastoma tumours showed allelic imbalance (AI) at this locus. Clinical data concerning N-myc amplification and DNA content were correlated with these results in the same patients. Allelic imbalance was found only in tumours containing a single copy of the N-myc gene and exhibiting hyperdiploidy. All nine patients with AI of chromosome 5q were alive after a median follow-up period of 46 months, while 7 of 15 (47%) of those lacking AI at this locus had died (P = 0.018). Allelic imbalance at three additional loci on chromosome 5 was demonstrated in tumours that exhibited AI at the APC locus, suggesting that endoreduplication of chromosome 5 had occurred. Fluorescent in situ hybridisation (FISH) analysis of tumour tissue from one patient exhibiting AI demonstrated two, three, four or six copies of the APC gene per cell, consistent with this hypothesis. These data suggest that allelic imbalance of chromosome 5 is involved in at least a subset of neuroblastomas and influences survival in patients with neuroblastoma.     

Allelic imbalance of 7q32.3-q36.1 during tumorigenesis in Barrett's esophagus

Malignant transformation of Barrett's esophagus is characterized by three distinct premalignant stages: intestinal metaplasia (MET), low- (LGD), and high-grade dysplasia (HGD). We reported recently an increase in the frequency of loss of 7q33-q35 between LGD and HGD as determined by comparative genomic hybridization (P. H. J. Riegman et al., Cancer Res., 61: 3164-3170, 2001). Now the 7q32.3-q36.1 region was additionally characterized by allelotype analysis with 11 polymorphic markers in 15 METs, 20 LGDs, 20 HGDs, and 20 Barrett's adenocarcinomas from different patients. Low percentages of imbalance were determined in METs and LGDs, 7% and 10%, respectively, whereas HGDs and Barrett's adenocarcinomas revealed high percentages of loss, 75% and 65%, respectively. This difference in frequency between LGDs and HGDs appeared highly significant: P = 0.00007. The majority of imbalances were found at D7S2439 and D7S483, located on 7q36.1. These data suggest that markers from this area can be used as a diagnostic tool in Barrett's esophagus, i.e., to distinguish between watchful waiting and active treatment.     

Allelic imbalance and microsatellite instability in plasma DNA released from polyclonal pancreatic adenocarcinoma

Evidence of circulating soluble DNA in blood-stream of cancer patients has emerged. Because the plasma DNA is largely derived from cancer cells, genetic analysis of plasma DNA is important to understand the molecular events occurred in cancer patient. Seven microsatellite markers in the soluble plasma DNA from patients with pancreatic adenocarcinoma and other pancreato-biliary malignant tumors were examined for microsatellite instability (MSI) and allelic imbalance (AI). A variety of genetic alterations including MSI and AI were detected in the plasma DNA. Some alterations were detected before recurrence of the tumor was verified. Analysis of five primary pancreatic adenocarcinomas by microdissection revealed that the heterogeneous nature of pancreatic tumors is associated with both MSI and AI in the same tumor. The presence of altered plasma DNA including MSI and/or AI from the same pancreatic cancer patient may be important evidence for the presence of these alterations in heterogeneous primary tumors. Analysis of plasma DNA could become one of the diagnostic or therapeutic measures for this type of pancreatic adenocarcinoma.     

Allelic imbalance and microsatellite instability in BRCA1 associated breast and ovarian tumors

We have investigated the involvement of microsatellite instability (MSI) and allelic imbalance (AI) at chromosome 13q and 17 in 41 breast and 41 ovarian carcinomas and their association with BRCA1 and BRCA2 gene mutations. MSI was detected in 20% of ovarian and 7% of breast tumors. AI at the BRCA1 locus was detected in 59% and 32% of ovarian and breast tumors, respectively. At the BRCA2 locus, AI rates were 49% and 44% for ovarian and breast tumors, respectively. Germline BRCA1 mutations, identified in 5 (12%) ovarian tumors and in one (2%) breast tumor were not associated with MSI. In only 2/5 BRCA1 positive tumors loss of the wild-type allele was observed. We conclude that BRCA1 mutation status is not associated with MSI and that MSI found in a fraction of ovarian tumors may reflect possible mutations in one of the DNA mismatch repair genes.     

Advances for studying clonal evolution in cancer

The “clonal evolution” model of cancer emerged and “evolved” amid ongoing advances in technology, especially in recent years during which next generation sequencing instruments have provided ever higher resolution pictures of the genetic changes in cancer cells and heterogeneity in tumors. It has become increasingly clear that clonal evolution is not a single sequential process, but instead frequently involves simultaneous evolution of multiple subclones that co-exist because they are of similar fitness or are spatially separated. Co-evolution of subclones also occurs when they complement each other’s survival advantages. Recent studies have also shown that clonal evolution is highly heterogeneous: different individual tumors of the same type may undergo very different paths of clonal evolution. New methodological advancements, including deep digital sequencing of a mixed tumor population, single cell sequencing, and the development of more sophisticated computational tools, will continue to shape and reshape the models of clonal evolution. In turn, these will provide both an improved framework for the understanding of cancer progression and a guide for treatment strategies aimed at the elimination of all, rather than just some, of the cancer cells within a patient.