Search

DNA sequencing offers a powerful tool in oncology based on the precise definition of structural rearrangements and copy number in tumor genomes. Here, we describe the development of methods to compute copy number and detect structural variants to locally reconstruct highly rearranged regions of the tumor genome with high precision from standard, short-read, paired-end sequencing datasets. We find that circular assemblies are the most parsimonious explanation for a set of highly amplified tumor regions in a subset of glioblastoma multiforme samples sequenced by The Cancer Genome Atlas (TCGA) consortium, revealing evidence for double minute chromosomes in these tumors. Further, we find that some samples harbor multiple circular amplicons and, in some cases, further rearrangements occurred after the initial amplicon-generating event. Fluorescence in situ hybridization analysis offered an initial confirmation of the presence of double minute chromosomes. Gene content in these assemblies helps identify likely driver oncogenes for these amplicons. RNA-seq data available for one double minute chromosome offered additional support for our local tumor genome assemblies, and identified the birth of a novel exon made possible through rearranged sequences present in the double minute chromosomes. Our method was also useful for analysis of a larger set of glioblastoma multiforme tumors for which exome sequencing data are available, finding evidence for oncogenic double minute chromosomes in more than 20% of clinical specimens examined, a frequency consistent with previous estimates.

We recently reported that forced overexpression of the transcription factor E2F-1 in human HT-1080 fibrosarcoma cells resulted in corresponding high levels of thymidylate synthase (TS) and resistance to 5-fluoropyrimidines (D. Banerjee et al., Cancer Res., 58: 4292-4296, 1998). Because colorectal metastasis to the lung has higher TS levels than liver metastasis and is less responsive to treatment with 5-fluorouracil (R. Gorlick et al., J. Clin. Oncol., 16: 1465-1469, 1998), it was, therefore, of interest to measure E2F-1 expression in these tumors. In contrast to marginally increased levels of dihydrofolate reductase and topoisomerase I in lung metastasis as compared with liver metastasis, lung tumors had a 5-fold increase in E2F-1 expression as compared with liver tumors, corresponding to the relative levels of TS in these metastases. These data indicate that there exists a close correlation between E2F-1 and TS levels and provide a rationale for targeting this transcription factor, ie., E2F-1, for the treatment of certain cancers.

We have previously described a type I transforming growth factor (TGF)-beta receptor (TbetaR-I) polymorphic allele, TbetaR-I(6A), that has a deletion of three alanines from a nine-alanine stretch. We observed a higher than expected number of TbetaR-I(6A) homozygotes among tumor and nontumor DNA from patients with a diagnosis of cancer. To test the hypothesis that TbetaR-I(6A) homozygosity is associated with cancer, we performed a case-control study in patients with a diagnosis of cancer and matched healthy individuals with no history of cancer and who were identical in their gender and their geographical and ethnic background to determine the relative germ-line frequencies of this allele. We found nine TbetaR-I(6A) homozygotes among 851 patients with cancer. In comparison, there were no TbetaR-I(6A) homozygotes among 735 healthy volunteers (P < 0.01). We also observed an excess of TbetaR-I(6A) heterozygotes in cancer cases compared to controls (14.6% versus 10.6%; P = 0.02, Fisher's exact test). A subset analysis revealed that 4 of 112 patients with colorectal cancer were TbetaR-I(6A) homozygotes (P < 0.01). Using mink lung epithelial cell lines devoid of TbetaR-I, we established stably transfected TbetaR-I and TbetaR-I(6A) cell lines. We found that, compared to TbetaR-I, TbetaR-I(6A) was impaired as a mediator of TGF-beta antiproliferative signals. We conclude that TbetaR-I(6A) acts as a tumor susceptibility allele that may contribute to the development of cancer, especially colon cancer, by means of reduced TGF-beta-mediated growth inhibition.

Comparative genomic hybridization analysis was performed to identify chromosomal imbalances in 24 human malignant mesothelioma (MM) cell lines derived from untreated primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. The most frequent underrepresented segments were 22q (58%) and 15q1.1-21 (54%); other recurrent sites of chromosomal loss included 1p12-22 (42%), 13q12-14 (42%), 14q24-qter (42%), 6q25-qter (38%), and 9p21 (38%). The most commonly overrepresented segment was 5p (54%). DNA sequence amplification at 3p12-13 was observed in two cases. Whereas some of the regions of copy number decreases (i.e., segments in 1p, 6q, 9p, and 22q) have previously been shown to be common sites of karyotypic and allelic loss in MM, our comparative genomic hybridization analyses identified a new recurrent site of chromosomal loss within 15q in this malignancy. To more precisely map the region of 15q deletion, loss of heterozygosity analyses were performed with a panel of polymorphic microsatellite markers distributed along 15q, which defined a minimal region of chromosomal loss at 15q11.1-15. The identification of frequent losses of a discrete segment in 15q suggests that this region harbors a putative tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many MMs.

Our previous cytogenetic studies of malignant mesotheliomas (MMs) revealed losses from 6q15-21 in approximately 40% of cases, suggestive of recurrent loss of function of a putative tumor suppressor gene(s) located in this chromosome region. To more precisely define the critical region of molecular genetic loss within 6q, we have constructed a high-resolution deletion map of this chromosome arm in 46 MMs. We analyzed 32 microsatellite markers to detect loss of heterozygosity in tumor DNAs. Allelic losses from 6q were observed in a high percentage (61%) of cases. Partial deletions of 6q were identified in 11 cases, and these were used to define four nonoverlapping regions of chromosomal loss: a region involving 6q14-21 (approximately 9 cM; 7 of 11 cases with partial deletions), a region within 6q16.3-21 (approximately 8 cM; 9 cases), a region within 6q21-23.2 (approximately 10 cM; 8 cases), and a distal region located at 6q25 (approximately 13 cM; 9 cases). Most cases exhibited losses from more than one of these regions. We conclude from these data that genomic losses involving 6q in MM are more frequent than previously recognized cytogenetically and that the deletions fall into four discrete locations, suggesting the existence of multiple tumor suppressor loci in 6q that may contribute to the pathogenesis of this malignancy.

Previous cytogenetic analysis has revealed frequent losses of chromosome 1p21-22 in human malignant mesothelioma, suggesting that the loss or inactivation of a tumor suppressor gene's) residing at this site may contribute to the tumorigenic conversion of mesothelial cells. To more precisely define the location of the target gene, primary tumor specimens and cell lines from 50 malignant mesotheliomas were examined for loss of heterozygosity using short tandem repeat polymorphism (STRP) markers. Nineteen STRP markers established by the Cooperative Human Linkage Center were selected for the initial screening of the entire short arm of chromosome 1. Thirty-seven cases (74%) showed allelic losses at least at one locus in 1p. Thirty-six of these cases showed losses of 1p21-22, including 23 with partial deletions involving this region. To obtain a higher resolution map of this region, another 13 STRP markers from the Genethon map were used to define the shortest region of overlapping deletions to a 4-cM segment flanked by the loci D1S435 and D1S236. The chromosomal location of the critically deleted region was confirmed to be within 1p22 by karyotypic and fluorescence in situ hybridization analyses.

To determine whether p16 is altered in human malignant mesothelioma (MM), molecular analysis of multiple 9p loci was performed on 40 cell lines and 23 primary tumors from 42 MM patients. We identified homozygous deletions of p16 in 34 (85%) cell lines and a point mutation in 1 line. Down-regulation of p16 was observed in 4 of the remaining cell lines, 1 of which displayed a DNA rearrangement of p16. Homozygous deletions of p16 were identified in 5 of 23 (22%) primary tumors; no mutations or rearrangements were found in these specimens. Four cell lines displayed a single homozygous deletion proximal to or distal to p16; 4 others had 2 nonoverlapping deletions, one involving p16 and the other involving a region proximal to this locus. These data indicate that alterations of p16 are a common occurrence in MM cell lines and, to a lesser extent, in primary tumors. Furthermore, deletions of 9p21-p22 outside of the p16 locus may reflect the involvement of other putative tumor suppressor genes that could also contribute to the pathogenesis of some MMs.

Allelic loss on the short arm of chromosome 3 (3p) is considered to be one of the early detectable events in the pathogenesis of renal cell carcinoma (RCC). Conflicting reports, however, suggest that this event may be absent in some renal tumors. The present study attempts to further define subgroups of renal tumors associated with 3p deletions. In addition, we have also attempted to identify late genetic events associated with tumorigenesis and tumor progression. Eighty-two primary renal tumors (69 RCC and 13 oncocytic tumors) were analyzed by restriction fragment length polymorphism analysis directed at chromosomes 3, 11p, 17p, and 18q. Results were correlated with histopathological information. Deletions of 3p were seen in nonpapillary RCC of all cell types, but were absent in oncocytic and most papillary tumors. Among the 60 nonpapillary RCC, significant correlations were seen between deletion of 17p and tumor grade (P = 0.037), P stage (P = 0.027), and nodal metastases (P = 0.042). We therefore conclude that 3p deletions, although not specific to any cell type or histological pattern of RCC, are seen in a majority of clear cell nonpapillary RCC but are absent in oncocytic and most papillary tumors. Additional allelic losses on chromosome 17p are associated with advanced disease and, therefore, may be related to tumor progression. Further studies on larger series of patients with extended follow-up will be necessary to investigate the prognostic value of molecular genetic markers in RCC.

Previous DNA analyses have demonstrated that 9p13-p22 is a frequent site of chromosomal loss in leukemia, glioma, melanoma, and lung and bladder carcinomas. Recent cytogenetic studies have revealed recurrent alterations of 9p in malignant mesothelioma (MM). We have performed gene dosage studies of 23 MM cell lines, using probes for several 9p21-p22 loci (IFNB, IFNA/IFNW, D9S3, D9S126, D9S169, and D9S171), to identify a common region of deletion. Homozygous and/or hemizygous deletions were identified in 19 (83%) cell lines. Homozygous losses (10 cell lines; 43%) occurred most often at the D9S171 and IFNA/IFNW loci. In 8 cell lines, 2 or more of the 9p loci examined were found to be homozygously lost; 2 others displayed homozygous losses only at the D9S171 locus. Results from our deletion mapping analysis suggest that D9S171 is located between IFNA/IFNW and D9S126. The data presented here indicate that allelic loss from 9p21-p22 is a common occurrence in MM and further delineate the location of a putative 9p tumor suppressor gene(s) to a region between IFNA/IFNW and D9S171. These MM cell lines may facilitate efforts to define an even smaller critically deleted region, leading to the eventual cloning and characterization of this gene.

Detailed cytogenetic analyses were carried out on primary tumor specimens and cell lines from 23 patients with pleural malignant mesothelioma (MM). Clonal abnormalities were identified in 20 of 23 MM. In 3 cases, karyotypic data were compiled from harvests of both short-term cultures (1-3 days), and primary cultures grown on murine feeder layers for several weeks. The karyotypes obtained with these 2 different culture methods were very similar, although polyploid versions of abnormal clones were found only in the long-term cultures. In addition, while short-term cultures from 9 tumor biopsies usually exhibited near-diploid clones, cell lines derived from 11 tumors tended to have higher ploidies. Each of the cytogenetically abnormal MM displayed multiple clonal alterations. The 2 most frequent changes were chromosomal losses of specific regions in 1p (17 cases) and 9p (16 cases). The shortest regions of overlap of these losses were at 1p21-p22 and 9p21-p22, respectively. Other common abnormalities included losses of 3p21 (13 cases) and 6q15-q21 (9 cases), and numerical losses of chromosomes 14, 16, 18, and 22 (each observed in 10-13 tumors). In many of the MM examined, most or all of these recurrent changes occurred in combination, suggesting the involvement of a pathogenetic cascade in this cancer. The pattern of recurrent chromosomal losses suggests that these regions represent the locations of tumor suppressor genes whose loss/inactivation may have a pivotal role in MM tumorigenesis.

The human homologue of the murine double minute 2 gene (MDM2), a p53-binding protein which may act as a regulator of p53 protein function, has recently been cloned. Initial studies of this gene in a variety of human tumors have shown frequent gene amplification in most types of sarcomas, including osteosarcomas. Amplification of the MDM2 gene may produce a functional inactivation of the p53 protein. To examine possible clinical or pathological correlates of MDM2 gene amplification in osteosarcoma, we studied 28 specimens on 26 patients with high grade osteosarcoma (16 primary, 11 metastatic, and 1 local recurrence) for MDM2 gene amplification by Southern blot analysis, using two MDM2 complementary DNA probes isolated by polymerase chain reaction. Four specimens (14%) showed amplification, including 3 metastases and 1 local recurrence. None of the primary osteosarcoma specimens had detectable MDM2 gene amplification. None of the specimens tested showed MDM2 gene rearrangement. In the present series, MDM2 gene amplification was detected significantly more frequently in metastatic or recurrent osteosarcomas than it was in primary osteosarcomas (P = 0.02). Our data suggest that MDM2 gene amplification may be associated with tumor progression and metastasis in osteosarcoma. Further investigation is warranted on the potential clinicopathological correlates of MDM2 gene amplification in osteosarcoma.

A putative tumor suppressor gene, p53, has been shown to be altered in a variety of human tumor types. The primary mechanism of p53 inactivation is believed to be mutation of one allele followed by loss of the second allele. Malignant mesothelioma is a tumor that has been highly associated with exposure to asbestos fibers, which are known to cause chromosomal abnormalities in mesothelial cells. We have examined four mesothelioma cell lines for genetic abnormalities in p53. Cytogenetic analysis revealed that two of the four tumors had abnormalities (numerical and/or structural) of chromosome 17 (the locus of the p53 gene). Restriction fragment length polymorphism analysis using a chromosome 17p-specific probe (pYNZ22) revealed that two tumors had loss of heterozygosity in the region of 17p13. The relative level of p53 mRNA expression was examined by Northern analysis, with one tumor showing negligible expression of p53 mRNA. The complementary DNA of p53 was generated from the three tumors showing detectable mRNA expression, and the region between codons 70 and 319 was amplified by the polymerase chain reaction and sequenced. DNA single-base substitutions were detected in two of the tumor cell lines, each resulting in amino acid substitutions. One tumor had an arginine to histidine substitution at position 175, and one tumor had a glycine to aspartic acid substitution at position 245. The observed mutations took place in regions of high cross-species sequence homology, indicating that these regions may be functionally important. The correlation of chromosomal loss in 17p on the cytogenetic and molecular level along with p53 mRNA expression and DNA sequence data indicate that genetic alterations in p53 could be a feature of malignant mesotheliomas and may reveal an important role of asbestos fibers in tumor suppressor gene inactivation.

We used cytogenetic and restriction fragment length polymorphism (RFLP) analysis methods to define genetic alterations and also correlate the changes with histopathology in renal cortical tumors. The study series is comprised of 50 renal tumors in 4 histological categories: (a) clear cell, nonpapillary, renal cell carcinoma (RCC) (n = 32); (b) nonclear cell, nonpapillary RCC (n = 10); (c) papillary RCC (n = 3); and (d) oncocytic tumors (n = 5). Successful karyotypes were obtained from 28 tumors (56%), of which 17 (61%) were abnormal. Abnormalities of chromosome 3p were seen in 9 tumors, which included unbalanced translocations and terminal or interstitial deletions. Abnormalities of chromosome 5 were identified in 11 tumors, 8 of which were due to unbalanced translocations between 3p and 5q, resulting in an extra copy for the region 5q22----ter. In addition, trisomy or tetrasomy of chromosome 17 was seen in 6 (5 with normal chromosome 3 and one with 3p deletion), trisomy or more copies of chromosome 7 in 8 (4 with 3p deletion, 2 with trisomy or tetrasomy 17, and 2 with trisomy alone), and trisomy 12 in 3 (all with trisomy 17) tumors. Furthermore, relative deficiency of chromosome 17p was seen in 3 (all with deletion 3p) and chromosome 18 in 4 (all with deletion 3p) tumors. RFLP analysis with four chromosome 3 specific probes detected 3p deletions in 19 tumors with the most common breakpoint located between 3p14-21. The 19 3p deletions detected by RFLP included tumors that also showed rearrangement of 3p by cytogenetics (n = 4) and those that showed normal karyotypes (n = 3) in addition to cytogenetic failures (n = 12). Deletions of 17p were seen in 5 of 31 informative cases. Thus, deletions of 3p were seen in a total of 24 tumors by cytogenetic and/or RFLP analysis, 21 of which were clear cell, nonpapillary RCC, whereas 3 had a minor clear-cell component. Oncocytic and nonclear, nonpapillary tumors, on the other hand, did not demonstrate 3p deletions by either technique, whereas trisomy 17 was seen in 3 of the 3 papillary tumors. The loss of alleles from chromosome 17p and 18 and an increased dosage of gene or genes on chromosomes 5q and 7 as seen in high-stage tumors of various histological subtypes may be associated with progression of disease.

In a cytogenetic analysis of 9 gastric and lower esophageal adenocarcinomas, we detected nonrandom rearrangements involving the region 11p13-15 in 8, thus identifying for the first time a specific chromosomal lesion in these tumors. In addition, rearrangements involving 3p21, translocations among the D group chromosomes, and i(5p) were each observed in more than half of the cases. The overall pattern of aberrations encountered in adenocarcinomas of gastric and lower esophageal origin was similar, suggesting that the tumors arising at these anatomical sites are biologically related. We also encountered cytogenetic evidence for gene amplification in the form of homogeneously staining regions and double-minute chromosomes in primary as well as metastatic lesions, which is consistent with amplification of a number of cellular oncogenes in these tumors detected by others and us at the molecular level. These cytogenetic findings are discussed in relation to nonrandom chromosome abnormalities and gene amplification reported in other types of adenocarcinoma.