A case of transitional cell carcinoma of the bladder with a del(9)(q11q21.2)

Monosomy for chromosome 9, as well as loss of heterozygosity for markers on this chromosome, has been detected in a high percentage of transitional cell carcinomas (TCC) of the bladder. We report a case of a TCC of the bladder with an interstitial del(9)(q11q21.2) that could be indicative of the presence of a putative tumor-suppressor gene related to bladder tumor progression. To elucidate the role of chromosome 9 in bladder tumors, it would be interesting to study a possible loss of heterozygosity in this chromosome region.     

Encouraging results of split-liver transplantation

Two sisters affected with renal cell carcinoma (RCC) is an extremely rare finding, and may indicate a hereditary pattern or the presence of other predisposing factors. We describe here 2 sisters presenting with clear cell renal cell cancer. Examination for von Hippel-Lindau (VHL)-related features and tuberous sclerosis (M. Bourneville) was negative and both had a normal constitutional karyotype. Cytogenetic analysis of the tumor tissue of both patients showed a translocation involving chromosomes 3 and 5, resulting in loss of 3p sequences and gain of part of 5q. The 5q breakpoints were similar, but the breakpoints at 3p appeared to differ. Allelic imbalance analysis supported our observations. Microsatellite analysis revealed that both sisters inherited different chromosome 3 parental alleles. For chromosome 5, 3 different haplotypes could be deduced, but the chromosome 5 alleles overrepresented in the different tumor tissues were from different parental origin. The development of the 2 RCCs in these 2 sisters thus cannot be explained by the inheritance of a mutated VHL gene located at 3p25, nor by the inheritance of other gene defects at chromosomes 3p or 5q. Although the chance that 2 sisters develop sporadic RCC is very low, in the presented case it is probably coincidental or related to another genetic predisposition.     

Cloning and characterization of a novel Rab-family gene, Rab36, within the region at 22q11.2 that is homozygously deleted in malignant rhabdoid tumors

Malignant rhabdoid tumors (MRTs) are rare, pediatric soft-tissue tumors. Homozygous deletions at chromosome 22q11.2 are a recurrent cytogenetic characteristic of MRTs, an indication that this locus may harbor one or more genes conferring tumor-suppressor activity. We constructed a deletion map of the relevant part of 22q11.2 from a panel of seven MRT cell lines, and isolated a novel gene from the center of the region. As it showed a high degree of sequence homology to genes of the Rab family, we designated it Rab36. The protein encoded by Rab36 was localized at the Golgi body. Sequencing of Rab36 cDNAs from three cell lines that retained at least one allele of this gene revealed no nonsense or frameshift mutations. Experiments to induce over-expression of Rab36 by transfection to an MRT cell line similarly failed to justify designation of this gene as a tumor suppressor that would contribute to tumorigenesis by a loss-of-function mechanism.     

Molecular biology of colorectal cancer

Colorectal cancer is a significant cause of morbidity and mortality in Western populations. This cancer develops as a result of the pathologic transformation of normal colonic epithelium to an adenomatous polyp and ultimately an invasive cancer. The multistep progression requires years and possibly decades and is accompanied by a number of recently characterized genetic alterations. Mutations in two classes of genes, tumor-suppressor genes and proto-oncogenes, are thought to impart a proliferative advantage to cells and contribute to development of the malignant phenotype. Inactivating mutations of both copies (alleles) of the adenomatous polyposis coli (APC) gene--a tumor-suppressor gene on chromosome 5q--mark one of the earliest events in colorectal carcinogenesis. Germline mutation of the APC gene and subsequent somatic mutation of the second APC allele cause the inherited familial adenomatous polyposis syndrome. This syndrome is characterized by the presence of hundreds to thousands of colonic adenomatous polyps. If these polyps are left untreated, colorectal cancer develops. Mutation leading to dysregulation of the K-ras protooncogene is also thought to be an early event in colon cancer formation. Conversely, loss of heterozygosity on the long arm of chromosome 18 (18q) occurs later in the sequence of development from adenoma to carcinoma, and this mutation may predict poor prognosis. Loss of the 18q region is thought to contribute to inactivation of the DCC tumor-suppressor gene. More recent evidence suggests that other tumor-suppressor genes--DPC4 and MADR2 of the transforming growth factor beta (TGF-beta) pathway--also may be inactivated by allelic loss on chromosome 18q. In addition, mutation of the tumor-suppressor gene p53 on chromosome 17p appears to be a late phenomenon in colorectal carcinogenesis. This mutation may allow the growing tumor with multiple genetic alterations to evade cell cycle arrest and apoptosis. Neoplastic progression is probably accompanied by additional, undiscovered genetic events, which are indicated by allelic loss on chromosomes 1q, 4p, 6p, 8p, 9q, and 22q in 25% to 50% of colorectal cancers. Recently, a third class of genes, DNA repair genes, has been implicated in tumorigenesis of colorectal cancer. Study findings suggest that DNA mismatch repair deficiency, due to germline mutation of the hMSH2, hMLH1, hPMS1, or hPMS2 genes, contributes to development of hereditary nonpolyposis colorectal cancer. The majority of tumors in patients with this disease and 10% to 15% of sporadic colon cancers display microsatellite instability, also know as the replication error positive (RER+) phenotype. This molecular marker of DNA mismatch repair deficiency may predict improved patient survival. Mismatch repair deficiency is thought to lead to mutation and inactivation of the genes for type II TGF-beta receptor and insulin-like growth-factor II receptor. Individuals from families at high risk for colorectal cancer (hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis) should be offered genetic counseling, predictive molecular testing, and when indicated, endoscopic surveillance at appropriate intervals. Recent studies have examined colorectal carcinogenesis in the light of other genetic processes. Telomerase activity is present in almost all cancers, including colorectal cancer, but rarely in benign lesions such as adenomatous polyps or normal tissues. Furthermore, genetic alterations that allow transformed colorectal epithelial cells to escape cell cycle arrest or apoptosis also have been recognized. In addition, hypomethylation or hypermethylation of DNA sequences may alter gene expression without nucleic acid mutation.     

Identification of two distinct deletion targets at 11q23 in cutaneous malignant melanoma

Karyotypic and molecular data indicate that genetic alterations of the long arm of chromosome 11 (11q) are involved in the pathogenesis of malignant melanoma as well as of other malignancies. We have shown previously, by analysis of loss of heterozygosity (LOH), that a tumor-suppressor gene playing an important role in malignant melanoma is likely to be located within a 51-cM region at 11q23. Its loss appeared to be a late event in tumor progression and an indicator of a less favorable clinical outcome. To further test this hypothesis on a larger set of tumors and to refine the region(s) of common allelic loss, we analyzed 21 polymorphic microsatellite repeats on 11q. A PCR-based assay for LOH was used to study normal and tumor tissues from 53 individuals with primary cutaneous malignant melanoma or metastatic disease. Our findings indicate that in cutaneous malignant melanoma there are at least 2 distinct regions of common allelic loss on 11q, one of them centered around marker APOC3 at 11q23.1-q23.2 delineated by markers D11S1347 and D11S4142 and spanning approximately 5 Mb and a second 3-Mb region around marker D11S925 at 11q23.3 delineated by markers D11S528 and D11S1345. Both regions have been described as deletion targets or as being included within larger allelic deletions detected in several other common tumor types. Thus, these 2 putative melanoma-suppressor loci are likely to harbor tumor-suppressor genes relevant to tumorigenesis of melanoma and a number of other common human malignancies.     

Detailed deletion mapping of the long arm of chromosome 6 in adult T-cell leukemia

Previously, we have found that the loss of heterozygosity (LOH) was frequently observed on chromosome 6q in acute/lymphoma-type adult T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for ATL may be present on chromosome 6q. To further define a region containing this gene, we performed fine-scale deletional mapping of chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly informative microsatellite markers. LOH was found in 9 samples (40. 9%) at 1 or more of the loci examined. Of the 9 samples, 8 shared the same smallest commonly deleted region flanked by D6S1652 and D6S1644 (6q15-21). The genetic distance between these two loci is approximately 4 cM. These results suggest that a putative tumor-suppressor gene on chromosome 6q15-21 probably plays a very important role in the evolution of acute/lymphomatous ATL. Our map provides key information toward cloning the gene.     

Unbalanced t(3;12) in a case of juvenile myelomonocytic leukemia (JMML) results in partial trisomy of 3q as defined by FISH

Juvenile myelomonocytic leukemia (JMML) is a rare disorder of early childhood, to which no recurrent chromosome rearrangement has been yet associated. We report a case where leukemic cells harbored a 46,XX,der(12)t(3;12) (q21 approximately 22;p13.33) karyotype, resulting in partial trisomy of 3q. The origin of chromosome material translocated to chromosome 12 was assessed by chromosome painting using a whole chromosome 3-specific probe. The breakpoint regions were defined by FISH using YAC probes from 3q and 12p chromosomal regions. Interestingly, partial trisomy of 3q has been detected in a previously reported JMML case, consequent to the presence of a der(15)t(3;15)(q13.1;q26). The involvement of a similar chromosome 3 rearrangement in these two JMML cases suggests the hypothesis that either the resulting duplication of some gene/s on 3q or the loss of heterozygosity (LOH) of some gene/s on 3p may be involved in one of the steps leading to JMML. On the other hand, it cannot be ruled out that the relevant mutation in our case might be consequent to the particular breakpoints at bands 3q21 approximately 22 and 12p13.3, that may alter the structure and/or expression of the involved gene/s.     

MEN1 gene analysis in sporadic adrenocortical neoplasms

Adrenocortical tumors occur as sporadic tumors, as part of the multiple endocrine neoplasia type 1 (MEN1) syndrome or as part of other hereditary disorders. We recently cloned the MEN1 gene, a tumor-suppressor gene located on chromosome 11q13. Subsequently, we showed that sequential somatic inactivation of both alleles of the MEN1 gene contributes to the development of some sporadic endocrine neoplasms (parathyroid, enteropancreatic neuroendocrine, bronchial carcinoid, and pituitary tumors). We now studied whether somatic inactivation of the MEN1 gene contributes to the pathogenesis of sporadic adrenocortical neoplasms. Seven adrenocortical carcinomas, 2 adrenocortical carcinoma cell lines, and 11 aldosterone-secreting, 8 cortisol-secreting, and 5 nonsecreting benign adrenocortical tumors were studied. Seven tumors (5 of 5 carcinomas, 2 of 21 nonsecreting benign adenomas; P < 0.001) exhibited loss of heterozygosity on 11q13. All 33 tumors and cell lines were screened for mutation throughout the MEN1 open-reading frame and adjacent splice junctions. None exhibited a mutation within the MEN1-coding region. We conclude that somatic MEN1 mutation within the MEN1-coding region does not occur commonly in sporadic adrenocortical tumors, although the majority of adrenocortical carcinomas exhibit 11q13 loss of heterozygosity.     

Absence or reduction of Fhit expression in most clear cell renal carcinomas

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.     

Mapping of a breast cancer tumor suppressor gene locus to a 4-cM interval on chromosome 18q21

DPC4 and DCC, putative tumor suppressor genes implicated in the genesis of several types of human cancer, lie on the long arm of human chromosome 18. We examined 200 primary breast cancers for allelic losses on chromosome 18, using 15 microsatellite markers distributed along the long arm. Allelic loss was detected most frequently (29-30%) at loci mapped to 18q21. Deletion mapping of the 34 tumors showing partial or interstitial deletions identified a commonly deleted region within the 4-cM interval flanked by D18S474 and D18S487 at 18q21.1-q21.3. Although this interval included the DPC4 and DCC genes, we excluded DPC4 from candidacy when polymerase chain reaction-single-strand conformation polymorphism analysis of each exon failed to detect abnormalities in any of the 54 breast cancers that exhibited loss of heterozygosity involving 18q. Allelic loss on 18q was found more frequently in tumors of the solid tubular histological type (24 of 55, 44%) than in other types (24 of 113, 21%) (P = 0.0049). The results suggest that a tumor suppressor gene located within the 4-cM region at 18q21, either DCC or another gene not yet identified, may play a role in the development of some sporadic breast cancers, particularly those of the solid tubular type.     

Linkage of familial Wilms' tumor predisposition to chromosome 19 and a two-locus model for the etiology of familial tumors

Familial predisposition to Wilms' tumor (WT), a childhood kidney tumor, is inherited as an autosomal dominant trait. For most WT families studied, the 11p13 gene WT1 and genomic regions implicated in tumorigenesis in a subset of tumors can be ruled out as the site of the familial predisposition gene. Following a genome-wide genetic linkage scan, we have obtained strong evidence (log of the odds ratio = 4.0) in five families for an inherited WT predisposition gene (FWT2) at 19q13.3-q13.4. In addition, we observed loss of heterozygosity at 19q in tumors from individuals from two families in which 19q can be ruled out as the site of the inherited predisposing mutation. From these data, we hypothesize that alterations at two distinct loci are critical rate-limiting steps in the etiology of familial WTs.     

Hot spots for molecular genetic alterations in lung cancer

Lung cancers are a heterogeneous group of tumors broadly classified as small cell or non-small cell lung cancers. In each case, numerous DNA mutations precede tumor formation, resulting in the activation of growth stimulatory genes and the loss of tumor suppressor genes. The known cellular functions of the tumor suppressor genes most commonly affected in lung cancer are reviewed herein, including the retinoblastoma (Rb) gene on chromosome 13q14, the p53 gene on 17p13, and the cyclin-dependent kinase inhibitor (CDKN2) gene on 9p21. The chromosomal locations for other potential tumor suppressor genes are on chromosomes 3p, 9p, and 11p. Candidate genes in these regions include the von Hippel-Lindau (VHL) gene at 3p25, the ubiquitin-activating enzyme homologue (UBE1L at 3p21, the genes for the dinucleoside polyphosphate hydrolase FHIT and receptor protein-tyrosine phosphatase gamma PTPRG at 3p14.2, the genes for tropomyosin beta (TM1) and a talin homologue (talin) at 9p21, and the H-ras gene at 11p15.     

Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identified as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identified apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identification of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.     

Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung

Gene amplification is a common genetic change in human cancer cells. Previously, we provided the first evidence for gene amplification at chromosome band 3q26 in squamous cell lung carcinoma. In this study, the following analyses were performed: (a) we evaluated biopsies and paraffin-embedded tissues of 16 additional squamous cell lung carcinomas for gene amplification using reverse chromosome painting. Of the 16 tumors, 3 tumors showed an amplification of the entire long arm of chromosome 3, and 3 tumors showed various amplifications on 3q, all of which involved chromosome band 3q26; (b) we tested eight genes encompassing region 3q25-qter in two different tumors to identify amplified genes on chromosome 3q. The genes SI, BCHE, and SLC2A2 were amplified in both tumors; and (c) we analyzed 15 additional paraffin-embedded tissues to determine the amplification frequency of these genes. Of the 15 squamous cell lung carcinomas, 6 showed amplification for at least 1 of the genes, with BCHE and SLC2A2 as the genes most frequently amplified. Together, our reverse chromosome painting data and our PCR analysis indicate gene amplification at 3q26 in 40% of all squamous cell lung carcinomas with BCHE and SLC2A2 as possible target genes of the amplification unit in squamous cell lung carcinoma.     

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.     

Allelic deletions on chromosome 11q13 in multiple endocrine neoplasia type 1-associated and sporadic gastrinomas and pancreatic endocrine tumors

Endocrine tumors (ETs) of pancreas and duodenum occur sporadically and as a part of multiple endocrine neoplasia type 1 (MEN1). The MEN1 tumor suppressor gene has been localized to chromosome 11q13 by linkage analysis but has not yet isolated. Previous allelic deletion studies in enteropancreatic ETs suggested MEN1 gene involvement in tumorigenesis of familial pancreatic ETs (nongastrinomas) and sporadic gastrinomas. However, only a few MEN1-associated duodenal gastrinomas and sporadic pancreatic nongastrinomas have been investigated. We used tissue microdissection to analyze 95 archival pancreatic and duodenal ETs and metastases from 50 patients for loss of heterozygosity (LOH) on 11q13 with 10 polymorphic markers spanning the area of the putative MEN1 gene. Chromosome 11q13 LOH was detected in 23 of 27 (85%) MEN1-associated pancreatic ETs (nongastrinomas), 14 of 34 (41%) MEN1-associated gastrinomas, 3 of 16 (19%) sporadic insulinomas, and 8 of 18 (44%) sporadic gastrinomas. Analysis of LOH on 11q13 showed different deletion patterns in ETs from different MEN1 patients and in multiple tumors from individual MEN1 patients. The present results suggest that the MEN1 gene plays a role in all four tumor types. The lower rate of 11q13 LOH in MEN1-associated and sporadic gastrinomas and sporadic insulinomas as compared to MEN1 nongastrinomas may reflect alternative genetic pathways for the development of these tumors or mechanisms of the MEN1 gene inactivation that do not involve large deletions. The isolation of the MEN1 gene is necessary to further define its role in pathogenesis of pancreatic and duodenal ETs.     

Combined molecular genetic studies of chromosome 22q and the neurofibromatosis type 2 gene in central nervous system tumors

Monosomy of chromosome 22 or deletions of 22q have been described in meningiomas and astrocytic tumors, the incidence of which is increased in Type 2 neurofibromatosis. Recently, the gene for neurofibromatosis Type 2 (NF2) has been identified at Chromosome 22q12, and a tumor suppression role has been suggested. Because there have been only a few studies of the NF2 gene on central nervous system tumors other than vestibular schwannomas, we investigated the potential role of NF2 as a tumor suppressor gene in a group of sporadic meningiomas and astrocytomas. Forty-four tumors (26 meningiomas and 18 astrocytic tumors of different grades) were screened for NF2 mutations for the entire 17 exons by the polymerase chain reaction-single-strand conformation polymorphism method. In addition, 37 tumors and their respective constitutional deoxyribonucleic acid were analyzed for loss of heterozygosity of 22q alleles by four polymorphic microsatellite markers. Seven inactivating mutations were found in Exons 4, 5, 6, and 10 in 7 of 26 (27%) meningiomas, but none were found in astrocytic tumors. Altogether, 69% of meningiomas and 20% of astrocytic tumors revealed a loss of heterozygosity of 22q markers. All tumors with NF2 mutations showed concurrent loss of alleles on 22q, thus fulfilling Knudson's criteria for tumor suppressor genes in meningiomas. We conclude that inactivation of the NF2 gene is involved in the pathogenesis of a proportion of meningiomas but not in astrocytic tumors. Because many meningiomas and some astrocytic tumors had allelic loss of 22q but intact NF2, there is a possibility that other tumor suppressor genes exist on 22q and may be involved in the pathogenesis of central nervous system tumors.