Mapping allelic deletions on the short arm of human chromosome 3 in kidney neoplasms]

Allelic deletions along the short arm of human chromosome 3 were mapped in 57 pairs of DNA samples from tumor and normal tissue of renal carcinoma patients in order to locate potential tumor suppressor genes. Twenty highly polymorphic microsatellite markers were used for deletion mapping. Allelic deletions were found in most of the samples (91%). Extended terminal deletions (56%) prevailed over shorter internal and multiple deletions and dominated (65%) in the most aggressive histopathological kidney cancer subtype, clear-cell carcinoma. Frequency analysis of loss of heterozygosity allowed detection of the human chromosome 3 regions most essential for renal carcinomas: the region adjacent to the gene VHL (3p26-p25), the region of homozygous deletions AP20 (3p22-p21.33), and a new region between markers D3S2420 and D3S2409 (3p21.31, 2.2 Mbp).     

Allele Deletion Mapping of the Short Arm of Human Chromosome 3 in Renal Carcinoma

Allelic deletions along the short arm of human chromosome 3 were mapped in 57 pairs of DNA samples from tumor and normal tissue of renal carcinoma patients in order to locate potential tumor suppressor genes. Twenty highly polymorphic microsatellite markers were used for deletion mapping. Allelic deletions were found in most of the samples (91%). Extended terminal deletions (56%) prevailed over shorter internal and multiple deletions and dominated (65%) in the most aggressive histopathological kidney cancer subtype, clear-cell carcinoma. Frequency analysis of loss of heterozygosity allowed detection of the human chromosome 3 regions most essential for renal carcinomas: the region adjacent to the gene VHL(3p26–p25), the region of homozygous deletions AP20 (3p22–p21.33), and a new region between markers D3S2420 and D3S2409 (3p21.31, 2.2 Mbp).     

Loss of heterozygosity of the L-myc oncogene in human breast tumors

Summary Recent studies suggest that loss of heterozygosity may play an important role in various human neoplasia. Cytogenetic abnormalities detected in primary breast tumors led us to examine breast tumor DNAs for deletions. In the present study, we demonstrate, using restriction fragment length polymorphism (RFLP) analysis at the L-myc proto-oncogene (chromosome 1p32), a frequent loss of heterozygosity in primary breast tumor DNAs (55 out of 152 informative tumor DNAs). Most of these deletions appear to be limited to chromosome 1p. No correlation was observed between this genetic alteration and several parameters of each patient's history or characteristics of the tumor. However, a significantly (P = 0.011) shorter survival period after relapse was observed for patients with loss of heterozygosity at L-myc in primary tumor DNAs compared with patients with tumor DNAs lacking this alteration.     

Deletion of YNZ22 and ALU-VPA/MYCL1 loci in human colonic adenocarcinoma and postoperative prognosis

Since deletions of the short arm of chromosome 17 are the most common genetic defects in human colorectal carcinoma (CC), we tested the YNZ22 locus (D17S30, 17p13.3) for loss of heterozygosity (LH) in adenocarcinoma and in the normal colonic mucosa of 49 CC patients, and studied the association of LH with clinicomorphological features of the tumor. Allele frequency distribution of YNZ22 did not differ for the patients and healthy people. LH in YNZ22 in the tumor was found in 33% (13/39) of all informative cases, its frequency being thrice higher in men than in women (chi 2 = 5.21, p = 0.022). The defect was associated with moderate or poor histological differentiation (P2 = 0.0055) and polyploidy > 3n (P2 = 0.0035) of tumor cells and with high incidence of post-surgery relapse or metastasis. Analysis of both YNZ22 and Alu-VpA/MycL1 (1p34.3) loci in the tumor allowed reliable relapse prognosis in 76% of the CC patients. The probability of post-surgery relapse or metastasis was estimated at no less than 67% for patients with LH in at least one of the two loci in the tumor, and at somewhat more than 20% for patients without LH.     

Human breast cancer: frequent p53 allele loss and protein overexpression

A sample of 114 primary breast tumors and corresponding constitutional DNA were tested for loss of heterozygosity (LOH) of the YNZ22 and p53 genes, both located in the 17p13 region. Loss of the p53 allele was found in 28 of 44 primary breast carcinomas (64%). In contrast LOH in only 26 of 61 tumors (43%) was detected with the variable number of tandem repeats (VNTR) probe YNZ22 mapping at 17p13.3 close to the p53 locus at 17p13.1. Among 19 tumors informative for both probes allele loss at 17p13.3 never occurred without p53 involvement. These data suggest, that p53 is the target of 17p13 allelic deletions in human breast cancer. Immunohistochemistry showed overexpression of the p53 protein in 25 of 50 cases (50%) presumably reflecting activating point mutations. Overexpression was not correlated with allele loss but seemed to be closely related to the presence of point mutations in this study. No homozygous deletions or rearrangements of the p53 gene were detected. This would argue for an important role of heterozygous p53 mutations in human breast cancer.     

Allelotype of human thyroid tumors: loss of chromosome 11q13 sequences in follicular neoplasms.

Two classes of genes are the targets of mutations involved in human tumorigenesis: oncogenes, the activation of which leads to growth stimulation, and tumor suppressor genes, which become tumorigenic through loss of function, often through allelic deletion. To obtain evidence for a role for tumor suppressor genes in thyroid tumorigenesis, we examined DNA from 80 thyroid neoplasms for loss of heterozygosity in multiple chromosomal loci using 19 polymorphic genomic probes. None of the informative thyroid tumors studied had allelic loss detected with probes for chromosome 2q (D2S44), 3p (D3F15S2, D3S32), 3q (D3S46), 4p (D4S125), 6p (D6S40), 8q (D8S39), 9q (D9S7), 12p (D12S14), 13q (D13S52), 17p (D17S30), or 18q (D18S10). One of eight of the follicular adenomas had a 10q deletion detected with marker D10S15, and one of 26 had a 10q deletion detected with D10S25. One of two of the follicular carcinomas had an 11p deletion in the H-ras locus. The most significant findings were on chromosome 11q13, the site containing the putative gene predisposing to multiple endocrine neoplasia type I. Four of 27 follicular adenomas had loss of heterozygosity for probes in this region. Allelic deletions were detected with the following probes: D11S149, PYGM, D11S146, and INT2. None of 13 informative papillary carcinomas and none of two follicular carcinomas had loss of heterozygosity detectable with these 11q13 markers. Allelic loss is a relatively infrequent event in human thyroid tumors. Deletions of chromosome 11q13 are present in about 14% of follicular, but not papillary, neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of Human Cadherin Genes to Chromosome Regions Exhibiting Cancer-Related Loss of Heterozygosity

This report presents the chromosomal localization of cadherin genes. Cadherins are cellular adhesion molecules. Since disturbance of intracellular adhesion is important for invasion and metastasis of tumor cells, cadherins are considered prime candidates for tumor suppressor genes. A variety of solid tumors show loss of heterozygosity of the long arm of chromosome 16, which is indicative of the potential location of tumor suppressor genes. Refined and new localizations of six cadherin genes (CDH3, 5, 8, 11, 13, and 15) to the long arm of chromosome 16 are shown. CDH15 was localized to 16q24.3, in a region that exhibits loss of heterozygosity in a number of sporadic breast cancer tumors. Previous localization of CDH13 (H-cadherin) to 16q24 suggested this gene as a tumor suppressor candidate in the 16q24.3 loss of heterozygosity region; however, refined mapping presented in this report localizes CDH13 proximal to this region. A human EST homologous to the chicken cadherin-7 was partially sequenced and found to represent a new human cadherin. This cadherin mapped to chromosome 18q22–q23, a region that exhibits loss of heterozygosity in head and neck squamous cell carcinomas. CDH16 was localized to 8q22.1, a region exhibiting loss of heterozygosity in adult acute myeloid leukemia.     

Chromosome 9p deletions in cutaneous malignant melanoma tumors: the minimal deleted region involves markers outside the p16 (CDKN2) gene.

We have analyzed 12 microsatellite markers on chromosome 9p in 54 paired cutaneous malignant melanoma (CMM) tumors and normal tissues. Forty-six percent of the tumors, including two in situ CMMs, showed loss of heterozygosity (LOH) at 9p. Only one tumor was homozygously deleted for 9p markers. The smallest deleted region was defined by five tumors and included markers D9S126 to D9S259. Loss of eight or more markers correlated significantly with worse prognosis (P < .002). Among the primary tumors, 87.5% of those with large deletions have a high risk of metastasis, as compared with only 18% of those without deletions or with loss of fewer than 8 markers (P < .001). It was not possible to demonstrate homozygous deletions of p16 in any of the CMM tumors. In four tumors, the LOH for 9p markers did not involve p16. The reported data suggest the existence of several tumor suppressor genes at 9p that are involved in the predisposition to and/or progression of CMM and exclude p16 from involvement in the early development of some melanoma tumors.     

Loss of heterozygosity on chromosome 5 in Iranian esophageal cancer patients

There is a high incidence of esophageal squamous cell carcinoma (ESCC) in Iran. Non-functionality of some tumor suppressor genes has been reported in esophageal cancer. Loss of heterozygosity on chromosome 5 has also been reported in esophageal carcinomas. We assessed loss of heterozygosity along a region of the long arm of chromosome 5 (5q), from 5q23.1 to 5q23.2, by PCR amplifying DNA fragments of tumor tissues from patients with ESCC and their corresponding normal samples. The PCR products were electrophoresed on 6% non-denaturing polyacrylamide gels, and band intensity was shown by silver staining. Of 40 patients with ESCC, 27, 25 and 36% of informative cases showed allelic losses at microsatellite markers D5S1384, D5S1478 and D5S1505, respectively. Two of the 40 patients studied had microsatellite instability at marker D5S1384. Based on the fact that loss of heterozygosity with more than 22% incidence for a specific marker cannot be regarded as a random event, we add support to previous reports concerning the presence of tumor suppressor genes in this chromosome region and that they affect esophageal cancer development. According to the data in NCBI UniSTS, the PCR product size of human DNA with primers of the D5S1505 marker ranges from 243 to 275 bp, containing about 20 repeats of the TAGA tetranucleotide, while the amplicon size of one allele of one of our cases was 207 bp, with about 10 repeats of the TAGA tetranucleotide, which would be the shortest sequence reported so far.     

Deletions in human chromosome arms 11p and 13q in primary hepatocellular carcinomas

Normal liver and hepatocellular carcinoma (HCC) genotypes were compared at loci on most of the human chromosomes with probes that detect restriction fragment length polymorphisms. Six of fourteen tumors exhibited loss of heterozygosity of one or more markers on 11p. Ten patients were informative for loci on 13q, and 5 of these 10 exhibited loss of heterozygosity for one or more of the 13q markers. Altogether, 9 of the 14 patients showed loss of a polymorphic allele for one or more loci on either 11p or 13q. A survey of loci on 16 additional chromosomes indicated that the deletions were not due to a general loss of heterozygosity in HCCs. Quantitative densitometry showed that each of the 10 deletions resulted in hemizygosity (no reduplication) of the remaining allele in tumor tissue. In contrast to hereditary embryonal tumors, in which reduplication of the remaining chromosome is the rule, simple deletion appears to be the primary mechanism responsible for the loss of heterozygosity in these adult, nonhereditary HCCs. These data show that HCCs arising in hepatitis B virus carriers are a genetically heterogeneous group of tumors, some of which may arise through 13q alterations, some through 11p alterations, some with both chromosomes altered, and some with both intact.     

Distal deletion of chromosome Ip in ductal carcinoma of the breast.

By use of recombinant DNA probes that correspond to genetic loci residing on human chromosome 1, DNA samples from 37 ductal breast carcinomas and constitutional DNA from the same individuals were tested for loss of heterozygosity. A high frequency (41%) of reduction to homozygosity was detected with the probe p1-79, which recognizes the highly polymorphic locus D1Z2, localized on 1p36. Loss of heterozygosity at other chromosome 1 loci was much less common, not exceeding a frequency of 10%, and was never observed in the absence of the D1Z2 loss. Somatic loss of heterozygosity at D1Z2 was more frequent in patients with a strong family history of breast cancer (60%), in patients with early diagnosis (before 45 years of age) (70%), and in those with multiple tumors or tumor foci (50%) than in patients with none of the characteristics of hereditary tumors (21%). No associations were observed between loss of heterozygosity and prognostic factors. These results suggest that inactivation of a tumor suppressor gene located on the distal portion of chromosome 1p, alone or combined with other genetic changes, may represent a fundamental step in the pathogenesis of ductal carcinoma of the breast.     

Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines

Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.     

A group of schwannomas with interstitial deletions on 22q located outside the NF2 locus shows no detectable mutations in the NF2 gene

Schwannomas are tumors arising mainly at cranial and spinal nerves. Bilateral vestibular schwannoma is the hallmark of neurofibromatosis type 2 (NF2). The NF2 gene has been cloned and comprehensive analysis of its mutations in schwannomas shows that up to 60% of tumors carry inactivating mutations. Thus, the genetic mechanism behind the development of more than 40% of schwannomas without NF2 mutations is unknown. We have therefore studied tumor tissue from 50 human schwannomas by allelotyping and have found chromosome 22 deletions in over 80% of the cases. We detected 14 cases (27%) that revealed partial deletions of one copy of chromosome 22, i.e., terminal and/or interstitial deletions. We sequenced the NF2 gene in seven of these tumors and detected only one case with mutations. The deletion mapping of chromosome 22 in tumors with partial deletions indicates that several regions, in addition to the NF2 locus, harbor genes involved in schwannoma tumorigenesis. Our findings suggest that heterogeneity in the mechanisms leading to the development of schwannomas probably exists. These findings are in agreement with the recent analysis of schwannomas from familial and sporadic cases of schwannomatosis and point to a possible role of an additional gene, which, in cooperation with the NF2 tumor suppressor, causes schwannomas. Received: 12 November 1998 / Accepted: 1 March 1999     

Deletions of the YNZ22and Alu-VpA/MycL1Loci and Post-Surgery Prognosis in Human Colorectal Adenocarcinoma

Since deletions of the short arm of chromosome 17 are the most common genetic defects in human colorectal carcinoma (CC), we tested the YNZ22locus (D17S30, 17p13.3) for loss of heterozygosity (LH) in adenocarcinoma and in the normal colonic mucosa of 49 CC patients, and studied the association of LH with clinicomorphological features of the tumor. Allele frequency distribution of YNZ22did not differ for the patients and healthy people. LH in YNZ22in the tumor was found in 33% (13/39) of all informative cases, its frequency being thrice higher in men than in women (²= 5.21, p= 0.022). The defect was associated with moderate or poor histological differentiation (P ₂= 0.0055) and polyploidy >3n(P ₂= 0.0035) of tumor cells and with high incidence of post-surgery relapse or metastasis. Analysis of both YNZ22and Alu-VpA/MycL1(1p34.3) loci in the tumor allowed reliable relapse prognosis in 76% of the CC patients. The probability of post-surgery relapse or metastasis was estimated at no less than 67% for patients with LH in at least one of the two loci in the tumor, and at somewhat more than 20% for patients without LH.     

Search for putative suppressor genes in meningioma: significance of chromosome 22

Summary Cytogenetic and molecular studies of various solid tumors have indicated that a series of different chromosomal regions may be deleted in the tumor genome. Usually, losses of heterozygosity are observed and, from this finding, the presence of specific genes acting as tumor suppressors has been deduced. In particular tumors, however, only a single chromosome site appears to be affected. Therefore, we have carried out a study of human meningioma, investigating 7 such putative suppressor regions by applying twelve site-specific DNA markers. In 6 out of 19 tumors, we exclusively found loss of heterozygosity for markers of the long arm of chromosome 22; none of the tumors showed statistically significant additional allelic losses for the regions 1p, 3p, 5p, 5q, 11p, 13q, 17p. Our data support the long-standing observation that only losses of or within chromosome 22 are associated with the development of meningiomas. Other suppressor regions are apparently not involved.     

Equal parental origin of chromosome 22 losses in human sporadic meningioma: no evidence for genomic imprinting.

Inactivation of tumor suppressor genes can occur either by mutation at the gene locus or by loss of part or all of the chromosome region containing the gene. The latter is most frequently detected by DNA markers as loss of heterozygosity in the tumor tissue. In several reports, the paternal homologue was preferentially retained in embryonal tumors associated with loss of particular chromosomal regions, suggesting genomic imprinting of the corresponding tumor suppressor loci. To explore the generality of these findings and the possible role of genomic imprinting in adult tumors of the nervous system, we have determined the parental origin of chromosome 22 loss in sporadic meningioma. Of nine cases studied, five tumors retained the maternally derived chromosome 22 homologue while four retained the paternally derived chromosome 22. Thus, in contrast to the embryonal tumors, the meningioma locus on chromosome 22 is inactivated by random mutation in sporadic adult meningiomas.     

Identification of New Members of the Gas2 and Ras Families in the 22q12 Chromosome Region

Monitoring of loss of heterozygosity in human tumors has suggested that the 22q12 region may contain another tumor suppressor gene(s) in addition to the NF2 gene. The genomic sequencing of a 40-kb region bounded by the EWS and BAM22 genes and containing a CpG-rich region has identified two new genes in the q12 region of chromosome 22. One of them, GAR22, is closely related to mouse Gas2, a gene isolated as being negatively regulated by serum and growth factors and exhibiting a high expression in growth arrested mouse fibroblasts. The other, RRP22, belongs to the Ras superfamily, in which it defines a new subgroup. Its expression appears to be strictly limited to the central nervous system. Growth-arrest-specific and Ras-related genes are likely candidates to be involved in tumorigenic processes. Although no mutation was observed in a small set of meningiomas and schwannomas, alteration of these new genes should now be searched in other tumors with frequent allelic losses on chromosome 22 not associated with NF2 mutation.     

Cervical carcinoma progression-associated genetic alterations on chromosome 6

To identify the loci associated with progression of cervical carcinoma, chromosome 6 regions were tested for loss of heterozygosity. Detailed analysis with 28 microsatellite markers revealed a high frequency of allelic deletions for several loci of the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16-21, 6q23-24, 6q25, 6q27) arms of chromosome 6. Examination of 37 microdissected carcinoma and 22 cervical dysplasia specimens revealed allelic deletions from the HLA class I-III genes (6p22-21.3) and subtelomeric locus 6p25 were found in more than 40% dysplasia specimens. With multiple microdissection of cryosections, genetic heterogeneity of squamous cervical carcinoma was analyzed, and clonal and subclonal allelic deletions from chromosome 6 were identified. Half of the tumors had clonal allelic deletion of D6S273 (6p21.3), which is in a Ly6G6D (MEGT1) intron in the HLA class III gene locus. The frequency of allelic deletions from the chromosome 6 long arm was no more than 20% in dysplasias. Allelic deletions from two loci, 6q14 and 6q16-21, were for the first time associated with invasion and metastasis in cervical carcinoma.     

Loss of heterozygosity of the tumor suppressor gene Tg737 in the side population cells of hepatocellular carcinomas is associated with poor prognosis

Analysis of loss of heterozygosity (LOH) is a useful method for finding genetic alterations in tumor and precancerous lesion tissues. In this study, we analyzed LOH of the tumor suppressor gene Tg737 in side population cells of human hepatocellular carcinomas. Side population cells were sorted and identification by flow cytometry from suspensions of hepatocarcinoma or normal liver cells generated from 95 hepatocellular carcinoma and normal tissues, respectively. DNA was extracted from the two groups of side population cells and peripheral blood specimens. Five microsatellite markers on the Tg737 gene were used to analyze the frequency of loss of heterozygosity in the side population cells of the hepatocellular carcinoma. Twenty-four (25.30%) tumor samples had a large deletion in more than three microsatellite markers. The highest frequency of loss of heterozygosity was observed with the G64212 marker (78.75%) and the SHGC-57879 marker (75.95%). Statistical analysis of the correlation between loss of heterozygosity of Tg737 and clinicopathological features indicated a strong correlation between the two markers associated with the highest frequency of loss of heterozygosity and survival. The results indicate that loss of heterozygosity of the tumor suppressor gene Tg737 may play an important role in the carcinogenetic mechanism of liver cancer stem cells. In addition, the independent association between loss of heterozygosity at the SHGC-57879 and G64212 markers and worsened short-term survival in patients could be used as a novel prognostic predictor. Further studies of side population cells may contribute to the establishment of novel therapeutic strategies for hepatocellular carcinoma.