Allele loss from chromosome 17 in ovarian cancer

In a number of human cancers genes capable of suppressing tumorigenicity have been identified and in some instances cloned. Successful isolation of such tumour suppressor genes has depended upon either the mapping of a locus which confers susceptibility to a specific tumour, or the finding of specific allele loss in the tumour cells of heterozygous individuals. In ovarian cancer it is known that a small proportion (approximately 5%) of tumours are due to inheritance (Lynch et al., 1989). However, as yet the locus responsible has not been mapped. The only incidence of allele loss in ovarian tumours reported is on the short arm of chromosome 11 using a c-Ha-ras I probe to detect an RFLP (Lee et al., 1989), and on 3p and 6 in a small number of cases (Ehlen & Dubeau (1990)). We describe here the results of analysis of 19 tumours for allele loss using a probe for a hypervariable locus on the long arm of chromosome 17. Approximately 77% (10/13) of tumours from informative patients showed complete or partial allele loss at this locus. Using a probe for the short arm of chromosome 17, 31% (4 of 13 informative patients) demonstrated allele loss at this position. These results suggest that possible involvement of more than one chromosomal locus in the development of ovarian cancer.     

The locus of the polymorphic epithelial mucin (PEM) tumour antigen on chromosome 1q21 shows a high frequency of alteration in primary human breast tumours

Tumour and blood leukocyte DNAs from sporadic breast cancer patients were examined for chromosome 1 loss of heterozygosity using a probe for a polymorphic epithelial mucin, PEM, which is expressed in greater than 92% of breast carcinomas as well as in normal lactating breast tissue. Expression is detected by the monoclonal antibodies (MAbs) HMFG-1, -2 and SM-3 which react with epitopes in the 20 amino-acid repeat unit of the core protein. The PEM probe has been mapped to the chromosome band 1q21, a region that is often incriminated in chromosomal rearrangements in breast tumours. Loss of heterozygosity or alteration at the PEM locus was detected in 34% of the 70 informative patients examined. Twenty of the 24 individuals showed loss of an allele, whereas 4 showed gain of an additional allele or amplification of an existing allele. Twenty-eight percent of informative cases exhibited alterations at the MS32 locus, 1q42-43, and 20% had alterations at the short arm locus MS1 at 1p33-35. These findings identify the long arm of chromosome 1 and in particular the region around the PEM gene for localization of a gene whose loss or alteration may, in some tumours, contribute to the progression of disease in breast cancer patients.     

Molecular genetic investigation of sporadic renal cell carcinoma: analysis of allele loss on chromosomes 3p, 5q, 11p, 17 and 22.

To investigate the role of tumour-suppressor genes on the short arm of chromosome 3 in the mechanism of tumorigenesis in non-familial renal cell carcinoma, we analysed 55 paired blood-tumour DNA samples for allele loss on chromosome 3p and in the region of known or putative tumour-suppressor genes on chromosomes 5, 11, 17 and 22. Sixty-four per cent (35/55) of informative tumours showed loss of heterozygosity (LOH) of at least one locus on the short arm of chromosome 3, compared with only 13% at the p53 tumour-suppressor gene and 6% at 17q21. LOH at chromosome 5q21 and 22q was uncommon (2-3%). Detailed analysis of the regions of LOH on chromosome 3p suggested that, in addition to the VHL gene in chromosome 3p25-p26, mutations in one or more tumour-suppressor genes in chromosome 3p13-p24 may be involved in the pathogenesis of sporadic renal cell carcinoma (RCC). We also confirmed previous suggestions that chromosome 3p allele loss is not a feature of papillary RCC (P < 0.05).     

p53 allele losses, mutations and expression in breast cancer and their relationship to clinico-pathological parameters.

The p53 locus on the short arm of chromosome 17 at 17p 13.1 was examined for loss of heterozygosity, mutation, mRNA and protein expression in 60 primary breast cancers. Allele loss around the p53 locus was detected in 19/45 informative tumours (42%). p53 mutations in the evolutionarily conserved exons 5 to 9 were detected in 17/60 (28%) by amplification mismatch and confirmed by direct DNA sequencing. p53 mRNA expression was detected by Northern blot in 36/59 (61%) of tumours, and p53 protein expression using antibody 1801 on frozen-tissue sections in 13/44 of the tumours examined. p53 mutation was significantly associated with oestrogen-receptor-poor tumours (p less than 0.01) and hence with poor prognosis, but not with other clinical or pathological parameters. There was no statistical correlation between loss of heterozygosity around the p53 locus at 17p13.1 and p53 mutation. Furthermore, p53 mutation was not associated with p53 expression detected by immunohistochemical staining with antibody 1801 or as p53 mRNA. In addition, events on 17p (allele losses, p53 mutation, p53 expression) were independent of c-erbB-2 expression. In breast cancer, by contrast with colorectal, lung and ovarian cancer, there appears to be no clear association between p53 DNA abnormalities and p53 expression.     

Allele loss at the c-Ha-ras1 locus in human ovarian cancer

Recent reports have shown allele loss at the c-Ha-ras1 locus on the short arm of chromosome 11 in some types of tumors. To determine whether loss of heterozygosity occurs at the c-Ha-ras1 locus in uncultured human ovarian carcinomas we used Southern blot analysis to study DNA from 17 pairs of ovarian tumors and matched white blood cell samples from the same patients. In one of these 17 tumors, the c-Ha-ras1 locus was rearranged, and in five tumor DNAs from ten informative patients, a c-Ha-ras1 allele was lost. This loss, of relatively high incidence, appears to be an important characteristic of human ovarian cancer and may provide a useful tool for understanding its biological behavior.     

Clonal allele loss in gastrointestinal cancers

Using a panel of DNA probes for hypervariable DNA regions we screened 52 gastrointestinal carcinomas for clonal allele losses on chromosomes 1, 5, 7, 12, 16 and 17. A total of 24/35 informative cases of colorectal cancers showed loss of constitutional heterozygosity at a locus on chromosome 17p, while 9/31 cases informative for a locus on 5q showed allele loss. Loss of sequences at 5q was linked to allele loss at 17p with a single exception. In gastric cancers loss of heterozygosity most frequently occurred at 1q (5/10 tumours) and at 12q (6/11 tumours). Gastrointestinal tumours show consistent chromosomal losses and the loci involved are different in gastric and colorectal cancers.     

Loss of alleles on chromosome 18 and on the short arm of chromosome 17 in polyploid colorectal carcinomas

The zygosity of 19 colorectal carcinomas (either near-diploid or polyploid) from patients known to be heterozygous for RFLPs located on chromosome 18 or on the short arm of chromosome 17 has been examined. In most cases, at least one allele was significantly under-represented. The reason for the absence of complete loss of heterozygosity was investigated for 5 polyploid tumors. It was shown that the diploid component which, in these tumors, is essentially composed of non-neoplastic cells, remains heterozygous as the polyploid component invariably loses heterozygosity. The results strongly suggest that many colorectal carcinomas originate from a single cell which had lost at least part of either chromosome 18 or of one short arm of chromosome 17, or both.     

Polymerase chain reaction allelotyping of human ovarian cancer.

We have used a set of microsatellite polymorphisms (MSPs) to examine the location and frequency of allele loss throughout the genome in a panel of 25 human epithelial ovarian tumours. When more than one MSP was employed per arm, mean informativity was 85.2% (range 64-100%). The average fractional allelic loss was 0.28 (range 0-0.65). A high frequency of allele loss was seen at 5q (40%), 9q (48%), 11p (43%), 14q (46%), 15q (40%), 17p (61%), 17q (64%), 19p (45%) and Xp (40%), confirming previous findings at some sites, but also suggesting the existence of new tumour-suppressor genes in regions (9q, 14q, 15q) which have not previously been studied in ovarian cancer. For 9q and 14q, partial loss of the arm was more common than loss of heterozygosity for all loci. There was a significant relationship between allele loss affecting the short arm of chromosome 17 and allele loss affecting 17q (P < 0.001). No other relationship was detected between allele losses at different sites. Polymerase chain reaction allelotyping is suitable for the examination of very small tumour samples and tumours in which classical karyotyping is problematic.     

Loss of heterozygosity at selective sites on chromosomes 13 and 17 in human breast carcinoma.

Four chromosomal regions were tested for loss of constitutional heterozygosity in primary tumours from 85 Icelandic breast cancer patients. Loss of heterozygosity and other types of gene rearrangements were observed in 37% of informative cases at the retinoblastoma locus, RB1, on chromosome 13q. Allele losses on chromosome 17 were tested with two polymorphic probes on 17p and two on 17q. Loss of heterozygosity or other types of genetic rearrangement were detected in 43.5% of cases on 17p near the p53 gene and 40.5% on 17q. In our study abnormalities at the RB1 locus and on chromosome 17 frequently occurred together, indicating that the coincident inactivation of more than one tumour suppressor gene may, in some cases, play a part in tumour formation. No significant correlation was found between these losses and clinico-histological parameters. Family history of breast cancer was found to be more common among patients with RB1 deletions and this trend was strengthened in cases where there were deletions at both the RB1 locus and on chromosome 17.     

Frequent loss of heterozygosity on chromosome 18 in ovarian adenocarcinoma which does not always include the DCC locus.

Inactivation of the DCC gene on chromosome 18 owing to loss of heterozygosity is a common finding in colorectal cancer. Because both ovarian and colon cancer are features of Lynch syndrome II, which has been provisionally mapped to chromosome 18, we hypothesized that loss of heterozygosity at the DCC locus may also occur in ovarian neoplasia. Fifty-two sporadic ovarian adenocarcinoma tumours were analysed by Southern blotting for loss of heterozygosity (LOH) at six chromosome 18 loci. Overall, tumours from 31 patients (60%) showed allelic loss at one or more of these loci. A similarly high level of LOH, 66%, was found at D17S5 (17p13.3). In contrast, moderate levels of LOH, of 31%, 39% and 33%, were found at MYCL1 (1p32), D1S57 (1p) and D14S20 (14q32.33) respectively. However, analysis of partial chromosome deletions in 11 patients indicates that the smallest region of overlap appears to exclude the DCC gene but to be between the D18S5 and D18S11 loci. This suggests that another locus, as well as or apart from DCC, may be involved.     

Loss of heterozygosity in Wilms' tumour involves two distinct regions of chromosome 11

Pairs of tumour and normal DNA samples from 38 Wilms' tumour patients have been investigated for loss of heterozygosity using 12 probes from chromosome 11. Allele loss was detected in only 11 cases (31%). Densitometric analysis showed that allele loss was not due to non-disjunction or hemizygous deletion, but rather to mitotic recombination or non-disjunction plus reduplication. Although the development of homozygosity sometimes involved the whole of the short arm of chromosome 11, in a few tumours allele loss was restricted to band 11p15 or 11p13 and distal sequences. This suggests mutations in two distinct regions play an important role in Wilms' tumorigenesis. There was no apparent correlation between loss of heterozygosity and tumour stage, age of presentation, or prior exposure to chemotherapy.     

Assignment of common allele loss in osteosarcoma to the subregion 17p13

Human osteosarcomas frequently show loss of alleles on chromosome 17 as well as those on chromosome 13 that harbors the retinoblastoma gene, indicating concerted operation of another tumor-suppressing gene on chromosome 17. To assign the affected gene to a defined region of chromosome 17, we performed mitotic recombination/deletion mapping by the use of 10 polymorphic loci on chromosome 17. Of 37 tumors studied, 28 (75.7%) showed loss of heterozygosity on chromosome 17. The affected regions varied among tumors, ranging in extent from a whole chromosome to a distal segment of the short arm. However, allele loss in one region, notably in 17p13 between D17S1 and D17S30, was common to all 28 tumors, suggesting the presence of a tumor-suppressing gene in this defined region.     

Allelic loss on the short arm of chromosome 11 in non-small-cell lung cancer.

Forty-eight samples of primary non-small-cell lung cancer (NSCLC) and normal tissue from the same patients were analyzed for allelic deletions on chromosome 11p. Five polymorphic loci were assessed to determine the incidence of 11p sequence deletions and to define hot-spots of deletions. Information was obtained from all patients in at least one locus. Our data show that the deletions observed were not randomly scattered over the short arm of chromosome 11. Rather, 2 hot-spots of deletions were observed: one in the area of the genes for catalase and beta-FSH corresponding to band 11p13, the other close to the IGF-II locus corresponding to band 11p15. A high incidence of loss of heterozygosity (LOH) was found with the probe for catalase (21/29), a locus flanking the centromeric region of the Wilms' tumor locus. Most of the samples exhibiting LOH of one or more of the alleles analyzed remained heterozygous for at least one other chromosome 11p allele. Furthermore, duplication of the intensity of the remaining allele was rarely observed. Our results indicate that LOH on the short arm of chromosome 11 is a common event in NSCLC and that the chromosomal region containing the Wilms' tumor locus is most commonly involved.     

Deletion of the c-Ha-ras-1 allele in human skin cancers

Previous studies have shown that c-Ha-ras-1 and other genes located on the short arm of chromosome 11 are frequently lost in a number of human tumors. We investigated whether similar losses of the c-Ha-ras-1 allele occurred in human squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs). DNAs were isolated from 35 pairs of skin tumors (25 BCCs and 10 SCCs) and matching normal skin from the same patients and analyzed for c-Ha-ras gene polymorphism by Southern blot hybridization. Sixteen BCC patients and 1 SCC patient were constitutionally heterozygous for the c-Ha-ras gene in their normal skin DNA. Of these 17 patients, five patients (four with BCC and one with SCC) (29%) showed loss of one of the c-Ha-ras alleles in their tumor DNA. One of the constitutionally heterozygous BCC patients exhibited deletion of the 6.6-kb c-Ha-ras allele and an extra copy of the 7.8-kb allele. In summary, loss of heterozygosity at the c-Ha-ras locus occurred frequently (29%) in the 17 human skin cancers studied. However, our finding that 90% of the patients with SCC, as opposed to 36% of the patients with BCC, had only one of the c-Ha-ras alleles in their normal skin tissue requires further study. Whether c-Ha-ras homozygosity has any bearing on genetic susceptibility to SCC remains to be established.     

Atm heterozygosity does not increase tumor susceptibility to ionizing radiation alone or in a p53 heterozygous background

Ataxia-Telangiectasia (A-T) is an autosomal recessive human disease characterized by genetic instability, radiosensitivity, immunodeficiency and cancer predisposition, because of mutation in both alleles of the ATM (ataxia-telangiectasia mutated) gene. The role of Atm heterozygosity in cancer susceptibility is controversial, in both human and mouse. Earlier studies identified deletions near the Atm gene on mouse chromosome 9 in radiation-induced lymphomas from p53 heterozygous mice. To determine whether Atm was the target of these deletions, Atm heterozygous as well as Atm/P53 double heterozygous mice were treated with ionizing radiation. There were no significant differences in tumor latency, progression and lifespan after gamma-radiation in Atm heterozygous mice compared with their wild-type control counterparts. Deletions were found on chromosome 9 near the Atm locus in radiation-induced tumors, but in 50% of the cases the deletion included the knockout allele, and the expression of Atm was maintained in the tumors indicating that loss of heterozygosity on chromosome 9 is not driven by Atm, but by an alternative tumor suppressor gene located near Atm on this chromosome. We conclude that Atm heterozygosity does not confer an increase in tumor susceptibility in this context.     

Definition and refinement of chromosome 8p regions of loss of heterozygosity in gastric cancer.

Loss of heterozygosity at several chromosomal loci is a common feature of the malignant progression of human tumors. These regions are thought to harbor one or more putative tumor suppressor gene(s) playing a role in tumor development. Allelic losses on the short arm of chromosome 8 (8p) have been reported as frequent events in several cancers, and three commonly deleted regions have been defined at 8p11.2-12, 8p21-22, and 8p23.1. To evaluate the possible involvement of these regions in gastric cancer, we used eight microsatellite markers to perform an extensive analysis of allele loss at 8p21-22 in 52 cases of primary gastric adenocarcinoma. We found that 44% of tumors showed allelic loss for at least one marker at 8p21-22. The critical region of loss was found to be between markers LPL and D8S258, which displayed loss of heterozygosity in 39% and 33% of cases, respectively. This region is centromeric to the LPL locus and centered on the D8S258 locus. We conclude that 8p22 deletion is a frequent event in gastric cancer and suggest the presence of a putative tumor suppressor gene near the D8S258 locus. Initial steps were taken toward the identification of this gene, which is likely to play an important role in the pathogenesis of gastric cancer and of other tumors as well.     

Molecular genetic analysis of chromosome 11p in familial Wilms tumour.

In the family reported here, a mother and both of her children developed a Wilms tumour, and all three tumours were of the relatively rare monomorphous epithelial histopathological subtype. Using restriction fragment length polymorphism analysis, both sibs were shown to inherit the same maternal allele from the 11p13 region but different maternal alleles from the 11p15 region. Using a combination of single-strand conformation polymorphism (SSCP) and polymerase chain reaction (PCR) sequencing techniques, no mutations were identified in the WT1 tumour-suppressor gene from the 11p13 region, but a novel polymorphism was identified in exon 1. mRNA expression studies using the insulin-like growth factor II (IGF-II) gene, located in 11p15, showed that there was no relaxation of imprinting at this locus. There was also no evidence of loss of heterozygosity on the long arm of chromosome 16. These findings indicate that the WT1 and IGF-II genes, together with the long arm of chromosome 16, are not directly implicated in tumorigenesis in this Wilms family, but that a recombination event has occurred on the short arm of chromosome 11.     

Presence of two members of c-erbA receptor gene family (c-erbA beta and c-erbA2) in smallest region of somatic homozygosity on chromosome 3p21-p25 in human breast carcinoma

The loss of heterozygosity of genes on the short arm of chromosome 3 (3p) in human breast carcinomas occurs in a region involved in other malignancies, including renal cell carcinoma, lung cancers, and von Hippel-Lindau disease. This finding suggests the presence of a gene(s) that plays a crucial role in multiple cancers. In our study of 84 informative (heterozygous) primary breast tumors, 30% showed losses of heterozygosity on chromosome 3. The shortest region of homozygosity in primary human breast tumor is located between the DNF15S2 and RAF1 loci in the 3p21-p25 region on the short arm of chromosome 3. This region includes at least two members of the c-erbA steroid/thyroid hormone receptor family (c-erbA beta and c-erbA2) that may be of special relevance to breast cancer. Furthermore, tumors with a loss of heterozygosity of genes on chromosome 3 were previously reported to have frequent allelic deletions on chromosome 11p and amplification of the c-myc proto-oncogene. These results highlight the occurrence of multiple genetic alterations in breast tumors.     

Preferential loss of maternal alleles in sporadic Wilms' tumour

Loss of heterozygosity at loci on the short arm of chromosome 11 has been reported in 31% (11/38) of Wilms' tumours in our series. Lymphoblastoid cell lines were prepared from the parents of 10/11 of the patients showing allele loss in their tumours. In 9 of the cases, where the parental origin of the alleles could be followed, it was the paternal alleles which were retained in the tumour. This preferential loss of the maternal alleles implies a role for genomic imprinting in the pathogenesis of Wilms' tumour.     

PCR-RFLP studies on chromosome 3p in formaldehyde-fixed,paraffin-embedded cervical cancer tissues

Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p2l-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.