Allelic imbalance study of 16q in human primary breast carcinomas using microsatellite markers

The high incidence of allelic imbalance on the long arm of chromosome 16 in breast cancer suggests its involvement in the development and progression of the tumor. Several loss of heterozygosity (LOH) studies have led to the assignment of commonly deleted regions on 16q where tumor suppressor genes may be located. The most recurrent LOH regions have been 16q22.1 and 16q22.4-qter. The aim of this study was to gain further insight into the occurrence of one or multiple “smallest regions of overlap” on 16q in a new series of breast carcinomas. Hence, a detailed allelic imbalance map was constructed for 46 sporadic breast carcinomas, using 11 polymorphic microsatellite markers located on chromosome 16. Allelic imbalance of one or more markers on 16q was shown by 30 of the 46 tumors (65%). Among these 30 carcinomas, LOH on the long arm of chromosome 16 was detected at all informative loci in 19 (41%); 13 of them showed allelic imbalance on the long but not on the short arm, with the occurrence of variable “breakpoints” in the pericentromeric region. The partial allelic imbalance in 11 tumors involved either the 16q22.1-qter LOH region or interstitial LOH regions. A commonly deleted region was found between D16S421 and D16S289 on 16q22.1 in 29 of the 30 tumors. The present data argue in favor of an important involvement of a tumor suppressor gene mapping to 16q22.1 in the genesis or progression of breast cancer.     

Loss of heterozygosity on chromosome arm 16q in breast cancer metastases

One of the main genetic abnormalities associated with breast carcinogenesis is the loss of genetic material from chromosome arm 16q. Different groups have identified two regions (16q22.1 and 16q24-ter) that are frequently deleted in primary tumors, suggesting the presence of tumor suppressor genes in these regions. Little is known about the late stages of tumor progression in this respect, and we, therefore, analyzed biopsy specimens of breast cancer metastases for deletions in these critical regions of 16q. We examined fine needle cytopunctures from 24 metastases, each with lymphocyte DNA, for allelic imbalance on 16q by means of polymerase chain reaction (PCR) with 15 highly polymorphic markers. All the metastatic samples showed deletion of at least one informative locus on 16q. The loss of heterozygosity (LOH) pattern often indicated the loss of a complete long arm of chromosome 16 (13 cases); nevertheless, in the remaining 11 samples, partial LOH patterns were observed. A small region of overlap (SRO2) in 16q22.1 was frequently involved, whereas another (SRO1) in 16q24-ter was affected in only two cases. A third region of LOH in 16q22.2-q23.2 was found in 6/11 samples. These results suggest that at least three different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer. Loss of material from the third region could be a major event in the genesis of metastases. Genes Chromosom. Cancer 19:185–191, 1997. © 1997 Wiley-Liss Inc.     

Mapping of a new target region of allelic loss to a 2-cM interval at 22q13.1 in primary breast cancer

Allelic losses on chromosome arm 22q are frequently observed in human meningiomas and in carcinomas of the colon, ovary, and breast. Among 140 primary breast cancers we examined for loss of heterozygosity (LOH) at 16 polymorphic loci on the long arm of chromosome 22, 56 (40%) showed LOH for at least one locus. Eleven of these tumors had retained heterozygosity for markers proximal to the NF2 locus but showed LOH for markers distal to NF2. Deletion mapping indicated a new common region of deletion, 2-cM in extent, at q13.1 between Interleukin 2 receptor β (IL2RB) and D22S279. Our results raise the possibility that one or more tumor suppressor genes associated with breast cancer may exist at 22q13.1. Comparison of these results with clinicohistological data indicated that allelic losses on 22q tend to occur more frequently in tumors of malignant histological types. Genes Chromosomes Cancer 21:108–112, 1998. © 1998 Wiley-Liss, Inc.     

Lower frequency of allele loss on chromosome 18q in human breast cancer than in colorectal tumors

Inactivation of tumor suppressor genes is thought to be a critical step in tumorigenesis. TheDCC (deleted in colorectal carcinoma) gene, located on the long arm of chromosome 18, has been shown to be frequently deleted in colorectal tumors. To investigate the involvement of allelic deletions on chromosome 18q in breast cancer tumorigenesis we analyzed 28 primary breast tumors and 28 colorectal, tumors (24 carcinomas, 4 adenomas) with four different polymorphic DNA markers detecting RFLPs on chromosome 18q. In breast cancer we found loss of heterozygosity (LOH) in 4 of 27 (15%) informative cases whereas 15 of 25 (60%) colorectal tumors showed allelic deletions. In all cases of allelic loss theDCC locus or its proximal vicinity (locus SSAV1) were involved. LOH on chromosome 18q occurs both in breast and colorectal cancer, yet the frequency of these deletions in breast tumors is lower than in colorectal tumors. Moreover, in breast cancer these mutations were only detected in large and undifferentiated tumors.Abbreviations LOH Loss of heterozygosity     

Defining regions of loss of heterozygosity of 16q in breast cancer cell lines

The loss of heterozygosity (LOH) of chromosome 16 was assessed in 21 breast cancer cell lines and two nontumorigenic breast epithelial cell lines by typing microsatellite markers distributed on this chromosome. In addition, dual-color fluorescence in situ hybridization was used to metaphase spreads of these cell lines using chromosome 16 paint and region specific probes. Eleven of the cell lines had LOH for chromosome 16, two for the entire chromosome, three for the long arm, and six had LOH for restricted regions of the long arm. The results supported evidence that there are two predominant regions of LOH, 16q22.1 and 16q24.3. The cell lines with chromosome 16 LOH can be used for screening candidate tumor suppressor genes at 16q in breast cancer.     

Mapping of a new target region of allelic loss to a 6-cM interval at 21q21 in primary breast cancers

A detailed analysis of loss of heterozygosity (LOH) in breast cancers was performed with 11 microsatellite markers on the long arm of chromosome 21. Among the 142 tumors examined, 44 (31%) showed LOH at one or more loci. Peak LOH frequency was observed on band 21q21. Deletion mapping identified a new commonly deleted region in a 6-cM interval of 21q21 between loci D21S1432 and D21S1437, and raised the possibility that one or more tumor suppressor genes associated with breast cancer may exist in this region. Comparison of these alterations with clinicopathological parameters revealed an association of LOH on 21q with loss of progesterone receptor (P = 0.0013). Genes Chromosomes Cancer 23:244–247, 1998. © 1998 Wiley-Liss, Inc.     

Different mechanisms of chromosome 16 loss of heterozygosity in well- versus poorly differentiated ductal breast cancer

Loss of heterozygosity (LOH) at the long arm of chromosome 16 is a frequent genetic alteration in breast cancer. It can occur by physical loss of part of or the entire chromosomal arm, resulting in a decrease in copy number or loss followed by mitotic recombination. Comparative genomic hybridization (CGH) demonstrated that well-differentiated breast tumors showed significantly more physical loss of 16q than did poorly differentiated ones and that this difference was already discernable in the preinvasive stage. However, polymorphic markers detected no difference in the frequency of 16q LOH between invasive tumors of different histological grade. Here, by combining data on LOH (n=52), fluorescence in situ hybridization (n=18) with chromosome 16-specific probes, and CGH (n=34), we show a preference in well-differentiated grade I tumors for physical loss of chromosome arm 16q, whereas in poorly differentiated grade III tumors LOH is accompanied by mitotic recombination. This clarifies the discrepancies observed between CGH and LOH for 16q in breast cancer. These different somatic genetic mechanisms may reflect the presence of multiple tumor suppressor genes that are the target of LOH at chromosome arm 16q.     

Mapping the chromosome 16 cadherin gene cluster to a minimal deleted region in ductal breast cancer

The cadherin family of cell adhesion molecules has been implicated in tumor metastasis and progression. Eight family members have been mapped to the long arm of chromosome 16. Using radiation hybrid mapping, we have located six of these genes within a cluster at 16q21-q22.1. In invasive lobular carcinoma of the breast frequent LOH and accompanying mutation affect the CDH1 gene, which is a member of this chromosome 16 gene cluster. CDH1 LOH also occurs in invasive ductal carcinoma, but in the absence of gene mutation. The proximity of other cadherin genes to 16q22.1 suggests that they may be affected by LOH in invasive ductal carcinomas. Using the mapping data, microsatellite markers were selected which span regions of chromosome 16 containing the cadherin genes. In breast cancer tissues, a high rate of allelic loss was found over the gene cluster region, with CDH1 being the most frequently lost marker. In invasive ductal carcinoma a minimal deleted region was identified within part of the chromosome 16 cadherin gene cluster. This provides strong evidence for the existence of a second 16q22 suppressor gene locus within the cadherin cluster.     

Loss of heterozygosity on chromosome arms 5q,IIp, IIq,I3q,and I6p in Human Testicular Germ Cell Tumors

To identify common regions of deletion in human testicular germ cell tumors (TGCTs), we have screened tumors from 33 patients for loss of heterozygosity (LOH) using Southern blot analysis with 39 polymorphic markers covering 21 chromosome arms. Losses in more than 2 tumors and occurring at a frequency of > 10% were found on chromosome arms 5q, 11p, 11q, 13q, and 16p, the highest being on chromosome arm 5q (19%). It is suggested that tumor suppressor genes on 5q among others may be involved in testicular tumorigenesis and that LOH in this region requires further investigation. No losses were found on 12q and 17p despite the fact that the most common cytogenetic abnormality in TGCTs is an i( 12p) and that the TP53 gene on 17p is the most frequently mutated gene in human cancers. The level of allelic imbalance varied considerably from one chromosome region to another (0–80%) and did not generally reflect the pattern of LOH. It tended to be high in overrepresented regions of the genome, 1q, 7p, and 12p. The tumor from one patient had a seminomatous component and a less differentiated component. We provide evidence for a common origin of both components and show that it is likely that this tumor has progressed from the seminoma to the less differentiated histology. © 1995 Wiley-Liss, Inc.     

Frequent loss of chromosome arm Ip DNA in parathyroid adenomas

Two molecular defects have been described in parathyroid adenomas: rearrangement and overexpression of the PRADI/cyclin DI oncogene and allelic loss of chromosome II DNA, often including the multiple endocrine neoplasia type I (MENI) putative tumor suppressor gene region. In an effort to identify additional parathyroid tumor suppressor genes, we examined 25 parathyroid adenomas for tumor-specific allelic loss of polymorphic DNA loci located near known or candidate tumor suppressor genes. Control leukocyte DNA from all 25 patients was heterozygous for I or more of the 9 chromosome I markers examined. Allelic loss at I or more of these informative loci on chromosome I was observed in 10 of 25 (40%) adenomas. Although many tumors lost extensive regions on chromosome I, all but one of these tumors had allelic loss of distal I p (I p32-pter); four tumors also lost loci on Iq. Allelic loss at IIqI3, the site of the MEN I gene, was detected in 5 of 2I (24%) informative cases, including 3 with Ip loss. In contrast, allelic loss was rarely observed at loci on 99 and Iop and was not observed at loci on 3p, 3q. 4p, 5q, I2q, I4q, I8q, 22q, or Xp. In summary, clonal allelic loss of loci on chromosome arm Ip is a frequent feature of parathyroid adenomas, implying that inactivation of a tumor suppressor gene(s) on Ip commonly contributes to their pathogenesis. © 1995 Wiley-Liss, Inc.     

Allelic losses on 18q21 are associated with progression and metastasis in human prostate cancer

We analyzed normal/tumor DNA pairs obtained from 46 patients with prostate cancers (stage B, 16 cases; C, 10 cases; D1, 4 cases; and endocrine therapy-resistant cancer-death, 16 cases) for loss of heterozygosity using 32 microsatellite markers on chromosome 18. Seventeen of the 46 cases (37%) showed loss of heterozygosity (LOH) for at least one locus on the long arm. Detailed deletion mapping in these tumors identified a distinct commonly deleted region within a 5-cM interval on 18q21.1. There was a statistical correlation between the frequency of LOH on 18q and clinical stage (χ² = 12.3; P = 0.0064). LOH on 18q was observed more frequently in Stage D1 cases (4/4; 100%) than in Stage B+C cases (5/26; 19%; P = 0.0046, Fisher's exact test). In 8 of 9 (89%) cancer-death patients from whom DNAs were available from both primary and metastatic tumors, the primary tumors had either no detectable abnormality of chromosome 18 or the region involving loss of heterozygosity was limited while the metastatic foci showed more frequent and extended allelic losses on this chromosome. No abnormalities were detected in the DCC and DPC4 genes when their exons were analyzed separately by single strand conformation polymorphism assay. These results suggest that inactivation of one or more putative tumor suppressor genes on 18q21 other than DCC and DPC4 plays an important role in the progression of human prostate cancer. Genes Chromosomes Cancer 20:140–147, 1997. © 1997 Wiley-Liss, Inc.     

Allelic loss of chromosomal arm 8p in breast cancer progression.

Loss of heterozygosity (LOH) of chromosomal arm 8p has been reported to occur at high frequency for a number of common forms of human cancer, including breast cancer. The objectives of this study were to define the regions on this chromosomal arm that are likely to contain breast cancer tumor suppressor genes and to determine when loss of chromosomal arm 8p occurs during breast cancer progression. For mapping the tumor suppressor gene loci, we evaluated 60 cases of infiltrating ductal cancer for allelic loss using 14 microsatellite markers mapped to this chromosomal arm and found LOH of 8p in 36 (60%) of the tumors. Whereas most of these tumors had allelic loss at all informative markers, five tumors had partial loss of 8p affecting two nonoverlapping regions. LOH for all but one of the tumors with 8p loss involved the region between markers D8S560 and D8S518 at 8p21.3-p23.3, suggesting that this is the locus of a breast cancer tumor suppressor gene. We then studied LOH of 8p in 38 cases of ductal carcinoma in situ (DCIS) with multiple individually microdissected tumor foci evaluated for each case. LOH of 8p was found in 14 of the DCIS cases (36%), including 6 of 16 cases of low histological grade and 8 of 22 cases of intermediate or high histological grade. In four of these DCIS cases, 8p LOH was seen in some but not all of the multiple tumor foci examined. These data suggest that during the evolution of these tumors, LOH of 8p occurred after loss of other chromosomal arms that were lost in all tumor foci. Thus, LOH of 8p, particularly 8p21.3-p23, is a common genetic alteration in infiltrating and in situ breast cancer. Although 8p LOH is common even in low histological grade DCIS, this allelic loss often appears to be preceded by loss of other alleles in the evolution of breast cancer.     

Hemizygous deletions of chromosome band 16q24 in Wilms tumor: detection by fluorescence in situ hybridization

Loss of heterozygosity (LOH) for markers on chromosome arm 16q in Wilms tumor has been linked to an increased risk of treatment failure. We therefore postulated that fluorescence in situ hybridization (FISH) with probes from this region might enhance current strategies for identifying high-risk patients at diagnosis. In a blinded comparative pilot study of 19 Wilms tumor samples from 18 patients with favorable histology, FISH and DNA polymorphism analysis yielded concordant results in 14 cases, either retention (n = 6) or loss (n = 8) of chromosome arm 16q markers. Discordant findings in 4 of the 5 remaining cases resulted from detection of LOH, but no loss by FISH. Two of these cases, directly comparable at marker D16S422, appeared to have tumor-specific uniparental disomy, in that 2 copies of D16S422 and the 16 centromere were evident, despite LOH. In 2 other cases, the discrepancies could be explained by LOH confined to loci distal to the D16S422 locus. In the fifth case, FISH detected 2 distinct populations of tumor cells, one characterized by normal diploidy and the other by monosomy 16, whereas DNA polymorphism analysis failed to indicate LOH altogether. Thus, FISH confirmed the presence of allelic loss (hence, the possible location of biologically important tumor suppressor genes) on the distal long arm of chromosome 16 in cases of favorable-histology Wilms tumor, with the advantages of technical simplicity, successful analysis of samples that were otherwise uninformative by analysis of DNA polymorphisms, and the addition of internal controls for chromosomal aneusomy. We suggest that combined analysis of the chromosome 16q region in Wilms tumor by FISH and DNA polymorphism analysis would improve evaluations to identify high-risk patients who might benefit from alternative therapy.     

Allelic losses at loci on chromosome 10 are associated with metastasis and progression of human prostate cancer

DNA samples from tumors and paired normal tissues from 48 patients with prostate cancer (stage B, 16 cases; stage C, 14 cases; stage D, 18 cases) were examined with 26 polymorphic markers spanning chromosome 10. Allelic losses were observed in 17 of the 46 cases (37%) that were informative with at least one of the markers. Detailed deletion mapping identified two distict commonly deleted regions on the long arm of chromosome 10 (10q22–q24:7cM and 10q25.1:17cM) and one on 10p, suggesting that at least three tumor suppressor genes associated with prostate cancer are present on this chromosome. We observed loss of heterozygosity more frequently in tumors from fatal cases (stage D, 8/16, 50%) than in localized tumors (stage B, 0/16, 0%; P = 0.001 or stage B + C, 5/30, 17%; P = 0.02 Fisher's exact test). All metastatic tissues showed allelic loss at one or more loci on 10q. In five of the nine patients from whom DNAs were available from both metastatic and primary tumors, the primary cancer foci had no detectable abnormality of chromosome 10, while the metastatic foci showed allelic loss on chromosome 10. These results suggested that inactivation of one or more tumor suppressor genes on chromosome 10 plays an important role in late stages of prostate cancer. Genes Chromosom Cancer 17:245–253 (1996). © 1996 Wiley-Liss, Inc.     

Chromosome 8p alterations in sporadic and BRCA2 999del5 linked breast cancer

Chromosomal losses involving the short arm of chromosome 8 are frequent in a variety of tumour types, including breast cancer, suggesting the presence of one or more tumour suppressor genes in this region. In this study, we have used 11 microsatellite markers to analyse loss of heterozygosity (LOH) at chromosome 8p in 151 sporadic breast tumours and 50 tumours from subjects carrying the BRCA2 999del5 mutation. Fifty percent of sporadic tumours compared to 78% of BRCA2 linked tumours exhibit LOH at one or more markers at 8p showing that chromosome 8p alterations in breast tumours from BRCA2 999del5 carriers are more pronounced than in sporadic breast tumours. The pattern of LOH is different in the two groups and a higher proportion of BRCA2 tumours have LOH in a large region of chromosome 8p. In the total patient material, LOH of 8p is associated with LOH at other chromosome regions, for example, 1p, 3p, 6q, 7q, 9p, 11p, 13q, 17p, and 20q, but no association is found between LOH at 8p and chromosome regions 11q, 16q, 17q, and 18q. Furthermore, an association is detected between LOH at 8p and positive node status, large tumour size, aneuploidy, and high S phase fraction. Breast cancer patients with LOH at chromosome 8p have a worse prognosis than patients without this defect. Multivariate analysis suggests that LOH at 8p is an independent prognostic factor. We conclude that chromosome 8p carries a tumour suppressor gene or genes, the loss of which results in growth advantage of breast tumour cells, especially in carriers of the BRCA2 999del5 mutation.


Keywords: chromosome 8; BRCA2; LOH; breast cancer     

Frequent loss of heterozygosity in the region of the D7S523 locus in advanced ovarian cancer

Loss of heterozygosity (LOH) of the long arm of chromosome 7 occurs frequently in many types of primary cancers. We analyzed 22 primary ovarian cancers for LOH of chromosome arm 7q using a set of 16 microsatellite markers in order to determine the location of a putative tumor suppressor gene (TSG). Eleven samples (50%) showed LOH at least at one locus on chromosome arm 7q. We identified the smallest commonly deleted region to be at 7q31.1, which includes D7S523. LOH of chromosome arm 7q was more frequent in advanced stages (III–IV) (7/9, 78%) than in early stages (I–II) (4/13, 31%) of ovarian cancer (P<0.05). These data suggest that alteration of a TSG at 7q31.1 gene plays an important role in advanced ovarian cancer. Genes Chromosom. Cancer 19:1–5, 1997. © 1997 Wiley-Liss, Inc.     

Allelotype of uterine cancer by analysis of RFLP and microsatellite polymorphisms: Frequent loss of heterozygosity on chromosome arms 3p, 9q, 10q,and 17p

Cancers in which mutations have been identified in putative tumor suppressor genes, such as the TP53 gene, the retinoblastoma (RBI) gene, the adenomatous polyposis coli (APC) gene, and the Wilms tumor (WTI) gene, frequently show loss of the corresponding allele on the homologous chromosome. To identify locations of tumor suppressor genes involved in uterine cancer, we examined loss of heterozygosity (LOH) by using genomic probes detecting RFLPs in 35 uterine cancers at 29 loci throughout the genome, and with highly informative microsatellite markers in 21 uterine cancers at nine putative or known tumor suppressor gene loci. High frequencies of allelic loss found at loci on 3p (71%), 9q (38%). 10q (35%). and 17p (35%) suggest that tumor suppressor genes involved in uterine carcinogenesis exist in these regions. There were no significant differences in frequencies of LOH between cancers of the uterine cervix and cancers of the uterine endometrium at any of the loci tested. Genes Chrom Cancer 9:119-123 (1994).© 1994 Wiley-Liss, Inc.     

Detection of frequent allelic loss of 6q23–q25.2 in microdissected human breast cancer tissues

Detection of allelic loss in human breast cancer is hindered by the fact that breast cancer tissues are frequently infiltrated by stromal and inflammatory cells. For this study, we carefully microdissected infiltrating breast cancer tumor cells from contaminating normal cells and analyzed the DNA from these samples for allelic loss on the long arm of chromosome 6 by using a panel of 15 dinucleotide repeat markers. We found 53 of the 66 cases studied (80%) to have allelic loss of either the entire chromosomal arm (37 cases) or a portion of the chromosomal arm (16 cases). One common region that was identified for all tumors with deletions of 6q was the area between markers D6S310/314 and D6S473/255, consistent with a tumor suppressor gene locus at 6q23–6q25.2. The use of tissue microdissection allowed the detection of allelic loss in this chromosomal region in human breast cancer at a much higher frequency than was previously recognized. Genes Chromosom Cancer 16:35–39 (1996). © 1996 Wiley-Liss, Inc.