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.     

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.     

Numerical chromosome alterations in colorectal carcinomas detected by fluorescence in situ hybridization

This study concerns DNA ploidy, numerical changes of chromosomes 7, 8, 10, 17 and 18, and allelic losses at chromosomes 17p13.3 (flanking the p53 gene) and 18q21 (location of the DCC gene) in 31 freshly resected colorectal tumours. Cytological smears were used to determine DNA ploidy by image analysis, and chromosome numbers by fluorescence in situ hybridization (FISH) using chromosome-specific pericentromeric -satellite DNA probes. Allelic losses were assessed by Southern blotting and by the polymerase chain reaction loss of heterozygosity method. Approximately 50% of the tumours were aneuploid. There was heterogeneity with respect to chromosome numbers, but gains and losses of chromosomes, or both, were detected in all carcinomas examined, including 10 that were nonaneuploid by image analysis. Trisomy 7 was found in 74% of the tumours, and monosomy of chromosome 18 in 32%. Allelic loss at chromosome 17p13.3 was evident in 13 of 26 informative cases, and only one case exhibited monosomy 17. In comparison monosomy 18 was found in 10 cases; 7 of them corresponded to approximately half of the cases with allelic loss within the DCC gene, and the other three were noninformative. These findings indicate that the loss of one chromosome 18 is an important mechanism producing allelic deletion of the DCC gene in colorectal carcinomas. Our data also suggest that monosomy 18 is a useful indicator for studying colorectal cancer progression on a cell by cell basis.     

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.     

A detailed deletion map of chromosome 20 in human oral squamous cell carcinoma

Loss of heterozygosity (LOH) on the long arm of chromosome 20 (20q) has been detected in several human cancers. However, little is known about LOH on chromosome 20 in oral squamous cell carcinoma (OSCC). To determine which loci of chromosome 20 were involved in OSCC tumorigenesis, 41 cases of OSCC were examined for LOH state on chromosome 20 at 17 microsatellite loci by PCR-LOH assay. LOH occurred in 41.5% of tumors in at least one locus. Among the 17 loci, D20S48 on 20p11.2 and RPN2 on 20q12-13.1 exhibited higher frequencies of LOH, 27.6% and 31.4%, respectively. The LOH incidence was significantly higher in tumors in which the primary site was on gingiva compared with other oral sites (p=0.012). Our results indicate that allelic deletions on 20q12-13.1 and 20p11.2 may play roles in OSCC carcinogenesis, and suggest that allelic deletions on 20q might have some relation with the primary site of OSCC.     

Identification of a I-cM region of common deletion on 13q14 associated with human prostate cancer.

Frequent allelic losses on chromosome arm 13q are observed in carcinomas of the head and neck, breast, ovary, and pituitary gland. We analyzed 59 primary prostate tumors (stage B, 18 patients; C, 12 patients; D1, 4 patients; and endocrine therapy-resistant cancer death, 25 patients), as well as 18 metastatic tissues from 14 of the 25 cancer death patients for loss of heterozygosity (LOH) using 35 microsatellite markers on chromosome arm 13q. Of the 59 primary tumors, 31 (53%) showed LOH involving at least one locus. Detailed deletion mapping identified a distinct commonly deleted region in the I-cM interval flanked by D13S153 and D13S273 on 13q14 and this region overlapped a part of the RB1 gene. Paired DNAs were available from both primary and metastatic tumors in the 14 cases of cancer death; among those pairs, we detected LOH on 13q in seven (50%) primary tumors, and in all metastatic foci (P = 0.0029). Moreover, the regions lost in metastatic tissues were more extensive than those seen in the corresponding primary tumors. These results suggest that inactivation of a putative tumor suppressor gene(s) including the RB1 gene on 13q14 plays an important role in human prostate cancer.     

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.     

Genetic alterations on 3p, 11q13, and 18q in nonfamilial and MEN 1-associated pancreatic endocrine tumors.

Pancreatic endocrine tumors occur sporadically and as part of the multiple endocrine neoplasia type 1 (MEN 1) and von Hippel-Lindau (VHL) syndromes. The MEN1 locus on 11q13 and a candidate tumor suppressor locus on 3p are known to be hemi- or homozygously mutated in a subset of these tumors. Chromosome arm 18q harbors the SMAD4/DPC4 tumor suppressor gene that is frequently deleted and inactivated in tumors of the exocrine pancreas. We have analyzed 22 nonfamilial and 16 MEN 1-associated pancreatic endocrine tumors for loss of heterozygosity (LOH) at 3p, 11q13, and 18q. LOH at 3p was revealed in 45% and 36% of tumors from 31 patients with nonfamilial and MEN 1-associated disease, respectively. The corresponding proportions for 11q13 were 55% and 91%, and for 18q 27% and 25%, respectively. A striking relation between LOH at 11q13 and 3p and a malignant phenotype was found for the nonfamilial tumors. None of the six benign tumors (all of them insulinomas) had allelic loss at 3p or 11q13, whereas 92% (P < 0.01) of the malignant tumors (including malignant insulinomas) had such deletions. Besides the 11q13 abnormality, more than half of the MEN 1-associated tumors had additional genetic lesions affecting 3p or 18q. LOH analysis of several tumors from two MEN 1 patients suggested different clonal origin of the lesions. Sequencing of the SMAD4/DPC4 gene did not identify mutations in coding regions or at exon/intron boundaries in tumors with LOH at 18q. The data indicate involvement of tumor suppressor genes on 3p and 18q, in addition to the MEN1 gene at 11q13, in the tumorigenesis of both nonfamilial and MEN 1-associated pancreatic endocrine tumors.     

Definition of a region of loss of heterozygosity at chromosome 11q in cervical carcinoma.

Chromosomal deletions at segment 11q23-q24 have been identified in a variety of human epithelial tumors, including cervical carcinoma (CC), indicating the presence in this region of at least a tumor suppressor gene (TSG) involved in the development of these neoplasms. To localize the 11q deletion target more precisely, 54 primary cervical carcinomas were examined for loss of heterozygosity (LOH) using a panel of microsatellite DNA markers mapping to 11p.15 and spanning region 11q23-qter. Nineteen tumors were found to have LOH at chromosome 11q. The highest frequency of LOH was observed at locus APOC-3, located in 11q23.1-q23.2, which was deleted in 42% of the informative cases. In contrast, LOH was infrequent at distal 11q in current series of CC. The smallest common region of loss included APOC-3 and was defined distally by marker D11S925 in region 11q23. The present data strongly suggest that the 11q suppressor gene(s) involved in cervical tumorigenesis is likely to be located at chromosome region 11q22-q23.     

Deletion mapping of 18q in conventional renal cell carcinoma

Loss of heterozygosity (LOH) is frequently associated with the inactivation of tumor suppressor genes. 18q LOH has been frequently reported in colorectal cancer and lung cancer; however, allelic loss on 18q has not been investigated in renal cell carcinoma (RCC). We evaluated LOH on 18q using nine microsatellite markers in 126 with conventional RCC (cRCC). LOH was observed in more than one 18q microsatellite locus in 24 cRCC (19%). We found the highest frequency of LOH (13.5%) at 18q21.3, where the DCC gene is located. We also assessed the relationship between LOH frequency and patient clinical parameters. Patients with a family history of cancer had a significantly higher frequency of 18q LOH than those without such a history (P=0.0017). No associations were found with other parameters, including gender, tumor grade, tumor stage, smoking status, and body mass index. The results suggest that inactivation of tumor suppressor genes at 18q21.3, including DCC and SMAD4 as candidates, may be involved in the tumorigenesis of some conventional RCCs.     

Loss of heterozygosity on 7q31 occurs early during breast tumorigenesis

Although human breast tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumor suppressor genes, the identity of the genetic alterations that are critical in the early stages of the breast tumorigenic process remains obscure. A high frequency (27–41%) of loss of heterozygosity (LOH) occurrence has been shown at the MET locus on chromosome band 7q31 and this specific alteration is associated with poorer survival. Here, we report that restriction fragment length polymorphism (RFLP) analysis on 221 informative (heterozygous) primary breast tumors and 57 informative relapses (13 local recurrences and 44 distant metastases) revealed a similar frequency of 7q31 LOH as tumors progress from primary cancer to relapse, in marked contrast to other changes such as 11p15.5 LOH. This finding suggests that inactivation of a putative tumor suppressor gene located in 7q31 is a very early event in breast tumorigenesis. Our results also show that metastatic potential is an induced phenomenon that occurs at a relatively early stage, rather than a marker of tumor progression.     

A comparison of the genetic pathways involved in the pathogenesis of three types of colorectal cancer

Patterns of allele loss (loss of heterozygosity, LOH) have been studied in order to investigate the genetic pathways involved in the pathogenesis of three types of colorectal cancer (CRC): sporadic CRC without replication errors (RER−) (32 cases); sporadic RER+ CRC (23 cases); and ulcerative colitis-associated CRC (UCACRC) (16 cases). Each tumour was assessed for allele loss at ten microsatellite markers which map close to known or putative tumour-suppressor genes: APC (5q21–q22); DCC (18q21.1); 1p35–p36; p16 (9p21); 22q; 8p; E-cadherin (16q22.1); β-catenin (3p22–p21.3); RB1 (13q14.1–q14.2); and HLA. Overall, high frequencies of allele loss (>30 per cent) were found near DCC (42 per cent), p16 (38 per cent), 22q (37 per cent), 1p35–p36 (34 per cent) and APC (31 per cent), and low frequencies (<20 per cent) near RB1 (16 per cent) and E-cadherin (13 per cent). LOH near β-catenin, HLA, and on 8p occurred at frequencies between 20 and 30 per cent. The overall frequency of allele loss did not differ among the three tumour groups, but some variation was seen at individual loci. There was a significantly higher frequency of LOH at 1p35–36 in RER+ tumours compared to RER− tumours. Allele loss at this site was also associated with a more advanced Dukes' stage at presentation. In addition, RER− tumours showed a higher frequency of allele loss at p16 than RER+ tumours. No significant difference existed at any locus between the frequency of LOH in sporadic CRC and in UCACRC. Pairwise analysis showed a negative association between LOH at APC and DCC, and between LOH at chromosome 22p and p53 overexpression. Thus, there may be specific differences between the mutation spectra of RER+ and RER− CRCs, but there are large degrees of overlap among the underlying genetic pathways of these cancers and UCACRCs. © 1998 John Wiley & Sons, Ltd.     

Mutation at chromosome 11q23 in human non-familial breast cancer: A microdissection microsatellite analysis

Allelotypic evaluation of loss of heterozygosity (LOH) has been instrumental in the identification of tumour suppressor genes. Here we report a high incidence of LOH at chromosome 11q23 in non-familial breast cancers with in situ, invasive, and metastatic tumour cells microdissected from archival haematoxylin and eosin (H & E) sections for polymerase chain reaction (PCR)-LOH analysis at polymorphic microsatellite loci. Ninety-four cases of non-familial breast cancer were examined at the D11S29 microsatellite locus on chromosome 11q23. Eighty-three cases (88 per cent) were informative and 35 cases overall (42 per cent) had LOH at this locus, comprising 23 per cent of in situ, 36 per cent of invasive, and 28 per cent of metastatic cancers. The DNA from those cancer cells with LOH was amplified at microsatellite loci D11S554 (11p12–p11.2) and D11S534 (11q13). In 19 of 67 cases overall (28 per cent), LOH occurred solely at 11q23. There was an association between LOH at 11q23 and tumour size ≥2 cm (P<0.01) in the overall results and the invasive cancers. The data revealed heterogeneity for LOH at D11S29 in in situ, invasive, and metastatic cells from the same case. In general, however, there was concordance between LOH (or its absence) in in situ and invasive disease. We conclude that the distal part of the long arm of chromosome 11 contains a region involved in breast carcinogenesis and that there is molecular heterogeneity at this chromosomal region in individual breast cancer cells.     

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.     

Novel somatic mutations in the BRCA1 gene in sporadic breast tumors

Germline mutations in two major susceptibility genes BRCA1 and BRCA2 contribute to the majority of inherited breast and ovarian cancers. Besides the germline mutation, tumor progression depends on the loss of a wild-type allele. Allelic losses in the BRCA1 and BRCA2 loci have also been detected in a high proportion of sporadic breast tumors, suggesting the role of these genes in the development of non-inherited breast cancer. Forty unselected breast tumors were analyzed for the loss of heterozygosity (LOH) at BRCA1 and BRCA2 regions and tumors with allelic deletions were screened for the presence of acquired genetic alterations in respective genes. 21.1% of 38 informative tumor samples carried LOH at the BRCA1 locus whereas 33.3% of 39 informative samples showed LOH at the BRCA2 locus. Pathogenic truncating mutations in the BRCA1 gene were found in two tumor samples with allelic losses, whereas no mutations were identified in the BRCA2 gene. Mutations were not detected in non-tumor samples from the same individuals, which indicated that the BRCA1 allele was inactivated by somatic mutations in tumor tissue. The c.1116G>A (1235G>A) nonsense mutation (p.W372X) belongs to the genetic abnormalities detected infrequently in hereditary tumors; the c.3862delG (3981delG) frameshift mutation (p.E1288fsX1306) is a novel gene alteration. The occurrence of inactivating somatic mutations in sporadic breast tumors suggested the role of the BRCA1 gene in tumorigenesis in at least a minor group of patients with non-familial breast cancer.     

Three distinct commonly deleted regions of chromosome arm 16q in human primary and metastatic prostate cancers

Human prostate cancers frequently show loss of heterozygosity (LOH) at loci on the long arm of chromosome 16 (16q). In this study, we analyzed prostate cancer specimens from 48 patients (Stage B, 20 cases; Stage C, 10 cases; cancer death, 18 cases) for allelic loss on 16q, using either restriction fragment length polymorphism (RFLP)- or polymerase chain reaction (PCR)-based methods. Allelic losses were observed in 20 (42%) of 48 cases, all of which were informative with at least one locus. Detailed deletion mapping identified three distinct commonly deleted regions on this chromosome arm: q22.1–q22.3, q23.2–q24.1, and q24.3-qter. On the basis of a published sex-averaged framework map, the estimated sizes of the commonly deleted regions were 4.7 (16q22.1–q22.3), 17.2 (16q23.2–q24.1) and 8.4 cM (16q24.3-qter). Allelic losses on 16q were observed more frequently in the cancer-death cases (11 of 18; 61%) than in early-stage tumor cases (9 of 30; 30%; P < 0.05). In 7 of 11 patients from whom DNA was available from metastatic cancers as well as from normal tissues and primary tumors, the primary cancer foci had no detectable abnormality of 16q, but the metastatic tumors showed LOH. These results suggest that inactivation of tumor suppressor genes on 16q plays an important role in the progression of prostate cancer. We also analyzed exons 5–8 of the E-cadherin gene, located at 16q22.1, in tumor DNA by means of PCR-single strand conformation polymorphism and direct sequencing, but we detected no somatic mutations in this candidate gene. Genes Chromosom Cancer 17:225–233 (1996). © 1996 Wiley-Liss, Inc.     

Study of allelic losses on 3p, 6q, and 17p in human urothelial cancer.

Forty-eight transitional cell carcinomas of the bladder and three transitional cell carcinomas of the renal pelvis were examined for loss of heterozygosity (LOH) on chromosomes 3p, 6q, and 17p. The most frequent allelic loss was seen on 17p (18/36, 50%) followed by 6q (6/22, 27%), and 3p (5/22, 23%). In cases with LOH at more than one locus, the same DNA sample often varied in degree of signal reduction for missing alleles. This observation indicates that LOH studies can serve to detect intratumor heterogeneity. No correlation was found between allelic losses at these chromosome arms and tumor grade and stage. Allelic losses on 6q were associated with tumors having a solid growth pattern; in this kind of tumors, allelic losses on 3p were associated with invasion.     

Infrequent genetic alterations of the PTEN gene in Japanese patients with sporadic prostate cancer

Prostate cancer is a major cause of cancer death among elderly men in America, Europe, and Japan. However, the molecular mechanism of carcinogenesis is not yet well characterized. Frequent loss of heterozygosity (LOH) on chromosome 10q was reported in prostate cancer, and a candidate tumor suppressor gene, PTEN, was isolated on chromosome band 10q23.3. To investigate the genetic alterations of PTEN, we examined 45 primary prostate cancer specimens. LOH at the PTEN locus was observed in two (11.1%) of 18 tumors. However, no mutations were observed in any of the primary prostate cancers. These data suggest that mutation of the PTEN gene does not play a major role in prostate carcinogenesis of Japanese patients. Received: February 6, 1998 / Accepted: July, 3, 1998