Preferentially deleted chromosome region 9p21 in large hepatocellular carcinomas

The role of somatic deletions in chromosome 9 and chromosome 22 loci in hepatocellular carcinomas (HCC) was studied. Twenty-one paired HCC and adjacent tumor-free liver tissue samples were examined for loss of heterozygosity at six chromosome 9 and ten chromosome 22 loci. Among informative cases, the highest LOH rates were observed at 9p21 (40% or 4/10 at IFNA) and 9q23 (23% or 3/13 at D9S318). Our observed LOH rate at 9p21 was significantly higher than the background level previously reported for the same tumor type. Clinical data indicate that chromosome 9p21 deletions occurred preferentially in larger tumors (>5 cm diameter). However, a sequence analysis of the MTS1 gene coding region in cases of 9p21 LOH did not reveal any change, suggesting another tumor suppressor gene as the LOH target.     

Loss of heterozygosity on chromosome 1 and 9 and hormone receptor analysis of metastatic malignant melanoma presenting in breast

Malignant melanoma (MM), the most common metastatic solid tumor to involve the breast, may present as a diagnostic problem, frequently requiring the use of ancillary studies for accurate diagnosis. The implication of hormonal interplay is strong since metastatic MM to the breast is seen nearly always in women. However, the role of hormonal status as a predisposing factor in the development of this entity is largely unresolved. A number of chromosomal loci, including 1p36 and 9p21-22, appear to harbor critical genes important to melanoma tumorigenesis, and additionally chromosome 9q22.3-31. We wanted to know if metastatic MM in breast showed chromosome 1p and 9p genetic alterations (loss of heterozygosity) similar to those that occur in primary cutaneous MM, and whether additional 9q LOH changes are present. Hormonal receptor status of the metastatic MM was also determined. We identified 20 patients with known MM metastatic to the breast, which we analyzed with the following genetic markers: D9S12 (9q22.3), D9S171 (9p21), IFNA (9p22), and D1S450 (1p). Visually directed microdissection was performed on archival histologic slides containing both tumor and adjacent normal breast epithelium, followed by single-step DNA extraction and polymerase chain reaction (PCR) amplification for evaluation of loss of heterozygosity (LOH) for the above-listed markers. Immunohistochemical (IHC) stains for estrogen receptor (ER) and progesterone receptor (PR) was performed on 10 of the cases. Twelve of the 20 cases contained DNA suitable for PCR amplification following direct visualization microdissection. Four of 8 (50%) informative cases showed LOH at 9p21 with D9S171. Ten cases were heterozygous for IFNA, with 2 cases (20%) showing LOH at this locus. These particular cases also showed LOH at 9p21. One of 9 (11%) informative cases showed LOH for D1S450 (1p36). Five cases were heterozygous for D9S12, and 2 (40%) showed LOH in the tumor at 9q22.3. IHC stains for ER and PR were negative in the 10 tumors studied. Metastatic MM presenting as a breast mass is an interesting entity often requiring IHC studies for diagnosis, particularly when the histologic features simulate breast carcinoma or when no primary tumor is known. These tumors are ER and PR negative. Metastatic MM involving the breast shows similar genetic allelic losses on chromosome 9p21-22 (50%) and 1p36 (11%), as previously described in primary cutaneous MM. Additional LOH was observed at the 9q22.3-31 locus (40%). We suggest this locus to be investigated for harboring potential genes important in the tumorigenesis of cutaneous MM.     

Identification and mapping of novel tumor suppressor loci on 6p in diffuse large B-cell non-Hodgkin's lymphoma.

Allelic deletions have been thought to be indicators of the presence of tumor suppressor genes (TSGs). As indicated by this allelotype study using 39 highly informative microsatellite markers distributed among all autosomal chromosomes, frequent loss of heterozygosity (LOH) has been found at 6p in B-cell non-Hodgkin's lymphoma. To identify the commonly deleted regions (CDRs), we performed fine deletion mapping using 26 highly polymorphic microsatellite markers on 6p. The most frequent LOH occurred at D651721, where 9 of 18 of the informative cases (50%) had allelic losses. Seventeen of 32 cases (53%) exhibited LOH at least at one locus on 6p. Ten of these 17 cases showed interstitial deletions, and their LOH patterns indicated two CDRs on 6p; one between D6S1721 and D6S260 (at 6p23-24), and the other between D6S265 and D6S291 (at 6p21). The genetic distance of both CDRs was 6 cM. The CDKN1A (p21) gene is reported to be located within the interval of the CDR at 6p21, but no mutation of the gene was found in these 32 patients. These data suggested that these two loci might harbor novel putative TSGs responsible for the pathogenesis of malignant lymphoma. We have constructed a contig of yeast artificial chromosome (YAC) clones spanning the most frequent CDR at 6p23-24. This YAC contig can be used for fine physical mapping of the region and cloning of candidate TSGs.     

Clonal relationships between epidermotropic metastatic melanomas and their primary lesions: a loss of heterozygosity and X-chromosome inactivation-based analysis.

Loss of heterozygosity (LOH) has previously been demonstrated at multiple chromosome microsatellites in primary and metastatic melanomas. Epidermotropic metastases of melanoma are unique in their varied histopathologic appearance, which can mimic a primary lesion. Our objective was to compare LOH profiles in primary and epidermotropic metastatic melanoma to delineate their clonal relationship. We examined the pattern of allelic loss in the primary melanomas of nine patients in addition to the 21 corresponding epidermotropic metastatic melanomas (average 2.3 metastases per patient). DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection. Eight DNA microsatellite markers on six different chromosomes were analyzed: D1S214 (1p), D6S305 (6q), D9S171 (9p), D9S157 (9p), IFNA (9p), D10S212 (10q), D11S258 (11q), D18S70 (18q). In addition, X-chromosome inactivation analysis was performed in tumors from four women. LOH was seen in 67% (6/9) of primary melanomas and 81% (17/21) of epidermotropic metastatic melanomas. The most frequent allelic losses in informative cases occurred at 10q (33%), 9p (22%), and 11q (22%) in primary melanomas, and at 10q (50%), 1p (44%), and 6q (39%) in epidermotropic metastatic melanomas. Primary lesions demonstrating LOH had concordant allelic loss in at least one locus in a corresponding epidermotropic metastatic melanoma in 83% (5/6) of cases. X-chromosome analysis showed nonrandom inactivation in 75% (3/4) and 71% (5/7) of primary melanoma and epidermotropic metastatic melanoma cases, respectively. Our LOH and X-chromosome inactivation analysis data suggest that epidermotropically metastatic melanomas are clonally related to their primary lesion in many cases. Our data also indicated that some cases diagnosed as epidermotropic metastatic melanoma might be divergent clones or new primaries rather than metastatic disease.     

Inverted papilloma of the urinary bladder: a molecular genetic appraisal.

Inverted papilloma of urinary bladder is an uncommon urothelial neoplasm. Its relationship to urothelial carcinoma is controversial. Little is known of the genetic abnormalities of inverted papilloma. To better understand its genetics, we analyzed 39 inverted papillomas, including 36 from men and three from women, for loss of heterozygosity (LOH). We examined four polymorphic microsatellite markers located on chromosome 9q32-33(D9S177), chromosome 9p22 (IFNA), chromosome 3p14.2 (D3S1300) and chromosome 17p13.1 (TP53), where genetic alterations occur frequently in urothelial carcinomas. Additionally, the status of inactivation of X-chromosome was examined in three female patients. The frequency of LOH in informative cases was 8% (3 of 37) for D9S177, 10% (4 of 38) for TP53, 8% (3 of 37) for IFNA and 8% (3 of 36) for D3S1300. In the analysis of X-chromosome inactivation, all three cases yielded informative results and one had nonrandom inactivation of X-chromosomes. The monoclonal origin demonstrated in the study of X-chromosome inactivation indicates the clonal process of inverted papilloma; however, the low incidence of LOH supports the view that inverted papilloma in urinary bladder is a benign neoplasm with molecular genetic abnormalities different from those of urothelial carcinoma.     

3p21, 5q21, and 9p21 allelic deletions are frequently found in normal bronchial cells adjacent to non-small-cell lung cancer, while they are unusual in patients with no evidence of malignancy.

Molecular cytogenetic and loss of heterozygosity (LOH) analyses of non-small-cell lung cancer (NSCLC) have shown frequent allelic deletions in a variety of chromosomes, such as 1p, 3p, 5q, 8p, 9p, 11p, 11q, and 17p. Allelic loss at 3p21, 9p21, and 5q21 has also been reported in premalignant epithelial lesions of the bronchus and in normal bronchial cells. These findings suggest that a tissue field of somatic genetic alterations precedes the histopathological phenotypic changes of carcinoma. LOH at chromosomal regions 3p21, 5q21, 9p21, and 17p (TP53) was looked for in the peritumoural normal bronchial cells from 30 archival surgically resected tumours. Microdissected normal bronchial cells from 20 benign cytological smears were also added to the study. Matched populations of lymphocytes, tumour cells, and normal bronchial cells adjacent to the tumour were microdissected from paraffin-embedded tissues, while matched populations of normal bronchial cells and inflammatory cells were microdissected from benign cytological smears (bronchial brushings). Polymerase chain reaction (PCR) amplification was performed utilizing the specific markers D5S346, D3S1300, D9S157, D9S171, and TP53. Within the NSCLC tumour cells, LOH was more frequently found at the 5q21 locus (72% of the informative cases), the 3p21 locus (47%), 9p21 (48%), and 17p (33%). Within the peritumoural normal bronchial cells, LOH at 5q21 was found in 37.5% of the cases, 22% showed LOH at 3p21, 27% at 9p21, and 13% at 17p (TP53). LOH was also detected in one case, in normal bronchial cells obtained from cytological smears at one locus (5q21). In conclusion, normal bronchial mucosa adjacent to NSCLC has frequent allelic losses at 3p21, 5q21, and 9p21, while LOH at these loci is unusual in normal bronchial cells obtained from cytological smears from patients with no evidence of malignancy. LOH at these loci may be present before the onset of the malignant growth. LOH studies may supplement the histopathological evaluation of bronchial cells to detect genotypic alterations in both cytological and biopsy specimens.     

Deletion mapping of the short arm of chromosome 3 in Merkel cell carcinoma

Little is known about the biology of Merkel cell carcinoma (MCC), also called small cell carcinoma of the skin. MCC has similarities with small cell lung cancer (SCLC): both are neuroendocrine malignancies with early metastasis to distant sites and a poor prognosis. Small cell lung cancer biopsies are known to have frequent losses on chromosome 3 in the region 3p21, yet MCCs have not been reported to have 3p deletions by karyotypic analysis. Considering the similarities between SCLC and MCC, we investigated 26 MCC tumours for loss of heterozygosity (LOH) on 3p. First, RFLP analysis was performed using PCR with nine primer sets from six loci. Second, 25 tumours were examined by microsatellite analysis for 3p markers D3S1289 and D3S1285 and SST on 3q. All 26 tumours were informative at one or more loci; of these, 18 (69%) demonstrated LOH for at least one marker on the short arm. For all informative loci the frequency of LOH was greater than 30% (range 33–75%). In a cell line derived from one tumour, it was possible to demonstrate rearrangement of chromosome 3 by in situ hybridisation. No LOH was seen in 15 informative cases for the 3q locus SST. A region 3p13-p21.1, centered on the marker D3S2, was deleted in all tumours demonstrating LOH, with a secondary deletion involving D3S30 detected in some tumours at 3p13. Our results indicate that LOH on 3p is a common occurrence in MCC; however, three tumours for which DNA was also available from a corresponding cell line suggest there may be a subset of MCC whose genesis is independent of deletions of 3p. Genes Chromosom Cancer 15:102–107 (1996) © 1996 Wiley-Liss, Inc.     

Molecular evidence supporting the neoplastic nature of odontogenic keratocyst: a laser capture microdissection study of 15 cases

AIMS: The bland histology of odontogenic keratocyst (OKC) belies its capacity for aggressive behaviour. Genetic alterations of OKC have not been well studied. We examined the frequency and pattern of allelic imbalance on five different chromosome regions from 15 patients with OKC. METHODS AND RESULTS: Laser-assisted microdissection was performed on formalin-fixed paraffin-embedded tissue. Polymerase chain reaction analysis of extracted DNA targeted five polymorphic DNA markers (D3S1285, D9S161, D11S1316, D13S290, and TP53) representing chromosome regions 3p14, 9p21, 11q23, 13q12.1 and 17p13, respectively. All 15 cases of OKC were informative at a minimum of three of five loci, with 11 informative on all five loci. Twelve of 15 cases (80%) demonstrated loss of heterozygosity (LOH). Seven cases (47%) showed LOH at more than two DNA loci. The frequency of LOH was 5/11 (45%) at D3S1285, 3/15 (20%) at D9S161, 4/14 (29%) at D11S1316, 8/14 (57%) at D13S290 and 3/15 (20%) at TP53. CONCLUSIONS: The majority of OKCs harbour chromosomal abnormalities. This finding supports the supposition that OKCs are neoplastic. Furthermore, OKCs harbour allelic loss at some of the same loci identified in squamous cell carcinoma. This may aid in explaining the rare occurrence of squamous cell carcinoma arising in OKC.     

Genome-wide analyses on loss of heterozygosity in head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor survival rate. Identifying the tumor suppressor gene (TSG) loci by genomic studies is an important step to uncover the molecular mechanisms involved in HNSCC pathogenesis. We therefore performed comprehensive analyses on loss of heterozygosity (LOH) using a genome-wide panel of 191 microsatellite markers in 22 HNSCC samples. We found 53 markers with significantly high LOH (>30%) on 21 chromosomal arms; the highest values of those were observed on 3p, 9p, 13q, 15q, and 17p, corresponding to D3S2432 (67%), D9S921-D9S925 (67%) and GATA62F03 (86%), D13S1493 (60%), D15S211 (62%), and D17S1353 (88%), respectively. Fifteen hot spots of LOH were defined in 13 chromosomal arms: 2q22-23, 4p15.2, 4q24-25, 5q31, 8p23, 9p23-24, 9q31.3, 9q34.2, 10q21, 11q21-22.3, 14q11-13, 14q22.3, 17p13, 18q11, and 19q12 as loci reported previously in HNSCCs. Furthermore, we identified five novel hot spots of LOH on three chromosomal arms in HNSCC at 2q33 (D2S1384), 2q37 (D2S125), 8q12-13 (D8S1136), 8q24 (D8S1128), and 15q21 (D15S211). In conclusion, our comprehensive allelotype analyses have unveiled and confirmed a total of 20 possible TSG loci that could be involved in the development of HNSCC. These results provide useful clues for identification of putative TSGs involved in HNSCC by fine mapping of the suspected regions and subsequent analysis for functional genes.     

Genetic Changes in Chromosomes 1p and 17p in Thyroid Cancer Progression

Little is known about the genetic alterations that occur during the progression of thyroid neoplasms. To understand better the biology of thyroid tumors, we investigated several genetic loci in benign and malignant thyroid neoplasms. Forty-one thyroid tumors (6 adenomas, 16 papillary, 14 follicular, and 5 anaplastic carcinomas) were studied. Normal and tumor cells were microdissected from paraffin-embedded tissues. DNA was used for polymerase chain reaction-based loss of heterozygosity (LOH) analysis with the following markers: D1S243 (1p35–36), D1S165 (1p36) and D1S162 (1p32), TP53 (17p13), and INT-2 (11q13). Immunohistochemistry for Ki-67 was performed. The Ki-67 labeling index (LI) was the percentage of positive tumor cells. LOH at 1p was seen in 2 of 5 (40%) informative cases of anaplastic carcinoma (2 of 2 at D1S162 and 1 of 2 at D1S165) and in 2 of 11 (18%) informative cases of follicular carcinoma (2 of 7 at D1S243, 2 of 7 at D1S165, and 1 of 6 at D1S162). One anaplastic (20%) and two follicular carcinomas (14%) had LOH in at least two of the 1p loci analyzed. None of the adenomas and papillary carcinomas had LOH at these loci. LOH at 17p and 11q13 were infrequent. Ki-67 LI was 1.4, 7, 16, and 65% in adenomas, papillary, follicular, and anaplastic carcinomas, respectively. Allelic loss at 1p may occur in aggressive types of thyroid carcinoma and may be a marker of poor prognosis. LOH at 1p may represent a late genetic event in thyroid carcinogenesis. LOH at 17p and 11q13 (MEN gene locus) is uncommon in thyroid neoplasms.     

Renal cell carcinoma: allelic loss at chromosome 9 using the fluorescent multiplex-polymerase chain reaction technique

Loss of tumor-suppressor genes on both arms of chromosome 9 appears to be common in many types of cancer. Chromosome 9q is often partially deleted in bladder cancer, lung cancer, and basal cell carcinoma. However, little data are available on allelic loss on chromosome 9 in renal cell carcinoma (RCC). One hundred and nine nonpapillary RCCs were studied for loss of heterozygosity (LOH) at 13 loci on chromosome 9 by using the fluorescent multiplex-polymerase chain reaction method to compare DNA from tumor samples and peripheral blood lymphocytes. At the loci tested, LOH was found in from 2.3% (9q31, D9S938) to 17% (9q22, 1AJL) of informative cases, and 27 (24.8%) of the 109 RCCs had LOH at 1 or more loci of chromosome 9. LOH was more often detected at 9q22 within the PTCH gene (17%) when compared with LOH at the other 12 loci (P = 0.0172). Regarding the relationship with clinical parameters, however, there were no statistically significant associations between this LOH and tumor stage or grade. Among the 109 tumors, 6 (5.5%) showed replication errors. Our results suggest that LOH of the PTCH gene may be related to the development of nonpapillary RCC, although the clinical relevance has not been not clarified.     

Loss of heterozygosity of chromosome 13 in Merkel cell carcinoma

We have examined a series of 24 Merkel cell carcinoma (MCC) DNAs for loss of heterozygosity (LOH) at eight loci on chromosome 13. All patients were heterozygous for at least one locus. Overall, 18 of 24 (75%) patients showed LOH, among whom 10 patients demonstrated LOH at all informative loci. A single common region of loss was identified in all cases and included the marker D13S233 (13q14.3), which maps close to the retinoblastoma susceptibility gene RB1. The RB1 protein was not detected by Western blot analysis in any of nine MCC cell lines tested. These data indicate that 13q losses are the most common chromosomal losses observed to date in MCC and the likely target of these deletions is the RB1 locus. Genes Chromosom. Cancer 20:93–97, 1997. © 1997 Wiley-Liss, Inc.     

Fine allelotyping of Erbb2-induced mammary tumors in mice reveals multiple discontinuous candidate regions of tumor-suppressor loci

Loss of heterozygosity (LOH) at human chromosome bands 1p32-36 and 10q23-26 is frequent in various human tumors, including breast cancers, and is thought to reflect the loss of tumor-suppressor genes (TSGs). To map such genes, high-resolution LOH analysis was performed on 93 Erbb2-induced mammary tumors from (BALB/c x C57BL/6) F1 MMTV/Erbb2 transgenic mice. A panel of 24 microsatellite markers specific to the region of mouse chr4, homologous to human 1p31-36, and 16 markers specific to the mouse chr19 region, homologous to human 10q23-26 were used. In addition, lower-density mapping was performed on the remaining portion of mouse chr4 [homologous to human 9p13, 9p21-24, 9q21-22, 9q31-34 (12 markers)] and chr19 [homologous to 9q21, 9p24, 11q12-13 (9 markers)]. Several distinct, discrete, and discontinuous LOH regions flanked by areas of heterozygosity were identified, 22 on chr4 and 14 on chr19. Among these, 13 were mapped in the region of homology with human 1p31-36 (between D4Mit153 and D4Mit254) and 9 in the region of homology with human 10q23-26 (between D19Mit46 and D19Mit6). Although several LOH loci span a large interval, many are relatively short (1-4 Mb), and a few span an interval of <1 Mb. This allelotyping represents the highest density of LOH loci yet mapped in these chromosomal regions. The presence of numerous LOH regions in alternation with regions of heterozygosity, consistent with mitotic recombination as a mechanism for generating such a mosaic pattern, suggests the presence of several TSGs in these regions and should facilitate their identification.     

Loss of heterozygosity of the BRCA1 and FHIT genes in patients with sporadic breast cancer from Southern Brazil

Aim: The evaluation of allelic losses at the FHIT and the BRCA1 genes and at three other loci at the 17q region in a series of 34 sporadic breast cancer cases from Southern Brazil. METHODS: The samples were evaluated for loss of heterozygosity (LOH) at the FHIT and the BRCA1 genes and at three other microsatellite markers at 17q, and the findings were correlated with clinicopathological parameters. RESULTS: The BRCA1 intragenic marker, D17S855, had the highest frequency of LOH, detected in 10 of 24 informative cases, followed by the D17S579 (six of 23 informative cases), D17S806 (five of 21 informative cases), and D17S785 markers (five of 21 informative cases). LOH at the FHIT intragenic marker, D3S1300, was found in six of 25 informative cases. In four of the six cases with LOH of the FHIT gene, there was concomitant loss of the BRCA1 intragenic marker. CONCLUSIONS: The frequency of allelic losses in the FHIT and BRCA1 loci in the Southern Brazilian population is similar to that described in the general population. No correlations were found when the total LOH frequency was compared with tumour size, grade, or presence of axillary lymph node metastasis. Further studies using larger sporadic breast cancer samples and additional markers would be useful to confirm these findings, in addition to establishing more specific associations with clinicopathological parameters in this specific population.     

胶质母细胞瘤14号染色体杂合性丢失的初步研究

目的 寻找胶质母细胞瘤（glioblastoma,GBM)14号染色体上可能存在肿瘤抑制基因的杂合性丢失（loss of heterozygosity,LOH)aqfa,为发现和定位肿瘤抑制基因提供线索和依据。方法 应用聚合酶链反应方法,采用荧光标记引物和377型DNA序列自动分析仪,分析了20例GBM患者14号染色体上14个微卫星多态性标记的LOH。结果 在50％（10／20）GBM患者的14号染色体上观察到LOH,在38．2％（81／212）可提供信息位点存在LOH。14p和14q的LOH率分别为32％（6／19）、50％（10／20）。在位于14q31-32．3的D14S65位点、14q21-24.1的D14S63－D14S74位点间区域检测到了较高LOH率,分别为57．1％、46．7％－47．1％。在所测位点均未检测到微卫星不稳定（microsatellite instability,MI)。结论 染色体14q上等位基因的丢失可能在GBM分子水平发病机理中起着重要作用,14q31-32.3的D14S65位点、14q21-24.1的D14S63－D14S74位点间区域可能存在与GBM相关的肿瘤抑制基因。     

A common region of allelic loss on chromosome region 3p25.3–26.3 in nasopharyngeal carcinoma

Loss of heterozygosity (LOH) of chromosome arm 3p has been commonly observed in carcinomas of various tissues, including those of nasopharyngeal carcinoma (NPC). To determine the frequency and extent of allelic loss in NPC, we investigated 16 loci on chromosome bands 3p21–26 in 24 tumor tissues by microsatellite analysis. LOH on 3p21–26 was found in 16 of 24 (66.7%) tumors. The highest frequency of allelic loss was found in two adjacent loci, D3S1620 (11/22, 50%) and D3S1560 (9/18, 50%). Eight cases showed LOH in one contiguous region and 5 cases in more than one region. Samples 1, 3, 4, 7, 8, 10, 16, 17, 18, 19, and 22 had a contiguous stretch of allelic loss between D3S1297 and D3S1597. The smallest common LOH/deletion region seems likely to lie between D3S1297 (3p26.3–26.2) and D3S1560 (3p25.3). The allelic loss map defined here will facilitate finer mapping of putative tumor suppressor gene loci and positional cloning of such genes, which may play a role in carcinogenesis of NPC. Genes Chromosomes Cancer 23:21–25, 1998. © 1998 Wiley-Liss, Inc.     

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.     

A novel candidate tumour suppressor locus at 9q32-33 in bladder cancer: localization of the candidate region within a single 840 kb YAC

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, implicating the presence of a tumour suppressor gene or genes on 9q. To define the location of a tumour suppressor locus on 9q in TCC, we screened 156 TCCs of the bladder and upper urinary tract by detailed deletion mapping using 31 microsatellite markers on 9q. Partial deletions of 9q were found in 10 TCCs (6%), and LOH at all informative loci on 9q was found in 77 TCCs (49%). In five low grade superficial bladder tumours, the partial deletion was localized to D9S195 located at 9q32-33, with retention of heterozygosity at all other informative loci including D9S103, D9S258, D9S275 and GSN. We constructed a yeast artificial chromosome (YAC) contig covering the deleted region in these five tumours and placed another four unmapped microsatellite markers on this contig map. Using these markers, we further defined the common deleted region to the interval between D9S1848 and AFMA239XA9. The region is covered by a single YAC (852e11), whose size is estimated to be 840 kb. Our data should expedite further fine mapping and identification of the candidate tumour suppressor gene at 9q32-33.      

Detection of loss of heterozygosity on chromosome 9q22.3 in microdissected sporadic basal cell carcinoma

Identification of loss of heterozygosity (LOH) at specific genetic loci in cancer cells suggests the presence of a tumor suppressor gene within the deleted region. A basal cell carcinoma (BCC) susceptibility gene, human homolog of drosophila patched (PTC), has been recently cloned and localized on chromosome 9q22.3. Mutation and deletion of this region has been reported in BCCs using frozen tumor tissue. The objective of this study was to test whether LOH of human PTC on chromosome 9q22 could be detected in archival sporadic BCCs. We studied 20 randomly selected sporadic BCCs by microdissection and polymerase chain reaction using paraffin-embedded, formalin-fixed material on glass slides. In all cases, analysis was performed with the polymorphic markers D9S53, D9S15, D9S287, and D9S303. The LOH frequencies were 30%, 42%, 56%, and 75% with D9S15, D9S287, D9S53, and D9S303, respectively. LOH at 9q22 was identified in 12 of 20 cases (60%) with at least one marker. Seven cases showed LOH with two markers, two cases with three markers, and one case showed LOH with all four markers. The results indicate that BCC LOH can be frequently identified in paraffin-embedded BCC after routine processing.     

Histogenesis of sarcomatoid urothelial carcinoma of the urinary bladder: evidence for a common clonal origin with divergent differentiation

The histogenesis of sarcomatoid urothelial carcinoma, a rare neoplasm with bidirectional epithelial and mesenchymal differentiation, has been a matter of controversy. To clarify its origin, we analysed the status of X-chromosome inactivation in sarcomatoid urothelial carcinomas from 10 female patients and examined losses of heterozygosity (LOH) in these specimens and in additional 20 tumours from male patients. Six polymorphic microsatellite markers where genetic alterations occur frequently in early or advanced stages of urothelial carcinomas, including D3S3050, D8S261, IFNA, D9S177, D11S569 and TP53, were investigated in the current study. The identical pattern of non-random X-chromosome inactivation in both carcinomatous and sarcomatous components was identified in five of eight informative female patients, and the remaining three informative cases showed a random, but concordant, pattern of X-chromosome inactivation. The concordant X-chromosome inactivation results in all eight informative cases support the concept of a monoclonal origin of both components of this biphasic neoplasm. Among the tumours demonstrating loss of heterozygosity, high incidences of an identical pattern of allelic loss between carcinomatous and sarcomatous components were identified in genetic alterations associated with early carcinogenesis: 86% at D8S261, 78% at D11S569, 75% at D9S177 and 57% at IFNA. In contrast, concordant LOH patterns were less frequently observed for microsatellites related to advanced carcinogenesis: only 40% at D3S3050 and 40% at TP53. The significant overlap of loss of heterozygosity supports a monoclonal cell origin and suggests that clonal divergence may occur during tumour progression and differentiation. Divergent patterns of discordant allelic loss of microsatellite markers imply that heterogeneous pathogenetic pathways may exist in the evolution of this enigmatic neoplasm.