Alterations in p53 predict response to preoperative high dose chemotherapy in patients with gastric cancer.

AIMS: To evaluate the usefulness of molecular markers in predicting histopathological and clinical response to preoperative high dose chemotherapy (HDCT) and survival of patients with advanced gastric cancer. METHODS: In a phase II trial, 25 patients with metastatic gastric cancer received preoperative tandem HDCT consisting of etoposide, cisplatin, and mitomycin, followed by autologous bone marrow transplantation to achieve surgical resectability. Samples before and after treatment, from normal and tumour tissue, were characterised histopathologically, and both p53 and BAX expression was analysed by immunohistochemistry. Pretreatment formalin fixed, paraffin wax embedded samples from normal and tumour tissue were microdissected, and the extracted DNA was preamplified using improved primer extension preamplification polymerase chain reaction. Detection of microsatellite instability (MSI) or loss of heterozygosity (LOH) was performed using markers for p53, BAX, BAT25, BAT26, D2S123, D17S250, and APC. Exons 5-9 of the p53 gene were sequenced directly on ABI 373. RESULTS: Four parameters were significantly associated with response to chemotherapy and prolonged overall survival: positive p53 immunostaining, positive p53 mutation status before chemotherapy, strong histological regression induced by preoperative HDCT, and surgical treatment. Patients's sex or age, tumour location or stage, lymph node status, Lauren classification, MSI, or LOH did not influence duration of survival significantly in this high risk population. CONCLUSION: Positive p53 immunostaining and p53 mutation status in pretreatment tumour biopsies might be useful molecular predictors of response and prognosis in patients with advanced gastric cancer treated by preoperative HDCT.     

Alterations of 9p in squamous cell carcinoma and adenocarcinoma of the lung: association with smoking, TP53, and survival

Tobacco smoke is well recognized as the major etiological contributor to lung cancer, yet the relationship between tobacco smoke exposure and a specific pattern of molecular abnormalities at somatic loci is less well characterized. We analyzed 100 primary tumors from patients undergoing surgical resection of squamous cell carcinoma and adenocarcinoma of the lung for loss of heterozygosity (LOH) and homozygous deletions at two microsatellite markers in a recombinogenic region of 9p13. We describe the relationship of alterations at these markers with tumor characteristics (both clinical and molecular), patient demographics, survival, and measures of tobacco-smoke exposure. Homozygous deletions in this region occurred in 25% (21/85) and LOH in 33% (28/85) of informative tumors examined. These alterations occurred more often in tumors with intense TP53 protein staining by immunohistochemistry, suggesting that inactivation of the TP53 pathway may contribute to these LOH events. Duration of smoking was greatest in patients with the homozygous deletion, intermediate in patients with LOH, and shortest in patients whose tumor did not demonstrate loss in these markers. Unexpectedly, LOH at 9p13 was a significant predictor of improved survival in patients, while the homozygous deletion was associated with the poorest patient survival. Together, these results suggest that TP53 alteration and long-term tobacco smoke exposure may contribute to genetic alterations at 9p13, and that the mechanism and biologic consequences of allele loss reflect individual biologic differences that determine the extent of loss (LOH or homozygous deletion), such that those patients with the deletion of this region face a more aggressive and deadly disease.     

Genetic alterations of the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q in human breast carcinomas.

Fifty-nine primary breast carcinomas and 11 metastases were examined to identify genetic alterations in the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q. Loss of heterozygosity (LOH) was frequently observed on chromosome arms 17p (p144D6 lost in 75%, pYNZ22.1 in 55%, and TP53 in 48% of the primary tumours), 13q (RBI lost in 40% of the primary tumours), and 17q (pRMU3 lost in 35%, pTHH59 in 29%, and NM23HI in 26% of the primary tumours). Loss of all the markers except p144D6 was observed even more frequently in the metastases. Pairwise comparisons for concordance of allele losses on 17p indicated that there might be two genes on 17p implicated in breast cancer development; the TP53 gene and a gene located close to the p144D6 and pYNZ22.1 markers. LOH of the RBI gene was associated with LOH of pYNZ22.1 and p144D6, but not with LOH of TP53. LOH of RBI and TP53 was associated with occurrence of ductal carcinomas, RBI and p144D6 losses with tumour size, and p144D6 losses with positive node status as well. LOH of TP53 and the three 17q markers NM23HI, pTHH59, and pRMU3 was most frequently observed in tumours from postmenopausal women. p144D6 losses occurred most frequently in progesterone receptor-negative tumours, whereas pTHH59 losses occurred most frequently in oestrogen receptor-negative tumours. LOH of the investigated loci was not associated with ERBB2 protooncogene amplification, with positive family history of breast cancer, or with survival.     

Clonality and genetic divergence in multifocal low-grade superficial urothelial carcinoma as determined by chromosome 9 and p53 deletion analysis

Multifocality and recurrence are clinically important features of urothelial carcinomas of the urinary bladder. Recent molecular genetic studies have suggested that multifocal urothelial carcinomas are monoclonally derived from an identical transformed progenitor cell. However, most of these studies investigated advanced and poorly differentiated tumors. The study presented focuses on early papillary tumors, including 52 superficial well-differentiated multifocal and recurrent bladder carcinomas from 10 patients. Microdissection separating urothelium from stromal cells was considered essential to obtain pure tumor cell populations. Genetic analysis was carried out by applying two different methods. Dual color fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 9 and 17 and gene-specific probes for chromosome loci 9q22, 9p21, and 17p13 was carried out in parallel to loss of heterozygosity (LOH) analyses applying 5 microsatellite markers on these chromosomes. Overall, deletions on chromosome 9p were found in 47 tumors (90%), at chromosome 9q in 36 tumors (69%) and at chromosome 17p in 3 tumors (6%). There was a very high correlation of the results between FISH and LOH analysis. Ten early superficial papillary tumors showed deletion of chromosome 9p without deletion of 9q, suggesting 9p deletions as a very early event in the development of papillary urothelial carcinoma. Although in four patients, all investigated tumors showed identical genetic alterations and one patient showed no genetic alterations at the loci investigated, in five patients, two or more clones with different deletions were found. In four of these patients, the results are compatible with clonal divergence and selection of different cell subpopulations derived from a common progenitor cell. However, in one patient different alleles in two markers at chromosome 9 were deleted, favoring an independent evolution of two recurring tumor cell clones. In summary, we could show that there is considerable genetic heterogeneity in early multifocal and recurring urothelial carcinoma and demonstrated the occurrence of two independent clones in at least one patient as an indicator of possible initial oligoclonality of bladder cancer.     

Excision repair cross-complementation group 1 (ERCC1) expression in advanced urothelial carcinoma patients receiving cisplatin-based chemotherapy

Cisplatin has been the cornerstone of the chemotherapy regimen for urothelial carcinoma. Excision repair cross-complementation group 1 (ERCC1) is a key component of the platinum-DNA repair machinery responsible for nucleotide excision repair. Recent reports have suggested that ERCC1 is a predictive and prognostic marker in solid cancers treated with platinum-based chemotherapy. We performed this study to determine whether or not immunohistochemical expression of ERCC1 can predict objective tumor response and cancer-specific survival in patients with advanced urothelial carcinoma treated with cisplatin-based chemotherapy. We performed a retrospective analysis of 89 patients with advanced or recurrent urothelial cancer, who had undergone treatment at Samsung Medical Center between May 2001 and August 2007. Pretherapeutic biopsy samples from 89 patients with a known tumor response were available. ERCC1 expression was assessed by immunohistochemistry. Of the 89 patients, ERCC1 expression was positive in 49 patients (55%). The overall response rate after chemotherapy was 68.5% (95% CI 54.8-74.8%). Among 61 patients who obtained a response, 27 were negative for ERCC-1 expression and 34 were positive (p = 0.61). Median duration of follow-up was 53.7 months (range 14.4-152.3 months). Progression-free survival (PFS) was 10.6 months for ERCC-1-negative patients and 8.4 months for ERCC-1-positive patients (p = 0.03); the difference in overall survival between patients with ERCC-1-negative tumors and ERCC-1-positive tumors (p = 0.73) was not statistically significant. Other than ERCC1 expression, there was no independent prognostic factor for PFS. These results suggest a negative contribution by ERCC1expression to PFS in metastatic urothelial carcinoma patients treated with cisplatin-based chemotherapy.     

Improved clonality analysis of multifocal bladder tumors by combination of histopathologic organ mapping, loss of heterozygosity, fluorescence in situ hybridization, and p53 analyses

The clonality status of multifocal bladder tumors is still controversially discussed with experimental evidence for both monoclonality and field cancerization. Methodologically, loss of heterozygosity (LOH) and genomic sequencing analyses are widely used in clonality analysis of malignant tumors. In the present study, we used LOH analysis and genomic sequencing in combination with fluorescence in situ hybridization (FISH) and extensive histopathologic whole-organ mapping to determine the clonal relationship of multifocal bladder cancer disease. Tissue sections (1 cm(2)) covering the entire urothelial lining were systematically dissected from 2 cystectomy specimens (cystectomy 1, no urothelial lesions, bladder infiltration by a leiomyosarcoma of the vaginal wall; cystectomy 2, multifocal pT3G3 tumors). The location of each sample was documented (bladder mapping). Urothelial cells were microdissected for LOH (chromosomes 9, 17p) and FISH analysis (CDKI2 (9p21), FACC (9q22), p53 (17p13.1), and centromeric probes for corresponding chromosome). Exons 5 to 9 of the p53 gene were sequenced in all tumor samples. No chromosomal alterations were detected in the cystectomy specimen without urothelial malignancies. The tumor-bearing bladder showed an increasing frequency of deletions with increasing malignancy of the investigated lesions. LOH analysis detected deletions only on chromosomes 9p and 17p. In contrast, FISH analysis revealed deletions of all investigated genes at chromosomes 9p, 9q, and 17p in all samples analyzed (preneoplastic and neoplastic tissue). An identical p53 mutation in codon 281 was found in all 7 analyzable tumor samples. Combination of molecular data with histopathologic bladder mapping suggested a monoclonal development of the multifocal lesions mostly via intraurothelial migration. Our data strengthen the results from recently published studies that patients with advanced urothelial carcinoma seem to have a monoclonal panurothelial disease in most cases. FISH showed a much higher sensitivity for detection of chromosomal losses than classical LOH analysis, especially in preneoplastic and small lesions. Combining 3 molecular approaches together with histopathologic organ mapping presents a valuable tool to determine the clonality status of multifocal bladder tumors.     

Loss of heterozygosity at chromosomes 3, 6, 8, 11, 16, and 17 in ovarian cancer: correlation to clinicopathological variables

Tumor specimens from 78 epithelial ovarian cancer patients were examined for loss of heterozygosity (LOH) at 11 microsatellite markers at chromosomes 3p14.2, 6q27, 8p12, 11p15.5, 11q23.1-q24, 16q24.3, and 17p13.1, to evaluate the involvement, possible clustering, and prognostic significance of these lesions in the progression of the disease. The LOH analysis was performed on polymerase chain reaction (PCR)-amplified DNA from sections of paraffin-embedded tumor and normal tissue pairs. In addition to primary tumors, specimens of metastatic tissues were studied from 19 patients. In the combined results from primary and metastatic tumors, LOH frequencies varied between 31% (6q27) and 69% (17p13.1). Only LOH at chromosomal regions 3p14.2 (D3S1300), 11p15.5 (D11S1318), 11q23.3-q24 (D11S1340 and D11S912), 16q24.3 (D16S476 and D16S3028), and 17p13.1 (D17S938) was associated with an adverse disease course. Our results indicate that LOH at 17p13.1 occurs independently from the other chromosomal sites studied, and is an early event in ovarian tumorigenesis. The LOH at 16q24.3, 11q23.3/q24, and 11p15.5 seems to occur later. The LOH at 11p15.5 and 11q23.3 was associated with reduced cancer-specific survival time; therefore, the studied markers could be located close to genes with influence on patient survival. Of the studied chromosomal regions, the most important tumor suppressor genes involved in the evolution of ovarian cancer appear to be located on chromosomes 11, 16, and 17. The genetic heterogeneity observed in primary and metastatic specimens demonstrates that there are multiple pathways involved in the progression of ovarian cancer.     

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.     

Frequent genetic alterations in flat urothelial hyperplasias and concomitant papillary bladder cancer as detected by CGH,LOH, and FISH analyses

Flat urothelial hyperplasia, defined as markedly thickened urothelium without cytological atypia, is regarded in the new WHO classification as a urothelial lesion without malignant potential. Frequent deletions of chromosome 9 detected by fluorescence in situ hybridization (FISH) have been previously reported in flat urothelial hyperplasias found in patients with papillary bladder cancer. Using comparative genomic hybridization (CGH) and microsatellite analysis, these hyperplasias and concomitant papillary tumours of the same patients were screened for other genetic alterations to validate and extend the previous findings. Eleven flat hyperplasias detected by 5-ALA-induced fluorescence endoscopy and ten papillary urothelial carcinomas (pTaG1-G2) from ten patients were investigated. After microdissection, the DNA of the lesions was pre-amplified using whole genome amplification (I-PEP-PCR). Loss of heterozygosity (LOH) analyses were performed with five microsatellite markers at chromosomes 9p, 9q, and 17p. CGH was performed using standard protocols. In 6 of 11 hyperplasias and 7 of 10 papillary tumours, deletions at chromosome 9 were simultaneously shown by FISH, LOH, and CGH analyses. There was a good correlation between FISH, LOH, and CGH analyses, with identical results in 6 of 10 patients. In addition to deletions at chromosome 9, further genetic alterations were detected by CGH in 9 of 10 investigated hyperplasias, including changes frequently found in invasive papillary bladder cancer (loss of chromosomes 2q, 4, 8p, and 11p; gain of chromosome 17; and amplification at 11q12q13). There was considerable genetic heterogeneity between hyperplasias and papillary tumours, but a clonal relationship was suggested by LOH and/or CGH analyses in 5 of 10 cases. These data support the hypothesis that flat urothelial hyperplasias can display many genetic alterations commonly found in bladder cancer and could therefore be an early neoplastic lesion in the multistep development of invasive urothelial carcinoma.     

Oligodendroglial tumors: refinement of candidate regions on chromosome arm 1p and correlation of 1p/19q status with survival

Loss of heterozygosity (LOH) on the chromosome arms 1p and 19q is frequent in oligodendroglial tumors and has been correlated with chemosensitivity and good prognosis in anaplastic oligodendrogliomas. The oligodendroglioma-associated tumor suppressor genes on 1p and 19q are as yet unknown. To narrow down candidate regions on 1p, we investigated oligodendroglial tumors from 89 patients for LOH at up to 30 polymorphic loci on 1p. In addition, all tumors were studied for LOH at 7 loci on 19q. Combined LOH on 1p and 19q was detected in 20 (83%) of 24 oligodendrogliomas, 15(63%) of 24 anaplastic oligodendrogliomas, 10 (56%) of 18 oligoastrocytomas, and 12 (52%) of 23 anaplastic oligoastrocytomas. Five tumors demonstrated partial deletions on 1p, which allowed to define 3 distinct candidate regions at 1p36.31-pter distal to D1S2633, 1p36.22-p36.31 between D1S489 and D1S2642, and 1p34.2-p36.1 between D1S2743 and D1S482, respectively. No partial deletions were detected on 19q. Combined LOH on 1p and 19q was associated with prolonged time to progression (TTP), longer overall survival (OS), and a higher 5-year survival rate. Depending on the presence or absence of combined LOH on 1p and 19q, patients with anaplastic oligodendroglial tumors treated with adjuvant radio- and/or chemotherapy showed a median TTP of 86 months versus 39 months, a median OS of 91 months versus 46 months, and a 5-year survival rate of 80% versus 36%, respectively. Similarly, LOH on 1p and 19q was associated with longer survival in patients with low-grade oligodendroglial tumors (TTP: 57 months versus 47 months; OS: 172 months versus 105 months; 5-year survival rate: 92% versus 70%). Thus, our results refine the location of putative oligodendroglioma suppressor genes on 1p and support the significance of LOH on 1p and 19q as a favorable prognostic marker.     

Incidence of loss of heterozygosity at p53 and BRCA1 loci in serous surface carcinoma

Serous surface carcinoma (SSC) is a neoplasm histologically indistinguishable from typical serous carcinomas that arise from the ovary but has a distinct clinical presentation. It is characterized by widespread peritoneal dissemination at presentation, but the ovaries are grossly normal in size and shape. If the carcinoma involves the ovaries microscopically, the tumor is confined to the surface or is minimally invasive. The recognition of this entity is important, because in some studies it appears to have a poorer prognosis than stage-matched serous cancers of the ovary. Loss of heterozygosity (LOH) of the p53 (17p) and BRCA1 (17q) tumor suppressor genes has been frequently identified in sporadic ovarian carcinomas. Although 17p LOH is correlated with common p53 gene mutations, inactivating mutations of the BRCA1 gene are uncommon in sporadic ovarian cases. In contrast, germline BRCA1 mutations are responsible for some hereditary forms of ovarian cancer, where it has been suggested that germline BRCA1 mutations confer a more favorable prognosis. In this study, 12 sporadic SSC were assessed for the presence of allelic deletions on the p53 and BRCA1 gene loci. DNA from both tumor and normal cells was obtained for LOH studies using tissue microdissection. Polymerase chain reaction (PCR) amplification was performed with the polymorphic DNA markers TP53 (17p13.1/p53 gene) and D17S579 (17q/BRCA1 gene). LOH in the p53 and BRCA1 loci was detected in 62.5% and 66.6% of the cases, respectively. In 50% of tumors informative for both markers, it is possible that an entire chromosome may be lost. In conclusion, we have shown that LOH of the p53 and BRCA1 loci is a frequent event in sporadic SSC, similar to what has been described in the usual form of serous ovarian carcinoma. Mutational analysis will be necessary to determine the exact role of these genes in this group of tumors.     

Chromosome 2p, 3p, 5q and 18q status in sporadic gastric cancer

AIM: The genetic make-up of gastric cancers in low-risk population groups from South Africa is largely unknown. The purpose of this study was to ascertain the incidence of microsatellite instability and loss of heterozygosity in this population. METHODS: Thirty-seven gastrectomy specimens for sporadic gastric cancer were analysed for the following clinicopathological parameters: age, gender, race, histopathological type, size of tumour, lymph node status and the presence/absence of Helicobacter pylori. DNA was then extracted from paraffin-embedded tissue and seven microsatellite markers in 2p, 3p, 5q and 18q loci were examined using automated DNA fluorescent technology. RESULTS: Only eight cases showed microsatellite instability (MSI) for one marker and were thus categorised as MSI-low. In the 3p region, loss of heterozygosity (LOH) was detected in 21.7-38.3% of informative cases, whilst in the 18q region CLOH ranged from 25 to 38.4%. LOH was not seen in the 2p locus and only one case showed LOH in the 5q region. When the molecular changes were compared with clinicopathological parameters, a statistically significant relationship did not emerge with any single parameter. CONCLUSIONS: This study shows that sporadic gastric cancer from a low-risk population in South Africa is MSI-low or MSI-stable, and that LOH in the 3p and 18q regions is similar to that seen in other low-risk populations from different geographical regions.     

Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer

Many molecular alterations are known to occur in urothelial carcinoma of the bladder, but their significance for tumor progression is poorly understood. Deletions of chromosome 8p are frequently found in several tumor types and are often associated with progressive disease. In all, 99 bladder tumors were screened for deletions at 8p using loss of heterozygosity (LOH) and multicolor fluorescence in situ hybridization FISH analyses. Allelic loss on chromosome 8p in at least one marker was found in 25/99 (25%) tumors. There was a significant correlation of 8p deletions with invasive tumor growth and a highly significant association with papillary growth pattern in patients with invasive disease. cDNA array analyses revealed that secreted Frizzled-related protein 1 (sFRP1), an antagonist of Frizzled receptors and Wnt pathway activation on chromosome 8p12-11.1, is frequently downregulated in bladder cancer. To investigate sFRP1 as a candidate for a putative progression-related gene on 8p, urothelial cell lines and primary urothelial carcinomas were screened for sFRP1 expression using quantitative real-time PCR, Northern blot, immunofluorescence and immunohistochemistry (IHC). Of the investigated bladder cancers, 38% showed loss of sFRP1 expression by quantitative RT-PCR. Evaluation of the protein expression by IHC using tissue microarrays containing 776 bladder cancers revealed loss or strong reduction of sFRP1 expression in 66% of cases. SFRP1 loss was associated with higher tumor stage and grade and shorter overall survival. In addition, loss of sFRP1 was an independent indicator of poor survival in patients with papillary but not with muscle invasive bladder cancer. There were neither mutations in the coding region of sFRP1 nor homozygous deletions at 8p12-11.21. However, promoter methylation was detected using methylation-specific PCR in 29% of cases. In conclusion, we could show a close correlation of chromosome 8p deletions and progression of papillary bladder tumors. The sFRP1 gene on chromosome 8p12-11.1 could be a candidate gene for the predicted, progression-related tumor suppressor gene in bladder cancer and could contribute to urothelial carcinogenesis.     

Loss of heterozygosity at 9q32-33 (DBC1 locus) in primary non-invasive papillary urothelial neoplasm of low malignant potential and low-grade urothelial carcinoma of the bladder and their associated normal urothelium

Tumour recurrence has a major impact on patients with non-invasive papillary urothelial tumours of the bladder. To explore the role of DBC1 (deleted in bladder cancer 1 locus), a candidate tumour suppressor gene located at 9q32-33, as prognostic marker we have performed loss of heterozygosity (LOH) testing in 49 patients with primary papillary urothelial tumours and associated normal urothelium. Data from the 38 tumours and 11 specimens of normal urothelium that were informative in the LOH study (D9S195 marker) showed that LOH in urothelium (45.4%) but not in non-invasive tumours (60.5%) was associated with tumour recurrence (p = 0.026) but not to grade or progression. Also, tumours whose normal urothelium had LOH were larger (p = 0.020) and showed cyclin D1 over-expression (p = 0.032). Non-significant increased expression of p53, p21Waf1, apoptotic index and tumour proliferation, and decreased expression of p27Kip1 or cyclin D3 also characterized tumours whose normal urothelium had LOH. The expression of these G1-S modulators, apoptotic index and tumour proliferation was more heterogeneous in papillary urothelial tumours, irrespective of having retained heterozygosity or LOH. Also, Bax expression decreased in papillary urothelial tumours having LOH (p = 0.0473), but Bcl-2 was unrelated to LOH status. In addition, FGFR3 protein expression decreased in LOH tumours (p = 0.036) and in those having LOH in their normal urothelium (p = 0.022). FGFR3 immunohistochemical expression was validated by western blot in selected cases. The survival analysis selected LOH in normal urothelium as a marker of disease-free survival (log-rank 5.32, p = 0.021), progression-free survival (log-rank 3.97, p = 0.046) and overall survival (log-rank 4.26, p = 0.038); LOH in tumours was significant in progression-free survival (log-rank 3.83, p = 0.042). It is concluded that LOH at the DBC1 locus in normal urothelium seems to be relevant in the prognosis of non-invasive papillary tumours of the bladder via selecting cases with increased proliferation, frequent alterations of the G1-S phase modulators, and decreased FGFR3 protein expression.     

Allelotypes as potential prognostic markers in ovarian carcinoma treated with cisplatin-based chemotherapy

We analyzed a series of ovarian carcinomas from patients treated with cisplatin-based chemotherapy for loss of heterozygosity (LOH) to determine the relationship between microsatellite alteration and prognosis. Patients with tumors that had lost alleles at loci 3p14-21, 12qter, or 17p13.3 showed significantly reduced survival compared to patients with tumors that retained both alleles at those loci. The 5-year mortality rates for patients exhibiting allele loss and patients with allele retention were 50 and 41%, respectively, for the 3p14-21 locus (P=0.0023); 57 and 24%, respectively, for 12qter (P=0.0256); and 55 and 40%, respectively, for 17p13.3 (P=0.0489). A statistically significant difference was also observed with respect to fractional allelic loss (FAL), which was a significant indicator for disease recurrence (P=0.0227). The prognosis of patients with high FAL values were significantly worse compared to those with low FAL values (P=0.0306). Our results suggested that LOH at loci 3p14-21, 12qter, or 17p13.3 was a significant predictor of poor survival in ovarian carcinomas treated with cisplatin-based chemotherapy. Furthermore, the association of FAL value with therapy response indicated that ovarian carcinomas with high levels of chromosomal alteration may be more resistant to this type of chemotherapy.     

Loss of Heterozygosity on Chromosome 11p15 during Histological Progression in Microdissected Ductal Carcinoma of the Breast

Microdissection of histologically identifiable components from formalin-fixed, paraffin-embedded tissue sections allows molecular genetic analyses to be correlated directly with pathological findings. In this study, we have characterized loss of heterozygosity (LOH) at chromosome 11p15 at different stages of progression in microdissected tumor components from 115 ductal carcinomas of the breast. Microdissected foci of intraductal, infiltrating, and metastatic tumors were analyzed to determine the stage of progression at which LOH at 11p15 occurs. LOH was detected in 43 (37%) of 115 cases. Foci of intraductal carcinoma could be microdissected from 85 cases, of which 30 (35%) showed LOH at some stage of progression. LOH was detected in the intraductal component in 26 of these 30 cases. Interstitial deletions were characterized by using a panel of 10 highly polymorphic markers. The smallest region of overlap (SRO) for LOH at 11p15 was bounded by the markers D11S4046 and D11S1758. LOH at 11p15.5 showed no correlation with estrogen receptor status, the presence of positive lymph nodes, tumor size, histological grade, or long-term survival. We conclude that 11p15 LOH usually occurs early in breast cancer development but less frequently does not develop until the infiltrating or metastatic stages of tumor progression.     

Allelic loss at 1p34, 13q12, 17p13.3, and 17q21.1 correlates with poor postoperative prognosis in breast cancer.

Allelic losses of tumor suppressor genes (TSGs), or the chromosomal regions harboring them, in tumor DNA may become useful postoperative prognostic indicators. To examine whether specific allelic losses might correlate with postoperative survival in a 5-year prospective follow-up, we tested tumors from a cohort of 264 breast cancer patients for allelic losses of 18 microsatellite markers representing either a known TSG or a region where genetic alterations are frequent in breast tumors. Patients whose tumors had lost an allele at 1p34, 13q12, 17p13.3, or 17q21.1 had significantly higher risks of postoperative mortality than those whose tumors retained both alleles at those loci (at 1p34, a 5-year mortality rate of 29% among patients with losses vs. 7% with retentions, P = 0. 0008; at 13q12, 31% vs. 10%, P = 0.0062; at 17p13.3, 24% vs. 13%, P = 0.026; and at 17q21.1, 31% vs. 13%, P = 0.0047). Furthermore, combined losses at 13q12 and 17p13.3 increased the predicted postoperative mortality risks by a factor of 9.6 (5-year mortality rate of 42% vs. 5% with retentions, P = 0.0001), and combined losses at 1p34 and 17p13.3 raised the predicted postoperative mortality risks by a factor of 8.6 (27% vs. 3%, P = 0.0064). We conclude that allelic losses at these loci can serve as negative prognostic indicators to guide postoperative management of patients. Genes Chromosomes Cancer 26:134-141, 1999.     

Allelotyping analysis at chromosome arm 8p of high-grade prostatic intraepithelial neoplasia and incidental, latent, and clinical prostate cancers

In this study, we used 7 informative microsatellite markers at 8p22, 23.1, and 23.2 in Japanese patients to compare frequency of loss of heterozygosity (LOH) in 53 lesions of high-grade prostatic intraepithelial neoplasia (HGPIN), 38 cases (38 lesions) of incidental prostate cancer (IPC), 31 cases (41 lesions) of latent prostate cancer (LPC), and 102 cases (168 lesions) of clinical prostate cancer (CPC). The frequency of LOH at 8p22-23.2 with at least 1 marker was 0%, 33%, 57%, and 51% in the HGPIN, IPC, LPC, and CPC cases, respectively. No statistically significant difference was found at 8p22-23.2 between the types of prostate cancer. However, the frequency of 8p22 deletion was significantly higher in CPC and LPC cases than in IPC cases (P = 0.0003) or lesions (P = 0.0017). The frequency of LOH at 8p22 and 8p23.1 loci in high-grade tumors was significantly higher than in low-grade tumors in both the LPCs/IPCs and CPCs (P < 0.05). Allelic loss at 8p22 was significantly more frequent in CPC than in IPC (P = 0.002) and in pT4 CPC than in earlier-stage CPC (P = 0.038). These findings suggest that deletion of 8p is an important event in both the initiation and metastasis of prostate cancer. The extremely high frequency of LOH at 8p22-23.1 in high-grade tumors suggests the existence of a novel putative tumor-suppressor gene associated with the progression of prostate cancer. These results should be useful in identifying the target gene of deletion at 8p.     

少突胶质细胞肿瘤染色体1p、19q和10q杂合性缺失与临床预后的关系

目的 探讨少突胶质细胞肿瘤微卫星变异的遗传和分子特性及其与临床预后的关系。方法 26例少突胶质细胞瘤和25例伴有少突胶质细胞瘤成分的胶质母细胞瘤成对的血和肿瘤标本DNA提取后,进行微卫星不稳定性分析染色体1、19q和10q杂合性缺失。结果 54％少突胶质细胞瘤检出1pLOH,58％检出19qLOH,35％检出10q LOH,其中50％间变性少突胶质细胞瘤出现10q LOH。1p／19q LOH相伴存,二者密切相关（P〈0．0001）;40％GBMO检出1pLOH,仅4例1p LOH伴10q LOH;60％检出19q LOH,64％检出10q LOH。结论 1P和19q LOH是少突胶质细胞瘤分子和遗传特性之一,并且与化疗敏感和预后好有关,10qLOH是少突胶质细胞瘤进展标志。1PLOH与长PFS有关,10qLOH与短PFS有关。GBMO分子表型不同于GBM。     

Lack of correlation between expression of retinoic acid receptor-beta and loss of heterozygosity on chromosome band 3p24 in esophageal cancer

Loss of heterozygosity (LOH) on chromosome arm 3p occurs frequently in human cancers, including esophageal cancer, suggesting that tumor suppressor genes may be located on this chromosome arm. The retinoic acid receptor-beta (RARB) gene is localized on chromosome band 3p24, and its expression is progressively lost during esophageal carcinogenesis. Furthermore, growth inhibition of esophageal cancer cell lines by all-trans retinoic acid has been associated with the constitutive and induced expression of RARB. We therefore assessed LOH on chromosome arm 3p and RARB expression in esophageal cancer to investigate the mechanism of altered RARB expression during carcinogenesis. We first analyzed LOH in 65 paired surgical specimens of normal mucosa and esophageal cancer by using 10 microsatellite markers, which resulted in 44 informative cases for subsequent study. LOH on chromosome band 3p24 was found to occur at an overall rate of 36.4% (16/44) by three markers (D3S1293, THRB, and D3S1283). LOH for these three individual markers was 14.0%, 47.4%, and 20.9%, respectively. Meanwhile, RARB expression was lost in 43.2% (19/44) of these 44 samples. The loss of RARB expression was not correlated with LOH on chromosome band 3p24 (gamma = -0.22, -0.069, and -0.02, P = 0.15, 0.78, and 0.9 for D3S1293, THRB, and D3S1283, respectively), although both altered RARB expression and LOH in esophageal cancer were statistically significant (P = 0.001 and 0.0001, respectively), indicating that the loss of RARB expression cannot be explained by LOH on 3p24.