Oligoclonality of early lesions of the urothelium as determined by microdissection-supported genetic analysis.

AIM: To contribute to the ongoing discussion of clonality of human urothelial cancer it was considered a valuable approach to analyze multiple areas from cystectomy specimens for deletions of chromosomes known to be involved early in bladder cancer development. MATERIAL AND METHODS: Thus, in 86 biopsies of 4 human cystectomies with different histological findings (maximal diagnosis: pT1G2, pTaG3, pT2G2, normal) loss of heterozygosity (LOH) was investigated as a deletion marker using markers of chromosomes 8p, 9p, 9q and 17p. Findings were compared to histology of the lesion. RESULTS: Findings indicate: (1) no changes in the markers investigated in the bladder with histologically normal urothelium in contrast to detection of LOH in normal urothelium of tumour-bearing bladders; (2) an accumulation of the number of LOH with increasing malignancy of lesions within one bladder, and (3) indications of oligoclonal neoplastic lesions in two of the urinary bladders investigated. CONCLUSIONS: The investigation of multiple lesions within one bladder presents a snapshot of genetic changes in differently advanced tumour stages. The hypotheses of tumour evolution and oligoclonality as derived from our LOH data need to be supported by deletion-independent clonality studies as X-chromosomal inactivation analysis.     

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.     

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.     

Histologic-genetic mapping by allele-specific PCR reveals intraurothelial spread of p53 mutant tumor clones

Common and clinically important features of urothelial carcinomas are multifocality and a high rate of recurrence. Molecular studies demonstrated that multifocal tumors are frequently composed of one tumor clone spreading throughout the urothelial tract. A combination of histologic and genetic mapping of cystectomy specimens from bladder cancer patients is a valuable tool to study bladder carcinogenesis and tumor cell spread by correlating urothelial morphologic features and defined genetic alterations. In the present study, the primary tumors of 14 cystectomy specimens were investigated for p53 protein overexpression by immunohistochemistry and p53 gene mutation by genomic sequencing. Seven tumors showed a strong nuclear staining for the p53 protein. In six of seven tumors, a p53 gene mutation was detected. Allele-specific PCR of defined p53 mutations was established in five of six cases with a p53 mutation. Subsequent screening of the entire urothelial lining of each cystectomy specimen by allele-specific PCR revealed p53-mutant cell clones in urothelial patches with carcinoma in situ and dysplasia, but also frequently in histomorphologically normal urothelium adjacent to the tumor. The pattern of tumor cell spread indicated a continuous intraurothelial growth of the p53-mutant clone. P53 immunohistochemistry visually confirmed the presence of mutant cells in most of these samples. We conclude that allele-specific PCR is a highly sensitive and reliable method for tracking specific p53 mutant clones in the urothelium. Moreover, the detection of p53-mutant cells in histologically normal or preneoplastic urothelial areas in four patients with invasive bladder cancer indicates an extensive intraurothelial tumor cell spread. The excellent correlation of immunohistochemically positive urothelial patches with the presence of a specific mutation highlights the biologic significance of p53-positive cells in the urothelium of tumor patients.     

Lasermicrodissection--an important prerequisite for the molecular-genetic analysis of bladder cancer

The past 10 years have shown that a crucial point in the molecular-genetic analysis of malignant tumors is the exact histopathological definition and separation of the investigated lesions. Laser microdissection has become an important tool allowing for the study of specific histological entities and defined preneoplastic lesions. Reliable detection of tumor-specific alterations can be compromised by the presence of normal cells. This requires microdissection of pure tumor cell populations (>80%), necessary for detecting chromosomal alterations by loss of heterozygosity analysis (LOH) and fluorescence in situ hybridization (FISH). The combination of microdissection and molecular methods could also be useful for studying multifocal lesions of the urothelial tract. This article describes in detail the use of laser microdissection, whole genome amplification by Improved Primer Extension Preamplification (I-PEP)-PCR and subsequent LOH, FISH, and sequencing analyses for the investigation of urothelial tumors and their precursors, e.g. dysplasias and hyperplasias. The combination of the described methods allows for a wide spectrum of molecular investigations in lesions with a limited number of tumor cells, and might help to understand the fundamental alterations involved in urothelial carcinogenesis.     

Frequent genetic alterations in simple urothelial hyperplasias of the bladder in patients with papillary urothelial carcinoma

In order to understand the origin of bladder cancer, very early urothelial lesions must be investigated in addition to more advanced tumors. Tissue from 31 biopsies of 12 patients with urothelial hyperplasias and simultaneous or consecutive superficial papillary tumors were used to microdissect urothelium from 15- microm sections of biopsies. The biopsies were obtained with the recently developed highly sensitive diagnostic method of 5-aminolevulinic acid-induced fluorescence endoscopy (AFE). Besides flat and papillary urothelial neoplasms, the method of photodynamic diagnostics also detects simple urothelial hyperplasias as fluorescent positive lesions. In addition, 12 fluorescence-positive biopsies showing histologically normal urothelium were investigated. Fluorescence in situ hybridization was done using a dual color staining technique of biotinylated centromeric probes of chromosomes 9 and 17 and digoxigenin-labeled gene-specific P1 probes for chromosomes 9q22 (FACC), 9p21(p16/CDKI2), and 17p13(p53). Ten of 14 hyperplasias (70%) showed deletions of chromosome 9. In 7 out of 8 patients with genetic alterations in the hyperplasias the genetic change was also present in the papillary tumor. Six out of 12 samples of microdissected normal urothelium also showed genetic alterations on chromosome 9. Microdissection of urothelial lesions, obtained during AFE, has led to the first unequivocal documentation of genetic changes in urothelial lesions diagnosed as normal in histopathology. Thus, this technical approach is important to provide insight into the earliest molecular alterations in bladder carcinogenesis.     

Loss of heterozygosity at 1p, 8p, 10p, 13q, and 17p in advanced urothelial cancer and lack of relation to chemotherapy response and outcome

Studies of urothelial tumors have identified structural abnormalities in a number of chromosomes. This study aimed to identify specific genetic changes of patients with advanced urothelial cancers, and relate these changes to increased chemotherapy sensitivity or good prognosis. We screened 56 muscle-invasive bladder cancer tumors for loss of heterozygosity (LOH) at chromosome 1p, 8p, 10p, 13q, and 17p with PCR using 6 microsatellite markers. All patients had recurrent locally advanced or metastatic disease. DNA was extracted after microdissection of the primary tumor and normal tissue from paraffin-embedded specimens. The PCR products were electrophoresed in an ABI Prism 377 DNA sequencer and the alleles from tumor DNA and normal tissue DNA were analyzed using the GeneScan program. The LOH findings were correlated with response to chemotherapy and survival. Allelic loss of specific markers was present in 26-50% of the informative tumors. The most frequent LOH was observed at 17p, supporting the notion that this region may contain genes of importance to urothelial cancer progression. The overall rate of response to chemotherapy was 48%, and ranged from 40% to 56% according to specific LOH changes. The median survival of all patients from start of chemotherapy was 5.8 months and ranged from 5.3 to 7.9 months for patients with specific LOH changes. Response and survival of patients with no lost markers was the same size, compared to patients with one, two, or more lost markers. Specific genetic changes were detected in a significant number of tumors from patients with advanced urothelial cancer. These changes were not predictive of response to chemotherapy or of the duration of survival.     

Molecular cytogenetic characterization of early and late renal cell carcinomas in von Hippel-Lindau disease

Deletions of 3p25, gains of chromosomes 7 and 10, and isochromosome 17q are known cytogenetic aberrations in sporadic renal cell carcinoma (RCC). In addition, a majority of RCCs have loss of heterozygosity (LOH) of the Von Hippel-Lindau (VHL) gene located at chromosome band 3p25. Patients who inherit a germline mutation of the VHL gene can develop multifocal RCCs and other solid tumors, including malignancies of the pancreas, adrenal medulla, and brain. VHL tumors follow the two-hit model of tumorigenesis, as LOH of VHL, a classic tumor suppressor gene, is the critical event in the development of the neoplastic phenotype. In an attempt to define the cytogenetic aberrations from early tumors to late RCC further, we applied spectral karyotyping (SKY) to 23 renal tumors harvested from 6 unrelated VHL patients undergoing surgery. Cysts and low-grade solid lesions were near-diploid and contained 1-2 reciprocal translocations, dicentric chromosomes, and/or isochromosomes. A variety of sole numerical aberrations included gains of chromosomes 1, 2, 4, 7, 10, 13, 21, and the X chromosome, although no tumors had sole numerical losses. Three patients shared a breakpoint at 2p21-22, and three others shared a dicentric chromosome 9 or an isochromosome 9q. In contrast to the near-diploidy of the low-grade lesions, a high-grade lesion and its nodal metastasis were markedly aneuploid, revealed loss of VHL by fluorescence in situ hybridization (FISH), and contained recurrent unbalanced translocations and losses of chromosome arms 2q, 3p, 4q, 9p, 14q, and 19p as demonstrated by comparative genomic hybridization (CGH). By combining SKY, CGH, and FISH of multiple tumors from the same VHL kidney, we have begun to identify chromosomal aberrations in the earliest stages of VHL-related renal cell tumors. Our current findings illustrate the cytogenetic heterogeneity of different VHL lesions from the same kidney, which supports the multiclonal origins of hereditary RCCs. Published 2001 Wiley-Liss, Inc.     

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.     

The spectrum of TP53 mutations in bladder carcinoma

The mutational spectrum for the TP53 gene was investigated in a large series of bladder tumors and bladder tumor cell lines. Tumors and cell lines were screened for the presence of TP53 point mutations by single-strand conformational polymorphism analysis followed by direct sequencing. Mutations were detected in 16 of 88 (18%) tumors and 4 of 14 cell lines (28%). In total, twelve missense mutations, one nonsense mutation, three deletions, and two insertions were identified by direct sequencing. Of the thirteen point mutations sequenced, only one was a transition at a CpG site, whereas five G:C → T:A transversions were found, suggesting a major role for exogenous mutagens in bladder tumorigenesis. Tumors were also examined for loss of heterozygosity (LOH) on chromosome arm 17p. LOH of one or more markers on 17p was detected in 31 % of tumors. All eight tumors with a TP53 mutation from patients informative at TP53 had LOH, whereas nine tumors with LOH at TP53 did not have an identified mutation. Three tumors had LOH on 17p at sites distal to the TP53 locus but retained both TP53 alleles, suggesting the involvement of another tumor suppressor gene on 17p in bladder tumorigenesis in some tumors. Genes Chrom Cancer 9:108-118 (1994).© 1994 Wiley-Liss, Inc.     

High-resolution whole-organ mapping with SNPs and its significance to early events of carcinogenesis

We attempted to identify deleted segments in two model tumor suppressor gene loci on chromosomes 13q14 and 17p13 that were associated with clonal expansion of in situ bladder preneoplasia using single nucleotide polymorphisms (SNPs)-based whole-organ histologic and genetic mapping. For mapping with SNPs, the sequence-based maps spanning approximately 27 and 5 Mb centered around RB1 and p53, respectively, were assembled. The integrated gene and SNP maps of the regions were used to select 661 and 960 SNPs, which were genotyped by pyrosequencing. Genotyping of SNPs was performed on DNA samples corresponding to histologic maps of the entire bladder mucosa in human cystectomy specimens with invasive urothelial carcinoma. By using this approach, we have identified deleted regions associated with clonal expansion of intraurothelial neoplasia; which ranged from 0.001 to 4.3 Mb (average 0.67 Mb) and formed clusters of discontinuous deleted segments. The high resolution of such maps is a prerequisite for future positional targeting of genes involved in early phases of bladder neoplasia. This approach also permits analysis of the overall genomic landscape of the involved region and discloses that a unique composition of noncoding DNA characterized by a high concentration of repetitive sequences may predispose to deletions.     

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.     

Patterns of chromosomal aberrations in urinary bladder tumours and adjacent urothelium

Bladder cancer is often characterized by recurrent and multifocal growth, and tumours are frequently accompanied by precancerous alterations of the surrounding urothelium. These findings have led to the hypothesis that cells from areas of genetically aberrant but morphologically non-cancerous or even unremarkable mucosa may be the source of bladder carcinomas. Fluorescence in situ hybridization (FISH) was performed using ten probes targeting five different chromosomes that are known to be frequently altered in bladder cancer (centromere 1, 8, 9, 11, 17 and 1p36, 8p23, 9p21, 11q13, 17p13) on paraffin-embedded tissue sections of 11 superficial bladder cancers. Copy number changes of the tumours were compared to those in the urothelium adjacent to the tumour. Eleven of 11 (100%) tumours and eight of 11 (73%) samples of adjacent urothelium showed copy number changes of at least one chromosome. The occurrence of similar patterns of chromosomal aberrations in the tumours and their associated urothelium supports the hypothesis of a clonal relationship. It is concluded that FISH analysis targeting five different chromosomes is more sensitive than conventional histology for distinguishing between neoplastic and normal cells of the urothelium.     

Analysis of chromosome 17p13 (p53 locus) alterations in gastric carcinoma cells by dual-color fluorescence in situ hybridization.

Chromosome 17 and p53 gene locus alterations were determined on 67 gastric carcinomas by dual-color fluorescence in situ hybridization, using probes for centromere 17 and the 17p13.1 (p53 locus). The results were compared with loss of heterozygosity (LOH) at 17p13.3, direct sequencing of exons 5 to 9 of p53, and nuclear overexpression of p53 protein. Deletion of p53 was found in 26 of 67 tumors (39%). All 26 also showed LOH at 17p13.3, frequently overexpressed p53 protein, and had polysomy 17. The functional loss of p53 gene in these tumors, 85% of which were of intestinal type, appears to be caused by both deletion of 17p13.1 and missense mutation of the remaining allele. There were 9 tumors that had neither deletion nor LOH but had a large proportion of cancer cells that overexpressed p53 election. Despite evidence of LOH, there was no p53 deletion in 11 tumors. Finally, 21 tumors, mostly of diffuse type, showed neither deletions, LOH, nor p53 overexpression. Our data suggest that in gastric cancer, deletion of 17p is principally responsible for the allelic loss at the p53 gene and that analysis of deletions by the dual-color fluorescence in situ hybridization is a sensitive and useful approach to clarify chromosomal aberrations.     

Molecular analysis of microdissected tumors and preneoplastic intraductal lesions in pancreatic carcinoma

Little or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16(INK4), DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at the p16(INK4) locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at the DPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions of p16(INK4) and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16(INK4) compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs.     

具有内翻生长特征的尿路上皮增生性病变的临床病理学研究

目的 研究伴内翻生长特征的尿路上皮增生性病变的临床病理特征,探讨免疫组织化学和多点荧光原位杂交在其鉴别诊断中的作用.方法 收集具有内翻生长特征的尿路上皮病变41例,分为内翻乳头状瘤、内翻生长型尿路上皮癌、旺炽型von Brunn细胞巢,用多点荧光原位杂交方法检测其3、7、17号染色体获得和9p21缺失;免疫组织化学EnVision法标记p53、CK20和Ki-67;并对12例进行随访.结果 (1)内翻乳头状瘤12例,平均1.2 cm,由互相连接的细胞索或巢在固有膜内生长,细胞索相对较细而宽窄一致,细胞巢外呈栅栏状、内为流水状排列,可见鳞状分化,无细胞学上的异型性,无或偶见核分裂象,4例可见少量表面外生乳头,被覆少于6层的正常尿路上皮.(2)内翻生长型尿路上皮癌24例,平均2.1 cm,结构似内翻乳头状瘤,但细胞索较粗并宽窄不一,细胞巢粗大并不规则状,可形成实体结构,瘤细胞轻至中度异形,核分裂象1～8个/10 HPF,3例表面均未见外生性乳头,但表层尿路上皮有明显异型增生,有少量外生乳头者外生成分形态符合低级别或低度恶性潜能.(3)旺炽型yon Brunn细胞巢5例,平均0.9 cm,表面被覆正常或增厚的黏膜组织,固有膜内见巢状分布、大小不等、排列紧密的尿路上皮团伴有囊腔形成,细胞均无异型性,无或偶见核分裂象.多点荧光原位杂交:79.1%(19/24)的内翻生长型尿路上皮癌存在染色体异常阳性,而内翻乳头状瘤和旺炽型von Brunn细胞巢无阳性染色体异常.免疫组织化学:CK20仅在2例内翻生长型尿路上皮癌中弱表达,内翻乳头状瘤和旺炽型von Brunn细胞巢均为阴性;16例内翻生长型尿路上皮癌和1例内翻乳头状瘤中有5%～50%的瘤细胞弱表达p53;内翻生长型尿路上皮癌中1%～5%表达Ki-67,内翻乳头状瘤和旺炽型von Brunn细胞巢均低于1%.随访:2例内翻生长型尿路上皮癌经多次复发后为浸润性癌,行全膀胱切除后仍发生远处转移.内翻乳头状瘤无复发.结论 伴内翻生长特征的尿路上皮增生性病变在良恶性病变中存在形态学上的重叠,但内翻生长型尿路上皮癌在形态及免疫组织化学上有独特特征.多点荧光原位杂交在鉴别诊断中有辅助作用.     

具有内翻生长特征的尿路上皮增生性病变的临床病理学研究

目的 研究伴内翻生长特征的尿路上皮增生性病变的临床病理特征,探讨免疫组织化学和多点荧光原位杂交在其鉴别诊断中的作用.方法 收集具有内翻生长特征的尿路上皮病变41例,分为内翻乳头状瘤、内翻生长型尿路上皮癌、旺炽型von Brunn细胞巢,用多点荧光原位杂交方法检测其3、7、17号染色体获得和9p21缺失;免疫组织化学EnVision法标记p53、CK20和Ki-67;并对12例进行随访.结果 (1)内翻乳头状瘤12例,平均1.2 cm,由互相连接的细胞索或巢在固有膜内生长,细胞索相对较细而宽窄一致,细胞巢外呈栅栏状、内为流水状排列,可见鳞状分化,无细胞学上的异型性,无或偶见核分裂象,4例可见少量表面外生乳头,被覆少于6层的正常尿路上皮.(2)内翻生长型尿路上皮癌24例,平均2.1 cm,结构似内翻乳头状瘤,但细胞索较粗并宽窄不一,细胞巢粗大并不规则状,可形成实体结构,瘤细胞轻至中度异形,核分裂象1～8个/10 HPF,3例表面均未见外生性乳头,但表层尿路上皮有明显异型增生,有少量外生乳头者外生成分形态符合低级别或低度恶性潜能.(3)旺炽型yon Brunn细胞巢5例,平均0.9 cm,表面被覆正常或增厚的黏膜组织,固有膜内见巢状分布、大小不等、排列紧密的尿路上皮团伴有囊腔形成,细胞均无异型性,无或偶见核分裂象.多点荧光原位杂交:79.1%(19/24)的内翻生长型尿路上皮癌存在染色体异常阳性,而内翻乳头状瘤和旺炽型von Brunn细胞巢无阳性染色体异常.免疫组织化学:CK20仅在2例内翻生长型尿路上皮癌中弱表达,内翻乳头状瘤和旺炽型von Brunn细胞巢均为阴性;16例内翻生长型尿路上皮癌和1例内翻乳头状瘤中有5%～50%的瘤细胞弱表达p53;内翻生长型尿路上皮癌中1%～5%表达Ki-67,内翻乳头状瘤和旺炽型von Brunn细胞巢均低于1%.随访:2例内翻生长型尿路上皮癌经多次复发后为浸润性癌,行全膀胱切除后仍发生远处转移.内翻乳头状瘤无复发.结论 伴内翻生长特征的尿路上皮增生性病变在良恶性病变中存在形态学上的重叠,但内翻生长型尿路上皮癌在形态及免疫组织化学上有独特特征.多点荧光原位杂交在鉴别诊断中有辅助作用.

Abstract：

Objective To study the clinieopathologie features of urothelial hyperplastie lesion with an endophytic growth pattern and the role of immunohistochemistry and muhitargeted fluorescence in situ hybridization(FISH)in the differential diagnosis.Methods Forty-one cases of urothelial lesions exhibiting endophytic growth patterns were reviewed and reclassified as inverted papilloma.urothelial carcinoma with an endophytic growth pattern,and florid von Brunn nest.The gains of chromosomes 3,7,and 17 and loss of 9p21 was detected by FISH,and performed immunohistochemical staining for CK20,p53,and Ki-67.Follow-up data of 12 cases were obtained.Results (1)Twelve inverted papillomas sized 1.2 cm in average.consisted of anastomosing cords and nests with uniform width distribution involving the lamina propfia,the central portion contained streaming cells with squamous metaplasia,and the periphery showed palisading.No or rare atypia and mitosis were found.Focal exophytic papillary component lined by less than 6 layers of normal urothelium were observed in 4 cases.(2)Twenty-four urothelial carcinomas with an endophytic growth pattern sized 2.1 cm in average,demonstrated the similar architecture with inverted papilloma,but exhibited thick columns and variable thickness ofthe cords,irregular size and shape of large nests with transition into solids.Mild to moderate cytologic atypia was shown,and mitotic figures ranged 1 to 8 per 10 HPFs.Exophytic papillary component was not observed in 3 cases.but the superficial urothelium showed dysplasia,while coexisted exophytie component in other eases was associated with low malignant potential or low grade tumor.(3)Five florid von Brunn nests sized 0.9 cm in average,had normal or hyperplastic urothelium,variable nests with cysts compacted in lamina propria,no cytologic atypia and mitosis.Twenty-one of 24(79.1%)urothelial carcinomas with an endophytie growth pattern displayed abnormally positive results by muhitargeted FISH,whereas all inverted papillomas and florid yon Brunn nests were negative.Immunohistochemically,CK20 Was weakly positive in 2 cases of urothelial carcinoma with an endophytic growth pattern,and negative in all inverted papillomas and florid yon Brunn nests.p53 weakly stained 5%to 50%nuclei of the tumor cells in 16 cafles of urothelial carcinomas with an endophytie growth pattem and 1 inverted papiHoma.1%-5%tumor ceUs expressed Ki-67 in urothelial carcinoma with an endophytic growth pattern,and less than 1%in inverted papiHoma and florid von Brunn nests.Follow-up study revealed that 2 cases of urothelial carcinoma with an endophytic growth pattern had developed invasive carcinoma,underwent cystectomy,and metastasized remotely.No recurrence occurred in cases of inverted papilloma.Conclusions Benign and malignant urothelial lesions with an endophytic growth pattern present histologie overlapping.Urothelial carcinoma with an endophytie growth pattern displays unique characteristics in morphology and immunohistochemistry.Multitargeted FISH analysis is helpful in the differential diagnosis.     

High-resolution deletion mapping of chromosome arm 17p in childhood primitive neuroectodermal tumors reveals a common chromosomal disruption within the Smith-Magenis region,an unstable region in chromosome band 17p11.2

Loss of heterozygosity (LOH) on chromosome arm 17p is the most common genetic aberration in childhood primitive neuroectodermal tumors (PNETs). To determine the frequency and extent of 17p deletions, 29 loci on 17p were investigated in 24 tumors by using restriction fragment length polymorphism (RFLP) and microsatellite analysis. LOH on 17p was found in 9 of 24 tumors. In all tumors with LOH, a continuous stretch from the telomere to chromosome band 17p11.2 was completely deleted, and no interstitial or terminal small-scale deletions were detected in the remaining 15 tumors. In four tumors with LOH on 17p, the chromosomal breakpoint was located between D17S953 and D17S805. To identify this deletion breakpoint on the cytogenetic map of chromosome 17 and to exclude uniparental disomy, we verified our data by using fluorescence in situ hybridization (FISH) analyses. By using two yeast artificial chromosome (YAC) clones that were positive for D17S689 and D17S953, the same breakpoint was confirmed in two specimens of cerebrospinal fluid (CSF) metastases by using FISH on interphase preparations. We demonstrate that, in most childhood PNETs with LOH on 17p, the breakpoint is close to, but not within, the centromere. It varies, and it occurs predominantly between the two markers D17S689 and D17S953, which is an unstable chromosomal region that is deleted or duplicated in the Smith-Magenis syndrome. Because LOH of 17p is associated with the formation of isochromosome 17q in the majority of PNETs, this study provides entry points to determine the molecular nature of this phenomenon. Genes Chromosom. Cancer 18:50–58, 1997. © 1997 Wiley-Liss, Inc.     

Utility of a multiprobe fluorescence in situ hybridization assay in the detection of superficial urothelial bladder cancer

We evaluated the performance of a multiprobe FISH (fluorescence in situ hybridization) assay for noninvasive detection of superficial urothelial carcinoma (UC) in the bladder, in comparison to urinary cytology. Voided urine samples from 74 patients with superficial UC were analyzed by both techniques. Urine samples from 19 patients with muscle-invasive tumors and from 19 healthy control subjects were also studied. For FISH analysis, labeled probes for chromosomes 3, 7, 9, and 17 were used to assess chromosomal abnormalities indicative of malignancy. We found a significant difference between the overall sensitivity of FISH and cytology in superficial UC detection (70.3 versus 35.1%, respectively; P < 0.0001). This significant difference was maintained when superficial UCs were broken down into low grade (52.8 versus 13.9%, respectively; P < 0.0005) and high grade (86.8 versus 55.3%, respectively; P < 0.0015) tumors. Overall specificity was 100% for cytology and 94.7% for FISH (difference not significant). Of patients with suspicious cytology, 69% were positive by FISH. Together, these findings suggest that FISH assay for chromosomes 3, 7, 9, and 17 has a higher sensitivity than cytology and a similar specificity in the detection of superficial UC--which could be useful for reducing some cystoscopies in the accurate follow-up usually performed in these patients.