The role of chromosome 8p22 deletion for predicting disease progression and pathological staging in prostate cancer.

The natural course of human prostate cancer is highly variable, and we still lack reliable tools to predict the patient's outcome. Recent publications suggest that the deletion of chromosome 8p22 has an important role for tumor progression in prostate cancer. Totally, 97 patients (41 Japanese and 56 Swedish) were studied to detect the status of chromosome 8p22 deletion by the fluorescence in situ hybridization (FISH) technique. Seventy-seven underwent surgery (59 radical prostatectomies or 18 lymph node dissections), and the specimens were prepared by touch biopsy. Fine-needle aspiration biopsies (FNAB) were obtained from another non-operative 20 cases. Disease progression was evaluated in 57 patients with a median follow-up of 59 months. 8p22 deletions were detected in 58 (60 %) of all cases. The frequency of 8p22 deletion did not significantly differ between different preparations of specimens (touch biopsy vs. FNAB) as well as between different races (Japanese vs. Swedish). Cases with more than pT3 tumors had a significantly higher frequency of 8p22 deletion than those with pT2 (p < 0.01). Multivariate analysis demonstrated that 8p22 deletion was the strongest parameter to predict disease progression (hazard ratio = 5.75; p = 0.0001). Studies on chromosomal deletions of 8p22 by the FISH technique may serve as a universal genetic marker to optimize the treatment strategy in patients with prostate cancer.

Deletions on chromosome 8p22 may predict disease progression as well as pathological staging in prostate cancer.

PURPOSE: A recent report demonstrated that the deletion of chromosome 8p22 could predict disease progression in stage III (capsular penetrating) prostate cancer. We studied if the status of chromosomal deletions of 8p22 could reflect pathological stage as well as patient prognosis, thereby serving as a diagnostic tool to optimize the treatment strategy in prostate cancer. EXPERIMENTAL DESIGN: A total of 97 patients (41 Japanese and 56 Swedish) were studied by the fluorescence in situ hybridization technique. Seventy-seven patients (23 pT2, 18 pT3, and 36 pN+ tumors) underwent surgery (radical prostatectomy or lymph node dissection). The specimens were prepared by touch biopsy. From another 20 cases, fine-needle aspiration biopsies were obtained. RESULTS: 8p22 deletions were detected in 47 (61%) and 11 (55%) specimens of 77 touch biopsies and 20 fine-needle aspiration biopsies, respectively. No significant difference was found in the frequency of 8p22 deletion between different preparations of specimens, as well as between different races (Japanese versus Swedish). The frequency of 8p22 deletion was statistically higher in patients with pT3 or more than in those with pT2 (P < 0.01). Disease progression was evaluated in 57 patients. The Cox proportional hazards model revealed 8p22 deletion to be the strongest parameter to predict disease progression (hazards ratio = 5.75; P = 0.0001). CONCLUSIONS: Studies on chromosomal deletions of 8p22 by fluorescence in situ hybridization technique may serve as a genetic marker to optimize the treatment strategy in patients with prostate cancer to the optimal treatment.

Characterization of chromosomal abnormalities in uroepithelial carcinomas by G-banding, spectral karyotyping and FISH analysis.

Chromosome analysis by G-banding, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) was performed on 24 short-term cultured transitional cell bladder carcinomas and 5 cell lines established from bladder carcinomas. Except for one tumor with an apparently normal chromosomal constitution, clonal chromosome abnormalities were detected in all examined cases by the combined approach. The application of SKY and FISH techniques improved the karyotypic descriptions, originally based on G-banding only, by identifying 32 additional numerical changes, by establishing the chromosomal origin of 27 markers and 2 ring chromosomes, by redefining 53 aberrations and by detecting 15 hidden chromosomal rearrangements. No recurrent translocation, however, was detected. The most prominent karyotypic feature was thus the occurrence of deletions and losses of whole chromosome copies indicating the importance of tumor suppressor genes in transitional cell carcinoma pathogenesis. Invasive carcinomas were karyotypically more complex than were low grade superficial tumors. Specific losses of material from chromosome 9 and from chromosome arms 11p and 8p, and gains of 8q and 1q seem to be early changes appearing in superficial tumors, whereas losses from 4p and 17p and the formation of an isochromosome for 5p were associated with more aggressive tumor phenotypes.

5q11, 8p11, and 10q22 are recurrent chromosomal breakpoints in prostate cancer cell lines.

Prostate cancer cell lines have been widely used as model systems characterizing pathogenetic, functional, and therapeutic aspects of prostate cancer development. However, their chromosomal compositions are poorly characterized. In this study, five prostate cancer cell lines-TSU-Pr1, JCA-1, NCI-H660, ALVA-31, and PPC-1-were investigated by G-banding, comparative genomic hybridization (CGH), and spectral karyotyping. The results were combined with our previous findings in the prostate cancer cell lines PC-3, DU145, and LNCaP. By comparative genomic hybridization (CGH), the most frequent losses were observed at 13q, 8p, 9p, and 4q, whereas gains were most commonly seen at 8q, 10q, and 18p. The composite karyotypes were characterized by multiple numerical and structural chromosomal aberrations. Recurrent breakpoints at 5q11, 8p11, and 10q22 were observed to participate in deletion and translocation events in five of the cell lines, suggesting the importance of tumor suppressor and/or oncogenes in these regions. ALVA-31 and PPC-1 shared nine identical derivative chromosomes, two of which have also been detected in PC-3. In addition, the identification of the same homozygous deletion at D10S541 and of an identical TP53 gene mutation in all three cell lines suggests a common origin of these cell lines.

Distinct deleted regions on chromosome segment 16q23-24 associated with metastases in prostate cancer.

Cadherins (CDH) are cell adhesion molecules and their dysfunctions have been implicated in the development of cancer metastases. Several cadherin genes are tandemly located on 16q, which is frequently deleted in prostate cancer. We therefore used 22 markers on 16q to localize important deleted regions in metastases of this tumor. We found 16q deletions in 24/32 (75%) tumors. All lymph node and brain metastases showed extensive deletions, while 52% of primary tumors displayed limited deletions. Commonly deleted regions (CDRs) on 16q23-24, CDR2 (D16S515-D16S516) and CDR4 (D16S520-D13S3028), were strongly associated with metastases and increased Gleason score. Reduced CDH1 (E-cadherin) expression was seen in 16/32 (50%) tumors, but the CDH1 gene is not within either of these two regions. Sequencing analysis for all 16 exons of the CDH1 gene did not reveal any mutations in 10 tumors, including three brain metastases with both 16q22.1 deletion and absent E-cadherin expression. Our results implicate other, yet unidentified genes on 16q23-24 to be the frequent targets of mutations and deletions in prostate cancer metastases.

Links between genetic and environmental factors and prostate cancer risk.

BACKGROUND: Genetic polymorphisms and expression of steroid receptors may explain why some individuals are more at risk of developing prostate cancer. Some risk factors often discussed are androgen stimulation, and vitamin A and D deficiency. Long CAG-repeats in exon 1 of the androgen receptor (AR) gene on the X chromosome seem to have a protective role against androgen overstimulation. Likewise, long vitamin D receptor alleles in the poly-A tract may prevent vitamin D stimulation. METHODS: Blood samples from 59 Swedish patients with sporadic prostate cancers, 59 with hereditary prostate cancer, and 34 Japanese prostate cancer patients were compared with benign controls. Tissue specimens from 37 Swedish and 23 Japanese prostate cancer patients with matching blood samples were investigated by immunohistochemical techniques. RESULTS: The number of CAG-repeats was identical in sporadic and hereditary prostate cancer patients, but the repeats were significantly shorter than in benign controls. Benign Japanese controls were similar to Swedish controls, but Japanese prostate cancers had longer repeats than did controls. Both the vitamin D and A receptor staining was stronger in Japanese than in Swedish prostate cancer specimens. Prostate cancer occurs approximately 5 years later in Japanese compared with Swedish men. CONCLUSIONS: Varying lengths of CAG-repeats of the androgen receptor cannot fully explain racial differences in clinical prostate cancer incidence. A larger content of vitamin A and D receptors may be linked to a delayed onset of clinical prostate cancer in Japanese men.

Characterization of chromosomal abnormalities in prostate cancer cell lines by spectral karyotyping.

Human prostate cancer is characterized by multiple gross chromosome alterations involving several chromosome regions. However, the specific genes involved in the development of prostate tumors are still largely unknown. Here we have studied the chromosome composition of the three established prostate cancer cell lines, LNCaP, PC-3, and DU145, by spectral karyotyping (SKY). SKY analysis showed complex karyotypes for all three cell lines, with 87, 58/113, and 62 chromosomes, respectively. All cell lines were shown to carry structural alterations of chromosomes 1, 2, 4, 6, 10, 15, and 16; however, no recurrent breakpoints were detected. Compared to previously published findings on these cell lines using comparative genomic hybridization, SKY revealed several balanced translocations and pinpointed rearrangement breakpoints. The SKY analysis was validated by fluorescence in situ hybridization using chromosome-specific, as well as locus-specific, probes. Identification of chromosome alterations in these cell lines by SKY may prove to be helpful in attempts to clone the genes involved in prostate cancer tumorigenesis.

Nitric oxide synthase activity in human renal cell carcinoma.

PURPOSE: Nitric oxide (NO) is generated in mammalian tissue by the conversion of L-arginine to L-citrulline. The reaction is catalyzed by nitric oxide synthase (NOS). NO has been suggested to have a dual role in tumor biology with both antitumor and tumor promoter activity. Furthermore, it has been proposed that NO contributes to interleukin-2-induced antitumor activity. Since interleukin-2 is used in the treatment of renal cell carcinoma (RCC) it was of interest to study the NOS activity in the human kidney and in RCC and its correlation to tumor grade. Furthermore, the effect of cytokine treatment on NOS activity and the effect of NO donor application was studied in cultured cells. MATERIALS AND METHODS: The effect of cytokine treatment on NOS activity and the effect of NO donor application on cell proliferation was studied in cultured human proximal tubular cells and in RCC cell lines HN4 and HN51. NOS activity was measured by the L-arginine to L-citrulline conversion assay. RESULTS: Calcium-dependent NOS activity was found in all non-malignant kidney tissues (486+/-63 pmol. min(-1) g(-1) tissue). The activity was significantly lower in RCC (24+/-6 pmol. min(-1) g(-1) tissue) and correlated with tumor grade; thus high grade tumors showed lower activity than low grade tumors. Calcium-independent NOS activity was not detected in non-malignant kidney tissue or in RCC tissue. In cultured proximal tubular cells and RCC cell lines HN4 and HN51, cytokine treatment induced a marked increase in NOS activity and NO exerted cytostatic effects on these cell lines. CONCLUSIONS: The NOS activity was higher in non-malignant kidney tissue than in RCC tissue and was inversely correlated with tumor grade. Furthermore, cytokine treatment induced a marked increase in NOS activity and NO exerted cytostatic effects on cultured proximal tubular cells and RCC cell lines.

Chromosome 16q24 deletion and decreased E-cadherin expression: possible association with metastatic potential in prostate cancer.

BACKGROUND: Deletion of chromosome 16q is a frequent aberration in prostatic carcinoma, indicating the existence of candidate tumor suppressor genes involved in the pathogenesis of prostate cancer. METHODS: Chromosome 16 numerical aberration and loss of 16q were studied by fluorescence in situ hybridization in 31 primary and 22 metastatic tumors from 53 patients. The results were compared with E-cadherin expression, tumor grade and stage, and DNA ploidy. RESULTS: Numerical chromosome 16 aberrations, 16q deletion, and loss of E-cadherin expression were found in 29%, 35%, and 29% of the primary tumors, respectively, and in 73%, 73%, and 73% of the metastases, respectively. High tumor grade and DNA aneuploidy were also found to have significant correlation with metastases. CONCLUSIONS: Deletion of chromosome 16q24 and/or loss of the E-cadherin function appears in a high frequency in metastases of prostate cancer. The strong correlations suggest that they may be important risk factors, contributing to the metastatic potential of the tumor.

Identification of two distinct deleted regions on chromosome 13 in prostate cancer.

Aberrations of 13q occur frequently in prostate cancer and this chromosome contains two known tumor suppressor genes, BRCA2 and Rb1. This study analysed 13q LOH, DNA ploidy, BRCA2 mutation and pRb expression in prostate cancers. In total, 13q deletions were found in 18 of 36 tumors but did not correlate with histological grade, stage or DNA ploidy. Two smallest regions of overlapping deletions were defined: one flanked by D13S218 and D13S153; the other flanked by D13S31 and D13S137. BRCA2 was less frequently deleted whereas Rb1 did have a high frequency of deletion. None of the two genes was located in any of these two regions. Furthermore, BRCA2 mutation was not found in the five tumors where deletions had involved the BRCA2 locus. Neither did the Rb1 deletion correlate with absent pRb expression. In addition, tetraploidy was found in 14 out of 25 tumors analysed and correlated with aberrant pRb expression. Our results indicate that 13q deletion is an early non-random event. Tumor suppressor genes other than BRCA2 or Rb1 may be the target of 13q deletions. Aberrant pRb expression may not reflect the two-hit Rb1 inactivation but may be involved in the tetraploidization of prostate cancer cells.

Molecular evidence for pap-G specific adhesion of Escherichia coli to human renal cells.

PURPOSE: To study the interaction between class II G-adhesin of Escherichia coli and human urogenital cells. MATERIAL AND METHODS: The adherence of two wild type P-fimbriated E. coli strains, both carrying a class II G-adhesion, and two constructed mutants (one class II G-adhesion knock-out mutant and one class switch mutant in which the papG gene was exchanged with a prsJ96 allele which is a representative of the class III G-adhesin) to human urogenital cells were examined by light microscopy and flow cytometry. RESULTS: The wild type E. coli strains adhered avidly to proximal tubular cells, but the isogenic mutant strains did only adhere in one of the experiments. A soluble receptor analogue inhibited bacterial attachment. CONCLUSIONS: These experiments strongly suggest that the papG class II tip adhesin of P-fimbriae is essential in the pathogenesis of human kidney infection.

Genotypic and phenotypic characterization of two newly established renal cell carcinoma cell lines.

Two new cell lines from human renal cell carcinoma are reported. Primary cell cultures from 75 consecutive cases of nephrectomy and metastatic surgery due to different stages of RCC during 4 years were studied. Two cell cultures could be propagated for more than 50 passages in vitro. HN4 was derived from a grade III clear cell carcinoma. HN51 originated from a metastatic brain lesion of a clear cell carcinoma grade III. Karyotype analysis of HN4 revealed triploidy with a clonal aberration, der(10)t(3;10)(q13;p12). HN51 also had a triploid pattern with different marker chromosomes but without any clonal aberration. Loss of heterozygosity studies revealed no loss of heterozygosity on 3p or other chromosomal markers in HN4 but LOH was found on one 3p marker and one 14q marker in addition to all 17 p and q markers in HN51. In vitro light microscopy showed distinctly different morphology in the two cell lines although they both had a typical epithelial growth pattern. Doubling times in vitro were low but slightly higher for HN51. Repeated tumorigenenic experiments in athymic mice only gave rise to subcutaneous tumors with HN51. On characterization by 2-dimensional gel electrophoresis, the two cell lines exhibited different polypeptide patterns with higher expression of proliferating cell nuclear antigen in HN51 and higher expression of glutathione-S-transferase in HN4 constituting the most prominent differences.

Chromosome imbalances in papillary renal cell carcinoma and first cytogenetic data of familial cases analyzed by comparative genomic hybridization.

We used comparative genomic hybridization to analyze 17 tumor samples from 11 patients with papillary renal cell carcinoma (RCC), including three patients with hereditary papillary RCC. Whereas the most frequent aberrations confirmed data obtained by banding analyses, copy number increases on 5q, which previously were considered characteristic of nonpapillary RCC, were identified in two cases. In two complex cases belonging to the same family, a characteristic pattern of chromosomal aberrations was found: five of the six imbalances present in the less complex case were included in the karyotype of the other case, suggesting a genetically determined mechanism resulting in genomic instability of specific chromosomes or chromosomal subregions and/or selection of specific mutations.

Mapping of chromosomal gains and losses in prostate cancer by comparative genomic hybridization.

Comparative genomic hybridization (CGH) allows detection of chromosomal imbalances in whole genomes in a comprehensive manner. With this approach, ten cases of prostate cancer (seven primary tumors and three metastases) were analyzed. Frequent chromosomal gains detected by CGH involved chromosome arms 7q, 8q, 9q, and 16p, and chromosomes 20 and 22, as well as frequent losses of chromosome arms 16q and 18q, in at least three of the ten cases. Overrepresentation of chromosome arm 9q has not been described in published reports. The CGH data were compared with results of a loss of heterozygosity (LOH) study, in which complete allelotyping was performed in the same prostate tumors with 74 different polymorphic markers. In general, a high concordance between the CGH and LOH results was observed (92%). Tumors revealing discrepancies by CGH and LOH analysis were investigated further by interphase cytogenetics, and the resulting picture regarding the genomic alterations is discussed in detail.

A group of NotI jumping and linking clones cover 2.5 Mb in the 3p21-p22 region suspected to contain a tumor suppressor gene.

The chromosomal region 3p21.2-p22 has been shown to be involved in the development of several forms of solid tumors. Such deletions, translocations, and rearrangements presumably result in the disturbance or loss of a critical gene function. Pulsed-field gel electrophoresis (PFGE), using NotI linking clones as a probe represent a powerful tool for analyzing such rearrangements. A NotI linking clone, AP20 (D3S1641), was localized by in situ hybridization to 3p21.3-p22. Two NotI jumping clones adjacent to this clone were isolated, clone J32-612 covering 0.5 Mb and clone J31-611 covering approximately 1 Mb. Clone J31-611 crosses the border of the deletion present in hybrid cell line MCH939.2, which contains a deleted 3p21 region. For these jumping clones, corresponding NotI linking clones, NLJ3 (D3S1642) and NL3-003, were isolated. Altogether, linking and jumping clones from the AP20 locus hybridize to NotI fragments totaling 2.5 Mb in length. These NotI-containing clones detect expressed sequences in several human tissues. Clone NLJ3 possesses homology to the human platelet-derived endothelial cell growth factor gene and may represent a new member of this gene family. Another clone (AP20) revealed 66% sequence similarity to rat skeletal muscle voltage-sensitive sodium channel subtype 2. Therefore, this group of clones will be useful not only for analyzing rearrangements in tumors, but also for the isolation of new genes from the 3p21.3-p22 region.

Transthyretin and cystatin C are catabolized in proximal tubular epithelial cells and the proteins are not useful as markers for renal cell carcinomas.

Ten human kidney specimens and thirty-two renal cell carcinomas were investigated for the presence of transthyretin mRNA and cystatin C mRNA using Northern blot analysis. Five of ten kidney specimens and 15 of 32 renal carcinomas were also immunohistochemically investigated for the presence of the corresponding proteins. Transthyretin mRNA could not be detected in any of the normal or neoplastic tissue preparations, whereas low amounts of cystatin C mRNA were found in nine of ten normal kidneys and in 24 of 32 renal cell carcinomas. Immunoreactive transthyretin and cystatin C were present in proximal tubular epithelial cells of all kidney specimens, whereas neither of the proteins was detected the tumour cells of the renal carcinomas. Immunoreactive cystatin C was, however, demonstrated in scattered monocyte/macrophage-like cells. We conclude that the presence of immunoreactive transthyretin and cystatin C in proximal tubular cells of the kidney is most likely due to reabsorption of the proteins from the primary urine. The small amounts of cystatin C mRNA in some of the normal and neoplastic renal preparations are probably due to cystatin C synthesis in macrophages. Transthyretin has been recommended as an immunohistochemical marker for renal cell carcinomas. Our results, however, clearly indicate that neither transthyretin nor cystatin C constitutes a useful marker for such neoplasms.

Hereditary papillary renal cell carcinoma: clinical studies in 10 families.

We recently described a 3-generation family with members affected with papillary renal cell carcinoma, an uncommon histological type of renal cell carcinoma. Possibly family 150 is an isolated occurrence, a reflection of some as yet unknown environmental factor. Alternatively, family 150 may represent a distinct class of inherited cancer. To distinguish between these 2 possibilities we sought additional families with papillary renal cell carcinoma and we identified 9 with members affected with papillary renal cell carcinoma. There were 29 affected male and 12 affected female subjects (ratio 2.41:1), including affected members of family 150. Papillary renal cell carcinomas were often detected incidentally in asymptomatic individuals or during screening of asymptomatic members of renal cell carcinoma families. The penetrance, the proportion of obligate gene carriers that showed clinical evidence of the disease, was reduced. The median survival of affected individuals was 52 years. The results support the concept that the predisposition to develop papillary renal cell carcinomas may be inherited and that hereditary papillary renal cell carcinoma constitutes a distinct class of inherited cancer.

Deletion mapping of chromosome 8p in prostate cancer by fluorescence in situ hybridization.

Double-target fluorescence in situ hybridization (FISH) was applied to 42 cases of prostate cancer and seven cases of histologically proven benign prostate hyperplasia for the detection of structural aberrations of chromosome 8. Cosmid probes for two chromosome 8p loci (LPL/8p22 and D8S7/8p23) were used in 34 specimens of malignant tumors obtained by the touch biopsy technique. Deletion was defined as when the number of cosmid signals was lower than the number of centromere signals in more than 35% of all nuclei observed. In total, thirty of the 42 (71%) specimens demonstrated any type of 8p deletion. Out of the 34 cases in which deletion mapping could be evaluated, distal deletion (D8S7) was detected in 17 (50%), of which 10 also showed deletion of LPL. Deletion of LPL was detected in 18 cases (53%), of which 8 (24%) retained the D8S7 (interstitial deletion). When the deletion pattern was graded as (1) no deletion (2) partial deletion (either D8S7 or LPL deleted) and (3) both deletions, the degree of deletion was well correlated with the tumor grade (P = 0.0009) and with stage (P = 0.0072, Fisher's Exact test). These data support the hypothesis that tumor suppressor gene(s) may be located in the chromosomal region 8p22, hence 8p deletions may play a crucial role in the pathogenesis of prostate cancer.

Nonrandom numerical aberrations of chromosomes 7, 9, and 10 in DNA-diploid bladder cancer.

Double-target in situ hybridization technique (ISH) was applied to 37 cases of bladder cancer to detect numerical aberrations of chromosomes 7, 9, 10, and 11 by centromeric DNA probes. Of 33 evaluable cases, 29 (88%) demonstrated chromosome aberrations. In 17 cases with diploid pattern as measured by flow-cytometric DNA analysis (FCM), 15 (88%) demonstrated chromosome aberrations. Of these, trisomy 7, monosomy 9, and trisomy 10 were detected in three, three, and one case, respectively, as a single chromosome aberration. In 14 (88%) of 16 cases with an aneuploid DNA pattern, chromosome aberrations for two or more chromosomes were demonstrated. A significant correlation was observed between grade of chromosome aberrations and tumor grade (p < 0.01, Fisher's test), between number of spots for chromosome 7 and peak value in FCM (p = 0.015, by Spearman rank order test). In eight cases, chromosome aberrations in the tumor were compared with the corresponding pattern in the grossly normal and histologically benign mucosa. Trisomy 10 and monosomy 9 were detected as chromosome numerical aberrations in the histologically normal mucosa, consistent with aberrations in the corresponding patients. We conclude that trisomy 7, monosomy 9, and trisomy 10 may be early events in the evolution of bladder cancer.