Thomas C. Gasser

Microarrays of bladder cancer tissue are highly representative of proliferation index and histological grade

The number of genes suggested to play a role in cancer biology is rapidly increasing. To be able to test a large number of molecular parameters in sufficiently large series of primary tumours, a tissue microarray (TMA) approach has been developed where samples from up to 1000 tumours can be simultaneously analysed on one glass slide. Because of the small size of the individual arrayed tissue samples (diameter 0.6 mm), the question arises of whether these specimens are representative of their donor tumours. To investigate how representative are the results obtained on TMAs, a set of 2317 bladder tumours that had been previously analysed for histological grade and Ki67 labelling index (LI) was used to construct four replica TMAs from different areas of each tumour. Clinical follow‐up information was available from 1092 patients. The histological grade and the Ki67 LI were determined for every arrayed tumour sample (4×2317 analyses each). Despite discrepancies in individual cases, the grade and Ki67 information obtained on minute arrayed samples were highly similar to the data obtained on large sections (p<0.0001). Most importantly, every individual association between grade or Ki67 LI and tumour stage or prognosis (recurrence, progression, tumour‐specific survival) that was observed in large section analysis could be fully reproduced on all four replica TMAs. These results show that intra‐tumour heterogeneity does not significantly affect the ability to detect clinico‐pathological correlations on TMAs, probably because of the large number of tumours that can be included in TMA studies. TMAs are a powerful tool for rapid identification of the biological or clinical significance of molecular alterations in bladder cancer and other tumour types. Copyright

High-Throughput Tissue Microarray Analysis of Cyclin E Gene Amplification and Overexpression in Urinary Bladder Cancer

Studies by comparative genomic hybridization revealed that the 19q13 chromosomal region is frequently amplified in bladder cancer. The cyclin E gene (CCNE), coding for a regulatory subunit of cyclin-dependent kinase 2, has been mapped to 19q13. To investigate the role of cyclin E alterations in bladder cancer, a tissue microarray of 2,317 specimens from 1,842 bladder cancer patients was constructed and analyzed for CCNE amplification by fluorescence in situ hybridization and for cyclin-E protein overexpression by immunohistochemistry. Fluorescence in situ hybridization analysis showed amplification in only 30 of the 1,561 evaluable tumors (1.9%). Amplification was significantly associated with stage and grade (P: < 0.0005 each). Immunohistochemically detectable cyclin E expression was strong in 233 (12.4%), weak in 354 (18.9%), and negative in 1, 286 of the 1,873 interpretable tumors. The majority (62.1%) of CCNE-amplified tumors were strongly immunohistochemistry-positive (P: < 0.0001). The frequency of protein expression increased from stage pTa (22.2%) to pT1 (45.5%; P: < 0.0001) but then decreased for stage pT2-4 (29.4%; P: < 0.0001 for pT1 versus pT2-4). Low cyclin E expression was associated with poor overall survival in all patients (P: < 0.0001), but had no prognostic impact independent of stage. It is concluded that cyclin E overexpression is characteristic to a subset of bladder carcinomas, especially at the stage of early invasion. This analysis of the prognostic impact of CCNE gene amplification and protein expression in >1,500 arrayed bladder cancers was accomplished in a period of 2 weeks, illustrating how the tissue microarray technology remarkably facilitates the evaluation of the clinical relevance of molecular alterations in cancer.

Multiprobe FISH for Enhanced Detection of Bladder Cancer in Voided Urine Specimens and Bladder Washings

The aim of this study was to evaluate the UroVysion (Vysis, Downers Grove, IL) fluorescence in situ hybridization (FISH) test for improved detection of bladder cancer in urinary specimens. Three groups of specimens were examined, including voided urine specimens (1) collected before resection of bladder cancer, (2) from cystoscopically negative bladders of patients with previous bladder cancer, and (3) from patients with benign prostatic hyperplasia (controls). FISH positivity was defined as more than 2 urothelial cells with an abnormal signal copy number of at least 1 of the 4 probes. FISH was positive in 1 of 27 control specimens and in 33 (73%) of 45 pTa, 12 (100%) of 12 pT1, and 13 (100%) of 13 pT2-4 tumors. The results were similar in a series of 68 bladder washings. In addition, FISH of voided urine specimens was positive in 5 of 10 patients with negative follow-up cystoscopy results. Subsequent recurrence was found in 4 of these patients but in none of 5 patients with FISH-negative results. Multiprobe FISH markedly improves the sensitivity and specificity of cytology for the detection of bladder cancer in urine specimens.

Chromosomal imbalances in papillary renal cell carcinoma: genetic differences between histological subtypes.

Papillary renal-cell carcinoma (RCC) is a renal carcinoma variant with distinct gross, microscopic, and cytogenetic features. Recently, a type 1 (pale cytoplasm, small-cell) and a type 2 (eosinophilic cytoplasm, large-cell) subtype of papillary RCC have been described. Chromosomal alterations associated with these tumor types were examined in 25 papillary RCCs by comparative genomic hybridization. Relative copy number gains were frequently detected at chromosomes 7p (56%), 7q (44%), 12q (28%), 16q (32%), 17p (56%), 17q (76%), and 20q (32%). Chromosomal regions that were most often lost included 1p (24%), 4q (36%), 6q (40%), 9p (36%), 13q (36%), Xp (28%), Xq (36%), and Y (73%). There were clinical and genetic differences between the subtypes of papillary RCC. Type 2 tumors were of higher nuclear grade (P = 0.0012) and higher stage (P = 0.01) and had a worse prognosis (P = 0.03) than type 1 tumors. The number of DNA gains per tumor, especially gains of 7p and 17p, was significantly higher in type 1 than in type 2 tumors (P < 0.01). These data suggest the existence of two distinct morphological and genetic subgroups of papillary RCC. Losses of chromosome Xp were associated with short patient survival (P < 0.01). Despite the small number of cases, this finding suggests that a gene on chromosome Xp may contribute to papillary RCC progression.

GreenLight Laser Vaporization of the Prostate: Single-Center Experience and Long-Term Results After 500 Procedures

BACKGROUND Long-term data of photoselective vaporization of the prostate (PVP) for treatment of lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH) is scanty. OBJECTIVE Evaluate the long-term efficacy and the complication rate in 80-watt (W) PVP. DESIGN, SETTING, AND PARTICIPANTS 500 consecutive patients with LUTS secondary to BPH underwent PVP between September 2002 and April 2007. The mean follow-up was 30.6+/-16.6 (5.2-60.6) mo. INTERVENTION All patients underwent 80-W PVP performed by seven surgeons. MEASUREMENTS We evaluated perioperative parameters, including operation time, delivered energy, changes of hemoglobin and serum sodium, catheterization, and hospitalization time as well as intraoperative and postoperative complications. Patients presenting for follow-up had data assessed on the International Prostate Symptom Score and quality-of-life questionnaire (IPPS-QoL), maximal flow rate (Q(max)), and post-voiding residual volume (Vres). RESULTS AND LIMITATIONS Mean patient age was 71.4+/-9.6 (46-96) yr, with a mean preoperative prostate volume of 56.1+/-25.3 (10-180) ml. Mean operation time was 66.4+/-26.8 (10-160) min, and mean energy delivery was 206+/-94 (2.4-619.0) kJ. Despite ongoing oral anticoagulation in 45% of the patients (n=225), no severe intraoperative complications were observed. Mean catheterization and postoperative hospitalization time was 1.8+/-1.2 (0-10) and 3.7+/-2.9 (0-35) d, respectively. The mean IPSS after 3 yr was 8.0+/-6.2, the QoL score was 1.3+/-1.3, the Q(max) was 18.4+/-8.0 ml/s, and the Vres was 28+/-42 ml. The retreatment rate was 6.8%. Urethral and bladder neck strictures were observed in 4.4% and 3.6% of the patients, respectively. Localized prostate cancer was diagnosed during follow-up in six patients. CONCLUSION PVP is a safe and effective procedure for treatment of LUTS secondary to BPH. Patients on ongoing oral anticoagulation can be safely operated on. PVP leads to an immediate and sustained improvement of subjective and objective voiding parameters. The late complication rate is comparable to that of transurethral electroresection of the prostate.

Ki67 LABELLING INDEX: AN INDEPENDENT PREDICTOR OF PROGRESSION IN PROSTATE CANCER TREATED BY RADICAL PROSTATECTOMY

The clinical course of prostate cancer is highly variable and cannot satisfactorily be predicted by histological criteria alone. Both tumour cell proliferation and neuroendocrine differentiation have been suggested as additional prognostic parameters, neuroendocrine differentiation being considered to enhance tumour cell proliferation. This study investigated the prognostic value of tumour cell proliferation [Ki67 labelling index (LI), MIB 1] and neuroendocrine differentiation and their relationship to each other. One hundred and thirty‐seven paraffin‐embedded radical prostatectomy specimens were examined. Neuroendocrine differentiation was found in 58 per cent of cases, but was not associated with pTN stage, Gleason score, Ki67 LI, or tumour progression. Ki67 LI was not significantly associated with pTN stage or with Gleason score. High grade (P=0·0005), advanced local stage (P=0·0004), positive lymph nodes (P=0·02), and high Ki67 LI (P=0·0203) were predictors of tumour progression if univariate analysis was performed, but Cox stepwise regression showed that only advanced local stage (P=0·0025) and Ki67 LI (P=0·0105) were independent predictors of tumour progression, the relative risk being 3·6 and 2·5, respectively. It is concluded that Ki67 is an important prognostic marker in prostate cancer with a potential for routine application.

Expression patterns of potential therapeutic targets in prostate cancer.

Androgen withdrawal is the only effective therapy for patients with advanced prostate cancer, but progression to androgen independence ultimately occurs in almost all patients. Novel therapeutic strategies targeting molecular mechanisms that mediate resistance to hormonal and chemotherapeutic treatment are highly warranted. Here, we aimed to evaluate the expression of potential therapeutic targets in advanced prostate cancer. A tissue microarray (TMA) containing samples from 535 tissue blocks was constructed, including benign prostatic hyperplasia as controls (n = 65), prostatic intraepithelial neoplasia (PIN; n = 78), clinically localized prostate cancers (n = 181), as well as hormone‐refractory local recurrences (n = 120) and distant metastases (n = 91). The expression of 13 different proteins was analyzed using immunohistochemistry (Bcl‐2, p53, ILK, Syndecan‐1, MUC‐1, EGFR, HER2/neu, HSP‐90, Ep‐CAM, MMP‐2, CD‐10, CD‐117 and Ki67). Significant overexpression in hormone‐refractory prostate cancer and metastatic tissue compared to localized prostate cancer was found for Ki67 (64% vs. 9%), Bcl‐2 (11% vs. 1%), p53 (35% vs. 4%), Syndecan‐1 (38% vs. 3%), EGFR (16% vs. 1%) and HER2/neu (16% vs. 0%). Overexpression of CD‐117 was restricted to 1 single metastasis. All other markers did not show relevant differences in expression between subgroups. Taken together, p53, Bcl‐2, Syndecan‐1, EGFR and HER2/neu are preferentially expressed in hormone‐refractory and metastatic prostate cancer. Selected inhibition of these targets might offer a strategy to treat advanced tumors and prevent further progression. Treatment decisions should not be based on findings in primary tumors but rather on tissues from recurrent or metastatic lesions.

Patterns of Chromosomal Imbalances in Advanced Urinary Bladder Cancer Detected by Comparative Genomic Hybridization

To identify genetic changes linked to bladder cancer progression we analyzed 90 invasive transitional cell carcinomas (37 pT1 and 53 pT2-4) by comparative genomic hybridization. The most frequent alterations included 1q+ (37%), 5p+ (24%), 6q- (19%), 8p-(29%), 8q+ (37%), 9p- (31%), 9q- (23%), 11p-(24%), 11q- (22%), 17q+ (29%), and 20q+ (28%). Interestingly, there were three groups of alterations that frequently occurred together (9p- and 11q13+/ 20q+ and 11q13+ or 17q+/1q+ and 3p+ or 11q-). These loci might carry genes that interact with each other in specific molecular pathways. There were remarkable genetic similarities between minimally and deeply invasive tumors of different histological grades, including a similar number of aberrations per tumor and an equal frequency of most individual alterations. However, deletions of 5q, 6q, and 15q and gains of 5p, 7p, and Xq were significantly more frequent in pT2-4 than in pT1 carcinomas. These loci may harbor genes that are important for bladder cancer progression.

Amplification of EIF3S3 Gene Is Associated with Advanced Stage in Prostate Cancer

Gain of the long arm of chromosome 8 (8q) is one of the most common gains found in the advanced prostate cancer by comparative genomic hybridization. We have previously identified a putative target gene for the 8q gain, EIF3S3, that encodes a p40 subunit of eukaryotic translation initiation factor 3 (eIF3). Here, we studied the frequency of the EIF3S3 amplification in different stages of prostate cancer and co-amplification of EIF3S3 and oncogene MYC. In addition, prognostic utility of the EIF3S3 copy number alteration was evaluated. The analyses were done with fluorescence in situ hybridization and tissue microarray technology. High-level amplification of EIF3S3 was found in 11 of 125 (9%) of pT1/pT2 tumors, 12 of 44 (27%) of pT3/pT4 tumors, and 8 of 37 (22%) of lymph node metastases as well as in 26 of 78 (33%) and 15 of 30 (50%) of hormone refractory locally recurrent tumors and metastases, respectively. The amplification was associated with high Gleason score (P < 0.001). One of the 79 tumors with EIF3S3 amplification had only two copies of MYC, whereas all tumors with amplification of MYC had also amplification of EIF3S3 indicating common co-amplification of the genes. Gain of EIF3S3 was associated with poor cancer-specific survival in incidentally found prostate carcinomas (P = 0.023). In the analyses of prostatectomy-treated patients, the amplification was not statistically significantly associated with progression-free time. In conclusion, amplification of EIF3S3 gene is common in late-stage prostate cancer suggesting that it may be functionally involved in the progression of the disease.

Multi-target fluorescence in situ hybridization in bladder washings for prediction of recurrent bladder cancer

The objective of this study was to evaluate the diagnostic value of chromosomal analysis by fluorescence in situ hybridization (FISH) for predicting recurrence of urothelial carcinoma (UC) after transurethral resection. One hundred and thirty‐eight patients (median age 68.5 years) with a history of UC were eligible for this prospective study. FISH was applied to cytospin specimens prepared from bladder washings taken during a negative control cystoscopy. The multi‐target FISH test UroVysion® (Abbott/Vysis) containing probes to the centromeres of chromosomes 3, 7, 17 and the 9p21 locus was used. UC recurrence was defined as a positive biopsy during follow‐up. The median follow‐up time was 19.2 (4–52) months. FISH was positive in 50 (36%) patients and negative in 88 (64%) patients. A recurrence occurred in 39% of the patients with a positive FISH test and in 21% of patients with a negative FISH test. FISH positivity according to manufacturers criteria, at the time of a negative cystoscopy, was not significantly associated with the risk of recurrence (p = 0.12). However, the sensitivity of the FISH test to predict recurrence was significantly improved by considering specimens with rare (≤10) tetraploid cells as negative (p < 0.006). In addition, presence of 9p21 deletion was significantly associated with recurrence (p < 0.01). Notably, positive standard cytology was an independent factor for subsequent recurrence in this study (p < 0.001). Taken together, multi‐target FISH may help to stratify the risk of recurrence of UC at the time of a negative follow‐up cystoscopy. Defining the optimal threshold for FISH positivity requires consideration of tetraploid pattern and 9p21 deletion. Our results also emphasize the paramount importance of conventional cytology for UC surveillance.