Daniel Ackermann

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.

Enlargement of Regional Lymph Nodes in Renal Cell Carcinoma is Often not Due to Metastases

Preoperative axial computerized tomography scans in 163 patients with renal cell carcinoma were reviewed to assess the predictive value for the diagnosis of regional lymph node metastases. Computerized tomography was falsely negative in 5 patients: 2 had metastatic lymph nodes in the renal hilus adjacent to the primary tumor measuring 2 and 2.5 cm., and 3 had micrometastases in nodes of less than 1 cm. In 43 patients enlarged lymph nodes with a diameter of 1 to 2.2 cm. (median 1.4 cm.) were diagnosed on the preoperative scan and this was confirmed at nephrectomy and pathologically. In 18 of these 43 patients (42%) histological study showed metastases of the renal cell carcinoma in the enlarged lymph nodes. In the other 25 patients (58%) the enlarged nodes showed only inflammatory changes and/or follicular hyperplasia. This finding was significantly more frequent in patients with tumor involvement of the renal vein and tumor necrosis (p = 0.0044). We conclude that the sensitivity of preoperative computerized tomography is good for the detection of enlarged lymph nodes in patients with renal cell cancer (95%). However, significant lymph node enlargement frequently may be caused by inflammatory changes, especially in the presence of tumor necrosis. This radiological finding should not be misinterpreted as metastatic disease, unless it has been proved cytologically by fine needle aspiration.

ENDOPYELOTOMY FOR PRIMARY URETEROPELVIC JUNCTION OBSTRUCTION: RISK FACTORS DETERMINE THE SUCCESS RATE

PURPOSE We prospectively assessed the feasibility, complications, and short-term and long-term results of endopyelotomy for primary ureteropelvic junction obstruction. MATERIALS AND METHODS In 80 consecutive patients primary ureteropelvic junction obstruction was diagnosed by excretory urogram or nephrostomogram, retrograde pyelography, diuresis renography and the Whitaker test in ambiguous cases. In all patients antegrade endopyelotomy was performed with a cold knife and an indwelling stent was left for 6 weeks. At 6 and 24 months postoperatively results were assessed clinically by an excretory urogram and/or diuretic renography and later by questionnaire and ultrasound. RESULTS The primary success rate was 89% (71 of 80 patients) after the first endopyelotomy and increased to 91% (73 of 80 patients) after 2 patients had a second endopyelotomy. After median followup of 26 months (range 1.5 to 72) 6 of the 73 initially successfully treated patients had relapse. Two were successfully re-treated by a second endopyelotomy, resulting in an overall success rate of 81% (65 of 80 patients) after 1 procedure and 86% (69 of 80 patients) after a second endopyelotomy in 4 patients. Mean preoperative pyelocaliceal volume decreased from 64 +/- 33 to 41 +/- 20 ml. (p = 0.0003) 6 months after endopyelotomy and did not change during the following 18 months. The probability of successful endopyelotomy was better in patients with a preoperative pyelocaliceal volume less than 50 ml. (87%) and worse in patients with a volume greater than 50 ml. (76%). A crossing vessel to the lower pole of the kidney causing persistent functional obstruction of the ureteropelvic junction was found in 6 of the 10 patients re-treated by open pyeloplasty (9) or nephrectomy (1). Preoperative mean renal function as determined by diuretic renography was significantly lower in patients with failed endopyelotomy than in successfully treated patients. Successfully treated patients showed no change in renal function 6 and 24 months postoperatively. CONCLUSIONS Endopyelotomy in primary ureteropelvic junction obstruction is a safe, minimally invasive procedure with a high primary success rate and a low relapse rate. Open pyeloplasty could be avoided in 86% of our patients. Endopyelotomy is less invasive, has less functional and esthetic sequelae than open pyeloplasty and does not compromise open surgery if that becomes necessary. We recommend endopyelotomy as first line treatment for patients with primary ureteropelvic junction obstruction.

High-throughput tissue microarray analysis of 11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinary bladder cancer

Gene amplification is a common mechanism for oncogene overexpression. High‐level amplifications at 11q13 have been repeatedly found in bladder cancer by comparative genomic hybridization (CGH) and other techniques. Putitative candidate oncogenes located in this region are CCND1 (PRAD1, bcl‐1), EMS1, FGF3 (Int‐2), and FGF4 (hst1, hstf1). To evaluate the involvement of these genes in bladder cancer, a tissue microarray (TMA) containing 2317 samples was screened by fluorescence in situ hybridization (FISH). The frequency of gains and amplifications of all genes increased significantly from stage pTa to pT1–4 and from low to high grade. In addition, amplification was associated with patient survival and progression of pT1 tumours. Among 123 tumours with amplifications, 68.3% showed amplification of all four genes; 19.5% amplification of CCND1, FGF4, and FGF3; and 0.8% co‐amplification of FGF4, FGF3, and EMS1. Amplification of CCND1 alone was found in 9% of the tumours, while EMS1 alone was amplified in 1.6% and FGF4 in 0.8%. Overall, the amplification frequency decreased with increasing genomic distance from CCND1, suggesting that, among the genes examined, CCND1 is the major target gene in the 11q13 amplicon in bladder cancer. Copyright

Intravesical Versus Intravesical Plus Intradermal Bacillus Calmette-Guerin: A Prospective Randomized Study In Patient With Recurrent Superficial Bladder Tumors

PURPOSE Intravesical instillation of bacillus Calmette-Guerin (BCG) induces various immunological reactions and decreases the recurrence rate of superficial bladder tumors. To determine whether additional immune stimulation with concomitant intradermal BCG applications could further lower the recurrence rate, 154 patients with superficial bladder tumors at high risk for recurrence were randomized to receive either 6 intravesical instillations of 120 mg. Pasteur strain BCG alone or combined with intradermal application. MATERIALS AND METHODS A total of 76 patients received intravesical and intradermal BCG, while 78 received intravesical BCG only. Median followup was 41 months (range 2 to 89) and 36 months (range 2 to 86), respectively. Both treatment groups were comparable regarding patient age and number of previous transurethral bladder tumor resections, as well as tumor recurrence rate, stage and grade before BCG therapy. RESULTS A highly significant decrease in the monthly tumor recurrence rate was observed in both arms after BCG compared to the pretreatment recurrence rates (p < 0.0001). Recurrence rate decreased from 0.73 +/- 1.07 (standard deviation) to 0.06 +/- 0.13 in the combined treatment group and from 0.71 +/- 0.90 to 0.074 +/- 0.17 in the intravesical treatment only group. However, we were unable to find any difference between the 2 groups regarding interval to initial recurrence or recurrence rates after BCG treatment. Changes in the purified protein derivative skin test performed before and after BCG therapy were not useful to predict response to treatment because 44% of our patients already had a positive test before treatment. Also, interpretation of the skin test was difficult and not always reliable. In the multivariate analysis, however, fever was an important prognostic factor. Patients with increased body temperature greater than 37.5C had a significantly lower recurrence rate than those without fever (37.5C or less) after BCG instillation (p = 0.009). Moreover, fever after BCG instillation was observed significantly more frequently in patients with a positive purified protein derivative skin test before treatment (p = 0.021). CONCLUSIONS The therapeutic benefit from intravesical BCG apparently was not substantially improved by simultaneous intradermal BCG vaccination. Fever following intravesical BCG instillation is an important prognostic factor regarding superficial bladder tumor recurrence. Fever occurs predominantly in patients who were previously sensitized to mycobacteria (by BCG vaccination or infection) as shown by a positive pretreatment purified protein derivative skin test. This finding suggests that previously sensitized patients respond significantly better to a single course of intravesical BCG.

Ileal bladder substitute: antireflux nipple or afferent tubular segment?

Spheroidal bladder substitutes made from double-folded ileal segments, similar to Goodwins cup-patch technique, are devoid of major coordinated wall contractions. This, together with the reservoirs direct anastomosis to the membranous urethra, prevents major intraluminal pressure peaks and assures a residue-free voiding of sterile urine. In order to determine whether, under these conditions, an afferent tubular isoperistaltic ileal segment of 20-cm length protects the upper urinary tract as efficiently as an antireflux nipple, 60 male patients who were subjected to radical cystectomy were prospectively randomised to groups in which a bladder substitute was formed together with either of these 2 antireflux devices. An analysis of the results obtained in 20 patients from each group who could be followed for more than 1 year (median observation time 30 and 36 months) showed no differences between the groups in metabolic disturbances, kidney size, reservoir capacity, diurnal and nocturnal urinary continence, the incidence of urinary tract infection or episodes of acute pyelonephritis. Later than 1 year postoperatively, intravenous urograms of the renoureteral units of 25% of the patients with antireflux nipples showed persistent but generally slight dilatation of the upper urinary tracts. This observation was significantly more frequent than it was in patients with afferent tubular segments. Urodynamic and radiographic studies showed that the competence of the antireflux nipples was secured by the raised surrounding intravesical pressure. This, however, also resulted in a transient functional obstruction, and a gradual rise of the basal pressure in the upper urinary tracts was recorded. In patients with afferent ileal tubular segments, contrast medium could be forced upwards into the renal pelvis when the bladder substitutes were overfilled. However, despite raised intravesical pressures, peristalsis in the isoperistaltic afferent tubular segment gradually returned contrast medium back to the reservoir. Our results suggest that the combination of an ileal low-pressure reservoir together with an afferent tubular isoperistaltic limb is at least as good as an antireflux nipple valve. Moreover, the use of the afferent ileal limb makes it possible to resect the distal and often diseased ureters together with the paraureteric lymphatics at a safe distance from the bladder tumor. This avoids also distal ischemic ureteric stenosis and makes possible a simple end-to-side ureterointestinal anastomosis with a small complication rate.

Prognosis after extracorporeal shock wave lithotripsy of radiopaque renal calculi: a multivariate analysis.

To obtain a better understanding of the prognostic factors influencing treatment outcome after extracorporeal shock wave lithotripsy (ESWL), a multivariate logistic analysis of the data from 246 patients has been undertaken. All of the patients were treated with the Dornier lithotriptor HM-3 for radiopaque renal calculi. Treatment success was defined as stone-free within 3 months of one ESWL session and without adjuvant measures after ESWL. In a first analysis, 210 patients with solitary and multiple calculi without adjuvant measures before ESWL were studied. Of 210 patients, 141 (67%) were free from stones after 3 months). Significant influences on the success rate were body mass index and stone number. In a second analysis only those 160 patients with solitary calculi were considered. In this group, age, body mass index, stone location, stone burden and serum calcium significantly influenced the prognosis. When patients with adjuvant measures were added to the analysis an increasing prognostic importance of the stone burden was seen. In patients with a small to medium stone burden (< 4.0 cm3), the number of stones seemed to be more important than the stone burden. Patients appear to have the best chance for successful ESWL when their body mass index is between 20 and 28, their age is between 40 and 60 years, their stones are in the renal pelvis and solitary, the stone burden is < 1.0 cm3, and when their serum calcium is normal.

Extracorporeal shock wave lithotripsy in situ or after push-up for upper ureteral calculi : a prospective randomized trial

A total of 110 patients with upper ureteral calculi was admitted to a prospective trial and randomly allocated to 2 groups: 1 group treated with in situ extracorporeal shock wave lithotripsy (ESWL) and 1 group treated with ureteral manipulation before ESWL. All patients had solitary upper ureteral calculi without urinary infection. The stones had to be smaller than 1 cm. and located more than 2 cm. lateral to the spine. ESWL was performed with the Dornier HM3 lithotriptor. One patient in the in situ ESWL group had to be treated twice because disintegration of the stone was insufficient after the initial treatment session. All other patients underwent only 1 treatment session. Because 16 patients were lost to followup, 94 were evaluable for the analysis of immediate and long-term results. For disintegration of the stones in situ ESWL needed significantly more shock waves (1,844 +/- 639 versus 1,297 +/- 473, p < 0.001) and a higher voltage (19.5 +/- 1.4 versus 18.7 +/- 0.9 kv., p < 0.001). There were no severe complications in either treatment group. At 3 months 44 of 46 patients (96%) after in situ ESWL and 45 of 48 (94%) after ureteral manipulation before ESWL were free of stones. In view of these results it is suggested that uncomplicated upper ureteral calculi (as defined previously) should be treated first with in situ ESWL, thus, avoiding an invasive procedure.

Cyclin D1 overexpression lacks prognostic significance in superficial urinary bladder cancer

The biological behaviour of urinary bladder neoplasms cannot be adequately predicted by histological criteria alone. Cyclin D1 is a cell‐cycle regulating protein known to be overexpressed in a proportion of bladder carcinomas. To evaluate the prognostic significance of cyclin D1 expression and its relationship with tumour phenotype, 392 bladder carcinomas were analysed by immunohistochemistry. Clinical follow‐up information was available in 337 patients with superficial bladder tumours (stages pTa/pT1). Cyclin D1 positivity was seen in 176 of 392 carcinomas. Cyclin D1 overexpression was strongly linked to papillary tumour growth, low stage, and low histological grade (p<0·005 each). Multivariate analysis showed that papillary tumour growth was the only parameter which was independently linked to cyclin D1 positivity. There was no significant difference in proliferative activity (Ki67 labelling index) between cyclin D1‐negative and ‐positive tumours. Cyclin D1 positivity was not linked to the risk of recurrence or tumour progression, either in pTa or in pT1 carcinomas. It is concluded that cyclin D1 positivity distinguishes a large subgroup of papillary bladder tumours, but there is no evidence of prognostic significance for increased cyclin D1 expression. Copyright