Analysis of HER2 expression in primary urinary bladder carcinoma and corresponding metastases

OBJECTIVE: To evaluate the expression of HER2 receptors (previously reported to be over-expressed in malignant urothelium) in both primary tumours and metastases of transitional cell cancer, using two different staining methods and two different scoring techniques, considering the potential use of these receptors as targets for planned systemic anti-HER2 nuclide-based treatment. MATERIALS AND METHODS: HER2 expression was evaluated with two different immunohistochemical methods in 90 patients with primary urinary bladder cancer tumours and corresponding metastases. Sections were first stained with the commercially available breast cancer test kit (HercepTest, Dako, Glostrup, Denmark). Parallel sections were then stained with a modified HercepTest procedure. Two different evaluation criteria were compared; the HercepTest score that requires > or = 10% stained tumour cells (as for breast cancer) and a proposed 'Target score' that requires > 67% stained tumour cells. The latter score is assumed to be preferable for HER2-targeted radionuclide therapy. RESULTS: Using the HercepTest kit, the Target score gave lower fractions of positive primary tumours and metastases than the HercepTest score. The modified HercepTest staining procedure and Target score gave high HER2 values in 80% of primary tumours and 62% of metastases, which is considerably more than that obtained with the HercepTest staining and score. There was a significant decrease in HER2 positivity with increasing distance from the primary tumour. In nine sentinel-node metastases assessed, all but one were HER2-positive. Considering all regional metastases, 74% were positive, and of distant metastases, 47%; 72% of the patients with positive primary tumours also expressed HER2 in their metastases. CONCLUSIONS: When combining the modified HercepTest with customised evaluation criteria, more HER2-positive tumours were diagnosed. The degree of HER2 down-regulation was significantly higher in distant than in regional metastases. HER2-targeted therapy may be an alternative or complementary to other methods in the future treatment of metastatic urinary bladder carcinoma.     

p53 expression in patients with advanced urothelial cancer of the urinary bladder

Study Type – Prognosis (inception cohort)
Level of Evidence 1b

OBJECTIVE

To test whether assessing p53 expression could improve the ability to predict disease recurrence and disease‐specific survival in a multi‐institutional cohort of patients with advanced urothelial carcinoma of the urinary bladder (UCB).

PATIENTS AND METHODS

The study comprised 692 patients with pT3–4 N0 or pTany N+ UCB treated with radical cystectomy and lymphadenectomy. The predictive accuracy (PA) was quantified using the 200 bootstrap‐corrected concordance index. The base model comprised age, gender, stage, grade, lymphovascular invasion, number of lymph nodes removed, number of lymph nodes positive, concomitant carcinoma in situ, and adjuvant chemotherapy.

RESULTS

p53 expression was altered in 341 (49.3%) patients. In multivariable analyses, p53 expression was independently associated with disease recurrence (hazard ratio, 1.66; P < 0.001) and cancer‐specific mortality (hazard ratio 1.65, P < 0.001). Overall, adding p53 did not significantly improve the PA of the base model (recurrence +0.7%, P = 0.085, and cancer‐specific mortality +1.2%, P = 0.050). In the subgroups of pT3N0 (280) and pT4N0 (83) patients, p53 slightly improved the PA of the base model by a statistically significant degree (recurrence +1.7% and +3.6%, respectively; cancer‐specific mortality +1.9% and +3.5%, respectively; all P < 0.001). In 329 patients with pTany N+ disease p53 status did not improve the PA of the base model.

CONCLUSION

While assessing p53 expression has limited utility in patients with lymph node‐positive UCB, it marginally improves prognostication in patients with advanced non‐metastatic UCB. Integration of p53 into a panel of biomarkers might be necessary to capture a more accurate picture of the biological potential of advanced UCB.     

膀胱移行细胞癌表皮生长因子受体基因扩增及其表达

运用免疫组织化学技术和分子杂交技术研究了膀胱移行细胞癌表皮生长因子受体（ＥＧＦｒ）表达状况以及ＥＧＦｒ基因扩增和其ｍＲＮＡ与受体蛋白表达的相关性。结果表明：４例正常膀胱粘膜ＥＧＦｒ染色阴性；５６例膀胱肿瘤中，２９例呈不同程度的阳性染色，并与肿瘤的分期、分级有显著性差异；１３例膀胱肿瘤中，ＥＧＦｒ基因未见扩增，５例ｍＲＮＡ过表达，７例ＥＧＦｒ过表达，但二者表达的量并无相关性。提示ＥＧＦｒ表达可作为膀     

The prognostic significance of epidermal growth factor receptor expression in breast cancer

The association between epidermal growth factor receptor (EGFR) expression, clinicopathological variables, silver-stained nuclear organizer region (Ag-NOR) counts, and patient survival was determined in 93 patients with operable breast cancer. The EGFR expression was found to be significantly associated with the presence and number of axillary lymph node metastases (P = 0.0429), but not with age, menopausal status, tumor size, histologic type or grade, or Ag-NOR counts. In a univariate analysis, a significant difference was also observed in the survival of patients stratified by tumor size (P = 0.0091), histologic grade (P = 0.0352), axillarly lymph node metastases (P = 0.0001), and EGFR expression (P = 0.0263). However, a multivariate analysis revealed that axillarly lymph node metastases was the only strong independent predictor ol'survival (P < 0.0001). When axillary lymph node metastases were excluded from the Cox model, the EGFR expression tended to be an independent prognostic factor (P = 0.0558). The results of this study thus indicate that the prognostic value of EGFR expression is limited because the EGFR expression is significantly associated with axillary lymph node metastases.     

Human DNA topoisomerase-IIalpha expression as a prognostic factor for transitional cell carcinoma of the urinary bladder

OBJECTIVE: To investigate the immunohistochemical expression of topoisomerase II-alpha (TII-alpha, a nuclear enzyme, the expression of which increases rapidly at the end of the S to G2/M phase and declines when mitosis ends) in bladder urothelial neoplasms (transitional cell carcinoma), and its correlation with grade, stage and survival. PATIENTS AND METHODS: Histological sections from 57 urothelial neoplasms were stained immunohistochemically for TII-alpha expression; the percentage of positive cells in the area of greatest staining was recorded as the TII-alpha index. RESULTS: TII-alpha nuclear staining was positive in all the samples except one. The mean (sd) TII-alpha index was 10.7 (5.9) in urothelial neoplasms of low malignant potential (grade 1), 15.5 (5.0) in low-grade (grade 2) and 42.1 (13.4) in high-grade urothelial carcinoma (grade 3). The mean TII-alpha index was 10.7 (5.9), 26.3 (14.8) and 44 (19) in stages pTa, pT1 and pT2, respectively. The TII-alpha index was significant for predicting death from cancer, independently of the stage or grade of the disease (P = 0.019, hazard ratio 1.1). CONCLUSIONS: A higher TII-alpha index indicates a greater probability of recurrence of disease and lower overall survival. Therefore TII-alpha expression has prognostic value in bladder carcinoma.     

Human epidermal growth factor receptor type 2 protein expression in Chinese metastatic prostate cancer patients correlates with cancer specific survival and increases after exposure to hormonal therapy

Aim： To investigate human epidermal growth factor receptor type 2 （HER2） protein expression and gene amplification in Chinese metastatic prostate cancer patients and their potential value as prognostic factors. Methods： Immunohistochemistry （IHC） was performed to investigate HER2 protein expression in prostate biopsy specimens from 104 Chinese metastatic prostate cancer patients. After 3-11 months of hormonal therapy, 12 patients underwent transurethral resection of the prostate （TURP）. HER2 protein expression of TURP specimens was compared with that of the original biopsy specimens. Of these, 10 biopsy and 4 TURP specimens with HER2 IHC staining scores ≥ 2＋ were investigated for HER2 gene amplification status by fluorescent in situ hybridization （FISH）. Results： Of the 104 prostate biopsy specimens, HER2 protein expression was 0, 1＋, 2＋ and 3＋ in 49 （47.1%）, 45 （43.3%）, 8 （7.7%） and 2 （1.9%） cases, respectively. There was a significant association between HER2 expression and Gleason score （P = 0.026）. HER2 protein expression of prostate cancer tissues increased in 33.3% of patients after hormonal therapy. None of the 14 specimens with HER2 IHC scores 〉 2＋ showed HER2 gene amplification. Patients with HER2 scores 〉 2＋ had a significantly higher chance of dying from prostate cancer than those with HER2 scores of 0 （P = 0.004） and 1＋ （P = 0.034）. Multivariate Cox regression analysis showed that HER2 protein expression intensity was an independent predictor of cancer-related death （P = 0.039）. Conclusion： An HER2 IHC score 〉 2＋ should be defined as HER2 protein overexpression in prostate cancer. Overexpression of HER2 protein in cancer tissue might suggest an increased risk of dying from prostate cancer. HER2 protein expression increases in some individual patients after hormonal therapy.     

Urinary insulin‐like growth factor 2 identifies the presence of urothelial carcinoma of the bladder

OBJECTIVE

To examine urinary insulin‐like growth factor 2 (IGF‐2) levels in patient urine samples and determine the potential of IGF‐2 as a marker for the presence of urothelial carcinoma of the bladder (UCB).

PATIENTS AND METHODS

The current gold standard for diagnosis of UCB is cystoscopy and cytological analysis. The identification of an accurate urine marker for UCB with the potential to replace unnecessary cystoscopy would benefit patients with UCB and others investigated after detecting haematuria. In the present study, we analysed 65 urine samples, and optimized an enzyme‐linked immunosorbent assay‐based approach to measure urinary levels of IGF‐2.

RESULTS

Based on a threshold of 5.4 ng/mL, patients with UCB have significantly elevated levels of urinary IGF‐2 (P = 0.009) and this difference remained significant after adjustment for age and sex (P = 0.04). Sensitivity and specificity values of 80% and 52%, respectively, were determined for urinary IGF‐2 alone and when combined with nuclear matrix protein 22 (NMP22; an approved biomarker for detection of UCB). There was a positive correlation between urinary IGF‐2 levels and NMP22 levels in patient urine samples and the combined assay improved the detection of UCB (sensitivity 85% and specificity 52%).

CONCLUSION

Substantiated evidence has identified IGF‐2 as a valuable marker for UCB. In addition, the novel observations of the present study have shown that aberrant levels of IGF‐2 occurring in the presence of UCB, can now be determined through a simple and inexpensive urine assay.     

Chemopreventive effects of cyclooxygenase-2 inhibitor and epidermal growth factor-receptor kinase inhibitor on rat urinary bladder carcinogenesis

OBJECTIVE: To examine the chemopreventive effects of a selective cyclooxygenase (COX)-2 inhibitor, meloxicam, and a selective epidermal growth factor (EGF)-receptor tyrosine kinase inhibitor, gefitinib (as a single agent) on a carcinogen-induced rodent bladder carcinogenesis model. MATERIALS AND METHODS: The study comprised 103 male Fisher-344 rats (8 weeks old); after initial carcinogen treatment for 8 weeks with 0.05%N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water, the rats were divided into five groups, i.e. group 1, control (vehicle only); group 2, gefitinib high-dose (15 mg/kg by gavage once daily); group 3, gefitinib low-dose (5 mg/kg); group 4, meloxicam high-dose (1.8 mg/kg by gavage once daily); and group 5, meloxicam low-dose (0.6 mg/kg). Twelve weeks later the rats were killed; after fixing the bladder in 10% formalin, the number and size of hyperplasia and carcinoma foci were recorded microscopically in sections stained with haematoxylin and eosin, submitted entirely as multiple strips. RESULTS: The incidence of carcinoma, confirmed microscopically, was: control 14/20 (70%); high-dose gefitinib, 7/20 (35%); low-dose gefitinib, 7/20 (35%); high-dose meloxicam 7/21 (33%); and low-dose meloxicam, 12/20 (60%). The mean numbers of carcinomas per bladder in groups 1-5 were 1.2, 0.5, 0.4, 0.5 and 1.1, respectively. The incidence and the mean number of carcinomas per bladder were significantly lower in the treatment groups (P < 0.05) than in the control group, except in the low-dose meloxicam group. There were no significant adverse effects. CONCLUSION: Both meloxicam and gefitinib have inhibitory effects on rat bladder carcinogenesis with no significant adverse effects. A combination of these drugs would be worth studying for their synergistic effects.     

Uridine 5'diphospho‐glucuronosyltransferase 1A expression as an independent prognosticator in urothelial carcinoma of the upper urinary tract

Objective

To determine the expression status of uridine 5'diphospho‐glucuronosyltransferase 1A, a major phase II drug metabolism enzyme, in upper urinary tract urothelial carcinoma, as well as to assess its prognostic significance.

Methods

We immunohistochemically stained for uridine 5'diphospho‐glucuronosyltransferase 1A in tissue microarray consisting of 99 upper urinary tract urothelial carcinoma samples and paired non‐neoplastic urothelial tissues. We also assessed the effect of uridine 5'diphospho‐glucuronosyltransferase 1A knockdown on urothelial cancer cell growth.

Results

Uridine 5'diphospho‐glucuronosyltransferase 1A was positive in 92.9% (27.3% weak [1+], 37.4% moderate [2+], 28.3% strong [3+]) of tumors, which was significantly (P < 0.001) lower than in benign urothelial tissues (98.8%; 3.5% 1+, 18.8% 2+, 76.4% 3+). All 37 (100%) non‐muscle‐invasive versus 55 (88.7%) of 62 muscle‐invasive tumors (P = 0.043) were immunoreactive for uridine 5'diphospho‐glucuronosyltransferase 1A. The rates of moderate‐to‐strong uridine 5'diphospho‐glucuronosyltransferase 1A expression and its positivity were also strongly associated with the absence of concomitant carcinoma in situ (P = 0.034) and lymphovascular invasion (P = 0.016), respectively. However, there were no statistically significant associations between uridine 5'diphospho‐glucuronosyltransferase 1A expression and tumor grade or pN/M status. Uridine 5'diphospho‐glucuronosyltransferase 1A loss in M0 tumors was strongly associated with lower progression‐free survival (P < 0.001) and cancer‐specific survival (P < 0.001) rates. Multivariate analysis further identified a strong correlation of uridine 5'diphospho‐glucuronosyltransferase 1A positivity with reduced cancer‐specific mortality (hazard ratio 0.28, P = 0.018). Meanwhile, uridine 5'diphospho‐glucuronosyltransferase 1A knockdown in urothelial cancer cells resulted in significant increases in their viability and migration.

Conclusions

These results suggest a preventive role of uridine 5'diphospho‐glucuronosyltransferase 1A signals in the development and progression of upper urinary tract urothelial carcinoma. Loss of uridine 5'diphospho‐glucuronosyltransferase 1A expression might serve as an independent predictor of poor prognosis in patients with upper urinary tract urothelial carcinoma.     

Improved cancer specific-survival in patients with carcinoma invading bladder muscle expressing cyclo-oxygenase-2

Study Type – Prognosis (case series) Level of Evidence 4

OBJECTIVE

To determine whether the expression of cyclo‐oxygenase (COX)‐2 has an influence on survival and on the response to chemotherapy in invasive bladder cancer.

PATIENTS AND METHODS

A population of 266 patients from a tertiary university centre with carcinoma invading bladder muscle without evidence of metastasis at time of cystectomy was analyzed retrospectively. COX‐2 expression was evaluated immunohistochemically with a monoclonal anti‐COX‐2 antibody. All pertinent clinical and pathological parameters were reviewed and correlated with risk factors influencing outcome, including disease‐specific and overall survival, as well as COX‐2 expression. Immunoreactivity was categorized as positive if COX‐2 staining was present in >5% tumour cells.

RESULTS

The expression of COX‐2 was not influenced by tumour stage, grade or nodal status, nor any other parameters. The risk factors that influenced disease‐specific survival in carcinoma invading bladder muscle on multivariate analysis were lymph node status (hazards ratio, HR = 2.46 for N1, P= 0.001, HR = 2.90 for N2, P < 0.001, HR = 5.19 for N3, P= 0.012), use of neoadjuvant chemotherapy (HR = 3.54; P= 0.004) or adjuvant chemotherapy (HR = 0.57, P= 0.014) and COX‐2 expression (HR = 0.64 if >5% cells had positive expression; P= 0.025). Kaplan–Meier analysis showed an increased disease‐specific survival (P= 0.0063), as well as longer recurrence‐free survival (P= 0.003), in patients with muscle‐invasive bladder tumours expressing COX‐2 in >5% of the cells. A tendency was also observed in a subgroup with positive nodes treated with adjuvant chemotherapy (P= 0.093).

CONCLUSIONS

The overexpression of COX‐2 is associated with a better recurrence‐free and disease‐specific survival in a large cohort of 266 patients with carcinoma invading bladder muscle treated by cystectomy. A trend for increased disease‐specific survival was also observed for patients with COX‐2 overexpression and positive nodes who received adjuvant chemotherapy. Potential of COX‐2 as a prognostic marker in bladder cancer should be considered.     

Prognostic role of ECOG performance status in patients with urothelial carcinoma of the upper urinary tract: an international study

Study Type – Prognosis (inception cohort series) Level of Evidence 2a What's known on the subject? and What does the study add? ECOG Performance Status has gained wide popularity as an integral part of the assessment of patients with upper urinary tract carcinoma. Our findings indicate that ECOG‐PS is strongly associated with perioperative and overall survival and should be considered carefully in our decision‐making process.

OBJECTIVE

• ? To evaluate the prognostic role of ECOG Performance status (ECOG‐PS) in a large multi‐institutional international cohort of patients treated with radical nephroureterectomy for upper tract urothelial carcinoma.

MATERIALS AND METHODS

• ? Data of 427 patients treated with radical nephroureterectomy at five international institutions in Asia, Europe and Northern America were collected retrospectively from 1987 to 2008.
• ? Logistic and Cox regression models were used for univariable and multivariable analyses.

RESULTS

• ? ECOG‐PS was 0 in 272 of 427 (64%) patients. The median follow‐up of the whole cohort was 32 months.
• ? The five‐year recurrence‐free (RFS), cancer‐specific (CSS) and overall (OS) survival estimates were 71.7%, 74.9% and 68.5%, respectively, in patients with ECOG‐PS 0 compared with 60.1%, 67.8%, and 51.4% respectively, in patients with ECOG‐PS ≥1 (P value 0.08 for RFS, 0.43 for CSS, and <0.001 for OS, respectively).
• ? On multivariable Cox regression analyses, ECOG‐PS was not an independent predictor of either RFS (hazard ratio 1.4; P = 0.107) or CSS (hazard ratio 1.2; P = 0.426) but was an independent predictor of OS (hazard ratio 1.5; P = 0.03).

CONCLUSIONS

• ? In this large multicentre international study, ECOG‐PS was not significantly associated with RFS and CSS.
• ? Conversely we find a strong association with survival 1‐month after surgery and OS. Further research is needed to ascertain the additive prognostic role of ECOG‐PS in well‐designed prospective multicentre studies.

     

Expression of angiopoietin-1 and -2, and its clinical significance in human bladder cancer

OBJECTIVE: To investigate the relationship between angiopoietin-1 and -2 expression and the clinicopathological variables and clinical outcome in patients with bladder cancer treated by surgical resection, as both have been recently identified as antagonistic angiogenic factors which regulate tumour growth. MATERIALS AND METHODS: The expression of angiopoietin-1 and -2 were assessed by immunohistochemistry in tissue sections from 52 transitional cell carcinomas of the bladder (33 grade 1, 15 grade 2, four grade 3, including two associated with carcinoma in situ; 22 were stage Ta, 19 T1 and 11 T2 tumours). Normal bladder specimens were also resected during each operation as controls. The expression angiopoietins were related to the clinicopathological variables of the tumours. RESULTS: Positive immunostaining was detected in 18 samples (35%) for angiopoietin-1 and in 23 (44) for angiopoietin-2. There was no significant difference in survival according to tumour angiopoietin-1 status in the patients, but in contrast the overall survival of patients with angiopoietin-2-positive tumours was significantly lower than for those with angiopoietin-2-negative tumours (P < 0.05). Positive angiopoietin-2 expression was significantly correlated with histological grade (P = 0.026), histological stage (P = 0.009) and poor prognosis (P < 0.05). On multivariate analysis, positive angiopoietin-2 expression was an independent negative predictor for survival (P = 0.042). CONCLUSIONS: These results suggest that angiopoietin-2 overexpression is associated with tumour progression, thereby indicating a poor prognosis for some patients treated by surgical resection for bladder carcinoma.     

Cyclooxygenase-2 promotes angiogenesis in pTa/T1 urothelial bladder carcinoma but does not predict recurrence

OBJECTIVE: To investigate the effect of cyclooxygenase-2 (COX-2) on microvessel density (MVD) and on the clinical prognosis in patients with non-muscle invasive urothelial carcinoma of the bladder, as COX-2 expression is significantly greater in epithelial tumours and there is increasing evidence that COX-2 might contribute to tumour neovascularization. PATIENTS AND METHODS: We assessed tumour samples from 110 patients undergoing transurethral resection for primary pTa/pT1 bladder carcinoma (pTa, 84; pT1, 26; grade 1, 22; grade 2, 81; grade 3, seven). Paraffin sections were assessed immunohistochemically using antibodies against COX-2, CD34 (endothelial cells) and CD105 (proliferating vessels). COX-2 expression was quantified by the number of stained cells (negative, +, ++) and the MVD calculated as vessels per field. RESULTS: Of the 110 tumours, 45 (41%) had no immunostaining for COX-2, 40 had faint staining with at least isolated positive cells (+) and 25 stained ++. COX-2 positive tumours had significantly greater vascularization for proliferating vessels. In COX-2 negative tumours the MVD was 22.1, identified by CD34 immunostaining, and 3.4 for proliferating vessels (CD105), whereas COX-2 positive tumours had a MVD of 18.3 (CD34), and of 5.8, respectively (CD105). Complete follow-up data were available in 91 patients; after a mean follow-up of 25 months, 18 (20%) had tumour recurrences. There was no significant difference in the recurrence rates or disease-free survival between COX-2-positive (19%, 25.6 months) or -negative patients (21%, 25.2 months). CONCLUSION: These results confirm the involvement of COX-2 in angiogenesis in bladder cancer, as COX-2 promoted blood vessel proliferation in the tumour zone, and indicate the usefulness of COX-2-inhibiting drugs in preventing and treating superficial bladder cancer.     

Expression of the epidermal growth factor receptor family in normal and malignant urothelium

OBJECTIVE: To compare the immunohistochemically assessed expression of the epidermal growth factor receptor (EGFR) family in normal and malignant bladder urothelium, and suggest new hypotheses about their function in the development and progression of transitional cell carcinoma (TCC). PATIENTS AND METHODS: EGFR, ERBB2, ERBB3 and ERBB4 were evaluated immunohistochemically in normal urothelium (NU, 15), primary non-metastasized invasive TCC (NMC, 19) and in primary invasive TCCs with corresponding metastases (MC, 51, both specimens). RESULTS: All NU samples expressed ERBB4, none expressed ERBB2 and two expressed EGFR; all staining was uniform throughout all cell layers. ERBB2 expression increased and ERBB4 decreased from normal samples to carcinomas. There was no difference between NMCs and MCs in ERBB2, ERBB3 and ERBB4, but the NMCs expressed more EGFR than both NU and MC samples. There were no associations with T category, grade or survival. All combinations of expression levels for the four receptors were detected, with no dominant profile. CONCLUSION: We hypothesise that: (i) ERBB4 is important for differentiation in NU; (ii) ERBB2 is up-regulated with carcinogenesis in the urinary bladder but does not discriminate between bladder cancer with or without metastases; (iii) EGFR may be a marker of indolent disease. A current hypothesis, that superficial layers of NU do not express EGFR and thus protect the basal cells from the mitogenic effect of urinary EGF, is challenged.