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Objectives: The primary endpoint in trials of perioperative systemic therapy for urothelial carcinoma is 5-year overall survival (OS). A shorter-term endpoint could significantly speed the translation of advances into practice. We hypothesized that disease-free survival (DFS) could be a surrogate endpoint for OS in upper tract urothelial carcinoma (UTUC) patients treated with radical nephroureterectomy (RNU)., Patients and Methods: The study included 2,492 patients treated with RNU with curative intent for UTUC., Results: 2/3-year DFS estimates were 78/73 %, and the 5-year OS estimate was 64 %. The overall agreements between 2- and 3-year DFS with 5-year OS were 85 and 87 %, respectively. Agreements were similar when analyzed in subgroups stratified by pathological stages, lymph node status, and adjuvant chemotherapy. The kappa statistic was 0.59 (95 % CI 0.55-0.63) for 2-year DFS/5-year OS and 0.64 (95 % CI 0.61-0.68) for 3-year DFS/5-year OS, indicating moderate reliability. The hazard ratio for DFS as a time-dependent variable for predicting OS was 11.5 (95 % CI 9.1-14.4), indicating a strong relationship between DFS and OS., Conclusions: In patients treated with RNU for UTUC, DFS and OS are highly correlated, regardless of tumor stage and adjuvant chemotherapy. While significant differences in DFS, assessed at 2 and 3 years, are highly likely to persist in OS at 5 years, marginal DFS advantages may not translate into OS benefit. External validation is necessary before accepting DFS as an appropriate surrogate endpoint for clinical trials investigating advanced UTUC patients.

Purpose: Tumor necrosis is associated with a poor oncological outcome in patients with upper tract urothelial carcinoma and other malignancies. We validated the association of tumor necrosis with pathological features and clinical outcomes in a large international cohort of patients with upper tract urothelial carcinoma treated with radical nephroureterectomy., Material and Methods: This retrospective study included 754 patients treated with radical nephroureterectomy at a total of 9 centers. Tumor necrosis was scored as greater than 10% of tumor area based on microscopic evaluation., Results: Tumor necrosis was present in 165 specimens (21.9%). The prevalence of tumor necrosis increased with advancing pathological stage, including 7%, 10.6% and 50% for T1, T2 and T3-4, respectively (p <0.001). Tumor necrosis was associated with features of aggressive upper tract urothelial carcinoma, such as high grade, lymph node metastasis, lymphovascular invasion, sessile tumor architecture and concomitant carcinoma in situ (p <0.002). Median followup in censored patients was 40 months (IQR 18 to 75). On univariate Cox regression analysis tumor necrosis was significantly associated with disease recurrence and cancer specific mortality (HR 2.4 and 2.7, p <0.001). However, on multivariate Cox regression analysis including patient age, stage, grade, lymph node status, lymphovascular invasion and adjuvant chemotherapy tumor necrosis was not associated with disease recurrence (HR 1.1, p = 0.49) or cancer specific mortality (HR 1.1, p = 0.51). Excluding 63 patients who received adjuvant chemotherapy and/or 49 with positive lymph nodes did not substantially change these results., Conclusions: In this large, multicenter international study tumor necrosis was associated with pathological features of biologically aggressive upper tract urothelial carcinoma. However, tumor necrosis was not an independent predictor of clinical outcomes., (Copyright © 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objectives: This paper studies the phenotypical characteristics of the lymphocytes present in mononuclear cell (MNC) preparations from peripheral blood (PBMNC), lymph nodes (LNMNC), tumor bladder (TBMNC), and tumor-free bladder (TFBMNC) from patients with infiltrated transitional cell carcinoma (TCC) of the bladder and PBMNC of healthy controls., Methods: Eight patients diagnosed with TCC of the bladder, according to UICC criteria, and 10 healthy controls were studied. For immunofluorescence staining, T lymphocytes were incubated with combinations of fluorescein (FITC, green)- and phycoerytrin (PE, red)-labelled monoclonal antibodies., Results: The percentage of NK cells in the LNMNC and TFBMNC was significantly decreased in comparison to that found in the PBMNC from these patients (p < 0.05). A significant enhancement of the expression of class II molecules of the major histocompatibility complex (MHC) by CD3+ T lymphocytes from TBMNC and TFBMNC specimens from bladder walls was found with respect to those quantified in CD3+ T lymphocytes from PBMNC (p < 0.05). In addition, the percentage of CD3+CD25+ T lymphocytes was significantly higher in PBMNC in TCC patients than in healthy controls (p < 0.05)., Conclusions: Our data clearly demonstrate that the presence of infiltrative TCC of the bladder is associated to an infiltration of in vivo activated T lymphocytes of the urinary bladder wall. This in vivo T lymphocyte activation has been considered an expression of the immune response against the tumor cells.

The immunomodulatory effect of prophylactic intravesical instillations of interferon alpha 2b (IFN-alpha-2b) on interferon gamma (IFN-gamma) and interleukin 4 (IL-4) production by peripheral blood mononuclear cells (PBMCs) from patients with superficial transitional cell carcinoma (STCC) of the bladder has been analysed. There were no significant differences in the production of IFN-gamma and IL-4 by PBMCs from untreated patients and healthy control subjects after 24 h of phytohaemagglutinin (PHA) stimulation. However, between 3 and 6 months after finishing the prophylactic intracavitary treatment with IFN-alpha-2b, PHA-stimulated PBMCs from patients with STCC of the bladder showed a significantly enhanced production of IFN-gamma and a significantly decreased production of IL-4. Both IFN-gamma and IL-4 returned to pretreatment levels 1 year after ending the treatment. In conclusion, prophylactic intravesical instillations of IFN-alpha-2b in patients with STCC of the bladder have an immunoregulatory effect on the production of IFN-gamma and IL-4 by PBMCs.

Background: The evolution of superficial transitional cell carcinoma (TCC) of the bladder poses a clinical management problem due to its tendency to recur after initial transurethral resection (TUR). Natural Killer (NK) cells are a distinct subset of lymphocytes that possess the ability to lyse tumor cells without prior sensitization in a nonmajor histocompatibility-restricted fashion. These cytotoxic cells constitute a relevant barrier against the local growth and systemic dissemination of neoplastic diseases., Methods: The immunomodulatory effect, after TUR of the tumor, of the prophylactic treatment with intravesical instillations of interferon-alpha-2b (IFN-alpha-2b) upon the NK activity of peripheral blood mononuclear cells (PBMNC) in 17 patients with superficial TCC of the bladder was analyzed using a 4-hour 51sodium chromate (51Cr)-release cytotoxicity assay against both NK-sensitive, or K562, and NK-resistant, or JY, tumor target cells., Results: There were no significant modifications of the NK activity of PBMNC during the 3 months of IFN-alpha-2b intracavitary treatment (P > 0.05). However, with respect to their levels before treatment, NK activity in PBMNC at 3 months posttreatment was enhanced significantly in the 14 patients who showed no sign of tumor recurrence in the 12 months of follow-up (P < 0.01). Six months after finishing the intracavitary IFN-alpha-2b instillations, the PBMNC NK activity in these patients had returned to the levels found before initiating the therapy (P > 0.05). This temporary enhancement of the PBMNC NK activity was not found in three patients with evidence of tumor recurrence at some time during the 12 months after treatment. In similar experimental conditions, significant enhancement of the NK activity in PBMNC was not observed in nine patients with superficial bladder TCC who were treated with intracavitary instillations of mitomycin C (MMC) after the TUR of the tumor and who were free of recurrence of the disease at 1 year of follow up., Conclusion: The prophylactic intracavitary treatment of superficial bladder TCC with IFN-alpha-2b may induce an immunomodulatory effect in the NK activity of PBMNC that appears to be associated with the clinical evolution of the disease.

Intravesical instillation of keyhole limpet hemocyanin (KLH) is a possible treatment for decreasing tumor recurrence after transurethral resection (TUR) in patients with superficial transitional cell carcinoma of the bladder (stages pTa-pT1, grades 1-3). Our study confirms the theory that instillation of KLH stimulates production of cytokines, resulting in their secretion in urine. Interleukin-1 (IL-1) stimulates the immune cascade through a domino effect and is produced mainly by activated macrophages. The instillation program was started 5-7 days after TUR of primary superficial cell carcinoma. 20 mg KLH in 20 ml of 0.9% NaCl was instilled into the bladder each week for 6 consecutive weeks and then monthly for 1 year. When KLH is instilled into the bladder, IL-1 alpha is secreted in the urine. A specific enzyme-linked immunosorbent assay (ELISA) was used for analysis. The ELISA for IL-1 alpha was established in our laboratory and showed a detection limit of 5 pg/ml. This IL-1 alpha ELISA deviation amounts to 3-7% within a series of measurements, and 5-15% from series to series. In the therapy group the IL-1 alpha secretion ranged from 0 to 30,905 pg/24 h and in the control group from 0 (collection period) to 2,472 pg/4 h. IL-1 alpha production increased significantly after KLH instillation in bladder cancer patients; however, the level varied considerably from patient to patient. Maximum production was achieved within a period of 4-8 h, decreasing within 24 h. There was a striking difference between the amount of IL-1 alpha produced over the 24-hour period in the control group and that of the KLH group. 8 of 14 patients (57%) who responded to KLH therapy had higher urine IL-1 alpha levels after 6 weeks of KLH treatment than those who failed to respond within 12 months, but the levels were not of statistical significance. The secretion of IL-1 alpha in urine is the biological response of the bladder to the antigen stimulus of KLH. No IL-2 was detected in the urine samples. It remains to be determined whether no IL-2 cytokine was present, or whether the amount was smaller than the minimal detection limit required for the ELISA.

The activation and proliferation of peripheral blood mononuclear cells (PBMNCs) are complex processes involving several surface molecules, cell secretion and response to cytokines. This paper investigates the immunomodulatory effect of prophylactic treatment with interferon alpha 2b (IFN-alpha 2b) upon the blastogenic response of PBMNCs from patients with superficial transitional cell carcinoma (STCC) of the bladder to mitogenic signals that interact with surface molecules [phytohaemagglutinin, PHA and anti-CD3 monoclonal antibodies, (MAbs)]. PBMNCs from the patients were studied prior to the transurethral resection (TUR) of the tumour, during the second month of prophylactic intravesical instillation of IFN-alpha 2b and 3 and 6 months after finishing the instillation treatment. The [3H]thymidine uptake of PBMNCs from 17 patients with STCC of the bladder after 5 days of PHA and anti-CD3 MAb stimulus was found to be significantly lower than that of healthy controls (P < 0.05). The addition of interleukin 2 (IL-2) to the culture medium did not correct this defective proliferative response to PHA and the anti-CD3 MAb. There were no significant differences between IL-2 production in PBMNCs from STCC patients after stimulation with PHA and in PBMNCs from healthy controls (P > 0.05). Patients without evidence of recurrence showed a significantly enhanced proliferative response in PBMNC to PHA and anti-CD3 MAb after intravesical prophylactic treatment with interferon-alpha 2b in the follow-up examinations 3 and 6 months after treatment (P < 0.01). However, three patients had evidence of tumour recurrence, and they showed no enhancement of the PBMNC proliferative response to these mitogens in the same examinations. In conclusion, the prophylactic intracavitary treatment of STCC with IFN-alpha 2b may induce a systemic immunomodulatory effect which is associated to the clinical evolution of the disease.

We have investigated the effect of interferon beta (INF beta) on the natural killer (NK) cytotoxic activity of peripheral blood mononuclear cells (PBMC) from patients with superficial and infiltrative transitional-cell carcinoma of the bladder (TCC) against both NK-sensitive and NK-resistant target cells. The normal NK activity found in PBMC from these patients can be significantly enhanced by short-term incubation (18 h) with INF beta (P < 0.05). The depressed NK cytotoxic activity found in PBMC from patients with infiltrative TCC can also be significantly enhanced, but not normalized, by short-term incubation with INF beta (P < 0.05). In kinetic studies we found that the maximal levels of the INF beta-promoted cytotoxic activity against NK-sensitive and against NK-resistant target cells in PBMC from TCC patients were reached after 18 h of culture. Short-term-INF beta-incubated PBMC from patients with TCC of the bladder also showed marked cytotoxic activity against NK-resistant target cells. The effector cells of the INF beta-induced cytotoxic activity in PBMC from patients with TCC were CD16+ CD3- NK cells. This cytotoxic inducer effect of INF beta synergized with that of interleukin-2. In conclusion, INF beta can enhance the NK activity of PMBC from patients with TCC of the bladder.

Background: Transitional cell carcinoma (TCC) of the bladder is associated with alterations in the immune system of the host. The authors demonstrated that in patients with bladder carcinoma there is a negative correlation between the levels of natural killer (NK) activity and the clinical evolution and pathologic stages of disease., Methods: The authors investigated the effect of various doses of recombinant interferon-alpha-2b (IFN-alpha-2b) for variable periods of culture on the nonmajor histocompatibility-restricted cytotoxic activity of peripheral blood mononuclear cells (PBMNC) with or without CD16 and CD3-depleted populations from patients with superficial (confined to the mucosa or lamina propria) and infiltrative (those infiltrating beyond the lamina propria) TCC of the bladder using 4-hour 51-sodium chromate (51Cr)-release cytotoxicity assays against both NK-sensitive (K562) and NK-resistant (JY) tumor target cells., Results: The normal NK activity detected in PBMNC from patients with superficial TCC of the bladder can be significantly enhanced by short-term (18-hour) incubation with recombinant IFN-alpha (P < 0.05). The depressed NK cytotoxic activity found in PBMNC from patients with infiltrative TCC can also be significantly enhanced, but not normalized, by short-term (18-hour) incubation with recombinant IFN-alpha (P < 0.05). Short-term recombinant IFN-alpha-incubated PBMNC from patients with superficial, but not infiltrative, TCC of the bladder also showed marked cytotoxic activity against NK-resistant target cells. By selection with CD16 or CD3 monoclonal antibodies and complement, it was also found that the precursor and effector lymphocytes of this recombinant IFN-alpha-promoted cytotoxicity belong to NK lineage. In kinetic studies, it was found that the maximal levels of the recombinant IFN-alpha-promoted cytotoxic activity against NK-sensitive and NK-resistant target cells in PBMNC from patients with TCC were reached after 18 hours of culture., Conclusion: Recombinant IFN-alpha can enhance the nonmajor histocompatibility-restricted cytotoxic activity of PBMNC from patients with TCC of the bladder.

We have investigated the effect of thymostimulin on the major histocompatibility (MHC) unrestricted cytotoxic activity of peripheral blood mononuclear cells (PBMNC) from patients with superficial transitional cell carcinoma (TCC) of the bladder. PBMNC from patients and healthy controls were incubated in the presence of thymostimulin for varying periods of time (2 or 18 h or 5 days), and were used as effectors against 51Cr-radiolabeled natural killer (NK)-sensitive (K-562) and NK-resistant (JY) target cells in cytotoxic assays. In 6 out of 14 patients analyzed, thymostimulin enhanced the cytotoxic activity of PBMNC against NK-sensitive target cells in a dose-dependent manner. This cytotoxic inducer effect of thymostimulin was maximal after 18 h of culture. Thymostimulin failed to induce lytic activity in PBMNC from TCC patients against NK-resistant target cells. We also found that thymostimulin could synergize with interleukin-2 in inducing non-MHC restricted cytotoxic activity in PBMNC from TCC patients. In conclusion, we have demonstrated that thymostimulin can enhance the natural killer cytotoxic activity of PBMNC from patients with TCC of the bladder.

Following complete transurethral resection, patients with superficial transitional cell carcinoma (TCC) were treated with intravesical interferon-alpha 2b. Ex vivo analysis of peripheral blood mononuclear cells (PBMNC) both during and after the treatment period showed an enhancement of natural killer (NK) activity which fell to basal levels upon cessation of therapy. Simultaneous analysis of T-cell activity revealed a similar pattern of variation in the response to the polyclonal mitogen phytohemagglutinin. Both findings point to an alteration in systemic immunoreactivity as a result of intravesical treatment. Further work will be aimed at determining whether this is relevant to the therapeutic value of such treatment and whether prolonged treatment will maintain these effects.

Incubation of peripheral blood mononuclear cells (PBMNC) from patients with transitional cell carcinoma of the bladder, with interferon (IFN-alpha 2b) causes dose-dependent enhancement of natural killer (NK) and cytotoxic activity. Increased lytic activity was also observed against both NK-sensitive and NK-resistant target cells and T lymphocytes from such patients showed altered activity upon incubation with IFN-alpha 2b. Furthermore, intracavitary instillation of IFN-alpha 2b is associated with infiltration of the bladder wall by NK cells and T lymphocytes. Future work will be aimed at investigating the possible prognostic value of these results as related to the therapeutic effect of intracavitary instillation of IFN-alpha 2b.

It has been shown that natural killer (NK) cells are involved in the immunosurveillance of tumors. In patients with transitional cell carcinoma (TCC) of the bladder, there is a negative correlation between the levels of NK activity in peripheral blood mononuclear cells (PBMNCs) and both the clinical evolution and the pathological stages of the disease. We have investigated the immunoregulatory effect of keyhole limpet hemocyanin (KLH) on the NK activity of patients with TCC of the bladder. We found that KLH enhances the cytotoxic activity of PBMNC from patients with superficial TCC against NK-sensitive target cells in a time-dependent manner. This KLH-enhanced cytotoxicity is not directed against NK-resistant target cells. However, KLH fails to normalize the depressed NK activity of PBMNCs from patients with infiltrative TCC. The present in vitro studies suggest that this immunoregulatory effect of KLH on NK cytotoxicity may be implicated in its therapeutic effect on superficial TCC of the bladder.

We studied the relationship of natural killer activity from peripheral blood mononuclear cells with clinical stage of disease and the different modalities of treatment in 67 untreated patients with transitional cell carcinoma of the bladder and 29 normal controls. Peripheral blood mononuclear cells from 39 patients with superficial bladder tumor (stages Ta and T1) showed a natural killer cell activity similar to that of controls (p greater than 0.05), while in 28 patients with infiltrating tumors (stages T2, T3 and T4) this activity was significantly depressed (p less than 0.01). This functional phenomenon cannot be ascribed to a deficient number of natural killer cells in patients with infiltrating tumors, since the amounts of HNK-1+ (Leu 7), CD16+ (Leu 11) and CD11b+ (OKM1) cells in peripheral blood mononuclear cells were similar in the 3 groups of subjects (p greater than 0.05). Furthermore, the natural killer activity of peripheral blood mononuclear cells was normal (p greater than 0.05) in patients who underwent transurethral resection of the tumors and intracavitary cytostatic therapy with doxorubicin who remained free of disease at least 6 months after treatment. However, in patients with superficial recurrent tumor a significant decrease in the natural killer activity of peripheral blood mononuclear cells was observed (p less than 0.05), which was more pronounced in those with infiltrating recurrence. Also, in the latter patients total cystoprostatectomy was associated with a relevant increase in the spontaneous level of peripheral blood mononuclear cells. We conclude that in patients with transitional cell carcinoma of the bladder there is a correlation of the levels of natural killer activity in peripheral blood mononuclear cells with clinical evolution and pathological stage of disease. The determination of this activity is useful to monitor patients with transitional cell carcinoma of the bladder.

A retrospective study was done on the long-term evolution of 48 patients diagnosed of transitional cell carcinoma of the bladder who were treated with full transurethral resection and intracavitary chemoprophylaxis with Thiotepa. All patients presented a minimum disease control period of 36 months, and were followed for 85.3 months on the average (38-156) months. The actuarial study of the relapse-free intervals turned out into a possibility of 89.3% of patients without a relapse by 5 years, and of 64.6% by 10 years. The actuarial study of tumor progress showed that 91.4% and 58.8% of the patients were free of progression by 5 and 10 years respectively. pT1-G3 tumors did not show greater relapse rate nor progression than the rest of cases, it is therefore convenient to consider other prognostic factors in the long-term evolution of these patients. 85.7% of patient deaths were due to direct consequence of the bladder tumor. These results and their clinical implications are analyzed.