The Impact of a New Interleukin-2-Based Immunotherapy Candidate on Urothelial Cells to Support Use for Intravesical Drug Delivery.

(1) Background: The intravesical instillation of interleukin-2 (IL-2) has been shown to be very well tolerated and promising in patients with bladder malignancies. This study aims to confirm the use of a new IL-2 containing immunotherapy candidate as safe for intravesical application. IL-2, produced in mammalian cells, is glycosylated, because of its unique solubility and stability optimized for intravesical use. (2) Materials and Methods: Urothelial cells and fibroblasts were generated out of porcine bladder and cultured until they reached second passage. Afterwards, they were cultivated in renal epithelial medium (REM) and Dulbecco's modified Eagles medium (DMEM) with the IL-2 candidate (IMS-Research) and three more types of human interleukin-2 immunotherapy products (IMS-Pure, Natural IL-2, Aldesleukin) in four different concentrations (100, 250, 500, 1000 IU/mL). Cell proliferation was analyzed by water soluble tetrazolium (WST) proliferation assay after 0, 3, and 6 days for single cell culture and co-culture. (3) Results: Proliferation assays showed that all IL-2 products induced very similar cultivation results and none of the IL-2 variants had a negative impact on the proliferation of urothelial cells and fibroblast in either concentration. (4) Conclusion: Human recombinant glycosylated IL-2 as well as human non-glycosylated IL-2 have no negative influence on the tissue cell proliferation of porcine urothelial cells and fibroblasts in vitro and represent a promising and innovative potential intravesical therapy candidate for patients in high need.

Inhibition of dendritic cell generation and function by serum from prostate cancer patients: correlation with serum-free PSA.

Tumor produces a number of immunosuppressive factors that block maturation of dendritic cells (DCs). Here, we demonstrated that endogenous factors presented in the serum of patients with prostate cancer (CaP) inhibited the generation of functionally active DCs from CD14+ monocytes in vitro. We have shown a significant inhibitory potential of serum obtained from patients with CaP and benign prostate hyperplasia benign prostatic hyperplasia (BPH) when compared with serum from healthy volunteers. As assessed by flow cytometry, expression of CD83, CD86, and CD40 molecules was strongly inhibited by CaP and BPH serum. In addition, these DCs were weak stimulators of allogeneic T cell proliferation when compared with DCs produced in the presence of healthy volunteer serum. Statistical analysis of the results revealed a positive relationship between the inhibition of expression of DC markers CD83 and CD80 and the levels of serum-free prostate-specific antigen (PSA). These data suggest that the DC system may be impaired in CaP patients.

Therapeutic approaches in metastatic renal cell carcinoma: local immunotherapy.

Since 1990, aerosol interleukin (IL)-2 has been used to treat pulmonary metastatic renal cell carcinoma (pmRCC). Inhalation therapy deposits a drug into the airways to achieve a high, local, clinical effect while avoiding serious systemic side effects. We report three studies to describe safety and efficacy of aerosol IL-2 in patients with pmRCC. In a multicenter study, 24 patients received exclusive inhalation (study I) of natural IL-2 (three dose levels, 48 weeks) and response and toxicity were evaluated. The survival of high-risk patients (study II) with mainly inhaled IL-2 (n=94) was compared with that of patients receiving systemic IL-2 (n=103). In ten patients we analyzed in detail lung function and markers of airway inflammation before and during inhalational IL-2 therapy (study III). Study I: The response of exclusive inhalation was 33.3% at 3 months and 16.7% at 6 months. Treatment was well tolerated, cough being the most frequent adverse event. Study II: The probabilities of survival at 5 years were 21% for the inhalational group and 0% for the systemic group. Study III: Inhaled IL-2 induced a moderate decline of forced expiratory volume (FEV), while exhaled nitric oxide (NO) and sputum eosinophils rose accompanied by moderate cough and dyspnea. In conclusion, inhalational IL-2 combines good efficacy and improves tolerability. This is especially important for patients who are not able to benefit from systemic IL-2 therapy. Whether the local eosinophilic response additionally supports the antitumor effect remains a challenging question.

Renal cell carcinoma - innovative medical treatments.

PURPOSE OF REVIEW: This review highlights recent innovations of medical treatments in advanced renal cell carcinoma. Because of significant toxicity many patients do not benefit from standard systemic cytokine therapy using interleukin-2, interferon-alpha, or both. Their median progression-free survival is about 2 months and their overall survival is poor. Here we report improvements in patient outcome, focusing on overall survival, progression-free survival and quality of life to meet their needs. RECENT FINDINGS: Rating treatment results requires analysis of patient selection and risk stratification. In well-selected patients systemic interleukin-2 therapy as well as combinations of cytokines (interleukin-2 and interferon-alpha) improve outcome, induce long-term survival and, in a minority of patients, even produce long-lasting complete remissions. Within the group of patients who are not candidates for systemic cytokines, interleukin-2 for pulmonary metastases given locally by inhalation achieves long-term progression-free survival and long-term overall survival similar to high-dose systemic interleukin-2 but without the toxicity associated with it. In patients at high risk for metastatic disease progression-free survival is prolonged by autologous tumor vaccine as reported in a recent randomized study. However, in established metastatic disease no clear benefit has been demonstrated by any of the many recent vaccine protocols. Chemotherapeutic agents and thalidomide generally fail to improve patient outcome but produce significant and sometimes irreversible toxicity. SUMMARY: Cytokine-based immunotherapy can be considered standard in the treatment of metastatic renal cell carcinoma today. Long-term survival and sometimes even cure seems possible in patients using systemic and local (inhaled) applications of interleukin-2 alone or in combination with interferon-alpha. Noncytokine treatment approaches are scientifically interesting but still disappointing from a patient's perspective.

Total and Gleason grade 4/5 cancer volumes are major contributors of human kallikrein 2, whereas free prostate specific antigen is largely contributed by benign gland volume in serum from patients with prostate cancer or benign prostatic biopsies.

PURPOSE: We measured concentrations of human glandular kallikrein 2 (hK2), total prostate specific antigen (tPSA), free PSA (fPSA) and percent fPSA to evaluate their relationship to total prostate gland volume, benign prostatic hyperplasia (BPH) volume, total prostate cancer (PCa) volume (CaVol) and the volume of Gleason grades 4/5 cancer (CaVolGl4) in the serum of 256 patients with PCa undergoing radical retropubic prostatectomy and 185 with negative systematic sextant biopsies. MATERIALS AND METHODS: Free and total PSA was measured using the Delfia Prostatus (Perkin-Elmer, Turku, Finland) total/free PSA assay and hK2 was measured using a research immunofluorometric assay. Transrectal ultrasound was used to determine total prostate and BPH volume. Total CaVol and CaVolGl4/5 were calculated using a volumetric program in specimens from 158 men with pT2a/b and 98 with pT3a or greater PCa. The Pearson correlation was performed after logarithmic conversion of PSA and hK2 levels. Benign gland, and pT2a/b and pT3a or greater PCa cases were subdivided into small vs large prostate gland volumes (42 cc or less vs greater than 42 cc). RESULTS: Total prostate and BPH volumes correlated closely with free PSA (r = 0.64 to 0.65, p <0.0001) in 143 patients with negative biopsy and a prostate of greater than 42 cc. Correlations of hK2 and tPSA with total prostate and BPH volumes were weaker (r = 0.35 to 0.36 and 0.45 to 0.46, respectively). In pT2a/b and pT3a or greater PCa cases hK2 most closely correlated with CaVol (range 0.31 to 0.62, p = 0.0072 and <0.0001) and with CaVolGl4/5 (range 0.26 to 0.56, p = 0.021 and <0.0001, respectively). The tPSA level correlated significantly with CaVol and CaVolGl4/5 except in glands 42 cc or greater harboring pT2a/b PCa (p = 0.08). Free PSA correlated significantly with CaVolGl4/5 only in pT3a or greater PCa (p <0.05), and with CaVol in pT3a or greater PCa and in small prostates harboring pT2a/b PCa. CONCLUSIONS: Large benign prostate gland volume affects fPSA more than tPSA in serum. In PCa hK2 more closely correlates with total cancer volume and high grade PCa volume compared with tPSA or fPSA.

Inhibition of dendropoiesis by tumor derived and purified prostate specific antigen.

PURPOSE: Prostate specific antigen (PSA) is a serine protease produced by the prostate gland at high concentrations. Serum PSA may be significantly elevated in prostate cancer and benign prostatic diseases. It has recently become evident that, in addition to being a tissue and/or serum marker, PSA may also have biological effects. Despite the voluminous literature on this biomarker in the diagnosis of prostatic diseases relatively few reports have addressed the issue of the physiological function, biological role and immune effects of PSA in the context of prostate cancer development and progression. MATERIALS AND METHODS: Human dendritic cell (DC) cultures were generated from CD34+ hematopoietic precursors in the presence of PSA. The DC phenotype was assessed by flow cytometry and DC ability to induce T-cell proliferation was detected by allogeneic mixed lymphocyte reaction assay. DCs were also generated in co-cultures with LNCaP cells in the presence of antiPSA antibodies. The concentrations of PSA in cultures were determined by the AXSYM System (Abbott Laboratories, Wiesbaden, Germany). RESULTS: We noted that purified and LNCaP derived PSA inhibited the generation and maturation of DC (dendropoiesis) in vitro, which might have a crucial role in the induction and regulation of specific antitumor immune responses. The addition of active PSA to DC cultures significantly inhibited the generation and maturation of DC, as assessed by the levels of expression of CD83, CD80, CD86 and HLA DR. The ability of DC to induce T-cell proliferation, which depends on the expression of co-stimulatory and major histocompatibility complex molecules, was also suppressed in PSA treated DC cultures. CONCLUSIONS: The antidendropoietic effect of PSA in vitro suggests a new mechanism of prostate cancer induced immunosuppression and tumor escape, and provides novel evidence of the immunoregulatory properties of PSA.

Renal cell carcinoma: novel treatments for advanced disease.

PURPOSE OF REVIEW: IL-2 or IFN-alpha induce remissions and prolong life in patients carefully selected for a possibly toxic treatment. However, there is a need for better-tolerated and more effective therapies, especially in patients with co-morbidities and those resistant to systemic immunotherapy. Recent achievements in the treatment of advanced renal cell carcinoma highlight potentially significant improvements. RECENT FINDINGS: Cytoreductive surgery or radiation of metastases seems beneficial in well-selected patients, especially as immunotherapy is available. Immune cells within the tumour correlate with response and survival, indicating the importance of local immune modulation. Such modulation has allowed the introduction of well-tolerated treatments such as the inhalation of IL-2 to control lung metastases, which results in a significant survival benefit for high-risk patients, as suggested by a recent outcome study in 200 patients. Antibody-based tumour targeting against cG250, specifically expressed on renal cell carcinoma, seems to stabilize progressive metastatic disease and does not induce toxicity. Vaccination strategies are also well tolerated, but have not yet shown convincing results in advanced disease. Other approaches have not fulfilled expectations. Thalidomide has significant neurotoxicity and its efficacy was not confirmed in recent studies. Stem cell transplantation has significant toxicity, and cannot yet be recommended, but may have future potential. SUMMARY: Cytokine-based immunotherapy can now be considered standard in the treatment of metastatic renal cell carcinoma. There is good evidence that additional local procedures such as surgery, radiation or the inhalation of IL-2 improve response and survival in metastatic disease with moderate toxicity, resulting in a significant improvement for patients suitable for these approaches.

The role of human glandular kallikrein 2 for prediction of pathologically organ confined prostate cancer.

BACKGROUND: In recent studies serum levels of human glandular kallikrein 2 (hK2) demonstrated significant differences in pathologically organ-confined versus non-organ-confined prostate cancer (PCa). In this study we investigated whether hK2 adds independent information when considered together with traditionally used parameters to predict organ confined (pT2a/b) PCa. METHODS: Serum levels of hK2, total and free prostate-specific antigens (PSA) were obtained one day before radical prostatectomy in 245 consecutive men. These were included with clinical stage and biopsy Gleason grade into univariate analysis and multivariate logistic regression models. RESULTS: pT2a/b PCa was found in n = 148 patients. In univariate analysis all preoperative parameters demonstrated significant association with the presence of pT2a/b PCa. Using multivariate logistic regression model hK2 (P = 0.022), clinical stage (P < 0.0001), and Gleason grade (P < 0.0001) were independent predictors of pT2a/b PCa whereas PSA (P = 0.3) was not. In bootstrap corrected logistic regression based nomograms the addition of hK2 density marginally enhanced predictive accuracy when PSA, PSA density, clinical stage, and Gleason grade were considered (AUC = 0.879 without hK2 density and 0.883 with hK2 density). CONCLUSIONS: hK2 and hK2 density could independently predict pT2a/b PCa. However, improvement in predictive accuracy was marginal when nomograms based on traditional variables were complemented with this serum marker.

Prediction of tumor heterogeneity in localized prostate cancer.

Clinical T1 and T2 prostatic carcinoma is a heterogeneous tumor with respect to pathologic stage and outcome. In the authors' experience, 60% of patients have a pT2 prostatic carcinoma, and 2% to 4% have tumors less than 0.5 cm3 in volume. The latter group cannot be predicted by the use of preoperative parameters with a sufficient sensitivity and specificity. Quantitative analysis of six systematic biopsies, that is, reporting the number of biopsies with any Gleason grade 4 or 5 cancer or the number of biopsies with more than 50% Gleason grade 4 and 5 cancer, together with preoperative PSA levels can be used to predict the different pathologic stages and risk groups of patients with T1 or T2 prostatic carcinoma. CART analysis that using these preoperative parameters can predict the lymph node stage and the capsular penetration on each side of the prostate with a sufficient positive and negative predictive value and a sufficient specificity to avoid routine lymphadenectomy in approximately 80% of the patients classified as a low-risk group for having lymph nodes positive for disease. CART analysis also allows a solid identification of patients in whom the unilateral or bilateral nerve may be spared during surgery. These algorithms may be improved further by determining the HK-2 level in the blood or by including other molecular biologic markers in the analysis of the biopsies. Clinical T1 or T2 prostatic carcinoma is a heterogeneous but fairly predictable tumor.

Clinical use of urinary markers for the detection and prognosis of bladder carcinoma: a comparison of immunocytology with monoclonal antibodies against Lewis X and 486p3/12 with the BTA STAT and NMP22 tests.

PURPOSE: The noninvasive detection of urothelial carcinoma remains challenging. We prospectively evaluated urine markers for bladder carcinoma. We compared the NMP22 (Matritech, Cambridge, Massachusetts) and BTA Stat (Bard Diagnostics, Redmond, Washington) tests with immunocytology using mAbs 486p3/12 and BG7 against Lewis X antigen. MATERIALS AND METHODS: The NMP22 and BTA Stat tests were performed in urine samples and immunocytology with mAbs 486p3/12 and BG7 staining were performed in bladder washing specimens in 146 samples of 115 patients undergoing transurethral resection for suspected bladder carcinoma (70) or 45 undergoing followup cystoscopy for a history of bladder carcinoma (76). Bladder carcinoma was detected in 54 patients, including stages pTa in 25, pT1 in 20, pT2 in 8 and carcinoma in situ in 1, while 61 had no evidence of bladder carcinoma. The cutoff was 10 units per ml. for the NMP22, 30% positive cells for 486p3/12 and 5% positive cells for the Lewis X tests. RESULTS: BTA Stat was positive in 65 samples (44.5%) and NMP22 was positive in 69 (47.3%). Immunocytology with mAbs 486p3/12 and BG7 against Lewis X was positive in 52 (35.6%) and 109 (74.7%) samples, respectively. Sensitivity was 70.3% for BTA Stat, 68.5% for NMP22, 68.5% for 486p3/12 and 94.4% for Lewis X. Specificity was 70.6% for BTA Stat, 65.2% for NMP22, 83.6% for 486p3/12 and 36.9% for Lewis X. Area under the receiver operating characteristics curve was 0.6804 for NMP22, 0.7226 for Lewis X and 0.8002 for 486p3/12. False-positive results on BTA Stat in 2 of 22 patients (9%), on NMP22 in 2 of 25 (8%), on 486p3/12 in 3 of 11 (27%) and on Lewis X in 4 of 43 (9.3%) were associated with tumor recurrence. Furthermore, negative results on BTA Stat in 2 of 39 patients (2%), on NMP22 in 2 of 36 (0.5%), on Lewis X in 0 of 18 (0%) and on 486p3/12 in 1 of 50 (2%) was associated with tumor recurrence during followup. CONCLUSIONS: Immunocytology with mAbs against Lewis X showed higher sensitivity than all commercially available tests evaluated. Because of its high sensitivity and high negative predictive value, it may be useful for screening in a high risk population. Patients with a false-positive 486p3/12 test results are at increased risk for tumor recurrence compared with those with negative results.

Assessment of clinical and pathologic characteristics predisposing to disease recurrence following radical prostatectomy in men with pathologically organ-confined prostate cancer.

OBJECTIVE: To identify risk factors for biochemical failure after radical prostatectomy (RP) in men with pathologically organ-confined (OC) prostate cancer (PCa). METHODS: Clinical and pathological characteristics of 331 consecutive men with pT2N0 PCa treated solely with RP were used in Cox proportional hazard models to identify independent predictors of prostate specific antigen (PSA) failure (PSA > or = 0.1 ng/ml). All pathologic specimens were step sectioned at 3 mm. RESULTS: Twelve patients (3.6%) failed at a median follow-up of 26 months (range 0.2-99.6 months) and 120 men remained at risk 3 years after RP. In univariate Cox models PSA (P < 0.001), percentage of high-grade cancer (P < 0.001) total and high-grade cancer volume (P = 0.001 and P < 0.0001, respectively) and RP Gleason sum (P = 0.003) represented significant predictors of PSA failure. Clinical stage (P = 0.4), surgical margin status (P = 0.3), age (P = 0.2), and pathologic evidence of unilateral versus bilateral PCa (P = 0.6) failed to reveal significance. In receiver operator curve (ROC) analyses, high-grade cancer volume achieved highest outcome predictive accuracy (area under the curve (AUC 0.93)), which was not exceeded by Cox regression-based nomogram combining serum PSA, RP Gleason sum, margin status and pathologic evidence of unilateral versus bilateral PCa (AUC 091). Predictive accuracy of this multivariate nomogram was not enhanced by adding total cancer volume (AUC 0.93), high-grade cancer volume (AUC 0.90), or percentage of high-grade cancer (AUC 0.90). CONCLUSIONS: In pT2N0 PCa high-grade cancer volume appears to represent the most important pathologic factor for prediction of outcome following RP. However, similar predictive accuracy may be achieved by combining routinely available tumor characteristics.

Percent free prostate specific antigen is not an independent predictor of organ confinement or prostate specific antigen recurrence in unscreened patients with localized prostate cancer treated with radical prostatectomy.

PURPOSE: We studied the controversial relationship of percent free prostate specific antigen (PSA) with organ confined prostate cancer and PSA failure after radical prostatectomy. MATERIALS AND METHODS: We tested the characteristics of the percent free PSA monoclonal Immulite DPC Immunoassay (Diagnostic Products Corp., Los Angeles, California) for predicting organ confinement in 698 consecutive unscreened men treated only with radical prostatectomy between 1995 and 2000. In addition, we assessed the ability of percent free PSA to predict post-radical prostatectomy PSA failure, defined as PSA 0.1 ng./ml. or greater, in a subset of 581 men in whom followup was available. All statistical analyses were repeated for stage T1c cancer with PSA between 2 and 10 ng./ml. RESULTS: On univariate analyses percent free PSA achieved significance for predicting organ confined disease at all clinical stages (p <0.001) and for stage T1c cancer with PSA between 2 and 10 ng./ml. (p = 0.012). However, significance dissipated after controlling for total PSA, biopsy Gleason sum and clinical stage (p = 0.135 and 0.851, respectively). In univariate Cox models percent free PSA failed to predict PSA failure across stages (p = 0.341), as well as in stage T1c cancer (p = 0.93). In multivariate analyses controlling for traditional PSA biopsy grade and clinical stage (p <0.001), percent free PSA failed to contribute to predicting PSA failure (p = 0.342). In the stage T1c subset biopsy Gleason sum (p <0.001) and PSA (p = 0.018) remained significant, in contrast to percent free PSA (p = 0.237). CONCLUSIONS: Percent free PSA has no independent, statistically significant association with organ confined status or posttreatment PSA outcome in unscreened patients who undergo radical prostatectomy for localized prostate cancer.