Cyrill A. Rentsch

A Retrospective Analysis of 153 Patients Treated With or Without Intravesical Bacillus Calmette-Guerin for Primary Stage T1 Grade 3 Bladder Cancer: Recurrence, Progression and Survival

PURPOSE We retrospectively evaluated the long-term outcome in patients with newly diagnosed stage T1 grade 3 bladder cancer treated with transurethral resection with or without intravesical bacillus Calmette-Guerin (BCG). MATERIALS AND METHODS Of 153 patients with a median age of 67 years (range 36 to 88) and a male-to-female ratio of 4:1 we treated 92 with transurethral bladder resection and additional BCG, and 61 with transurethral bladder resection alone. BCG was administered intravesically as 120 mg. BCG Pasteur F dissolved in 50 ml. saline, retained for up to 2 hours weekly for 6 weeks and repeated as necessary. RESULTS Median followup was 5.3 years (range 0.4 to 18.2). Disease recurred in 70% of the patients treated with BCG and in 75% treated with transurethral resection alone. Median time to recurrence was 38 and 22 months for BCG and resection alone (p = 0.19). Tumor progressed in 33% of patients with BCG and in 36% with resection alone. Deferred cystectomy was performed in 29% of the patients with BCG and in 31% with resection alone. Overall and disease specific survival did not differ significantly. CONCLUSIONS Our results suggest that intravesical BCG therapy after transurethral bladder resection for stage T1 grade 3 bladder cancer may delay the time to recurrence and cystectomy but it does not substantially alter the final outcome. Our findings reflect the rule of 30% for stage T1 grade 3 cancer, namely approximately 30% of patients never have recurrence, 30% ultimately die of metastatic disease and 30% require deferred cystectomy.

Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancer.

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with > or = 10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 mug/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer.

URINARY INTERLEUKIN-8 AND 18 PREDICT THE RESPONSE OF SUPERFICIAL BLADDER CANCER TO INTRAVESICAL THERAPY WITH BACILLUS CALMETTE-GUERIN

PURPOSE We evaluate the predictive value of urinary cytokine levels of interleukin (IL) 8 and 18 for response in patients receiving intravesical bacillus Calmette-Guerin (BCG) for prevention of recurrences of superficial bladder cancer and treatment of carcinoma in situ. MATERIALS AND METHODS In 28 patients with superficial bladder cancer treated with BCG IL-8 expression in the urine during the first 6 hours after the first BCG instillation was determined. In 17 patients IL-18 levels were also evaluated during the first 12 hours after BCG instillation. IL-8 and 18 levels were determined by solid phase double ligand enzyme-linked immunosorbent assay. RESULTS In 12 of the 28 patients assessed for IL-8 expression disease recurred after a median followup of 66 months. Median IL-8 expression during the first 6 hours for these patients was 851 ng. (range 232 to 8,497). Median IL-8 expression during the first 6 hours in patients without recurrence was 4,200 ng. (range 432 to 12, 232). Of 8 patients with a followup of greater than 36 months 7 (88%) had no recurrent disease and IL-8 levels greater than 4,000 ng. Patients secreting more than 4,000 ng. IL-8 into the urine after BCG have a significantly higher chance of remaining disease-free (p <0.05), and those with elevated IL-18 expression have a significantly longer disease-free survival (p <0.05). After a median followup of 23 months (range 7 to 93) 6 of the 17 patients assessed for IL-18 expression had treatment failure. Median IL-18 expression in those patients during the first 12 hours was 2,632 pg. (range 860 to 8,298). Median IL-18 expression during the first 12 hours in patients without recurrence was 12,258 pg. (range 1,727 to 151,495). CONCLUSIONS In this study we confirmed the value of quantitative IL-8 expression in the urine during the first 6 hours after BCG instillation for superficial bladder cancer to predict freedom of disease. Furthermore, to our knowledge we report for the first time the potential value of IL-18 expression in the urine during the first 12 hours after BCG to predict freedom from disease. These findings may help improve the treatment of patients with superficial bladder cancer, especially by identifying those with a high risk of disease recurrence and progression after BCG therapy.

Preexisting BCG-Specific T Cells Improve Intravesical Immunotherapy for Bladder Cancer

Preexisting immunity to BCG improves treatment response to intravesical BCG therapy for bladder cancer in mouse models and humans. Bugs as Drugs Although still in its infancy for the treatment of many tumor types, immunotherapy has been and remains the standard of care for patients with bladder cancer. The concept behind this success is repeated instillations of bacillus Calmette-Guérin (BCG; the same bacteria used as a vaccine for Mycobacterium tuberculosis) in the bladder, inducing an immune response that fights the cancer. The clinical response rate in bladder cancer patients is 50 to 70%, higher than any other immunotherapy but still leaving room for improvement. Biot et al. provide new insight into the mechanisms behind BCG’s success in the bladder and suggest that parenteral BCG exposure may boost the success rate for bladder cancer treatment. Using a mouse model of bladder cancer, the authors show that a single instillation of BCG induced an immune response, but repetitive instillations were required for robust T cell trafficking to the bladder. Previous parenteral exposure to BCG overcame this requirement in the mice, inducing an enhanced innate immune response, accelerating T cell trafficking to the bladder, and improving the immune response to the tumor. Furthermore, bladder cancer patients who had preexisting immunity to BCG (secondary to their childhood M. tuberculosis vaccination) showed improved recurrence-free survival after standard BCG therapy relative to those patients without preexisting immunity. Thus, immunizing BCG-naïve cancer patients before therapy may provide a simple strategy that could improve therapeutic response. Therapeutic intravesical instillation of bacillus Calmette-Guérin (BCG) is effective at triggering inflammation and eliciting successful tumor immunity in patients with non–muscle invasive bladder cancer, with 50 to 70% clinical response. Therapeutic success relies on repeated instillations of live BCG administered as adjuvant therapy shortly after tumor resection; however, the precise mechanisms remain unclear. Using an experimental model, we demonstrate that after a single instillation, BCG could disseminate to bladder draining lymph nodes and prime interferon-γ–producing T cells. Nonetheless, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. Parenteral exposure to BCG before instillation overcame this requirement; after the first intravesical instillation, BCG triggered a more robust acute inflammatory process and accelerated T cell entry into the bladder, as compared to the standard protocol. Moreover, parenteral exposure to BCG before intravesical treatment of an orthotopic tumor markedly improved response to therapy. Indeed, patients with sustained preexisting immunity to BCG showed a significant improvement in recurrence-free survival. Together, these data suggest that monitoring patients’ response to purified protein derivative, and, in their absence, boosting BCG responses by parenteral exposure before intravesical treatment initiation, may be a safe and effective means of improving intravesical BCG-induced clinical responses.

Bacillus Calmette-Guérin Strain Differences Have an Impact on Clinical Outcome in Bladder Cancer Immunotherapy

BACKGROUND Whether the commonly used bacillus Calmette-Guérin (BCG) strains Connaught and Tice confer different treatment responses in non-muscle-invasive bladder cancer (NMIBC) is unknown. OBJECTIVES To compare clinical efficacy, immunogenicity, and genetics of BCG Connaught and Tice. DESIGN, SETTING, AND PARTICIPANTS A prospective randomized single-institution trial with treatment of 142 high-risk NMIBC patients with BCG Connaught or Tice. INTERVENTION Patients were randomized to receive six instillations of BCG Connaught or Tice. For experimental studies, BCG strains were compared in C57Bl/6 mice. Bladders and lymphoid tissues were analyzed by cytometry and the latter cultivated to detect live BCG. BCG genomic DNA was sequenced and compared with reference genomes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Recurrence-free survival was the primary end point of the clinical study. The Kaplan-Meier estimator was used for estimating survival and time-to-event end points. Nonparametric tests served for the analysis of the in vivo results. RESULTS AND LIMITATIONS Treatment with BCG Connaught conferred significantly greater 5-yr recurrence-free survival compared with treatment with BCG Tice (p=0.0108). Comparable numbers of patients experienced BCG therapy-related side effects in each treatment group (p=0.09). In mice, BCG Connaught induced stronger T-helper cell 1-biased responses, greater priming of BCG-specific CD8(+) T cells, and more robust T-cell recruitment to the bladder than BCG Tice. Genome sequencing of the BCG strains revealed candidate genes potentially involved in the differential clinical responses. CONCLUSIONS BCG strain may have an impact on treatment outcome in NMIBC immunotherapy. PATIENT SUMMARY We compared the efficacy of two commonly used bacillus Calmette-Guérin (BCG) strains for the treatment of NMIBC and found that treatment with BCG Connaught prevented recurrences more efficiently than BCG Tice. Comparison of the immunogenicity of the two strains in mice indicated superior immunogenicity of BCG Connaught. We also identified genetic differences that may explain the differential efficacy of the Connaught and Tice BCG strains. TRIAL REGISTRATION NCT00003779.

In vitro propagation and characterization of neoplastic stem/progenitor-like cells from human prostate cancer tissue

According to the cancer stem cell hypothesis, tumor growth is sustained by a subpopulation of cancer stem/progenitor‐like cells. Self‐renewal and high clonogenic potential are characteristics shared by normal stem and neoplastic stem/progenitor‐like cells. We investigated whether human prostate cancer specimens contain cells with these properties.

Toxicity and early treatment outcomes in low- and intermediate-risk prostate cancer managed by high-dose-rate brachytherapy as a monotherapy

PURPOSE To determine the acute and late genitourinary (GU) and gastrointestinal (GI) toxicity and present short-term biochemical no evidence of disease (bNED) rates after high-dose-rate brachytherapy (HDR-B) monotherapy. METHODS AND MATERIALS Between October 2003 and June 2006, 36 patients with low (28) and intermediate (8) risk prostate cancer (PCA) were treated by HDR-B monotherapy. All patients received one implant and four fractions of 9.5Gy within 48h for a total prescribed dose (PD) of 38Gy. Five patients received hormonal therapy (HT). Median age was 63.5 years and median followup was 3 years (range, 0.4-4 years). Toxicity was scored according to the CTCAE version 3.0. Biochemical failure was defined according to the Phoenix criteria. RESULTS Acute and late Grade 3 GU toxicity was observed in 1 (3%) and 4 (11%) patients, respectively. Grade 3 GI toxicity was absent. The three- year bNED survival rate was 100%. The sexual preservation rate in patients without HT was 75%. Late Grade 3 GU toxicity was associated with the planning target volume (PTV) V(100) (% PTV receiving > or =100% of the PD; p=0.036), D(90) (dose delivered to 90% of the PTV; p=0.02), and the urethral V(120) (urethral volume receiving > or =120% of the PD; p=0.043). The urethral V(120) was associated with increased PTV V(100) (p<0.001) and D(90) (p=0.003). CONCLUSIONS After HDR-B monotherapy, late Grade 3 GU toxicity is associated with the urethral V(120) and the V(100) and D(90) of the PTV. Decrease of the irradiated urethral volume may reduce the GU toxicity and potentially improve the therapeutic ratio of this treatment.

Deep Clonal Profiling of Formalin Fixed Paraffin Embedded Clinical Samples

Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.

Differential expression of TGFβ‐stimulated clone 22 in normal prostate and prostate cancer

The transforming growth factor‐β (TGFβ) superfamily and its downstream effector genes are key regulators of epithelial homeostasis. Altered expression of these genes may be associated with malignant transformation of the prostate gland. The cDNA array analysis of differential expression of the TGFβ superfamily and functionally related genes between patient‐matched noncancerous prostate (NP) and prostate cancer (PC) bulk tissue specimens highlighted two genes, namely TGFβ‐stimulated clone‐22 (TSC‐22) and Id4. Verification of their mRNA expression by real‐time PCR in patient‐matched NP and PC bulk tissue, in laser‐captured pure epithelial and cancer cells and in NP and PC cell lines confirmed TSC‐22 underexpression, but not Id4 overexpression, in PC and in human PC cell lines. Immunohistochemical analysis showed that TSC‐22 protein expression in NP is restricted to the basal cells and colocalizes with the basal cell marker cytokeratin 5. In contrast, all matched PC samples lack TSC‐22 immunoreactivity. Likewise, PC cell lines do not show detectable TSC‐22 protein expression as shown by immunoblotting. TSC‐22 should be considered as a novel basal cell marker, potentially useful for studying lineage determination within the epithelial compartment of the prostate. Conversely, lack of TSC‐22 seems to be a hallmark of malignant transformation of the prostate epithelium. Accordingly, TSC‐22 immunohistochemistry may prove to be a diagnostic tool for discriminating benign lesions from malignant ones of the prostate. The suggested tumour suppressor function of TSC‐22 warrants further investigation on its role in prostate carcinogenesis and on the TSC‐22 pathway as a candidate therapeutic target in PC.

Estrogen receptor β expression and androgen receptor phosphorylation correlate with a poor clinical outcome in hormone-naïve prostate cancer and are elevated in castration-resistant disease

Patients with advanced prostate cancer (PC) are usually treated with androgen withdrawal. While this therapy is initially effective, nearly all PCs become refractory to it. As hormone receptors play a crucial role in this process, we constructed a tissue microarray consisting of PC samples from 107 hormone-naïve (HN) and 101 castration-resistant (CR) PC patients and analyzed the androgen receptor (AR) gene copy number and the protein expression profiles of AR, Serin210-phosphorylated AR (pAR(210)), estrogen receptor (ER)β, ERα and the proliferation marker Ki67. The amplification of the AR gene was virtually restricted to CR PC and was significantly associated with increased AR protein expression (P<0.0001) and higher tumor cell proliferation (P=0.001). Strong AR expression was observed in a subgroup of HN PC patients with an adverse prognosis. In contrast, the absence of AR expression in CR PC was significantly associated with a poor overall survival. While pAR(210) was predominantly found in CR PC patients (P<0.0001), pAR(210) positivity was observed in a subgroup of HN PC patients with a poor survival (P<0.05). Epithelial ERα expression was restricted to CR PC cells (9%). ERβ protein expression was found in 38% of both HN and CR PCs, but was elevated in matched CR PC specimens. Similar to pAR(210), the presence of ERβ in HN patients was significantly associated with an adverse prognosis (P<0.005). Our results strongly suggest a major role for pAR(210) and ERβ in HN PC. The expression of these markers might be directly involved in CR tumor growth.