564 REAL TIME DIAGNOSIS AND GRADING OF BLADDER CANCER WITH PROBE-BASED CONFOCAL LASER ENDOMICROSCOPY: A PROSPECTIVE DIAGNOSTIC ACCURACY STUDY

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562 COULD HYALURONIC ACID REDUCE BACILLUS CALMETTE GUERIN (BCG) LOCAL SIDE EFFECTS? A PRELIMINARY REPORT Enrico Finazzi Agro*, Pierluigi Bove, Claudio Perugia, Giuseppe Vespasiani, Annalisa Maugliani, Roberto Miano, Rome, Italy INTRODUCTION AND OBJECTIVES: Bacillus Calmette Guerin (BCG) is considered the most effective treatment to increase disease-free interval and reduce progression of non muscle invasive bladder cancer (NMIBC). Although considered safe, BCG can produce local side effects leading to treatment cessation or interruption. Hyaluronic acid (HA) has been used for the treatment of radiation and chemical cystitis. Aim of this preliminary study was to investigate if the co-administration of HA may reduce lower local side effects of BCG. METHODS: 30 consecutive subjects undergoing BCG intravesical administration for high risk NMIBC were randomized (after informed consent) to receive BCG only (group A) or BCG and HA (group B). HA (Cystistat, Bioniche Pharma, Switzerland) was administered intravesically after every BCG administration, after BCG evacuation and bladder washing with saline. Patients were instructed to maintain HA in the bladder as long as possible after catheter removal. A 1 to 10 Visual Analog Scale (VAS) for bladder pain, International Prostate Symptom Score (IPSS ) and number of micturitions per day were evaluated in the two groups before and after six weekly BCG instillation. Patients were evaluated at three months by means of cystostopy and urine cytology. RESULTS: Only one out of 30 (3,3%) patients in group A dropped out from the protocol, for local side effects. Results are reported in table 1. Three patient in group A and 4 in group B presented with recurrent pathology at three month follow up. CONCLUSIONS: VAS for bladder pain was significantly lower after BCG administration in group B (patients treated with HA); IPSS and number of daily micturitions were not significantly lower in group B, but differences in their pre-post treatment change were significant. These very preliminary data seem to support a possible role of HA in reducing BCG local side effects. Nevertheless, randomized controlled trials should be performed before to draw any conclusion, even to exclude possible interferences of HA on BCG efficacy. Table 1 Mean (SD) VAS (1-10)

Group A Group B Pre-treatment

Group A Group B Post-treatment 4,2 (1,6)

p

.04

Group A Differences

Group B (post-pre)

p

1,5 (0,7) ⫺0,7 (1,6) .0001

4,9 (1,8)

.56

5,8 (1)

13,9 (4,4) 14,7 (4,0)

.60

17,5 (2,6)

15,3 (4)

.10

3 (3,5)

Number of daily micturitions 10,3 (2,2) 10,8 (2,4)

.53

11,5 (1,3)

10,9 (2,1)

.44

1,23 (1,7)

IPSS

4,5 (2)

p

0,53 (1,6) .02

0,13 (1)

.04

Source of Funding: The drug used in this study (Cystistat) was provided by Bioniche Pharma.

563 IL-17 PRODUCTION BY ␥␦ T CELLS IS IMPORTANT FOR THE ANTITUMOR EFFECT OF MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUERIN TREATMENT AGAINST BLADDER CANCER Takashi Dejima*, Ario Takeuchi, Fukuoka, Japan; Masatoshi Eto, Kumamoto, Japan; Tatsuya Nakatani, Osaka, Japan; Yasunobu Yoshikai, Seiji Naito, Fukuoka, Japan INTRODUCTION AND OBJECTIVES: Intravesical inoculation of Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been used for the treatment of bladder cancer. However the antitumor effector mechanisms remain elusive. Recent studies demonstrated that neutrophils infiltrated in the bladder after BCG treatment played a key role in the antitumor effect. Interleukin (IL)-17 (also known as IL-17A) is a T cell-derived proinflammatory cytokine, which is involved in various

pathogenesis where neutrophils are involved. However, at present, the mechanism of neutrophil infiltration after BCG treatment is not fully understood. METHODS: ␥␦ T cell-deficient (C␦ knock out (KO) and IL-17KO mice were kindly provided by Dr. S. Itohara and Dr. Y. Iwakura, respectively. The murine bladder cancer cell line, MB49, was kindly provided by Dr T. L. Ratliff. Mice were catheterized to receive an intravesical inoculate of 1 x 105 MB49 tumor cells on day 0. On days 1, 8, 15, and 22, mice were treated intravesically with either 3 x 106 CFU of BCG Connaught strain or PBS. Cytokines in the bladder were measured by ELISA. Cellular infiltrations in the bladder were analyzed by flow cytometry. For identification of IL-17-producing cells, intracellular cytokine staining method was used. RESULTS: Significant infiltration of neutrophils was observed from one week after starting BCG treatment, and it gradually increased during the observation period. IL-17 production was induced as early as 1 day after BCG injection. During the course of repeated BCG administration, similar level of IL-17 production was induced after each injection. Infiltration of neutrophils was significantly reduced in IL-17KO mice. The control wild type (WT) mice treated with BCG exhibited significantly longer survival compared to PBS-treated mice. On the other hand, there was no difference in the survival between BCG- and PBS-treated IL-17KO mice. The major source of IL-17A was ␥␦T cell population in the bladder. BCG-treated C␦KO mice showed significant reduction of IL-17 production and neutrophil infiltration compared with BCG-treated control mice. On the other hand, there was no difference in either IL-17 production or neutrophil count between CD4 or NK cell-depleted mice and the control mice. Survival of C␦KO mice was not improved by BCG treatment. CONCLUSIONS: ␥␦ T cells in the bladder have the anti tumor effect of intravesical BCG treatment via IL-17 production. Source of Funding: None

564 REAL TIME DIAGNOSIS AND GRADING OF BLADDER CANCER WITH PROBE-BASED CONFOCAL LASER ENDOMICROSCOPY: A PROSPECTIVE DIAGNOSTIC ACCURACY STUDY Jen-Jane Liu*, Timothy Chang, Stanford, CA; Shelly Hsiao, Palo Alto, CA; Ying Pan, Kathleen Mach, Jesse McKenney, Stanford, CA; Kristin Jensen, Palo Alto, CA; Joseph Liao, Stanford, CA INTRODUCTION AND OBJECTIVES: White light cystoscopy (WLC) has recognized shortcomings in bladder cancer diagnosis, particularly in differentiating nonpapillary urothelial carcinoma from inflammation. We recently described the feasibility and safety profile of probe-based confocal laser endomicroscopy (CLE), a new optical imaging technology for the urinary tract, which enables in vivo microscopy of suspicious bladder lesions. Real time optical biopsy with CLE could be a useful adjunct to overcome the shortcomings of WLC. In this study, we assess the diagnostic accuracy of CLE as an adjunct to WLC for the diagnosis and grading of bladder cancer. METHODS: Since 2010, consecutive patients scheduled for transurethral resection of bladder tumor at the VA Palo Alto Health Care System were recruited. Patients underwent WLC, CLE, and histologic confirmation of resected tissue. Both targeted (suspicious appearing) and random (normal appearing) biopsies were taken. Diagnostic accuracy for both cancer and grade was determined real time by the operative surgeon and offline in a blinded fashion after image processing. Using histology as the gold standard, the accuracy of WLC, CLE, and WLC ⫹ CLE were calculated. RESULTS: 57 patients and 135 biopsies were prospectively analyzed. Of 78 targeted biopsies, 50 contained cancer (17 low grade, LG, 33 high grade, HG). Overall accuracy for cancer diagnosis in targeted biopsies was 74% for WLC alone, 78% for WLC ⫹ CLE, and 76% for CLE alone (offline). Accuracy for WLC diagnosis of LG cancer was 82%; the real time addition of CLE improved accuracy to 100%. Offline analysis of LG cancer with CLE alone was 65% accurate. For

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HG cancer, accuracy with WLC alone and WLC ⫹ CLE was 52% and 42%, respectively. Offline review with CLE alone resulted in 67% accuracy for diagnosis of HG cancer. CONCLUSIONS: As an adjunct to WLC, CLE offers the potential for real time bladder cancer grading. Real time addition of CLE to WLC improved the diagnostic accuracy of LG cancer. Although LG cancer is largely a white light diagnosis, additional morphologic information from CLE enabled all LG lesions to be identified. Interestingly, the addition of CLE to WLC did not improve HG cancer diagnostic accuracy, and offline analysis with CLE was the most accurate. Offline CLE for HG cancer may help the observer avoid pitfalls of WLC such as potential bias from lesions that appear LG or normal on WLC. Future studies, including prospective multi-center studies, will be needed to validate the current findings. Source of Funding: Supported in part by NIH/National Cancer Institute R01 CA160986

565 PROBING THE METHYLATION STATUS OF DIFFERENT TUMOR SUPPRESSOR GENES FROM BLADDER CANCER PATIENTS Shumaila Bilgrami, shahid pervez, Karachi, Pakistan; Sohail Qureshi, Lahore, Pakistan; Farhat Abbas*, Karachi, Pakistan INTRODUCTION AND OBJECTIVES: Promoter methylation induced silencing of tumor suppressor genes has been implicated for various genes in bladder cancer. We analyzed the promoter methylation in a panel of tumor suppressor genes involved in apoptosis, DNA repair, cell cycle control and progression in non-invasive and invasive bladder cancer. METHODS: Promoter hypermethylation was investigated in the gene promoters of RASSF1a(Ras association domain family member 1), APC (Adenomatous Polyposis Coli), MGMT ( O-6-methylguanineDNA methyltransferase), p16 and p15 in 43 non-invasive and low grade, 33 invasive and high grade bladder cancer and 10 cases with normal bladder mucosa/benign urologic disease using real-time methylation-specific PCR with SYBR green. In addition to the 86 tissue samples, DNA methylation analyses were also carried out in 16 plasma samples from patients with invasive high grade bladder cancer. RESULTS: Promoter hypermethylation was frequently observed in RASSF1a, APC and MGMT genes (p-value⬍0.001) and was prominent in the invasive high grade bladder cancer tissues as compared to the non-invasive low grade group (p⬍0.001) and normal bladder mucosa (p-values 0.040, 0.000 and 0.003 for RASSF1a, APC and MGMT, respectively). When the data from16 plasma samples were compared to the corresponding tissue samples, only APC (p-value 0.006) and p16 (p-value 0.011) showed significance in paired-T test. CONCLUSIONS: Our results suggest that promoter methylation analysis can serve as a valuable tool for monitoring progression of bladder cancers and assessing their spread. Although the data on plasma samples is preliminary, it has encouraged us to interrogate the DNA methylation status of an expanded panel comprised of oncogenes and tumor-suppressor genes in a larger number of samples. Source of Funding: Grant support from “Higher Education Commission”, Pakistan

566 IDENTIFICATION OF GENETIC MARKERS ASSOCIATED WITH ´ RIN INTRAVESICAL RESPONSE TO BACILLUS CALMETTE-GUE THERAPY IN NON-MUSCLE-INVASIVE BLADDER CANCER Shaheen Alanee*, New York, NY; David Gallagher, Dublin, Ireland; Joseph Vijai, Robert Hamilton, Irina Ostrovnaya, Illana Garcia-Grossman, Ashley Regazzie, Jason Littman, NewYork, NY; Przybylo Jennifer, Palo Alto, CA; Christopher Manschreck, NewYork, NY; Mia Gaudet, Atlanta, GA; Matthew Milowsky, Chapel Hill, NC; Harry Herr, Kenneth Offit, Dean Bajorin, NewYork, NY INTRODUCTION AND OBJECTIVES: Bacillus Calmette-Gue´rin (BCG) intravesical therapy has an important role in the management of

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high-risk non-muscle-invasive bladder cancer (NMIBC). However, some patients fail to achieve response so markers predictive of BCG-refractory disease would enhance clinical management. This study examines the association between germline single-nucleotide polymorphisms (SNPs) and response to BCG therapy. METHODS: Saliva or blood samples were collected from patients with NMIBC diagnosed between 1984 and 2010 and treated at a single center. SNPs were selected based on reported associations with bladder cancer and BCG response, and genotyped using the Sequenom MassARRAY iPLEX. No response to BCG was arbitrarily defined as the presence of pathologically documented tumor in the bladder at 6 months after treatment. Logistic regression was used to test the association between the outcome of interest (persistent disease at 6 months) and individual SNPs, as well as clinical variables. A genetic score was created equal to the weighted sum of the number of risk alleles in significant SNPs, where weights were the coefficients from the multivariate logistic regression model. RESULTS: The cohort consisted of 158 patients (median age, 65 years) from our institution who had received intravesical BCG for NMIBC (35.2% stage T1, 32.7% stage Ta, 31.4% stage Tis), with 93% having high-grade disease. At 6 months follow up, 22 (13.9%) patients had shown no response to BCG. Of the 80 SNPs we genotyped, 11 SNPs were associated with lack of response to BCG on univariate analysis, and 8 of them were included in a genetic score. The score was highly significant predictor for lack of response to BCG, even after adjusting for stage (p⫽ 1.3e-09), and OR per unit of increase in genetic score was 2.4 (95% CI 1.7, 3.7) with a bootstrap-adjusted AUC of 90% (95%CI:84-97%) for predicting BCG refractory status. CONCLUSIONS: A SNP based model predictive of BCG-refractory bladder cancer was identified suggesting that host factors may play a substantial role in BCG resistance. A validation study on an independent dataset is planned to determine the clinical utility of these findings.

Source of Funding: None

567 NEW IMMUNOTHERAPY FOR NON MUSCLE INVASIVE BLADDER CANCER (NMIBC): EFFECTS OF IMMUNOMODULATOR P-MAPA Wagner Fa´varo*, Botucatu, Brazil; Athanase Billis, Iseu S. Nunes, Nelson Duran, Campinas, Brazil INTRODUCTION AND OBJECTIVES: Immunotherapy represents one of the approaches for the treatment of cancer. Compounds which are able to act as toll-like receptors (TLRs) agonists may represent promising candidates to be developed as medicines against cancer. BCG is used as a therapeutic tool for some cancer types, including the urothelial cancer. P-MAPA is an acronym for Protein Aggregate Magnesium-Ammonium Phospholinoleate-Palmitoleate Anhydride having significant in vivo antitumor. Thus, the aims of the hereby study were to characterize effects of the P-MAPA on TLRs in vitro and in vivo, as well as to verify its potential as adjuvant therapy for NMIBC. For its purpose, the efficacy of P-MAPA was compared versus BCG in the NMIBC mouse model. METHODS: Thirty female Fisher 344, 7 week old, rats were anesthetized and received 1.5 mg/kg dose of n-methyl-n-nitrosourea (MNU), intravesically every other week for 7 weeks. After MNU treat-