High-grade Muscle-invasive Bladder Research Articles

Outcome of radical cystectomy in remote area of Pakistan: Recep Tayyip Erdogan Hospital, Muzaffargarh (IHHN)

Background: Radical cystectomy with bilateral pelvic lymphadenectomy is the standard treatment for high grade/ muscle invasive urinary bladder tumor. Continent or incontinent urinary diversion is the part of this major operation. High volume centers perform routinely but low volume centers do less frequent. It might be because of availability of equipment and trained surgeon in remote area of Pakistan. Objectives: to assess the results of radical cystectomy at a low-volume center for high-grade muscle-invasive bladder tumours. For patients aged 50 to 75, this involves evaluating surgical complications, overall survival, and the absence of tumour recurrence throughout a one-year follow-up period. Study design: A Retrospective StudyDuration and place of study: Recep Tayyip Erdogan hospital, Muzaffargarh from October 2018 to August 2022 Methods: total Cases from both genders and 50 to 75 years old with High grade/ Muscle invasive urinary bladder carcinoma, incomplete resection during TURBT due to high volume of mass (irresistible) included. Performa designed for collection of information. All the patients underwent same procedure by same urologist. Patients were followed for one year after surgery. Results: All participants were followed for one year. 33.3% patients had paralytic ileus (CDC I), 13.3% had wound infection (CDC I) and 6.7% had urinary leakage (CDC IIIb). There was 86.6% overall survival rate. 6.7% lost to follow up. Mean operative time was 308 ±95 minutes. There was no recurrence noted throughout the study period. Conclusion: Good operative results can also achieve at low volume centers. High level of surveillance and expertise help to achieve better outcomes in remote. Keywords: cystectomy, radical cystectomy, bladder tumor, ileal conduit

BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL as urinary marker for bladder cancer: Final results of a German multicenter study

Introduction and objectiveBTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL are urinary-based rapid tests. This multicenter study is the first study comparing all available rapid tests on a large cohort of bladder cancer patients and healthy controls in one setting. MethodsIn total 732 urine samples (second morning urine) in a real-world assessment have been analyzed. We evaluated clinical samples from 464 patients with histologically confirmed urothelial tumors of the urinary bladder (17 solitary CIS, 189 low-grade, 187 high-grade nonmuscle invasive, 71 high-grade muscle invasive), 77 patients with No Evidence of Disease (NED), and from 191 healthy controls. Urine samples were analyzed by the BTA stat®, NMP22® BladderChek®, UBC® Rapid Test point-of-care (POC) system using the concile Omega 100 POC reader, and CancerCheck® UBC® rapid VISUAL. Sensitivities and specificities were calculated by contingency analyses. ResultsAll investigated urinary markers detected more pathological concentrations in urine of bladder cancer patients compared to tumor-free patients. The calculated diagnostic sensitivities for BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, CancerCheck® UBC® rapid VISUAL, and cytology were 62.4%, 13.4%, 58.2%, 28.6%, 36.2% for low-grade, 83.4%, 49.5%, 84.5%, 63.1%, 71.2% for high-grade nonmuscle invasive, and 95.8%, 35.2%, 76.1%, 50.7%, 67.7% for high-grade muscle-invasive bladder cancer. The specificity was 67.9%, 95.5%, 79.4%, 94.4%, and 83.7%, respectively. The area under the curve (AUC) after receiver operating characteristics (ROC) analysis for high-grade non–muscle-invasive tumors was 0.757, 0.725, 0.819, 0.787, and 0.774, respectively. ConclusionsThe analysis of more than 700 urine samples offers an objective view on urine-based rapid diagnostics. Elevated pathological concentrations of markers in urine of bladder cancer patients were detected in all investigated tests. The highest sensitivities for high-grade non–muscle-invasive tumors were calculated for BTA stat® and UBC® Rapid Test, whereas NMP22® BladderChek®, and cytology showed the highest specificities. BTA stat® and UBC® Rapid Test have the potential to be used as a clinical valuable urinary protein biomarker for the detection of high-grade non–muscle-invasive bladder cancer patients and could be included in the management of these tumors.

The preliminary efficacy of "quadri-combination" therapy on the treatment of bladder cancer

To investigate the safety and efficacy of "quadri-combination" therapy including maximal transurethral resection of bladder tumor (mTURBT), combined with systemic chemotherapy and immunotherapy, concurrent radiotherapy, and immune maintenance therapy. The clinical data of 8 patients with bladder cancer who could not tolerate or refused radical cystectomy at the Department of Urology, Peking University Cancer Hospital from November 2019 to October 2021 were retrospectively analyzed. There were 5 males and 3 females with a mean age of 69 years. The Eastern Cooperative Oncology Group(ECOG) score was 0 in 6 cases and 1 in 2 cases. There were 5 cases of high-grade urothelial carcinoma (1 case of T3b; 2 cases were T2; 2 cases of T1 stage, with multiple tumors and repeated recurrence), 1 case of high-grade urothelial carcinoma with carcinoma in situ (T1/Tis stage), 1 case of high-grade urothelial carcinoma with squamous differentiation (T3b stage), and 1 case of high-grade urothelial carcinoma with glandular differentiation (T2). All patients underwent "quadri-combination" therapy.The patient's tolerance, success rate of bladder preservation and prognosis were evaluated.The median follow-up time was 22.5 (12-35) months. One patient with high-grade muscle-invasive bladder cancer (T2) received mTURBT, albumin-bound paclitaxel and durvalumab combined therapy for 3 cycles, concurrent radiotherapy, and immune maintenance therapy for 18 months, and the tumor recurrence was found. The pathology was high-grade urothelial carcinoma. Salvage radical cystectomy combined with pelvic lymph node dissection is recommended. The remaining 7 patients were regularly reexamined, and no recurrence or metastasis was found.The 2-year progression-free survival rate was 80%, and the success rate of bladder preservation was 87.5%(7/8). Treatment-related adverse reactions were resolved by symptomatic treatment, and patients' compliance and tolerance were acceptable.The "quadri-combination" bladder-preserving therapy is feasible and well tolerated, but further studies are needed.

Primary High Grade Muscle Invasive Bladder Urothelial Carcinoma with Metastasis to the Penis Sparing the Urethra- A Case Presentation and Review of Literature

Background: Metastatic transitional cell carcinoma in the penis is extremely rare. Symptoms like penile pain, swelling, or priapism should raise the suspicion, when they happen in a known case of invasive bladder cancer. Case presentation: We report an unusual case of penile pain and swelling in a 69-year-old man, known to have invasive bladder cancer that was initially treated with radiotherapy. Although both penile ultrasound and MRI suspected hematoma or scarring rather than malignancy, the penile biopsy confirmed metastatic transitional cell carcinoma. Conclusion: Metastatic urothelial carcinoma in the penis is a rare presentation with poor prognosis, where treatment options are very limited and mainly palliative.

Hsa-miR-30a-3p overcomes the acquired protective autophagy of bladder cancer in chemotherapy and suppresses tumor growth and muscle invasion

Bladder cancer (BC) is the second most common urologic cancer in western countries. New strategies for managing high-grade muscle-invasive bladder cancer (MIBC) are urgently required because MIBC has a high risk of recurrence and poor survival. A growing body of evidence indicates that microRNA has potent antitumorigenic properties in various cancers, and thus, therapeutic strategies based on microRNA may show promising results in cancer therapy. Analysis of The Cancer Genome Atlas (TCGA) database indicated that hsa-miR-30a-3p is downregulated in human BC. Our in vitro investigation demonstrated that hsa-miR-30a-3p suppresses the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 and reduces the cell invasive potential of BC cells. Furthermore, hsa-miR-30a-3p directly targets ATG5, ATG12, and Beclin 1; this in turn improves the chemosensitivity of BC cells to cisplatin through the repression of protective autophagy. In a tumor-xenograft mice model, hsa-miR-30a-3p suppressed muscle invasion. Cotreatment with hsa-miR-30a-3p enhanced the antitumor effect of cisplatin in reducing tumor growth in BC. The current study provides a novel strategy of using hsa-miR-30a-3p as an adjuvant or replacement therapy in future BC treatment.

Fibroblast growth factor receptor 3 gene (FGFR3) mutations in high-grade muscle-invasive urothelial bladder cancer in a Brazilian population: evaluation and prevalence.

ABSTRACTObjective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer.Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software.Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient.Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.

Upregulation of IFN\u0263-mediated chemokines dominate the immune transcriptome of muscle-invasive urothelial carcinoma

Tumor inflammation is prognostically significant in high-grade muscle-invasive bladder cancer (MIBC). However, the underlying mechanisms remain elusive. To identify inflammation-associated immune gene expression patterns, we performed transcriptomic profiling of 40 MIBC archival tumors using the NanoString nCounter Human v.1.1 PanCancer Panel. Findings were validated using the TCGA MIBC dataset. Unsupervised and supervised clustering identified a distinctive immune-related gene expression profile for inflammation, characterized by significant upregulation of 149 genes, particularly chemokines, a subset of which also had potential prognostic utility. Some of the most enriched biological processes were lymphocyte activation and proliferation, leukocyte adhesion and migration, antigen processing and presentation and cellular response to IFN-γ. Upregulation of numerous IFN-γ-inducible chemokines, class II MHC molecules and immune checkpoint genes was detected as part of the complex immune response to MIBC. Further, B-cell markers linked to tertiary lymphoid structures were upregulated, which in turn is predictive of tumor response to immunotherapy and favorable outcome. Our findings of both an overall activated immune profıle and immunosuppressive microenvironment provide novel insights into the complex immune milieu of MIBC with inflammation and supports its clinical significance for predicting prognosis and immunotherapeutic responsiveness, which warrants further investigation. This may open novel opportunities to identify mechanisms for developing new immunotherapeutic strategies.

High Grade Muscle-Invasive Urinary Bladder Cancer in A 36 Year Old Male Patient: A Case Report & Review of Literature

We are reporting a case of urothelial bladder cancer in a 36 year old male patient with no history or exposure to any risk factors. The incidence of urothelial bladder cancer is very low in young individuals i.e. below 40 years of age with reported rate of incidence around 0.1-0.4 %. Most of the times, these young individuals present with non-muscle invasive bladder cancer with low grade and low stage. As the age increases, the incidence of high grade bladder cancer increases along with it. The index case presented with high grade muscle invasive bladder cancer at the time of diagnosis without any known risk factors. The 5-year survival of urothelial bladder cancer is better in young patients (93.8 %) as compared to older people(85.1 %). Cigarette smoking is responsible for development of bladder cancer in majority of patients followed by exposure to occupational carcinogens. Role of genetic alterations in development of bladder cancer is still under research and process of urothelial bladder carcinogenesis is unanswered in young individuals.

Racial differences in aneuploidy in high-grade muscle-invasive bladder cancer.

400 Background: Bladder cancer is marked by racial disparities in stage at presentation, treatment, and survival. It is unknown how somatic tumor genomes differ by race. Tumor aneuploidy, defined as aberrant counts of chromosome arms, is pervasive in tumors including bladder cancer. Its associations with race are incompletely understood. We sought here to characterize the relation of race with aneuploidy in high-grade muscle-invasive bladder cancer. Methods: To quantify how aneuploidy differs by race, we leveraged the cohort of patients with high-grade, muscle-invasive bladder cancer from The Cancer Genome Atlas. Chromosome arm gains and losses were identified based on Affymetrix SNP 6.0 arrays (Taylor 2018). We focused on aneuploidy burden defined as the count of chromosome arms altered by gains and losses per tumor, which was based on both p and q chromosome arms of all autosomes except for acrocentric autosomes (q arms only) and ranged from 0 to 34 altered chromosome arms. We used multivariable linear regression to obtain mean differences and 95% confidence intervals (CIs) in the number of altered chromosome arms between self-reported racial groups, adjusting for demographics (sex, age at diagnosis, smoking status) and tumor characteristics (grade, histology). We also evaluated associations between race and number of altered chromosome arms stratified by papillary/non-papillary histology and sex. Results: Of 362 participants, 315 self-identified as White (87%), 25 as Asian (7%), and 22 as Black (6%). 73% were men, 21% were current smokers, and 52% former smokers. Median age at diagnosis was 69 years (interquartile range [IQR]: 61 to 76). Asians and Blacks tended to be younger at diagnosis than Whites, with more never-smokers among Asians. More profiled tumors were of papillary histology among Asians (44%), and fewer among Blacks (18%), than among Whites (30%). Aneuploidy burden was high overall (median, 14 altered chromosome arms; IQR: 8 to 19); only 17% of tumors had five or fewer altered chromosome arms. Compared to Whites, aneuploidy burden overall was similar in tumors from Asians (adjusted mean difference, –2.3 fewer altered chromosome arms; 95% CI: –5.4 to 0.9) and Blacks (–1.7; 95% CI: –5.1 to 1.7). Asian race appeared more strongly associated with lower aneuploidy burden in non-papillary tumors (–3.9 altered chromosome arms; 95% CI: –7.7 to –0.1) than in papillary tumors (–0.3; 95% CI: –5.6 to 5.0), and in men (–3.5; 95% CI: –7.0 to 0.1) than in women (–0.8; 95% CI: –6.8 to 5.2). Conclusions: These findings suggest potential differences in aneuploidy burden between Asians and Whites among high-grade muscle-invasive bladder cancers. They also highlight a need for validation in cohorts that are more racially diverse and have a well-defined source population (study base) with detailed data on cancer risk factors and histopathology.

Abstract A13: Entinostat enhances antitumor immune responses and cooperates with PD-1 blockade in bladder cancer mouse models

Abstract Bladder cancer harbors a high frequency of somatic mutations that occur randomly across the genome. A fraction of these mutations can be expressed and processed into peptides called neoantigens. When presented on the surface of tumor cells by the major histocompatibility complexes (MHC), these neoantigens can invoke an adaptive immune response. In general, a high neoantigen burden is associated with a significant increase in overall survival. However, tumor cells can avoid the detection of immune cells by silencing the expression of the neoantigens. The post-translational acetylation of histone tails is catalyzed by histone acetyltransferases (HATs) and is counterbalanced by histone deacetylases (HDACs), which mediate their removal. Histone acetylation is associated with open chromatin and active transcription. HDAC inhibition would, therefore, promote histone acetylation and restore the expression of suppressed genes. Many of the frequently mutated genes in bladder cancer are involved in chromatin remodeling, such as KMT2D, KDM6A, and KMT2C, suggesting the contribution of epigenetic dysregulation to disease progression. Therefore, therapy that targets epigenetic processes such as HDAC inhibition is a rational treatment approach for bladder cancer. Here, we evaluated the antitumor efficacy and mechanisms of entinostat, a selective HDAC1 and HDAC3 inhibitor, in a preclinical murine model of high-grade muscle-invasive bladder cancer, BBN963. In this study, BBN963 cells were implanted subcutaneously into immunocompetent C57BL/6 (B6) mice or immunodeficient NOD/scid/gamma (NSG) mice. Entinostat exhibited robust in vivo activity in B6 mice implanted with BBN963 cells with a number of complete responses. However, the effect was much weaker in BBN963 cells grown in NSG mice. RNAseq analysis demonstrated increased immune gene signature expression in entinostat treated tumors from B6 (but not NSG) mice. Flow cytometry verified the increased numbers of CD8+ effector memory T cells. Most interestingly, the expressions of predicted neoantigens were almost completely lost in the entinostat-treated tumors in B6 mice but were unchanged in tumors from NSG mice. Additionally, the in vivo antitumor effect of entinostat in the BBN963 cells lacking MHC class 1 was significantly reduced. Finally, the combination of anti-PD1 therapy and entinostat robustly out-performed each agent alone in inhibiting BBN963 tumor growth with 67% incident of complete response. These data support the hypothesis that entinostat mediates antitumor effect through enhanced neoantigen expression and antigen-driven immune response. Citation Format: Andrew S. Truong, Mi Zhou, Takanobu Utsumi, Bhavani Krishnan, Kyle G. Stewart, Ryoichi Saito, Ujjawal Manocha, Sean T. Bailey, William Y. Kim. Entinostat enhances antitumor immune responses and cooperates with PD-1 blockade in bladder cancer mouse models [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A13.

Overexpression of long non-coding RNA TUG1 predicts poor prognosis and promotes cancer cell proliferation and migration in high-grade muscle-invasive bladder cancer.

Long non-coding RNA TUG1 is involved in the development and progression of a variety of tumors. Little is known about TUG1 function in high-grade muscle-invasive bladder cancer (MIBC). The aims of our study were to determine expression levels of long non-coding RNA TUG1 in tumor tissue, to evaluate its relationship with clinico-pathological features of high-grade MIBC, and to describe its function in MIBC cells in vitro. TUG1 expression levels were determined in paired tumor and adjacent non-tumor bladder tissues of 47 patients with high-grade MIBC using real-time PCR. Cell line T-24 and siRNA silencing were used to study the TUG1 function in vitro. We observed significantly increased levels of TUG1 in tumor tissue in comparison to adjacent non-tumor bladder tissue (P<0.0001). TUG1 levels were significantly increased in metastatic tumors (P=0.0147) and were associated with shorter overall survival of MIBC patients (P=0.0241). TUG1 silencing in vitro led to 34% decrease in cancer cell proliferation (P=0.0004) and 23% reduction in migration capacity of cancer cells (P<0.0001). We did not observe any significant effects of TUG1 silencing on cell cycle distribution and number of apoptotic cells. Our study confirmed overexpression of TUG1 in MIBC tumor tissue and described its association with worse overall survival in high-grade MIBC patients. Together with in vitro observations, these data suggest an oncogenic role of TUG1 and its potential usage as biomarker or therapeutic target in MIBC.

MP46-01 AKR1C3 ACTIVATION AND INTRACRINE ANDROGENS CONFER RESISTANCE TO ENZALUTAMIDE

INTRODUCTION AND OBJECTIVES: The metabolism of cancer cells is fundamentally different from that of their normal counterparts. The M2 isoenzyme of pyruvate kinase (PKM2), expressed primarily during embryogenesis, is switched back on and exerts two major effects during oncogenesis: (i) slowing down glycolysis thus shunting glycolytic intermediates for anabolic process necessary for cell proliferation; and (ii) interacting with transcriptional factors thus upregulating growth-promoting genes. The role of PKM2 in bladder cancer formation and progression is presently unknown. METHODS: Human specimens and cultured cells from normal urothelia, low-gradenon-invasive and high-grademuscle-invasive bladder cancerwereassessed forPKM2expressionby immunohistochemistryand immunoblotting. Selected cancer cell lines over-expressing PKM2 were treated with shikonin, a naphthoquinone inhibitor of PKM2 found in medicinal plants, alone or in combination with rapamycin, an mTOR inhibitor, and their effects on cell proliferation, metabolism, apoptotic, autophagic and unfolded protein responses were determined using biochemical and molecular approaches. Target-specific effects of shikonin were verified by transfection with PKM2-specifc small interfering RNA (siRNA). RESULTS: Compared to normal urothelia, both low-grade noninvasive and high-grade muscle-invasive bladder cancer markedly over-express PKM2. Accompanying this is the hyper-phosphorylation of tyrosine residue at codon Y105 and nuclear translocation of PKM2. Cultured bladder cancer cell lines including UroTsa, RT4, RT112, J82, T24 and UMUC3 also express significantly more PKM2 than primarycultured urothelial cells. Exposure of RT112 and T24 to shikonin results in a dose-dependent inhibition of proliferation, reduced oxygen consumption and marked upregulation of signals indicative of apoptosis, autophage and unfolded protein response. A greater inhibition of cell proliferation is observed when shikonin is combined with rapamycin, suggesting a synergism between PKM2 and mTOR inhibition. Enforced expression of siRNA of PKM2 produces a similar inhibitory effect on cell line proliferation, establishing target-specific effects of shikonin. CONCLUSIONS: These data demonstrate for the first time an important role of PKM2 overexpression in bladder cancer formation and progression, and suggest a potentially unconventional way to therapeutically inhibit bladder cancer via specific alteration of tumor-promoting metabolism.

MP45-19 UPREGULATION OF ISOENZYME M2 OF PYRUVATE KINASE IN BLADDER CANCER: IMPLICATION IN TUMOR INITIATION, PROGRESSION AND THERAPEUTIC TARGETING.

INTRODUCTION AND OBJECTIVES: The metabolism of cancer cells is fundamentally different from that of their normal counterparts. The M2 isoenzyme of pyruvate kinase (PKM2), expressed primarily during embryogenesis, is switched back on and exerts two major effects during oncogenesis: (i) slowing down glycolysis thus shunting glycolytic intermediates for anabolic process necessary for cell proliferation; and (ii) interacting with transcriptional factors thus upregulating growth-promoting genes. The role of PKM2 in bladder cancer formation and progression is presently unknown. METHODS: Human specimens and cultured cells from normal urothelia, low-gradenon-invasive and high-grademuscle-invasive bladder cancerwereassessed forPKM2expressionby immunohistochemistryand immunoblotting. Selected cancer cell lines over-expressing PKM2 were treated with shikonin, a naphthoquinone inhibitor of PKM2 found in medicinal plants, alone or in combination with rapamycin, an mTOR inhibitor, and their effects on cell proliferation, metabolism, apoptotic, autophagic and unfolded protein responses were determined using biochemical and molecular approaches. Target-specific effects of shikonin were verified by transfection with PKM2-specifc small interfering RNA (siRNA). RESULTS: Compared to normal urothelia, both low-grade noninvasive and high-grade muscle-invasive bladder cancer markedly over-express PKM2. Accompanying this is the hyper-phosphorylation of tyrosine residue at codon Y105 and nuclear translocation of PKM2. Cultured bladder cancer cell lines including UroTsa, RT4, RT112, J82, T24 and UMUC3 also express significantly more PKM2 than primarycultured urothelial cells. Exposure of RT112 and T24 to shikonin results in a dose-dependent inhibition of proliferation, reduced oxygen consumption and marked upregulation of signals indicative of apoptosis, autophage and unfolded protein response. A greater inhibition of cell proliferation is observed when shikonin is combined with rapamycin, suggesting a synergism between PKM2 and mTOR inhibition. Enforced expression of siRNA of PKM2 produces a similar inhibitory effect on cell line proliferation, establishing target-specific effects of shikonin. CONCLUSIONS: These data demonstrate for the first time an important role of PKM2 overexpression in bladder cancer formation and progression, and suggest a potentially unconventional way to therapeutically inhibit bladder cancer via specific alteration of tumor-promoting metabolism.

Increased expression of L-selectin (CD62L) in high-grade urothelial carcinoma: A potential marker for metastatic disease

IntroductionL-Selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leukocytes with a primary function of directing leukocyte migration and homing of lymphocytes to lymph nodes. In a gene expression microarray study comparing laser-captured microdissected high-grade muscle-invasive bladder cancer (MIBC) without prior treatment and low-grade bladder cancer (LGBC) human samples, we found CD62L to be the highest differentially expressed gene. We sought to examine the differential expression of CD62L in MIBCs and its clinical relevance. MethodsUnfixed fresh and formalin-fixed paraffin-embedded human bladder cancer specimens and serum samples were obtained from the University of Connecticut Health Center tumor bank. Tumor cells were isolated from frozen tumor tissue sections by laser-captured microdissected followed by RNA isolation. Quantitative polymerase chain reaction was used to validate the level of CD62L transcripts. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to evaluate the CD62L protein localization and expression level. Flow cytometry was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine database. ResultsImmunostaining showed a uniformly higher expression of CD62L in MIBC specimens vs. LGBCs specimens. Further, CD62L localization was seen in foci of metastatic tumor cells in lymph node specimens from patients with high-grade MIBC and known nodal involvement. Up-regulated expression of CD62L was also observed by flow cytometric analysis of freshly isolated tumor cells from biopsies of high-grade cancers vs. LGBC specimens. Circulating CD62L levels were also found to be higher in serum samples from patients with high-grade metastatic vs. high-grade nonmetastatic MIBC. In addition, in silico analysis of Oncomine Microarray Database showed a significant correlation between CD62L expression and tumor aggressiveness and clinical outcomes. ConclusionThese data confirm the expression of CD62L on urothelial carcinoma cells and suggest that CD62L may serve as biomarker to predict the presence of or risk for developing metastatic disease in patients with bladder cancer.

Abstract 53: Increased expression of L-selectin (CD62L) in high grade bladder cancer: a potential biomarker for lymph node metastasis

Abstract Introduction and Objectives: L-selectin (CD62L) is a vascular adhesion molecule which is primarily expressed on leucocytes. Its principal function is to direct leucocyte migration and homing to lymph nodes (LNs). In a gene expression microarray study of laser captured micro-dissected high grade muscle invasive bladder cancer (MIBC) vs. low grade bladder cancer (LGBC) from human specimens, we found CD62L to be the highest differentially expressed gene. We sought to examine the differential expression of CD62L in MIBCs and its clinical relevance. Methods: Unfixed fresh and formalin fixed paraffin-embedded human bladder cancer specimens and serum samples were obtained from the UCHC tumor bank. Tumor cells were isolated from 5-10 µm sections of frozen tumor tissue (3 MIBC and 3 LGBC) by laser capture microdissection followed by RNA isolation. Preparation of cRNA and hybridization to Agilent Human GE 4x44K v2 microarray chips was performed by GenUS Biosystems (Northbrook, IL). qPCR was used to validate the level of CD62L transcripts. Immunohistochemistry and ELISA were performed to evaluate the CD62L protein localization and expression level. Flow cytometry was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine Database. Results: Two-hundred fifty genes were differentially expressed (≥ 2-fold) between the groups with 86 overexpressed in MIBC compared to LGBC. The most highly elevated transcript (34-fold) was L-selectin. The increased expression of CD62L transcript in high grade MIBC vs. LGBC specimens was validated by qPCR. Using 12 human bladder tumor samples representing high grade MIBC and LGBC, we found a uniformly higher expression level of CD62L in MIBC samples. Further, CD62L localization was seen in foci of metastatic tumor cells in LN specimens from patients with high grade MIBC and known nodal involvement. Up-regulated expression of CD62L was also observed by flow cytometry of freshly isolated tumor cells from biopsies of high grade MIBC vs. LGBC patients. Circulating CD62L levels were also found to be higher (640 ± 75 vs. 558 ± 34 ng/ml) in serum samples from patients with high grade metastatic vs. high grade non-metastatic MIBC. In addition, in silico analysis of Oncomine Microarray Database showed a significant correlation between CD62L expression and tumor aggressiveness and clinical outcomes. Conclusion: These findings suggest that elevated CD62L expression may serve as a marker of either occult metastatic disease or the propensity of a tumor to metastasize. Citation Format: Dharamainder Choudhary, Poornima Hegde, Shilpa Choudhary, Kevin Claffey, Pramod Srivastava, Carol C. Pilbeam, John Arthur Taylor. Increased expression of L-selectin (CD62L) in high grade bladder cancer: a potential biomarker for lymph node metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2014-53

Evaluation of risk of muscle invasion, perivesical and/or lymph node affectation by diffusion-weighted magnetic nuclear resonance in the patient who is a candidate for radical cystectomy

Abstract Introduction Preoperative staging of bladder cancer using imaging methods has serious limitations. The accuracy of the abdominal diffusion-weighted magnetic resonance (DW-MRI) to predict residual muscle invasion, perivesical and/or lymph node affectation in the cystectomy specimen is evaluated. Material and methods A prospective study was performed on 20 patients with high grade muscle invasive bladder cancer who received transurethral resection of the bladder (TURB) in a period of S ), specificity (sp), positive predictive value (PPV), negative predictive value (NPV) and accuracy (Ac) of the test were analyzed. The medians of the apparent diffusion coefficient (ADC) value (Mann–Whitney) were compared and the ROC curves study for DW-MRI and ADC was carried out. Results Distribution by categories was: pT0 1(5%), pT1 6(30%), pT2 2(10%), pT3 8(40%) and pT4 3(15%). There was an agreement in the T-pT assignment in 17(85%). In 7(35%) there was lymph node affectation (pN1-2). Consistency of the DW-MRI for muscle affectation was k = 0.89 (CI 0.67–1; S = 1.0, Sp = 0.86, PPV = 0.93, NPV = 1.0, Ac = 0.95), for perivesical fat affectation k = 0.6 (CI 0.25–0.95; S = 0.8, Sp = 0.8, PPV = 0.8, NPV = 0.8, Ac = 0.8) and for lymph node affectation k = 0.89 (CI 0.67–1; S = 0.86, Sp = 1.0, PPV = 1.0, NPV = 0.93, Ac = 0.95). Mean value of ADC was greater in G2 tumors (OMS1987) compared to G3 ( p = 0.08). Evaluation of DW-MRI imaging and ADC numerical value showed equivalent areas under the curve for muscle (0.93 and 0.9; Z = 0.7), fat (0.8 and 0.91; Z = 0.31) and lymph node (0.93 and 0.97; Z = 0.36) affectation, respectively. Conclusions DW-MRI allows for good pre-operative evaluation of the patient who is a candidate for cystectomy, especially for the prediction of muscle (

Evaluación del riesgo de invasión muscular, afectación perivesical y/o ganglionar mediante resonancia magnética nuclear balanceada con difusión en el paciente candidato a cistectomía radical

IntroductionPreoperative staging of bladder cancer using imaging methods has serious limitations. The accuracy of the abdominal diffusion-weighted magnetic resonance (DW-MRI) to predict residual muscle invasion, perivesical and/or lymph node affectation in the cystectomy specimen is evaluated. Material and methodsA prospective study was performed on 20 patients with high grade muscle invasive bladder cancer who received transurethral resection of the bladder (TURB) in a period of <1 month. The DW-MRI was performed before the radical cystectomy and the radiologist predicted muscle invasion, extravesical affectation and lymph node affectation, being blind to the histopathological study. Sensitivity (S), specificity (sp), positive predictive value (PPV), negative predictive value (NPV) and accuracy (Ac) of the test were analyzed. The medians of the apparent diffusion coefficient (ADC) value (Mann-Whitney) were compared and the ROC curves study for DW-MRI and ADC was carried out. ResultsDistribution by categories was: pT0 1(5%), pT1 6(30%), pT2 2(10%), pT3 8(40%) and pT4 3(15%). There was agreement in the T-pT assignment in 17(85%). In 7(35%) there was lymph node affectation (pN1-2). Consistency of the DW-MRI for muscle affectation was k=.89 (CI .67-1; S=1.0, Sp=,86, PPV=.93, NPV=1.0, Ac=.95), for perivesical fat affectation k=.6 (CI .25-.95; S=.8, Sp=.8, PPV=.8, NPV=.8, Ac=.8) and for lymph node affectation k=.89 (CI .67-1; S=.86, Sp=1.0, PPV=1.0, NPV=.93, Ac=.95). Mean value of ADC was greater in G2 tumors (OMS1987) compared to G3 (p=.08). Evaluation of DW-MRI imaging and ADC numerical value showed equivalent areas under the curve for muscle (.93 and .9; Z=.7), fat (.8 and .91; Z=.31) and lymph node (.93 and .97; Z=.36) affectation, respectively. ConclusionsDW-MRI allows for good pre-operative evaluation of the patient who is a candidate for cystectomy, especially for the prediction of muscle (<pT2 vs ≥pT2) and/or lymph node (N0 vs N1-2) affectation. Both are key points to choice the therapeutic attitude after the bladder TURB. Furthermore, the ADC coefficient also predicts tumor differentiation grade.

Prognostic significance of second mitochondria-derived activator of caspase expression in bladder cancer and target for therapy.

e15000 Background: Although the expression of second mitochondria-derived activator of caspase (Smac/DIABLO) has been reported in various cancers, little is known about its clinical significance in bladder cancer. The present study was designed to evaluate the relationship between progression of disease and Smac/DIABLO expression by clinical pathological analysis of patients with bladder cancer. Methods: The level of Smac/DIABLO expression was quantified by western blot analysis using non-fixed fresh frozen tissues derived from patients with bladder cancer. Results: All normal bladders expressed Smac/DIABLO. However, 64/84 ( 76% ) of bladder cancers expressed Smac/DIABLO and 24% were negative. In Ta and T1 superficial bladder cancers, 98% expressed Smac/DIABLO, whereas only 41% expressed Smac/DIABLO in muscle-invasive bladder cancers. Smac/DIABLO expression inversely correlated with the grade of bladder cancer. Patients with Ta and T1 superficial bladder cancer with higher Smac/DIABLO expression had a longer postoperative recurrence-free period than those with lower Smac/DIABLO expression after transurethral resection in the 5-year follow-up. Patients with invasive bladder cancer expressing Smac/DIABLO had a longer postoperative disease-specific survival than those without Smac/DIABLO expression after radical cystectomy in the 5-year follow-up. The cisplatin-resistant T24 bladder cancer line ( T24/CDDP ) and the adriamycin-resistant T24 line ( T24/ADR ) showed lower level of Smac/DIABLO expression, compared with the T24 parental line. Conclusions: The present study demonstrates for the first time that Smac/DIABLO expression was downregulated in bladder cancer, especially in high grade muscle-invasive bladder cancer, and that lower Smac/DIABLO expression in bladder cancer predicted a worse prognosis. In addition, the cisplatin-resistant T24/CDDP line and the adriamycin-resistant T24/ADR line expressed lower level of Smac/DIABLO expression. These results suggest that Smac/DIABLO expression in bladder cancer may be used as a prognostic parameter, and that low Smac/DIABLO expression in bladder cancer may be associated with resistance to chemotherapy.

Mid‐term oncological control after laparoscopic radical cystectomy in men: a single‐centre experience

• To assess the mid-term (3 years of follow-up) oncological control of laparoscopic radical cystectomy (LRC) for high-grade muscle-invasive bladder cancer in a well studied male population. • We assessed 40 men with bladder cancer (mean [range] age 66.5 [50-75] years) who underwent LRC and extended pelvic lymphadenectomy at our institution between April 2004 and September 2008. • Of the 40 patients, 13 (32.5%) had a complete laparoscopic procedure (ileal conduit: seven patients; neobladder: five patients; bilateral ureterostomy: one patient) and 27 (67.5%) had an LRC procedure only (ileal conduit: 15 patients; neobladder: 12 patients). • No major complications were observed intraoperatively. • The mean operating time was 407 min and the mean blood loss was 720 mL. Four patients (10%) required conversion to open surgery. The mean (range) hospital stay was 10.2 (7-25) days. One patient died of myocardial infarction in the postoperative period. • Pathological analysis showed organ-confined tumours (stage pT0/pT1/pT2/pT3a) in 22 patients (55%) and extravesical disease (pT3/pT4) in 18 (45%). Of the 40 patients, six (15%) had lymph node involvement. The mean (range) number of nodes removed was 19.9 (5-40). • At a mean (range) follow-up period of 36 (0-72) months, 26 patients were alive with no evidence of disease (disease-free survival rate 67%). • Laparoscopic radical cystectomy is a safe, feasible, and effective alternative to open radical cystectomy (ORC). The 3-year oncological efficacy was comparable with that of ORC.

Telomerase activity as a potential marker in preneoplastic bladder lesions.

To assess telomerase activity (involved in cell immortalization and detectable in most malignant tumours but not in normal somatic tissues) as a marker in cancer diagnosis. Tissue telomerase activity was assayed by two different techniques, the telomeric repeat amplification protocol-polymerase chain reaction (TRAP-PCR) and a telomerase PCR-enzyme linked immunosorbent assay. Malignant and inflammatory bladder lesions and their adjacent normal tissues were assessed for telomerase activity in a group of 18 patients, 14 of whom had urothelial carcinoma and four a nonspecific inflammatory lesion of the bladder. Eleven of the 14 tumour samples analysed were telomerase-positive and two of the three telomerase-negative tumour samples had a detectable 'telomerase inhibitor'. In the apparently normal tissues next to bladder tumours, four of the 14 specimens were telomerase-positive. Interestingly, these lesions were always next to high-grade muscle-invasive bladder tumours (pT2G3). Two of the four nonspecific inflammatory lesions (one of cystitis glandularis and one of severe dysplasia), known to be preneoplastic lesions, were also telomerase-positive. These results strongly suggest that the reactivation of telomerase may be an early event in bladder carcinogenesis, preceding morphological changes related to malignant transformation. Telomerase activity may therefore be useful both as an indicator of malignant potential in preneoplastic lesions, e.g. cystitis glandularis and severe dysplasia, and as a prognostic marker of bladder tumour relapse or progression.