- Email: [email protected]
MP88-20 EVALUATION OF THE BIOLOGICAL BASIS OF UROTHELIAL CARCINOMA WITH SQUAMOUS DIFFERENTIATION
THE JOURNAL OF UROLOGYâ
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measured the expression of all HAase family members in clinical specimens and evaluated their function. METHODS: Q-PCR was performed for measuring the mRNA levels of 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) ¼ 22; tumor (TBL) ¼ 37) and 160 urine specimens (BCa ¼ 52; normal ¼ 18; history of BCa ¼ 30; benign conditions ¼ 59). By stable transfection of normal urothelial (Urotsa) and BCa (T24, 253J-Lung, HT1376) cells, HYAL4 function was analyzed in vitro (proliferation, motility, invasion, immunoblotting, Q-PCR assays) and in vivo (s.c and orthotopic xenograft models). RESULTS: Among the 6 HAases, HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 2.7; 29.7 21.9; 26.7 21.9), when compared to NBL tissues (p<0.001). In multivariate analysis, in addition to HYAL1, HYAL4 was an independent prognosticator of metastasis and death due to BCa (p<0.001). HYAL1, HYAL4 and HYALP1 mRNA levels were also significantly elevated in BCa patients0 urine (p< 0.001). Urinary HYAL-4 mRNA levels had reasonable accuracy (72%) to detect high-grade BCa. Overexpression of HYAL4 in normal urothelial and BCa cells significantly increased their invasive potential and chemotactic motility (>3fold). This increase was due to up-regulation of metastasis promoters, and stem cell phenotype. In s.c. and orthotopic BCa models, HYAL4 expression increased both tumor growth and metastasis to lung and spleen. CONCLUSIONS: This is the first study that links HYAL4 to cancer biology. HYAL4 is a potential biomarker of high-grade BCa and metastasis and promotes BCa growth and progression. Source of Funding: Grant NCI/NIH 14R01CA72821; 5R01CA176691-02; Marie C. Hupe, Judith Knapp & Martin J.P. Hennig were fellows of the Biomedical Exchange Program and received funding from the German Academic Exchange Service (DAAD).
MP88-19 URINARY MICROBIOME PATTERNS APPEARS TO BE CORRELATIVE TO INTRAVESICAL RECURRENCE OF NON-MUSCLE INVASIVE BLADDER CANCER Yige Bao*, Chengdu, China, People’s Republic of; Hassan Razvi, Gregory Gloor, Gregor Reid, John Denstedt, Jeremy Burton, London, Canada INTRODUCTION AND OBJECTIVES: Several studies have recently reported association between various urinary conditions. The objective of this study was to describe the urinary microbiome among bladder cancer patients, observe longitudinal urinary microbiome changes during surgery and chemotherapy, and identify microbiome patterns that are associated with frequent bladder cancer recurrences. METHODS: After optimising urine sample collection, storage and processing methods for microbiome analysis, we carried out a prospective, multicenter cohort study, recruiting patients with newly diagnosed Ta, T1 or CIS transitional cell carcinoma of the bladder who underwent transurethral resection with or without intravesical instillation. Urine samples were collected at multiple time points before and after surgery as the patients were followed up for recurrence. Routine urine culture, enhanced culture, qPCR quantification and V4 16S rRNA gene sequencing were performed on urine samples for microbiome analysis. Urine samples were also analysed from gender and age-matched healthy volunteer controls. RESULTS: To eliminate “background” 16S rRNA amplification we found it necessary to collect at least 20 ml of urine and store the pellet in -80 C. We also needed to treat samples with PMA to bind any contaminating environmental DNA present. Ninety-nine patients were recruited from two medical centers and followed up for up to three
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
years. When compared to healthy individuals, bladder cancer patients had urinary microbiome with increased alpha (within sample) diversity with a decreased proportion of “typically” commensal species. Healthy individuals generally maintained a stable urinary microbiome during the longitudinal follow up, while cancer patients’ urinary microbiome were disrupted during periods following surgery and subsequent chemotherapy, though often a restored community profile was observed in the follow up. A common urinary microbiome pattern was identified among patients with frequent recurrences compared to patients with no recurrence. CONCLUSIONS: The urinary microbiome maybe indicative of likely recurrence among non-muscle invasive bladder cancer patients Source of Funding: W Garfield Weston Foundation National Natural Science Foundation of China, cat# 81500522
MP88-20 EVALUATION OF THE BIOLOGICAL BASIS OF UROTHELIAL CARCINOMA WITH SQUAMOUS DIFFERENTIATION Thomas Sanford*, Maxwell Meng, Sima Porten, San Francisco, CA INTRODUCTION AND OBJECTIVES: Squamous differentiation is found in up to 60% of patients with urothelial carcinoma of the bladder. There is conflicting evidence regarding clinical relevance of aberrant urothelial differentiation - both population-based and retrospective studies have reported conflicting results regarding the correlation between squamous differentiation and poor prognosis/advanced stage. The biology of these tumors is poorly understood but some suggest that urothelial carcinoma with extensive squamous differentiation may resemble pure squamous tumors with similar responses to chemotherapy, radiation, and higher rates of local recurrence. Our aim was to compare urothelial carcinoma with squamous differentiation with urothelial carcinoma without any variant histology at the genomic level to better elucidate targetable differences. METHODS: We reviewed pathology reports for 412 patients with urothelial carcinoma from The Cancer Genome Atlas Project (TCGA) to identify patients with any amount squamous differentiation. Copy number alteration data obtained via the GISTC algorithm was downloaded from cbioportal.com. Chi square analysis was used to compare chromosomal amplifications in samples with squamous differentiation compared with samples that had no amount of variant histology. RESULTS: 50 patients (12%) had a report of at least 1% squamous differentiation. Of these 50 patients with evidence of squamous differentiation, there were 15 patients in which there was a comment regarding the percent of involvement. On average, 17% of the sample had squamous differentiation. A total of 257 (62%) of patients had no variant histology. Our analysis showed there were multiple specific sites in the 3q21-26 chromosomal region that were amplified in samples with squamous histology compared with pure urothelial carcinoma histology. The specific sites found to be amplified included SOX2 and PIK3. CONCLUSIONS: Amplifications of chromosome 3q21-26 were enriched in patients with squamous differentiation compared to those with pure urothelial carcinoma of the bladder. Amplification of this region has been reported in squamous transformation in multiple sites, including lung cancer. This study shows that, similar to other cancers, SOX2 may be necessary for squamous transformation. Furthermore, squamous cancer, which has traditionally been chemo-resistant, may respond to agents that target amplifications of genes known to be found on the long arm of chromosome 3 including the PIK3-Akt pathway. Source of Funding: none
e1136
measured the expression of all HAase family members in clinical specimens and evaluated their function. METHODS: Q-PCR was performed for measuring the mRNA levels of 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) ¼ 22; tumor (TBL) ¼ 37) and 160 urine specimens (BCa ¼ 52; normal ¼ 18; history of BCa ¼ 30; benign conditions ¼ 59). By stable transfection of normal urothelial (Urotsa) and BCa (T24, 253J-Lung, HT1376) cells, HYAL4 function was analyzed in vitro (proliferation, motility, invasion, immunoblotting, Q-PCR assays) and in vivo (s.c and orthotopic xenograft models). RESULTS: Among the 6 HAases, HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 2.7; 29.7 21.9; 26.7 21.9), when compared to NBL tissues (p<0.001). In multivariate analysis, in addition to HYAL1, HYAL4 was an independent prognosticator of metastasis and death due to BCa (p<0.001). HYAL1, HYAL4 and HYALP1 mRNA levels were also significantly elevated in BCa patients0 urine (p< 0.001). Urinary HYAL-4 mRNA levels had reasonable accuracy (72%) to detect high-grade BCa. Overexpression of HYAL4 in normal urothelial and BCa cells significantly increased their invasive potential and chemotactic motility (>3fold). This increase was due to up-regulation of metastasis promoters, and stem cell phenotype. In s.c. and orthotopic BCa models, HYAL4 expression increased both tumor growth and metastasis to lung and spleen. CONCLUSIONS: This is the first study that links HYAL4 to cancer biology. HYAL4 is a potential biomarker of high-grade BCa and metastasis and promotes BCa growth and progression. Source of Funding: Grant NCI/NIH 14R01CA72821; 5R01CA176691-02; Marie C. Hupe, Judith Knapp & Martin J.P. Hennig were fellows of the Biomedical Exchange Program and received funding from the German Academic Exchange Service (DAAD).
MP88-19 URINARY MICROBIOME PATTERNS APPEARS TO BE CORRELATIVE TO INTRAVESICAL RECURRENCE OF NON-MUSCLE INVASIVE BLADDER CANCER Yige Bao*, Chengdu, China, People’s Republic of; Hassan Razvi, Gregory Gloor, Gregor Reid, John Denstedt, Jeremy Burton, London, Canada INTRODUCTION AND OBJECTIVES: Several studies have recently reported association between various urinary conditions. The objective of this study was to describe the urinary microbiome among bladder cancer patients, observe longitudinal urinary microbiome changes during surgery and chemotherapy, and identify microbiome patterns that are associated with frequent bladder cancer recurrences. METHODS: After optimising urine sample collection, storage and processing methods for microbiome analysis, we carried out a prospective, multicenter cohort study, recruiting patients with newly diagnosed Ta, T1 or CIS transitional cell carcinoma of the bladder who underwent transurethral resection with or without intravesical instillation. Urine samples were collected at multiple time points before and after surgery as the patients were followed up for recurrence. Routine urine culture, enhanced culture, qPCR quantification and V4 16S rRNA gene sequencing were performed on urine samples for microbiome analysis. Urine samples were also analysed from gender and age-matched healthy volunteer controls. RESULTS: To eliminate “background” 16S rRNA amplification we found it necessary to collect at least 20 ml of urine and store the pellet in -80 C. We also needed to treat samples with PMA to bind any contaminating environmental DNA present. Ninety-nine patients were recruited from two medical centers and followed up for up to three
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
years. When compared to healthy individuals, bladder cancer patients had urinary microbiome with increased alpha (within sample) diversity with a decreased proportion of “typically” commensal species. Healthy individuals generally maintained a stable urinary microbiome during the longitudinal follow up, while cancer patients’ urinary microbiome were disrupted during periods following surgery and subsequent chemotherapy, though often a restored community profile was observed in the follow up. A common urinary microbiome pattern was identified among patients with frequent recurrences compared to patients with no recurrence. CONCLUSIONS: The urinary microbiome maybe indicative of likely recurrence among non-muscle invasive bladder cancer patients Source of Funding: W Garfield Weston Foundation National Natural Science Foundation of China, cat# 81500522
MP88-20 EVALUATION OF THE BIOLOGICAL BASIS OF UROTHELIAL CARCINOMA WITH SQUAMOUS DIFFERENTIATION Thomas Sanford*, Maxwell Meng, Sima Porten, San Francisco, CA INTRODUCTION AND OBJECTIVES: Squamous differentiation is found in up to 60% of patients with urothelial carcinoma of the bladder. There is conflicting evidence regarding clinical relevance of aberrant urothelial differentiation - both population-based and retrospective studies have reported conflicting results regarding the correlation between squamous differentiation and poor prognosis/advanced stage. The biology of these tumors is poorly understood but some suggest that urothelial carcinoma with extensive squamous differentiation may resemble pure squamous tumors with similar responses to chemotherapy, radiation, and higher rates of local recurrence. Our aim was to compare urothelial carcinoma with squamous differentiation with urothelial carcinoma without any variant histology at the genomic level to better elucidate targetable differences. METHODS: We reviewed pathology reports for 412 patients with urothelial carcinoma from The Cancer Genome Atlas Project (TCGA) to identify patients with any amount squamous differentiation. Copy number alteration data obtained via the GISTC algorithm was downloaded from cbioportal.com. Chi square analysis was used to compare chromosomal amplifications in samples with squamous differentiation compared with samples that had no amount of variant histology. RESULTS: 50 patients (12%) had a report of at least 1% squamous differentiation. Of these 50 patients with evidence of squamous differentiation, there were 15 patients in which there was a comment regarding the percent of involvement. On average, 17% of the sample had squamous differentiation. A total of 257 (62%) of patients had no variant histology. Our analysis showed there were multiple specific sites in the 3q21-26 chromosomal region that were amplified in samples with squamous histology compared with pure urothelial carcinoma histology. The specific sites found to be amplified included SOX2 and PIK3. CONCLUSIONS: Amplifications of chromosome 3q21-26 were enriched in patients with squamous differentiation compared to those with pure urothelial carcinoma of the bladder. Amplification of this region has been reported in squamous transformation in multiple sites, including lung cancer. This study shows that, similar to other cancers, SOX2 may be necessary for squamous transformation. Furthermore, squamous cancer, which has traditionally been chemo-resistant, may respond to agents that target amplifications of genes known to be found on the long arm of chromosome 3 including the PIK3-Akt pathway. Source of Funding: none