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Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
Bladder Cancer Diagnostic-Initial Team Project
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Bladder Cancer Diagnostic-Initial Team Project

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A mini-project to find biomarkers for bladder cancer diagnosis. We narrowed down our list of viable candidates down to three that could be used in combination to provide sensitivity and specificity …

A mini-project to find biomarkers for bladder cancer diagnosis. We narrowed down our list of viable candidates down to three that could be used in combination to provide sensitivity and specificity values greater than 94%. Furthermore, we calculated long-term monitoring and payor costs as well as potential profit.

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  • Advantages of using miRs:
    - Appear stable ex vivo when stored at room temperature
    - Small size makes them less likely to fragment
    - Lack of polyadenylated tail
    - Secreted within exosomes, protect RNA from degradation
    - Possible to multiplex miR assays into custom combinations to detect specific diseases
    - Low abundance miRs are not reliably detected in urinary cells even though their expression may be UCC-specific
  • - Combined with cytopathology, 78.1% sensitivity in detecting TCC, up from 20.0% (low-grade) and 64.0% (high-grade) for cytology alone.
    - Drawbacks: Low specificity (58.6%) compared to 92% for cytology; as low as 33.3% in patients undergoing intravesical therapy.
  • Is this test stage-specific?
    Is this test specific for bladder cancer?
  • Typical cost $400, may cost up to $2000.
  • Surveillance and maintenance through monitoring procedures
  • Five year survival rates range from 98% for Stage 0 (in situ carcinoma) to 15% for Stage IV (NCI). Average 10 year survival rate is 51.5% for males and 42.4% for females (Cancer Research UK)
    - About 7 out of 10 (70%) bladder cancers are CIS when diagnosed (Cancer Research UK).
    -At least 75-80% of patients survive 5-years.
  • The role of IL-8 signaling in the tumor microenvironment. Tumor-derived IL-8 has the capacity to exert profound effects on the tumor microenvironment. For example, secretion of IL-8 from cancer cells can enhance the proliferation and survival of cancer cells through autocrine signaling pathways. In addition, tumor-derived IL-8 will activate endothelial cells in the tumor vasculature to promote angiogenesis and induce a chemotactic infiltration of neutrophils into the tumor site. Although IL-8 can promote cell invasion and migration, the capacity of IL-8 to induce tumor-associated macrophages to secrete additional growth factors will further increase the rate of cell proliferation and cancer cell invasion at the tumor site. The multiple effects of IL-8 signaling upon different cell types present within the tumor microenvironment suggests that targeting of CXC-chemokine signaling (including but not limited to IL-8) may have important implications to halt disease progression and assist in sensitizing tumors to chemotherapeutic and biological agents.
  • The role of IL-8 signaling in the tumor microenvironment. Tumor-derived IL-8 has the capacity to exert profound effects on the tumor microenvironment. For example, secretion of IL-8 from cancer cells can enhance the proliferation and survival of cancer cells through autocrine signaling pathways. In addition, tumor-derived IL-8 will activate endothelial cells in the tumor vasculature to promote angiogenesis and induce a chemotactic infiltration of neutrophils into the tumor site. Although IL-8 can promote cell invasion and migration, the capacity of IL-8 to induce tumor-associated macrophages to secrete additional growth factors will further increase the rate of cell proliferation and cancer cell invasion at the tumor site. The multiple effects of IL-8 signaling upon different cell types present within the tumor microenvironment suggests that targeting of CXC-chemokine signaling (including but not limited to IL-8) may have important implications to halt disease progression and assist in sensitizing tumors to chemotherapeutic and biological agents.
  • Transcript

    • 1. ACDS Laboratories A new biomarker for bladder cancer diagnosis Mirza Ahmed Katy Chun Sagar Desai Nik Sanyal August 29, 2013
    • 2. Who We Are Dedicated to the advancement of biomarker development to address unmet medical needs and improve patient health
    • 3. What is Bladder Cancer? Stages of bladder cancer http://www.urologyhealth.org/urology/articles/images/anato my_bladdercancer.jpg 1. Reasonably large population - Fourth most common cancer in men and eighth most common cancer in women5 - 72,570 new cases in the US in 20132 2. Importance of early detection - 96.4% 5-year relative survival rate if diagnosed in stage 0 or 1 - 15,210 deaths in the US in 20132 3. Costly expenses - Highest lifetime treatment costs per patient of all cancers 3 - “The natural history of bladder cancer is one of recurrence and progression necessitating long-term evaluation and monitoring”5 2 National Cancer Institute, Bladder Cancer 3 Sievert et al., World J Uro. 2009 5 Gilbert et al., Urology. 2003
    • 4. Current clinical flow for the diagnosis of bladder cancer
    • 5. Sensitivity and specificity - Sensitivity (true positive rate) measures the proportion of actual positives that are correctly identified by the test4 - Specificity (true negative rate) measures the proportion of negatives that are correctly identified by the test4 4 Altman & Bland, BMJ, 1994
    • 6. Current diagnostic methods - Voided urine cytology Presence of abnormal (cancerous) cells in urine Issues: low sensitivity (43.8%), results not available immediately 5 - Cystoscopy Endoscopy via urethra Issues: Invasive, dependent on operator skill,inability to detect recurrences in the upper urinary tract rigid cystoscopy - painful and needs general anesthesia, may lead to scarring flexible cystoscopy - does not allow removal of tissue5 Cystoscopy of the bladder http://www.mayoclinic.com/images/image_popup/r 7_cystoscopy.jpg 5 Gilbert et al., Urology. 2003
    • 7. Demand for a better biomarker - Need exists for biomarker that is: - High sensitivity and specificity - Easily detected using a non-invasive procedure - cost-effective - Several candidate biomarkers 6,7 , but most have drawbacks 6 Urquidi et al., Urology. 2012 7 Liu et al., BMC Cancer. 2013
    • 8. Selected Biomarkers 1) miRNA - 135b/15b/1224-3p 2) DD23 antigen 3) Protein markers - VEGF, ApoE & IL-8
    • 9. 1. MicroRNA A combination of 3 miRNAs - 135b/15b/1224-3p - appear as promising stable biomarkers8 Assay Urinary samples (n = 121) from patients with bladder cancer and age-matched controls (n = 53) were analyzed using real-time PCR for changes in expression levels. Clinical validity Sensitivity: 94% Specificity: 51% Overall accuracy: 86% 8 Miah et al., British Journal of Cancer. 2012
    • 10. 2. DD23 antigen Anti-DD23 antibody shows increased sensitivity of urinary based detection of UCC when combined with cytology5 Assay Urinary samples (n = 151) from patients with a history of bladder cancer were evaluated for DD23 antigen expression using alcohol- fixed urinary cytology Clinical validity Sensitivi ty (%) Specifici ty (%) Cytology 43.8 92.0 Cytology + DD23 78.1 58.6 5 Gilbert et al., Urology. 2003
    • 11. 3. Protein markers A combination of 3 proteins - VEGF/ApoE/IL-8 - appear as a highly accurate biomarker10 - Vascular endothelial growth factor (VEGF) - Apolipoprotein E (ApoE) - Interleukin-8 (IL-8) Assay Voided urine samples from individuals without previous history of UCC (n = 63) and individuals with newly diagnosed UCC (n=64) were quantified by Enzyme-Linked Immunosorbent Assay (ELISA) Clinical validity Sensitivity: 90% Specificity: 97% Overall accuracy: 93% 10 Goodison et al., PLOSone. 2012
    • 12. Scientific assessment Elevated levels of biomarkers in bladder cancer individuals 10 Goodison et al., PLOSone. 2012
    • 13. VEGF (Vascular Endothelial Growth Factor) Induces blood vessel formation (Angiogenesis) which helps sustains tumor growth. Frequently upregulated during bladder6 , prostate12 , breast cancer 13 . Anti-cancer drug Bevacizumab (Avastin) targets VEGF receptor binding. ApoE (Apolipoprotein E) A type of lipoprotein found in chylomicron intermediate-density lipoprotein (IDL). In breast cancer, ApoE is upregulated via HIF1α14 . IL-8 (Interleukin-8) Signaling factor produced by endothelial cells and macrophages. Promotes angiogenesis during hypoxia and environmental stress. Promotes cell proliferation in lung 15 and colon cancer16 . Scientific assessment VEGF, ApoE and IL-8 in cancer Waugh DJ, Wilson C. Clin Cancer Res. 2008 Nov 1;14(21) 6 Urquidi et al., Urology. 2012 12 Pratheeshkumar et al., PLoS One, 2012 13 Marty and Pivot, Eur. J. Cancer, 2008 14 Papi et al, PLoS One, 2013 15 Luppi et al, Lung Cancer, 2007 16 Ning et al, Int. J. Cancer, 2011
    • 14. Scientific assessment Advantages of ELISA - Rapid - Low detection limits - Low per sample cost - High-throughput (96 or 384-well format) biology.arizona.edu/immunology/activities/elisa /graphics/elisa_plate.gif Epitomics Inc.
    • 15. Revised clinical flow for the diagnosis of bladder cancer
    • 16. Patient Value Early, highly-accurate, non-invasive diagnosis - Early stage diagnosis increases have long-term survival rates greater than 80%2 - Accurate diagnosis allows appropriate treatment and prevents unnecessary surgery - Avoid the need for invasive cystoscopy Easy, long-term monitoring - Cost-effective - Can be performed in outpatient setting 2 National Cancer Institute, Bladder Cancer
    • 17. Economic Value Economic analysis: Current prices Cytology Average cost = $100 17 Cystoscopy Current Medicare reimbursement = $379 non-facility18 17 Lotan and Roehrborn, J. Urology. 2002 18 CMS.gov, Centers for Medicare and Medicaid Services, Cytology
    • 18. Current pricing for the diagnosis & monitoring of bladder cancer $100 + $379 = $479 $100 + $379 = $479
    • 19. Economic Value Economic analysis: Recurrences and monitoring 80% of bladder cancer patients survive 5-years19 . Long-term monitoring of bladder cancer patients: - Cytology and cystoscopy: Every 3 months during first 2 years (8 procedures) Every 6 months during next 2 years (4 procedures) Every 12 months during subsequent years 20 1initial + 8 + 4 + 1 = 14 procedures/patient/5 years 19 Mayo Clinic, Cancer. 2011 20 Hong and Loughlin, Urology. 2008
    • 20. - Average amount of diagnosis and monitoring procedures/patient in first 5 years = 14 times Total costdiag/monitoring = (cost of cytology+cystoscopy) X 14 = $479 X 14 = $6,706 - 72,570 new cases/year - Approximately 80% of new cases or 58,056 will survive 5 years = 58,056 X 14 = 812,784 procedures Total costdiag/monitoring = $479 X 812,784 procedures = $389,323,536 Economic Value Economic analysis: Current prices
    • 21. Economic value Economic analysis: Pricing/cost estimate Assay development + Instrumentation Costs $100,000* x 3 proteins = $300,000 (Source: ImmunoChemistry Technologies, LLC.) Reagent cost per sample: 3 proteins X 16 replicates21 = 48 wells/urine sample 4 samples/kit or $156/4 samples = $39/urine sample ELISA test price to payor: $156 (Source: ImmunoChemistry Technologies, LLC.) 21 Burd, Clin Microbiol Rev. 2010
    • 22. Projected prices for the diagnosis & monitoring of bladder cancer $479 + $156 = $635 $156 Price of ELISA kit = $156
    • 23. Economic value Economic analysis: Projected savings Estimated cost of procedure with biomarker = $156 Initial cost of diagnosis = $479 + $156 = $635 Diagnosis and monitoring costs over 5 years = $635 + (13 X $156) = $2663/patient For 5 year survivors, total cost of monitoring = 58,056 X $2663 = $154,603,228 Therefore, diagnosis and monitoring cost savings = $389,323,536 - $154,603,228 = $234,720,308
    • 24. Market assessment Economic analysis: Projected value Unit demand = Expected number of tests carried out per year = 812,784/5 years = 162,556 tests/year Price that payors would pay for test = $156 Expected annual revenue = = $156/test X 162,556 tests = $25,358,736 Total Costs = Assay development cost + cost of reagents Assay development cost = $300,000 Cost of reagents = $39 X 162,556 = $6,339,684 Total Cost = $6,339,684 + $300,000 = $6,639,684 Potential Profit = $25,358,736 - $6,639,684 = $19,019,052
    • 25. Investment Recommendation - High clinical validity and accuracy as a biological indicator - Non-invasive and easy long-term monitoring - Cost-effective - High unit demand Based on our opportunity analysis, we would strongly recommend investing in this biomarker
    • 26. APPENDIX
    • 27. Signs and symptoms Signs and symptoms - Hematuria (blood in urine) - Urgent need to empty bladder - Frequent urination - Needing to strain (bear down) when urinating - Pain during urination Source: National Cancer Institute, What You Need to Know About Bladder Cancer,
    • 28. Bladder anatomy -Inner layer: Inner lining of bladder. Composed of transitional cells, 7 cell layers thick. Capable of stretching to accommodate urine. -Middle layer: Smooth muscle. Contraction of muscle squeezes urine out during urination. - Outer layer: External layer containing fat, fibrous tissue and blood vessels.
    • 29. Transitional Cell Carcinoma - 90% of bladder cancer occurs in transitional cells (NCI) - Known as transitional cell carcinoma (TCC) or urothelial cell carcinoma (UCC). (World Health Organization)
    • 30. Potential Biomarkers Potential biomarkers: 1) Protein assays 2) miRNA 3) DD23 monoclonal antibody 4) YAP-1 5) Metallothionein 6) Prefoldin
    • 31. IL8 (Interleukin-8) & Cancer - A chemokine (signalling factor) produced by Endothelial cells and macrophages. “Many studies have shown overexpression of IL-8 by tumor cells, often induced in response to chemotherapeutic interventions or environmental stresses such as hypoxia.” “The induction of IL-8 signaling activates multiple upstream signaling pathways that (a) impinge on gene expression via regulation of numerous transcription factor activities, (b) modulate the cellular proteome at the level of translation, and/or (c) effect the organization of the cell cytoskeleton through posttranslational regulation of regulatory proteins. As a consequence of the diversity of effectors and downstream targets, IL-8 signaling promotes angiogenic responses in endothelial cells, increases proliferation and survival of endothelial and cancer cells, and potentiates the migration of cancer cells, endothelial cells, and infiltrating neutrophils at the tumor site. Accordingly, IL-8 expression correlates with the angiogenesis, tumorigenicity, and metastasis of tumors in numerous xenograft and orthotopic in vivo models. ” Waugh DJ, Wilson C. Clin Cancer Res. 2008 Nov 1;14(21)
    • 32. IL8 (Interleukin-8) & Cancer - A chemokine (signalling factor) produced by Endothelial cells and macrophages. “Many studies have shown overexpression of IL-8 by tumor cells, often induced in response to chemotherapeutic interventions or environmental stresses such as hypoxia.” “The induction of IL-8 signaling activates multiple upstream signaling pathways that (a) impinge on gene expression via regulation of numerous transcription factor activities, (b) modulate the cellular proteome at the level of translation, and/or (c) effect the organization of the cell cytoskeleton through posttranslational regulation of regulatory proteins. As a consequence of the diversity of effectors and downstream targets, IL-8 signaling promotes angiogenic responses in endothelial cells, increases proliferation and survival of endothelial and cancer cells, and potentiates the migration of cancer cells, endothelial cells, and infiltrating neutrophils at the tumor site. Accordingly, IL-8 expression correlates with the angiogenesis, tumorigenicity, and metastasis of tumors in numerous xenograft and orthotopic in vivo models. ” Waugh DJ, Wilson C. Clin Cancer Res. 2008 Nov 1;14(21)
    • 33. APOE (Apolipoprotein E) & Cancer -APOE : “ class of apolipoprotein found in the chylomicron and Intermediate-density lipoprotein (IDLs) that is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.[1]” http://en.wikipedia.org/wiki/Apolipoprotein_E “TAF secreted TGFβ induces TNFα expression in breast CSCs. TNFα binds TNFR1 on breast CSCs and activates the PPARα/HIF1α interplay which up- regulates miR130b expression. The interplay is counterbalanced by PPARγ via miR17-5p up- regulation. In turn, the PPARα/HIF1α interplay regulates CAIX, ApoE, IL6 and SLUG expression.” Source: Papi et al PLoS One. 2013;8(1):e54968
    • 34. VEGF (Vascular Endothelial Growth Factor) & Cancer VEGF: Vascular endothelial growth factor, a signalling protien that induces angiogenesis. VEGF is upregulated during hypoxia. Source: Vishnu et al, Onco Targets Ther. 2011; 4: 97–113.
    • 35. Role of APOE , IL8, VEGF in bladder cancer Source: Waugh DJ, Wilson C. Clin Cancer Res. 2008 Nov 1;14(21)
    • 36. Biomarker Selection - We selected the 3-protein (VEGF, ApoE and IL8) assay as a biomarker. - Advantages over current protocol: - -
    • 37. Performance of urinary biomarker combinations Source: Goodison S, Chang M, Dai Y, Urquidi V, Rosser CJ. PLoS One. 2012;7(10)
    • 38. Cost of cystoscopy + cytology = $11846/cancer (Kamat et al., 2010) Mean number of subsequent tumor episodes = 3.8 (Soloway et al., 2003) Total cost of initial assessment and surveillance/maintenance = $11846/cancer X 1 initial cancer = $11846 $11846/cancer X 3.8 subsequent cancer episodes = $45014.80 = $56860.80
    • 39. 3 Protein marker - Pricing assessment 1. Source: BDBiosciences IL-8 kit. (~$490/kit) 2. Refer to assay development doc. Cheaper price/kit (~$156/kit) 3. 2 x 96-well plates/kit. How many replicates for each patient sample for each protein? 2 to 4 replicates (Source: Burd, Clin Microbiol Rev. 2010 July; 23(3): 550–576. CLSI/NCCLS. 2003. Evaluation of the linearity of quantitative measurement procedures: a statistical approach.Approved guideline. CLSI document EP6-A. Clinical and Laboratory Standards Institute, Wayne, PA.)

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