ACDS Laboratories has developed a new 3-protein biomarker for bladder cancer diagnosis using VEGF, ApoE, and IL-8. The biomarker provides high sensitivity of 90% and specificity of 97% in a study of 127 patients, outperforming current methods. It can be detected through a non-invasive urine test using ELISA, addressing the need for a simple, accurate, and cost-effective diagnostic. Economic analysis shows the biomarker could save over $200 million annually in the US through reducing invasive procedures and long-term monitoring costs compared to current protocols. Based on the strong clinical and economic value, ACDS recommends investing in developing this biomarker.

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

27. APPENDIX

28. 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,

29. 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.

30. 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)

31. Potential Biomarkers
Potential biomarkers:
1) Protein assays
2) miRNA
3) DD23 monoclonal antibody
4) YAP-1
5) Metallothionein
6) Prefoldin

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. 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)

34. 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

35. 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.

36. Role of APOE , IL8, VEGF in bladder
cancer
Source: Waugh DJ, Wilson C. Clin Cancer Res. 2008 Nov 1;14(21)

37. Biomarker Selection
- We selected the 3-protein (VEGF, ApoE and IL8) assay as a biomarker.
- Advantages over current protocol:
-
-

38. Performance of urinary biomarker
combinations
Source: Goodison S, Chang M, Dai Y, Urquidi V, Rosser CJ. PLoS One. 2012;7(10)

39. 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

40. 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.)