Emerging Prognostic Biomarkers in Urothelial Carcinoma

Emerging Prognostic Biomarkers in Urothelial Carcinoma George J. Netto, M.D. Departments of Pathology, Oncology & Urology Johns Hopkins University Baltimore, MD USA

Overview Current Clinicopathologic Prognostic Parameters Molecular Pathways of Oncogenesis Emerging Prognostic Biomarkers Targets of Therapy

Bladder Carcinoma Epidemiologic Features In USA: 72,570 new cases 15,210 deaths in 2013 336,000 new cases worldwide Peak incidence: sixth decade. Male/Female: almost 3/1 4th most common in M and 12th most common in F Highest incidences in: Western Europe, N. America and Australia.

UrCa Disease Costs and Management Opportunities Major health cost burden per patient: - Frequent cystoscopy, high rate of recurrence etc - $ 4 Billion per year in USA alone; largest cost per pt for any type of tumor Unique amenability to applying molecular detection methods (e.g. UroVysion FISH, FGFR3 mutation) and molecular Rx delivery to target

Urothelial Carcinoma (UrCa) Two Phenotypes? Superficial non-muscle invasive UrCa (NMI-BC) : 70-80% Majority of UrCa (60-70%) present as non-invasive (pta) tumors at time of first Dx 50% will recur as non-invasive tumors and only 5-10% of will progress Mainstay of Rx: TURB +/- Intravesical Chemotherapy and Immune therapy BCG Muscle Invasive UrCa (MI-BC): 20-30% 15 % of MI UrCa have history of prior Superficial UrCa 80-90% are primary Muscle invasive UrCa Practically all are high grade Despite aggressive Rx (Cystectomy +/- Chemotherapy) <50% overall survival

Bladder Urothelial Carcinoma Two Divergent Molecular Pathways Superficial TCC H-RAS/FGFR3/mTOR Normal Urothelium Urothelial Hyperplasia PUNLMP Low Grade Papillary UrCa Falt CIS High Grade Papillary UrCa Invasive UrCa Muscle Inv TCC P53/RB

Clinico-Pathologic Prognostic Factors

Superficial Urothelial Carcinoma (Non-Muscle Invasive UrCa) pta/pt1 Pathologic Prognosticators WHO/ISUP 2004 Grade pt: pta vs pt1 Depth of Lamina propria Invasion: pt1a,b,c CIS, Prostatic duct involvement LVI? Size: >5 cm Multifocality/Extent: ureter, upper tract and urethral involvement Failed Intravesical Rx /Recurrence within 6 month Duration of Disease Soloway et al J Urol 2002 O Donnell et al Sem Oncol 2007

Non-Invasive Urothelial Carcinoma Recurrence/Progression WHO/ISUP Grade: Urothelial Papilloma: lowest risk of recurrence & no progression PUNLMP: 35% (25-47%) risk of recurrence, 4% risk of progression, 1% DOD LG UrCa: 50% (30-76%) recurrence rate, 10% progress, 5% DOD HG UrCa: most frequent recurrence rate ( 50-69%), 25-65% progress Flat CIS is an aggressive disease Chaux A et al Hum Pathol 2011 Lee et al Hun Pathol 2011 Ledbret et al J Urol 2000 Lopez-Beltran et al Eur Urol 2004

Sylvester et al. Eur Urol 2006 2596 pts from 7 EORTC trials Predictive Model Parameters: Number of Tumors Tumor Size : 3 cm Prior Recurrence Rate: 1 Rec/yr pt CIS WHO grade

Sylvester et al. Eur Urol 2006

Stein et al J Clin Oncol 2001 1054 pts uniformly Rx: Radical Cystectomy+LN ± Adj Chemo radiation 10.2 yr median F/U DFS 68% at 5yr and 60% at 10 yrs ptnm significant predictor (OC vs Non OC) in term of DFS and OS - OC LN neg group: 85% DFS at 5yr - Non OC LN neg group: 58% DFS at 5yr - LN positive group: 35% DFS at 5yr Stage Sub-grouping within OC and Non OC was not significant predictor of DFS or OS DFS OS

Urinary Bladder Urothelial Ca Molecular Pathways

Urothelial Hyperplasia LG URCa 70% Recurrence 70-80% Normal Urothelium ~15% 20-30% ~50% Dysplasia/CIS HG URCa Invasive URCa Metastasis Netto, G. J. Nature Rev. Urol. 2011.193

9q-/9p- Urothelial Hyperplasia LG URCa 70% Recurrence 70-80% Normal Urothelium ~15% 20-30% 9q-/9p- ~50% Dysplasia/CIS HG URCa Invasive URCa Metastasis Netto, G. J. Nature Rev. Urol. 2011.193

Lindgren et al PLoS 2012 Bladder Urothelial Ca Chromosomal Alterations

Bladder Urothelial Carcinoma Chromosomal Aberrations Chromosome 9: Genetic losses in Chromosome 9 are early events in both Superficial and MI-BCa LOH analysis, CGH and array CGH consistently detected deletions in both arms of Ch 9 (up to 80-90%) Potential TSG Loci: - 9p21: CDKN2A (encodes p16 and p14); deleted in up to 50% of UrCa - 9q34: TSC1

Bladder Urothelial Carcinoma Chromosomal Aberrations Chromosomal Gains: 3q,7p,17q gains 17q gains: HER2 amplification; TOPO2A Diagnostic Application: Numerical Ch 3, 7, 17 are exploited in UroVysion FISH assay (in addition to loss of p16 @ 9p21)

Moonen et al Eur Urol 2007 Red: Ch3 Green: Ch 7 Blue: Ch 17 Gold: 9p21 UroVysion positivity (i) at least 4 cells with gain of more than 1 chromosome of chrom 3,7,17 and/or (ii) at least 12 cells with heterozygous or homozygous deletion of 9p21

Skacel et al J Urol 2003: 120 urine cytology (instrumented and voided) All with concurrent TURBT (82 UrCa + 38 negative) Overall: Sensitivity 85% Specificity of 97% FISH sensitivity in Cytology Groups: 100% (suspicious Cyto) 89% (Atypical Cyto) 60% (Negative Cyto) 8/9 FISH positive pts with originally negative bx had subsequent positive biopsy within 12 Months and 1 had CIS at 15 months F/U Yoder et al Am J Clin Path 2007: 65% of Anticipatory Positive Dx with UrCa in 29 months

Sarosdy et al J Urol 2006 497 pts with hematuria from 23 centers UrCa in 10% of pts on TURB FISH: 69% overall sensitivity (84% excluding ptag1) 65% UrCa in smokers with hematuria with positive FISH vs 24% if FISH neg STAGE

9q-/9p- Urothelial Hyperplasia LG URCa 70% Recurrence 70-80% Normal Urothelium ~15% 20-30% 9q-/9p- ~50% Dysplasia/CIS HG URCa Invasive URCa Metastasis Netto, G. J. Nature Rev. Urol. 2011.193

9q-/9p- Urothelial Hyperplasia LG URCa 70% Recurrence 70-80% FGFR3/HRAS/PIK3CA-Akt Normal Urothelium ~15% 20-30% 9q-/9p- ~50% Dysplasia/CIS HG URCa Invasive URCa Metastasis Netto, G. J. Nature Rev. Urol. 2011.193

Bladder Urothelial Carcinoma RTK-HRAS Pathway

Schultz L et al Cancer 2010 Chaux A et al Urology 2013

Bladder Urothelial Carcinoma FGFR3-HRAS Pathway FGFR3 Role in Surveillance in NMI-BC FGFR3 alone or combined with RAS and PIK3CA detect early recurrence Zuiverloon et al. Clin Cancer Res 2010 Miyaki et al. Cancer Science 2010 PCR based assays for detecting FGFR3 mutations in voided urine (45% sensitivity) Positive urine sample associated with concomitant/future recurrence in 81% (90% in patients with consecutive samples) Superior to cytology (78% vs. 0%)

Rhijn et al. Eur Urol 2010 mg1 (Pos FGFR3 mutation/mib1 normal) : favorable prognosis mg2 (Neg FGFR3 mutation/mib1 normal OR pos FGFR3 mutation/mib1 High): intermediate prognosis mg3 (Neg FGFR3 mutation/mib1 High ): poor prognosis

Sylvester et al. Eur Urol 2006 2596 pts from 7 EORTC trials Predictive Model Parameters: Number of Tumors Tumor Size : 3 cm Prior Recurrence Rate: 1 Rec/yr pt CIS WHO grade

Rhijn et al. Eur Urol 2010 230 pts FGFR3 mutations related to favorable disease High MIB-1 correlated with pt1, high grade, and high EORTC risk scores EORTC risk scores independent predictors of recurrence and progression mg independent predictor of progression and DSS Adding mg to the multivariable model for progression increased predictive accuracy (74.9% to 81.7%) mg more reproducible than the pathologic grade (41 74%). mg1 (Pos FGFR3 mutation/mib1 normal) : favorable prognosis mg2 (Neg FGFR3 mutation/mib1 normal OR pos FGFR3 mutation/mib1 High): intermediate prognosis mg3 (Neg FGFR3 mutation/mib1 High ): poor prognosis

9q-/9p- Urothelial Hyperplasia LG URCa 70% Recurrence 70-80% FGFR3/HRAS/PIK3CA-Akt Normal Urothelium ~15% P53,Rb 8p-,11p-,13q-,14q- 20-30% 9q-/9p- ~50% Dysplasia/CIS HG URCa Invasive URCa Metastasis P53, Rb, 8p- 8p+,17p- Netto, G. J. Nature Rev. Urol. 2011.193

Superficial TCC RTK-HRAS Muscle Inv TCC p53 RB Netto, G. J. Nature Rev. Urol. 2011.193

George et al. JCO 2007 p53 gene and protein status show discordance in 35% of cases Exon 5 mutations demonstrated a wild-type protein Both p53 gene and protein status correlated with stage and outcome Combining p53 gene and protein status stratifies DFS: Wild-type gene and unaltered protein Either mutated gene or altered protein Mutated gene and altered protein BEST INTERMEDIATE WORST

Prognostc Biomarkers in UrCa Cooperative Effect of Cell Cycle Regulators Shariat S et al J Urol 2007 74 pts superficial (non muscle invasive) TURB P53,p21, prb, p27; IHC on TMA sections; 3.5 yr median F/U Alteration rates: p53 (34%) ; p21 (35%) ; RB (39%); p27(47%) Each marker significantly predicted progression Combination markers stratified pts into risk group SYNERGISTIC Increased risk of recurrence and progression with incremental number of altered markers Recurrence 0 PFS 1 0 4 1 3 2 4 3 2

Prognostc Biomarkers in UrCa Cooperative Effect of Cell Cycle Regulators Chatterjee et al JCO 2004 164 cystectomy P53,RB and p21 0 alteration: 23% 5 yr recurrence 1 alteration: 32% 5 yr recurrence 2 alteration: 57% 5 yr recurrence 3 alteration: 93% 5 yr recurrence p53,rb,p21

So Where do we stand?! Do we currently use ANY marker for PROGNOSTICATION in Bladder Cancer? Do we currently use ANY marker for THERAPY PREDICTION in Bladder Cancer?

Margulis et al JNCI 2009 713 radical cystectomy at six centers. High Ki-67 (>20%) labeling index independently associated with recurrence and DSS Addition of Ki-67 labeling index improved the accuracy of standard multivariate prediction model (by 2.9% for recurrence and 2.4% for DSS)

Genomics as Prognostic Tools

Two Genomic Circuits: FGFR3 mut/ampl; CCND1; PIK3CA mut; 9q (CDKN2A) deletions E2F3 ampl; RB1 del; PTEN del; CDKN2A; 5p gain P53/MDM2 alterations in both circuits at advanced Dz Lindgren et al. PLoS 2012

Lindgren et al PLoS 2012

Conclusions: Molecular understanding of bladder cancer oncogenesis has brought us within reach of our goals of stratifying management based on biomarkers More work remains.. Thank You!!!

Targeted Therapy for Bladder Ca.

Bladder Urothelial Carcinoma Targets of Rx Oncogenic pathways offer opportunities for targeted Rx: RTK-RAS-MAPK Angiogenesis mtor-pik3ca

UrCa TARGETED THERAPY Anti EGFR Randomized phase II trial Cetuximab Recombinant humanized murine monoclonal Ab Metastatic/recurrent non-resectable dz Gemcitabine & Carboplatin (GC) with or without Cetuximab Blocking extracellular EGFR domain inhibits downstream signal transduction pathway proliferation Anti-angiogenesis? Lapatinib in EGFR-positive and ERBB2-positive bladder tumor (phaseii/iii trial underway) Iyer G et al; Expert Rev in Anticancer Ther 2010 Wulfing C et al Cancer 2009

UrCa TARGETED THERAPY Tyrosin Kinase Inhibitors Phase II Cancer and Leukemia Group B trial (CALBG) Gefitinib: No OS or PFS advantage for GC+ Gefitinib Rx Vs GC alone Multitarget Agents: Phase II Sunitinib maintainance at MSKCC promising results Randomized trial underway Phase II Sorafenib (inhibitor RAF1, BRAF, PDGFRB, KDR, and FLT4) failed Philips GK et al Ann Oncol 2009 Bradely et al Clin Genitourin Cancer 2007

UrCa TARGETED THERAPY Anti Angiogenesis Bevacizumab: Recombinant humanized monoclonal anti-vegf Antibody First-line combination Rx with GC in patients with metastatic Dz Phase II study: two-thirds demonstrated objective response (14% CR) Phase III Cancer and Leukemia Group B trial (CALBG) underway Hahn et al JCO 2011 Elfiki AA et al Curr Oncol Rep 2009

UrCa TARGETED THERAPY mtor Inhibitors Evrolimus: Sirolimus-derived mtor inhibitor used in RCC Phase II trial underway in advanced Dz Iyer G et al; Expert Rev in Anticancer Ther 2010

Conclusions: Molecular understanding of Urothelial Ca oncogenesis has brought us within reach of our goals of stratifying management in Bladder Cancer pts based on biomarkers More work remains.. Thank You!!!

Superficial TCC RTK-HRAS Muscle Inv TCC p53 RB Netto, G. J. Nature Rev. Urol. 2011.193

Superficial UrCa Clinico-Pathologic PGx Urothelial Dysplasia * * Urothelial CIS * * * * * * O Donnell et al Sem Oncol 2007

Bladder Carcinoma Clinical Presentation/Evaluation 75% pts present with gross hematuria 10% with irritation symptoms: dysuria, urgency, frequency CIS Cystoscopy & TUR Bx: DX gold standard Understaging: 15-50% MP sampling Overstaging: muscularis propria vs muscularis mucosa (Large venules present in lamina propria)