The Search for Biomarkers in Bladder Cancer. CDDP and IO world

The Search for Biomarkers in Bladder Cancer. CDDP and IO world Joaquim Bellmunt, MD PhD Associate Professor of Medicine Harvard Medical School PSMAR-IMIM September 9 th, 208

Disclosures Advisory role: Genentech, Merck, Pfizer, GSK, BMS, Pierre-Fabre, Sanofi Aventis, Astellas, OncoGenex, Janssen, BioClin Speaker role: Pfizer, Merck, GSK, Novartis, Pierre-Fabre, Astellas, BioClin Research funding: Takeda, Pfizer, Novartis, Sanofi Aventis

Chemotherapy (CDDP) Biomarkers

Molecular determinants of response to cisplatin-based neoadjuvant chemotherapy Bellmunt J, Curr Opin Urol 203, 23:466 47

Molecular determinants of response to cisplatin-based neoadjuvant chemotherapy Bellmunt J, Curr Opin Urol 203, 23:466 47

SWOG TRIAL: COXEN-directed neoadjuvant chemotherapy Prospective validation of the COXEN biomarker to predict pt0 /pt Muscle-Invasive Bladder Cancer SWOG 870 criteria - T2-T4a N0M0, cisplatin eligible NCI-60 Cell Line Panel (IC50) 2 3 2 3 575A Sensitive 23 23 23 y 2 2 2 2 2 VMCUB Sensitive COXEN 2 y y y y y y R 2 2 2 2 2 2 2 2 2 GC UMUC6 Sensitive DD- 2 2 MVAC KU7 Resistant x 2 y y y y 2 2 UMUC Resistant x 2 y y 2 2 2 2 2 2 2 2 2 2 CRL2742 2 2 Resistant x 2 2 UMUC2 Resistant 2 2 253J-BV Resistant x 2 2 2 KK47 Resistant x 2 y y 2 2 2 2 2 2 2 UMUC9 Resistant 2 2 2 Cystectomy to assess pt0 or pt Pathology 2 2 TCCSUP 253JLaval 2 2 Sensitive 2 T24 2 Resistant Resistant 2 x x x2 2 2 2 2 2 2 2 COXEN Model Predicting response to chemotherapy 2 2 2 2 CUBIII UMUC3D 2 HT376 Theodorescu et al. Sensitive 2 Clin Can 2007;4407 3(5):4407 Resistant Resistant x x x 2 2 2 2 2 2 2 2 2 Cisplatin, Gemcitabine, Methotrexate, Doxorubicin, Vinblastine Human Bladder 2 2 Cancer Cell Lines Bladder Cancer patient samples y y y y 2 2 2 2 SW70 Resistant x 2 253J-P Resistant x 2 2 2 CRL7833 Resistant x 2 Gene 2 Expression 2 Model 2 CRL793 Resistant x 2 2 2 Propotion Surviving Propotion Surviving 0.0 0.2 0.4 0.6 0.8.0 0.0 0.2 0.4 0.6 0.8.0 Correlate with COXEN prediction pt0 /pt MVAC (N=6) Orntoft MVAC Kaplan-Meier Analysis P = 0.0469 Predicted Responders (5) Predicted Nonresponders (9) 0 2 24 36 48 60 72 84 96 Survival Time (Months) GC (N=4) Orntoft Cisplatin (MVAC) Match Kaplan-Meier Analysis Predicted Responders (4) Predicted Nonresponders (0) P = 0.0303 0 2 24 36 48 60 72 84 96 Survival Time (Months)

We developed a gene expression model (GEM) to predict the pathological node status in primary tumour tissue from three independent cohorts of patients who were clinically node negative.

Aggregate results RESEARCH ARTICLE Somatic A Phase IERCC2 Pharmacologic Mutations Study Correlate of with Necitumumab Cisplatin Sensitivity (IMC-F8), in Muscle-Invasive a Fully Human IgG Monoclonal Urothelial Carcinoma Antibody,2 5,2 Eliezer M.G.Van Andreas Bader Allen, David, Kent Brown W. Mouw, and3,4matthew, Philip Kim Winkler, Gopa Iyer6,7, Nikhil Wagle,2, Hikmat Al-Ahmadie6,8, Cong Zhu2, Irina Ostrovnaya9, Gregory V. Kryukov2, Kevin W. O Connor3, John Sfakianos5, Ilana Garcia-Grossman7, Jaegil Kim2, 0 7 2 Elizabeth A. Guancial, Richard Bambury, Samira Bahl, Namrata Gupta2, randomized trial in 4,560 The Mammary Prevention 3 (MAP.3) placebo-controlled ABSTRACT Deborah Farlow2, Angela Quhigh-risk, Sabina Signoretti,women Justine A. Barletta, postmenopausal showed a 65% reduction in invasive breast can6,8 2 2 2,2 Victor Reuter, Jesse Boehm, Michael Lawrence Getz, Few differences in adverse events cer with the use of exemestane at 35 months of, Gad median follow-up. 5,6 Philip Kantoff Bernardbetween H. Bochner, Toni K. Choueiri, Deanrisk:benefi F. Bajorint6,7, were,observed the arms, suggesting a promising balance with exemestane for 6,7,3 3,4,2 David B. Solit, Stacey Gabriel D Andrea, Levi A. Garraway use in chemoprevention. Yet,, Alan the MAP.3 design and implementation raise, concerns about limited data 6,7 and Jonathan E. Rosenberg maturity and not prospectively including key bone-related and other toxicities as study endpoints. Exemestane for prevention is juxtaposed against selective estrogen receptor modulators and the other aromatase inhibitors. Additional issues for prevention, including the influence of obesity, alternative dosing, and biomarker use in phase III trials, are addressed. Cisplatin-based chemotherapy is the standard of care for patients with muscle-invaabstract urothelial carcinoma. downstaging to pt0/ptis neoadjuvant SIGNIFICANCE: Thesive recently completed MAP.3Pathologic trial of exemestane for breast cancerafter prevention offers cisplatin-based is associated with survival, although molecular determinants a potential newchemotherapy standard for pharmaceutical riskimproved reduction in high-risk postmenopausal women. In of cisplatin response are We of performed whole-exome sequencing preaddition to describing key incompletely findings fromunderstood. the publication MAP.3 and related trials, our reviewon undertreatment tumor and germline DNA from 50 patients with muscle-invasive urothelial carcinoma takes a detailed analysis of the strengths and weaknesses of MAP.3 as well as the implicationswho for received neoadjuvant cisplatin-based chemotherapy followed cystectomy (25 pt0/ptis respondcancer Discov; 2(X); XXX XXX. 202 by AACR. future prevention research. ers, 25 pt2+ nonresponders ) to identify somatic mutations that occurred preferentially in responders. ERCC2, a nucleotide excision repair gene, was the only significantly mutated gene enriched in the cisplatin responders compared with nonresponders (q < 0.0). Expression of representative ERCC2 mutants in an ERCC2-deficient cell line failed to rescue cisplatin and UV sensitivity compared with wild-type ERCC2. The lack of normal ERCC2 function may contribute to cisplatin sensitivity in urothelial cancer, and somatic ERCC2 mutation status may inform cisplatin-containing regimen usage in muscle-invasive urothelial carcinoma. SIGNIFICANCE: Somatic ERCC2 mutations correlate with complete response to cisplatin-based chemosensitivity in muscle-invasive urothelial carcinoma, and clinically identified mutations lead to cisplatin sensitivity in vitro. Nucleotide excision repair pathway defects may drive exceptional response to conventional chemotherapy. Cancer Discov; 4(0); 40 53. 204 AACR. See related commentary by Turchi et al., p. 8. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. 2Broad Institute of MIT and Harvard, Cambridge, Massachusetts. 3Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. 4Harvard Radiation Oncology Program, Boston, Massachusetts. 5 Urology Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York. 6Weill Cornell Medical College, Cornell University, New York, New York. 7Genitourinary Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York. 8Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York. 9Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York. 0Division of Hematology/Oncology, Department of Medicine, University of Rochester Medical Center School of Medicine and Dentistry, Rochester, New York. Department of Pathology, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts. 2Massachusetts General Hospital Cancer Center and Department of Pathology, Boston, Massachusetts. 40 CANCER DISCOVERY"OCTOBER 204 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York. 3 Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). E.M. Van Allen and K.W. Mouw contributed equally to this article. L.A. Garraway and J.E. Rosenberg contributed equally to this article. Corresponding Authors: Jonathan E. Rosenberg, Genitourinary Oncology Service, Memorial Sloan Kettering Cancer Center, 275 York Avenue, New York, NY 0065. Phone: 646-422-446; Fax: 646-227-247; E-mail: rosenbj@mskcc.org; and Levi A. Garraway, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, D542, Boston, MA 025. Phone: 67-632-6689; Fax: 67-582-7880; E-mail: levi_garraway@dfci.harvard.edu doi: 0.58/259-8290.CD-4-0623 204 American Association for Cancer Research. www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on December 28, 204. 204 American Association for Cancer Research.

Markers of pathologic response to neoadjuvant cisplatin-based chemotherapy Plimack. GU-ASCO 205 DNA repair gene variants associated with pcr Van Allen EM,..Rosenberg J; GU-ASCO 205, Cancer Discov 204; Liu D, JAMA Oncol 206 ERCC2 mutations associated with pcr à Bladder sparing approach for those with somatic ERCC2 mutations planned to be prospectively investigated Groenendijk FH; Eur Urol 205) ERBB2 mutations are associated with pathologic response

Are the ERCC2 genomic test ready for prime 9me? ERCC2 is linked to pcr in only 40% of pa9ents pcr is seen in ERCC2 nega9ve pts Is not a hot spot muta9on. Not all the mut are drivers Tumor heterogeneity is an issue in all tumors We can only enrich the posi9ve predic9on ERCC2 altera9ons correlated with improved response and survival in discovery and valida9on sets ERCC2 n Sensi+vity for response Specificity for response PPV for response NPV for response Discovery: MSKCC DFCI 50 36% 00% 00% 6% Valida9on: Philadelphia (DDGC+AMVAC) 48 80% 93% 80% 93% Philadelphia Validation Cohort MSK/DFCI Discovery Cohort Liu, et al. JAMA Onc 206

Eur Urol. 207 Oct;72(4):544-554.

Limita+on: No clear effect on pathological response to NAC (observed primarily in the basal tumors ) Outcome after NAC varies by molecular subtype Patients with basal tumors appear to derive the most benefit from NAC Luminal non immune infiltrated tumors had the best prognosis, irrespective of the treatment strategy, implying that these patients do not appear to derive benefit from NAC. Luminal immune-infiltrated tumors were significantly worse than those with luminal non-infiltrated tumors. Patients with luminal-infiltrated tumors appear to have poor prognosis with and without NAC Patients with claudin-low tumors had the worst prognosis irrespective of treatment strategy in our analysis, suggesting also that these patients derived little or no benefit from NAC Eur Urol. 207 Oct;72(4):544-554.

Immunotherapy Biomarkers

Novel Biomarkers: Beyond PD Early data suggests the following may enrich for response to PD pathway inhibition: Higher mutational load TCGA Subtype (Luminal II) CD8 infiltration Immune related gene expression signatures (Nanostring) Peripheral expansion of certain TCR clones

Summary of FDA- Approved and Inves+ga+onal PD- L Assays in Urothelial Carcinoma* Pembrolizumab Atezolizumab 2,3 Nivolumab 4 Durvalumab 5 Avelumab 6 Ab clone/ epitope 22C3 SP42 28-8 SP263 73-0 Cell type scored TCs and ICs ICs TCs TCs or ICs TCs Scoring method CPS: % of PD- L posi9ve TCs and ICs rela9ve to the total number of tumor cells % of PD- L expressing ICs % of PD- L expressing TCs % of PD- L expressing TCs or ICs % of PD-L expressing TCs FDA status for urothelial carcinoma NA Complementary NA NA NA PD- L thresholds under evalua+on % 0% IC2/3 ( 5%), IC ( % but <5%), IC0 (<%) % 5% 25% 5% * No head- to- head studies have been conducted and direct comparisons cannot be made between these studies. Bellmunt, J et al. N Engl J Med. 207; 2 Loriot Y, et al. Poster presenta9on at ESMO 206. Abstract 83P; 3 Ventana. Roche receives FDA Approval for novel PD- L biomarker assay [press release]. May 8, 206; 4 Sharma P, et al. Lancet Oncol. 207; 5 Powles T, et al. Poster presenta9on at ASCO GU 207. Abstract 286; 6 Patel M, et al. Poster presenta9on at ESMO 207. Abstract 777PD.

PD- L Expression as a Predictor of Checkpoint Blockade Sensi+vity in UC Powles, et al. Nature. 204. Phase I Atezolizumab Rosenberg, et al. Lancet. 206. Phase II Atezolizumab Balar, et al. Lancet. 207. Phase II Atezolizumab Massard, et al. J Clin Oncol. 206. Phase I Durvalumab Sharma, et al. Lancet Oncol. 206. Phase I/II Nivolumab Sharma, et al. Lancet Oncol. 207. Phase I/II Nivolumab Plimack, et al. Lancet Oncol. 207. Phase I Pembrolizumab Bellmunt, et al. N Engl J Med. 207. Phase III Pembrolizumab 5/8 studies reported posi9ve associa9on with PD- L staining

Opposite results in the cis-ineligible st line single arm trials Vuky J, et al. J Clin Oncol 36, 208 (suppl; abstr 4524) Balar AV, et al. J Clin Oncol 36, 208 (suppl; abstr 4523)

TCGA Subtype (mrna) NIVOLUMAB PATIENT SELECTION Interferon-γ genes are enriched in responders vs those with progressive disease (P<0.0) Puta+ve biomarker: TCGA groups? Complete Response a Partial Response Stable Disease Progressive Disease Discordant results with Atezolizumab study where: - Luminal II have high T eff and low stromal genes. - Basal subtypes in TCGA have more immune suppressed phenotypes (high T eff and High stromal), 00% Percentage 75% 50% 59. 4.8 30.9 30.4 39. 60.6 25% 0% 0 22.7 6.6 Cluster Luminal (Luminal (Cluster ) n=66 ) n=66 24.2 2.7 25.4 8.7 5. Cluster Luminal 2 (Luminal 2 Cluster Basal 3 (Basal ) Cluster Basal 4 (Basal 2 2) (Cluster 2) n=55 2) (Cluster n=23 3) (Cluster n=33 4) n=55 n=23 n=33 Courtesy of L Albiges Galsky et al. LBA 3. ESMO 206

Focusing on new genomic findings (ASCO, ESMO,TCGA ) TMB,MMR,DDR, TGFβ

Biomarkers beyond PD-L: Mutation load is associated with OS and RR with CI

Altera+ons in DNA Damage Response and Repair Genes as Poten+al Marker of Clinical Benefit From PD- /PD- L Blockade in Advanced Urothelial Cancers Min Yuen Teo, & J Rosenberg J Clin Oncol February 28, 208.

Somatic mutations and response to immunotherapy Teo et al. Somatic DDR mutations deletereous 26.9% à better PFS and OS after ICB Iyer et al. MMR-D UC à better response to ICB Immune checkpoint inhibition Somatic mutations Neoantigens Presentation by APCs Cytotoxic T-cell response Unleashed antitumor response Better clinical outcomes Presented by: Bishoy Faltas

Pembrolizumab indicated for treatment of patients with unresectable / metastatic solid tumors that are microsatellite instability-high (MSI-H) or mismatch repair deficient (dmmr) (also urothelial cancer) FDA, May 23 rd, 207 Presented by: Petros Grivas

TGFbeta papers Any new resistance mechanism?

The impact of checkpoint inhibition on patient outcome in muc is dictated by three core biological pathways: (i) pre-existing T-cell immunity and (ii) TMB are positively associated with outcome, whereas (iii) TGFβ is associated with lack of response and reduced survival The enrichment of the fibroblast TGFβ -response signature in nonresponding immune-excluded tumours, combined with results from Lund molecular subtyping and preclinical models showing that coinhibition of TGFβ and PD-L converted tumours from an excluded to an inflamed phenotype, support a model in which TGFβ signalling may counteract anti-tumour immunity by restricting the movement of T-cells in the TME.

Tumor mutational burden correlates with response to immune checkpoint blockade in multiple solid tumors, although in microsatellite-stable tumors this association is of uncertain clinical utility. Limited utility of TMB as a clinical biomarker for individual patients. WES of 249 tumors and matched normal w outcomes to immune checkpoint Therapy mul9ple cancer types. 7 published studies (n = 7) + 78 newly sequenced pre- Tto. Iden+fied genomic correlates of response beyond TMB: Soma9c events in individual driver genes, Certain global muta9onal signatures, Specific HLA- restricted neoan9gens. Osen interrelated Miao, D et al. Nat Genet. 208 Sep;50(9):27-28

.- Mutational burden and response to immune checkpoint therapy CR or PR had significantly higher TMB compared with PD. - Persisted within types TMB had poor predic9ve power to differen9ate CR or PR from PD in this cohort Higher muta9onal loads in pa9ents with stable disease with long compared to short dura9on of OS Clonal nonsynonymous MB strongly predicted CR or PR versus PD across cancer types and responses Subclonal muta9ons (> 50%), (=high intratumoral heterogeneity), were substan9ally more likely to have progressive disease than complete or par9al response

2.- Mutations in specific genes associated with response or resistance o IO checkpoint therapy Analysis limited to known hotspot Clonal driver altera9ons in PIK3CA, KRAS, and PBRM were enriched in pa9ents with CR or PR, while clonal driver muta9ons in EGFR were enriched in pa9ents with PD Aser correc9ng for TMB, KRAS and PIK3CA remained associated with complete or par9al response (did not pass FDR) Clonal hotspot muta9ons in PIK3CA, those with CR or PR had melanoma, HNSCC, anal cancer, or bladder cancer, whereas the majority of those with SD or PD had lung cancer

3.- Integrated analysis of mutational burden, intratumoral heterogeneity, and mutational signatures in melanoma, HNSCC, and bladder cancer In bladder cancer and HNSCC, analyses demonstrated associa9on of APOBEC- associated signatures (S2 and S3) with higher muta9onal burdens and greater likelihood of complete or par9al response. APOBEC mrna expression has also previously been associated with increased PD- L immunohistochemical staining and high tumor muta9onal burden in urothelial carcinoma Miao, D et al. Nat Genet. 208 Sep;50(9):27-28

Response- associated in silico predicted neoan+gens 87 predicted neoan9gens were generated by driver muta9ons. 8 of these driver neoan9gens occurred recurrently in pa9ents with CR or PR but not in pa9ents with PD in a human leukocyte an9gen (HLA)- dependent manner Miao, D et al. Nat Genet. 208 Sep;50(9):27-28

Future Directions: Microbiota as Biomarkers Ig-SEQ MICROBIOME CULTUROMICS TRANSCRIPTOMICS 6S RNA SEQUENCING SHOTGUN METAGENOMICS FUNCTION

Human Studies Analysis of 3 fecal samples of patients with MM treated with anti PD- q The gut microbiota of responders had a greater diversity q Responders had increased abundance of Clostridiales (specifically the Ruminoccocaceae family) q No association between oral microbiome and response to therapy

Immunotherapy or Chemotherapy for Locally Advanced and Metastatic UC Question: How do you identify for immunotherapy?

BLADDER TCGA 207 Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, et al Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell. 207 Oct 9;7(3):540-556

Molecular Characteriza+on q TCGA (N = 42, previously: N = 3) 58 SMGs (34 not previously, 6 not in Pan- Cancer) Median F/U - > 7.5 m Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, et al Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell. 207 Oct 9;7(3):540-556

Future treatment paradigm?? Framework for prospec9ve hypothesis tes9ng in clinical trials, as well as for valida9on in ongoing or completed clinical trials that test, or have tested, treatment strategies CLASSIFICATION INTO DIFFERENT MOLECULAR SUBTYPES Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, et al Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell. 207 Oct 9;7(3):540-556

Gene expression publicly available were combined and reanalyzed. The dataset contained 24 unique tumors encompassing non- muscleinvasive (NMIBC) and muscle- invasive BLCA (MIBC). Subtypes were reproduced on The Cancer Genome Atlas, UROMOL, and IMvigor20. hxps://doi.org/0.06/j.eururo.208.08.027

Take Home Messages Immunotherapy in Urothelial Cancer is here to stay New FDA approvals: Pembrolizumab and atezolizumab in st line cispla9n- unfit (requires PD- L+) First FDA approval in muc in the last 30 years Does PD- L expression guide treatment selec9on? Several ongoing phase III trials with chemo- IO or IO- IO combina9ons for pla9num- fit or unfit pa9ents Promising biomarker results but s9ll very preliminary

Prognos+c/predic+ve biological factors Currently, no biomarkers can be recommended in daily clinical prac+ce because they have no impact on predic+ng outcome, treatment decisions or monitoring therapy in bladder cancer