CIRCULATING TUMOR DNA AND CLINICAL OUTCOMES IN ADVANCED PROSTATE CANCER

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1 Urology Rounds Vancouver 27 th September2016 CIRCULATING TUMOR DNA AND CLINICAL OUTCOMES IN ADVANCED PROSTATE CANCER Dr. Alexander W Wyatt, D.Phil Senior Research Scientist, Vancouver Prostate Centre Assistant Professor, University of British Columbia

2 NATURAL HISTORY OF PROSTATE CANCER Huge disparity between local and metastatic disease 5 year survival rate for localized disease ~100% 5 year survival rate for metastatic disease ~28% (US, 2015)

3 KNOWN RISK FACTORS FOR PROSTATE CANCER DEVELOPMENT Increasing age African-American race Positive family history 1 relatives of men with prostate cancer have 2X risk of developing prostate cancer 1 Risk compounded in early onset cases Nordic twin studies suggest 42% of risk is due to genetic factors 1 Goldgar et al., J. Natl Cancer Inst. 86, (1994) 2 Lichtenstein, et al., N. Engl. J. Med. 343,78 85 (2000)

ANDROGEN RECEPTOR SIGNALING AND PROSTATE CANCER DEVELOPMENT: LOCALIZED DISEASE Androgen receptor Ligand-activated transcription factor Activated by testosterone, dihydrotestosterone Driver of PC

4 ANDROGEN RECEPTOR SIGNALING AND PROSTATE CANCER DEVELOPMENT: LOCALIZED DISEASE Androgen receptor Ligand-activated transcription factor Activated by testosterone, dihydrotestosterone Driver of PC development and progression 1,2 1 Basch E, Loblaw DA, Oliver TK, et al. J Clin Oncol 2014;32: Saad F, Chi KN, Finelli A, et al. Can Urol Assoc J 2015;9: Wyatt AW and Gleave ME. EMBO Mol Med 2015; 7:

5 LANDSCAPE OF GENOMIC ALTERATIONS IN LOCALIZED DISEASE TGCA published 333 primary prostate tumors in 2015 Confirmed the known landscape TP53, PTEN, RB1, SPOP ETS fusions

6 HEREDITARY PROSTATE CANCER GENES Challenging due to the late-onset nature of the disease and the high background rate of sporadic disease in the general population GWAS identified >100 common variants (>5%) explaining >30% of familial risk 1,2 Low risk: OR G84E missense mutation in HOXB13 in 5% of European prostate cancer families to 20-fold increased risk of developing PCa, especially early-onset Rare mutations in BRCA1, BRCA2, MSH2 account for small proportion of familial cases BRCA2 mutations associated with % PCa overall 4 1 International Consortium for Prostate Cancer Genetics, Human Genetics 132: pp 5 14 (2013) 2 Kote-Jarai Z et al., Nat Genet 43: (2011) 3 Ewing et al., N Engl J Med 366(2): (2012) 4 Kote-Karai et al., Br J Cancer 105(8): (2011)

7 ANDROGEN RECEPTOR SIGNALING, ANDROGEN DEPRIVATION AND CRPC Castration-resistant prostate cancer (CRPC) AR axis signaling despite low serum androgen levels Compensatory mechanisms (e.g., AR gene amplification, AR overexpression, AR mutation, extratesticular androgen production) 1 1 Karantanos T, Evans CP, Tombal B, et al. Eur Urol 2015;67: Wyatt AW and Gleave ME. EMBO Mol Med 2015; 7:

8 ENZALUTAMIDE IS A POTENT AR ANTAGONIST THAT IMPROVES OUTCOMES IN mcrpc HC APPROVAL TREATMENT MOA LINE OF TREATMENT PIVOTAL TRIAL 2005 Docetaxel Anti-mitotic taxane chemotherapy 2011 Cabazitaxel Second generation taxane designed to overcome docetaxel resistance 2011 Abiraterone Androgen biosynthesis inhibitor First TAX 327, Tannock 2004 Second Second TROPIC, de Bono 2010 COU-AA-301, de Bono Enzalutamide Antiandrogen Second AFFIRM, Scher Abiraterone Androgen biosynthesis inhibitor First COU-AA-302, Rathkopf Rad-233 Bone-targeting radiotherapy First ALSYMPCA, Parker Enzalutamide Antiandrogen First PREVAIL, Beer 2014 NA Sipuleucel-T Cell-based immunotherapy First IMPACT, Kantoff 2010 Abbreviations: HC, Health Canada; MOA, mode of action; NA, not applicable

RESISTANCE TO ANDROGEN RECEPTOR TARGETED THERAPIES IS INEVITABLE Primary Resistance: No PSA response or PSA progression from start of treatment in pivotal phase III trial of enzalutamide in patients

9 RESISTANCE TO ANDROGEN RECEPTOR TARGETED THERAPIES IS INEVITABLE Primary Resistance: No PSA response or PSA progression from start of treatment in pivotal phase III trial of enzalutamide in patients pretreated with docetaxel. AFFIRM 1 > 50% confirmed PSA fall: Enza 54% ; Placebo 2% (p < ) > 90% confirmed PSA fall: Enza 25%; Placebo 1% (p < ) Placebo Enzalutamide 1 out of 5 patients show primary resistance* Enzalutamide (n = 800) Placebo (n = 399) * Estimated from graph; Positive PSA change percentages indicate patients who never had PSA decline in response to treatment. de Bono JS, Fizazi K, Saad F, et al. Primary, secondary, and quality-of-life endpoint results from the phase III AFFIRM study of MDV3100, an androgen receptor signaling inhibitor. J Clin Oncol 2012;30(suppl): 4519

10 TREATMENT RESISTANCE IS INEVITABLE Treatment resistance and progression are inevitable AR-related Amplification AR ligand binding domain mutations AR truncated variant expression Non-AR related: PI3K/AKT, CDK/RB1, etc Molecular biomarkers that can predict individual outcomes are lacking Historically requires CRPC tissue Antonarakis ES, Lu C, Wang H, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371: Robinson D, Van Allen EM, et al. Integrative clinical genomics of advanced prostate cancer. Cell 2015; 161:

11 THE LIQUID BIOPSY PROMISES A MINIMALLY- INVASIVE TOOL TO SAMPLE CRPC TUMORS Wyatt AW and Gleave ME. Targeting the adaptive molecular landscape of castration-resistant prostate cancer. EMBO Mol Med 2015; 7:

12 CELL-FREE DNA cfdna is found freely in the circulation 4-6x higher cfdna concentration in cancer patients vs. healthy controls ctdna (tumor) can constitute <1% - 90% of total cfdna Genomic changes in ctdna are detectable in mcrpc patients CN gains/losses, mutations, rearrangements Reflect changes found in tissue biopsies E Heitzer, Genome Med, 5:30, 2013; JD Joseph Cancer Discov, 3:1020, 2013; S Carreira, Sci Transl Med, 6: 254ra125, 2014; A Azad, Clin Cancer Res, 21;2315, 2015

13 GROSS COPY NUMBER CHANGES ARE ROBUSTLY DETECTABLE IN CFDNA 65 mcrpc patients profiled by acgh Azad AA et al. Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: Biomarkers of Therapeutic Resistance in Castration-Resistant Prostate Cancer. Clin Cancer Res 2015; 21:

14 CELL-FREE DNA AS A POTENTIAL BIOMARKER FOR ENZALUTAMIDE RESPONSE 39 patients in our study commenced enzalutamide after providing cfdna

AR MUTATIONS AND COPY NUMBER STATUS INFLUENCE RESPONSE TO ABIRATERONE Patients with AR gain or T878A or L702H before abiraterone had a significantly worse overall survival [hazard ratio

15 AR MUTATIONS AND COPY NUMBER STATUS INFLUENCE RESPONSE TO ABIRATERONE Patients with AR gain or T878A or L702H before abiraterone had a significantly worse overall survival [hazard ratio (HR), 7.33; 95% confidence interval (CI), 3.51 to 15.34; P = )]. Carreira et al., Sci Transl Med Sep 17;6(254):254ra125. Romanel et al., Sci Transl Med Nov 4;7(312):312re10ZZZZ

16 STUDY DESIGN ENZA RESPONSE Hypothesis: genomic aberration (especially to the AR) detectable in cfdna predicts response, and reveals resistance mechanisms, to enzalutamide Wyatt AW, et al., JAMA Oncology 2016

17 PATIENT POPULATION AT BASELINE Characteristic n = 65 Age, median (range) 72 (53-92) Metastases, n (%) Bone Lymph node Visceral ECOG performance status Laboratory, median (range) Haemoglobin (g/l) LDH (U/L) ALP (U/L) Albumin (g/l) All prior systemic treatments, n (%) Abiraterone Docetaxel Bicalutamide Flutamide Nilutamide Other 62 (95) 29 (45) 9 (14) 47 (72) 18 (28) 122 (74-156) 236 ( ) 130 ( ) 39 (29-49) 40 (62) 27 (42) 60 (92) 11 (17) 15 (23) 20 (31)

18 COPY NUMBER ABERRATION AT BASELINE By acgh, 27/63 baseline samples harbored detectable changes in genome copy number AR amplification: 10/63 at baseline AR gain: 9/63 at baseline Could be amplifications masked by low ctdna

19 AR MUTATIONS AND BROAD LIGAND SPECIFICITY Lorente, D. et al. (2014) Switching and withdrawing hormonal agents for castration-resistant prostate cancer. Nat. Rev. Urol. doi: /nrurol

20 AR MUTATIONS IN CFDNA FROM PATIENTS TREATED WITH ENZALUTAMIDE Median allelic frequency of AR mutations at baseline was 8.74%, considerably above detection threshold At baseline: AR mutation Frequency Any 22% L702H 10% H875Y 10% T878A 8% W742L/C 6% Other 2% Multiple ( 2) 8% Experiments: K Beja

21 AR MUTATIONS IN CFDNA FROM PATIENTS TREATED WITH ENZALUTAMIDE L702H and T878A only detected in patients who received prior abiraterone (p<0.05) No AR mutations in samples with AR amplification Experiments: K Beja

22 GENOMIC CHANGES IN CFDNA AT BASELINE AND DISEASE PROGRESSION The detection of AR gain/amplification, multiple AR mutations, RB1 loss, MET gain and MYC gain was linked to adverse outcomes on enzalutamide After adjusting for the presence of ctdna, detection of RB1 loss (p=0.01) and MET gain (p=0.02) remained significantly associated with worse progression-free survival (PFS) (Cox proportional-hazards modeling). Trend towards shorter PFS for patients with multiple AR mutations (p=0.09)

GENOMIC CHANGES IN CFDNA AT BASELINE AND DISEASE PROGRESSION Among patients with AR aberrations, those with a heavily aberrant AR (amplification or 2 mutations) had median PFS of only 1.

23 GENOMIC CHANGES IN CFDNA AT BASELINE AND DISEASE PROGRESSION Among patients with AR aberrations, those with a heavily aberrant AR (amplification or 2 mutations) had median PFS of only 1.9 months compared to 4.4 months for patients with a single AR mutation (p=0.035; logrank). Patients with a single AR mutation did not appear to exhibit primary resistance to enzalutamide.

24 DYNAMIC CFDNA CHANGES AND ACQUIRED ENZALUTAMIDE RESISTANCE 9/30 (30%) had AR copy number gain and 13/30 (43%) had AR mutation. Compared to baseline samples, we detected emergence and/or regression of particular copy number changes and AR mutations in over half the cohort Suggests tumor clonal population changes and consistent with complex and dynamic intra-patient heterogeneity

25 DYNAMIC CFDNA CHANGES AND ACQUIRED ENZALUTAMIDE RESISTANCE Complete regression of clones bearing W742L/C mutations. Emergence of L702H positive clones in five patients, and an increase in L702H frequency in three patients that were positive at baseline. All 11 patients with L702H at either baseline or progression had received prior abiraterone (11/40 vs. 0/22 p=0.0274; Fishers exact test). Only one instance of F877L at progression High heterogeneity in patients with multiple mutations

CLINICALLY-ACTIONALBE MUTATIONS IN CRPC TISSUE BIOPSY STUDY Aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.

26 CLINICALLY-ACTIONALBE MUTATIONS IN CRPC TISSUE BIOPSY STUDY Aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 8% germline! 1: Robinson D, et al. Integrative clinical genomics of advanced prostate cancer. Cell 2015; 161:

27 CLINICALLY ACTIONALBE MUTATIONS IN CFDNA FROM PROGRESSING PATIENTS Recent study 1 of mcrpc tissue biopsies suggests a proportion of patients harbor specific genomic aberration that potentially confers sensitivity to novel investigative agents We performed deep targeted sequencing of 19 frequently mutated or clinically-actionable prostate cancer genes in our progression samples with sufficient remaining cfdna (n=14). Ion Ampliseq Custom DNA panel (Life Tech) 1: Robinson D, et al. Integrative clinical genomics of advanced prostate cancer. Cell 2015; 161:

28 CLINICALLY ACTIONALBE MUTATIONS IN CFDNA FROM PROGRESSING PATIENTS Somatic mutations and/or copy number changes were detected in all samples, at a remarkably high allelic frequency (median mutation frequency excluding AR = 19.7%). Experiments: K Beja

CLINICALLY ACTIONALBE MUTATIONS IN CFDNA FROM PROGRESSING PATIENTS We screened all patients at progression for germline BRCA1 and BRCA2 mutations Two patients harboured germline BRCA2

29 CLINICALLY ACTIONALBE MUTATIONS IN CFDNA FROM PROGRESSING PATIENTS We screened all patients at progression for germline BRCA1 and BRCA2 mutations Two patients harboured germline BRCA2 frameshift/nonsense mutations, and a third patient had a germline PALB2 frameshift mutation One patient (VC-022) with a BRCA2 mutation had matched cfdna sequenced, and loss-of-heterozygosity was evident in cfdna. Wyatt AW, et al., JAMA Oncology 2016

30 DNA REPAIR DEFECTS AND OLAPARIB IN METASTATIC PROSTATE CANCER Men with mpca and DNA repair defects have sustained response to PARP inhibition

31 PATIENT WITH LETHAL PROSTATE CANCER Chedgy et al. Clin Genitourin Cancer Apr;14(2):e233-6.

32 COMPLETE LOSS OF BRCA2 IN CTDNA Chedgy et al. Clin Genitourin Cancer Apr;14(2):e233-6.

33 DRAMATIC RESPONSE TO DNA DAMAGING AGENT Chedgy et al. Clin Genitourin Cancer Apr;14(2):e233-6.

34 CONCLUSIONS FROM ENZA STUDY Genomic aberrations are readily detectable in cfdna Specific aberrations were associated with adverse clinical outcomes on enzalutamide AR gain/amp; RB1 loss AR amplification marked the worst outcomes Single AR mutations were not predictive for enzalutamide outcomes L702H and T878A were observed exclusively in previously abiraterone treated patients and persist through enzalutamide treatment F877L was only observed in a single patient to date 8% of patients had 2 AR mutations Associated with poor outcome Clinically-actionable aberration is detectable in cfdna at progression on enzalutamide. Wyatt AW, et al., JAMA Oncology 2016

CURRENT METHODOLOGY OVERVIEW cfdna extraction from plasma using Qiagen kit Initial NGS library preparation using KAPA kit Target capture using Roche Nimblegen SeqCap EZ Choice custom probe set 73

35 CURRENT METHODOLOGY OVERVIEW cfdna extraction from plasma using Qiagen kit Initial NGS library preparation using KAPA kit Target capture using Roche Nimblegen SeqCap EZ Choice custom probe set 73 CRPC-related genes including AR pathway genes, prostate cancer drivers (e.g. TP53, SPOP), cell cycle drivers (e.g. CCND1, RB1, CDK4/6), DNA repair genes (e.g. BRCA1/2, FANC family genes, ATM, MSH2/6), PI3K pathway genes (e.g. PIK3CA, PTEN) Sequencing to minimum 500X with Illumina MiSeq or HiSeq Data analytics using tailored bioinformatics pipeline K Beja, G Vandekerkhove, E Warner, A Wong et al.

39 Patient 9172 liquid biopsy obtained August 2016 Sample type: cell-free DNA and matched leukocyte DNA Diagnosis: castration-resistant prostate cancer Methods: targeted sequencing was performed using a custom Roche NimbleGen SeqCap Comprehensive Cancer Design (72 prostate cancer related cancer genes) and Illumina technology. Copy number profile AR amplification PTEN deletion RB1 deletion TP53 deletion Relevant mutations PTEN splice site mutation at 30.7% (note deletion of other allele above, suggesting bi-allelic PTEN loss) TP53 missense mutation at 26.7% (note deletion of other allele above, suggesting bi-allelic TP53 loss) Complex FOXA1 rearrangement disrupting one allele Other notes: ctdna fraction appears to be high (even for CRPC), potentially indicating high tumor burden and/or progression

40 ABI-ENZA CROSSOVER TRIAL June 2016: >150 patients accrued cfdna from 135 baseline (median yield ~10-15ng/ml plasma) Deep sequencing of 62 patients presented at ASCO M. Annala, K. Beja, S. Parimi, G. Vandekerkhove, E. Warner, A Wong, M. Zulfiqar, D. Finch, C. Oja, J. Vergidis, M. Nykter, M. E. Gleave, K. N. Chi

41 ABI-ENZA CROSSOVER TRIAL: PRELIM RESULTS AR gene amplification in 17 patients Median time on treatment = 200 vs 420 days (p<0.05) PI3K and WNT pathway events are clearly evident Deletion of DNA damage repair components including evidence of bi-allelic BRCA2 loss BRCA2 loss median time on treatment = 120 vs 342 days (p<0.05) Current work: Expansion to all patients Temporal samples Exome sequencing ddpcr Wyatt and Chi; ASCO 2016 and unpublished

42 NEW DATA FROM ASCO ON GERMLINE DEFECTS A large consortium recruited 692 men with metastatic prostate cancer from 8 different institutions Unselected for family history, age, race Found 84 damaging germline mutations in 11.8% of men Significantly higher than normal pop (2.7% - EXAC) and localized prostate cancer (4.6% - TCGA, high-risk) 44% BRCA2, ATM 13.5 CHEK2 11%, BRCA1 7%. Patients with germline mutation were no more likely to have 1st degree relative with PCa. However, were significantly more likely to have 1st degree relative with other cancers Inherited mutations in DNA repair genes in men with metastatic castration-resistant prostate cancer. J Clin Oncol 34, 2016 (suppl; abstr 5009) Presented at ASCO 2016 by Dr. Peter Nelson.

43 UMBRELLA TRIAL INCLUDING A WEE1 INHIBITOR Umbrella trial: CDK4/6; WEE1, AKT, BRD,? To commence Fall 2016 Target = 300; 3 years Prospectively enrolled; 4 week TAT. Rubin, E. H. & Gilliland, D. G. (2012) Drug development and clinical trials the path to an approved cancer drug. Nat. Rev. Clin. Oncol.

44 CONCLUSIONS cfdna / ctdna profiling is a minimally-invasive tool to study mcrpc Aberrations detectable in ctdna are associated with resistance to systemic therapies ctdna provides a tool to enrich clinical trials with patients likely to respond Potentially most exciting for patients with DNA damage repair defects Should all metastatic PCa patients and families should be screened for germline defects regardless of family history or age?

45 ACKNOWLEDGEMENTS Wyatt Laboratory Kevin Beja Gillian Vandekerkhove Evan Warner Amanda Wong Edmund Chedgy (c/o Martin Gleave) Werner Struss (c/o Alan So) Vancouver Prostate Centre & UBC S Volik, B McConeghy, C Collins Martin Gleave Institute of Biosciences and Medical Technology, Tampere Matti Annala (joint with Wyatt lab) Matti Nykter BC Cancer Agency Arun Azad, Sunil Parimi, Leanne Seto, Lejla Gavranovic M Zulfiqar, D Finch, C Oja, J Vergidis Kim Chi

Sequencing Strategies in Metastatic Castration Resistant Prostate Cancer (MCRPC)

Sequencing Strategies in Metastatic Castration Resistant Prostate Cancer (MCRPC) Amit Bahl Consultant Oncologist Bristol Cancer Institute Clinical Director Spire Specialist Care Centre UK Disclosures Advisory