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Clinical Utility of Droplet Digital PCR on Liquid Biopsies from Patients with Castration Resistant Prostate Cancer
- 1. in partnership with Making the discoveries that defeat cancer Clinical Utility of Droplet Digital PCR on Liquid Biopsies from Patients with Castration- Resistant Prostate Cancer Daniel Wetterskog, PhD Senior Scientific Officer Institute of Cancer Research daniel.wetterskog@icr.ac.uk 4BIO Summit - 5th qPCR & Digital PCR Congress - 041217
- 2. Presentation outline • Introduction to prostate cancer • Androgen signaling • Anti-androgen therapies • Genomic aberrations of the Androgen Receptor (AR) • Monitoring treatment resistance • Liquid biopsy studies to monitor treatment resistance in CRPC patients • Abiraterone resistance – Target NGS • Abiraterone and Enzalutamide resistance - ddPCR • Design of clinical trial • Multiplex ddPCR assays for patient stratification based on AR aberrations 2
- 3. Androgen Signalling and Prostate Cancer Hypothalamus Anterior pituary Adrenal gland Testes LH LHRH ACTH Androgens Androgen responsive cell AR AR Androgen responsive genes such as PSA AR Transcriptional Activation
- 4. Anti-androgen therapies for Prostate Cancer Hypothalamus Anterior pituary Adrenal gland Testes LH LHRH ACTH Androgens Prostate Cancer Cell AR Androgen responsive genes such as PSA No transcription Abiraterone AR E E LHRH analogs Enzalutamide
- 5. Androgen receptor mutations in metastatic prostate cancer Prostate Cancer Cell Androgen responsive genes such as PSA Transcriptional activation L702H T878A E Alternative activators • Mutations in AR are first seen after anti- androgen treatments have been started • AR T878A • Activated by other molecules such as progesterone • Affects enzalutamide binding • AR L702H • Activated by prednisone which is a steroid often used as a co-treatment with abiraterone or taxanes T878A L702H T878A E
- 6. Androgen receptor amplification in metastatic prostate cancer Prostate Cancer Cell Androgen responsive genes such as PSA Transcriptional activation AR AR AR AR AR AR • Amplification of the AR gene is usually not detected in primary prostate cancer • Increased AR protein levels might circumvent androgen deprivation therapy AR AR AR AR AR AR AR
- 7. Monitoring treatment resistance in prostate cancer patients 7
- 8. Liquid biopsies : Plasma as a source of tumour DNA 8
- 9. Technical considerations for cell free DNA / liquid biopsy studies If protocol allows, collect more plasma samples, use techniques requiring less input 9 • Low amount of DNA • Unknown/low tumour content - ctDNA • Fragmented DNA Use assays with high sensitivity and/or methods to assess tumour content Adjust design of assays for short pieces of DNA
- 10. in partnership with Making the discoveries that defeat cancer Study 1) Targeted NGS Plasma AR and abiraterone-resistant prostate cancer, Romanel et.al, Science Translational Medicine, 2015
- 11. Targeted NGS Panel Design 11 • Targeted NGS panel covering 39,000bp (median coverage = 1434X using 6-10ng DNA) • Including common and dominant deletions and mutations found in mCRPC Chromosome and gene targets Bases covered Amplicons n= Amplicon bp length 8p23 including NKX3.1 10017 87 73-140 10q23 including PTEN 8060 37 64-133 CYP17A1 2315 21 82-134 FOXA1 1526 14 87-129 TP53 2036 19 93-128 SPOP 1682 16 72-127 21q22 including TMPRSS2-ERG 12005 107 75-137 AR 3478 30 78-137
- 12. AR aberrations : Mutual exclusivity between AR gained and mutated alleles
- 13. Association of AR gene status with PSA response upon Abiraterone treatment
- 14. Association of AR status with overall survival and progression-free survival in patients receiving Abiraterone
- 15. in partnership with Making the discoveries that defeat cancer Study 2) Droplet digital PCR Androgen receptor gene status in plasma DNA associates with worse outcome on enzalutamide or abiraterone for castration-resistant prostate cancer – Conteduca V, Wetterskog D et.al, Ann Onc, 2017
- 16. Droplet Digital PCR for determining AR status in plasma ddPCR for AR Copy number ddPCR for AR mutations HEX amplitude HEX amplitude
- 18. Method comparison of Targeted NGS and ddPCR for determining AR status Bias -0.018 SD of bias 0.046 Bias -0.20 SD of bias 0.81
- 19. Histogram of T Frequencyofhighestmaximumlikelihood 0 5 10 0200040006000800010000 Establishing cut-off for AR gain in liquid biopsies using ddPCR • Establishing the AR copy number value that optimally splits the cohort into two groups who have different prognosis of overall survival • Bootstrapping with replacement technique was used and iterated the search for the optimal cutpoint 30,000 times to estimate the measures of dispersion • A ddPCR AR copy number value above or equal to 2.01 was considered gain Frequencyofhighestmaximumlikelihood Copy numbers 0 5 10 10000 8000
- 20. Association of AR status with overall survival in patient treated with abiraterone or enzalutamide Pre-chemo Post-chemo
- 21. Association of AR status with survival in pre-chemo patients treated with enzalutamide
- 22. Preliminary Trial design - Plasma AR Analysis to DIrect the manaGement of Metastatic-CRPC (PARADIGM)
- 23. ddPCR multiplexing assay ddPCR for AR copy number and mutations
- 24. ddPCR multiplexing assay v2 ddPCR for AR copy number and mutations
- 25. ddPCR multiplexing assay v2 ddPCR for AR copy number and mutations
- 26. in partnership with Gert Attard Vincenza Conteduca Emily Grist Anna Wingate Karolina Nowakowska Anjui Wu Anu Jayaram Paolo Cremaschi Samanta Salvi Marina Perry Larissa Mendes Mariana Pereira Treatment resistance group
- 27. in partnership with ICR cancer biomarkers team Suzanne Carreira Jane Goodall Roberta Ferraldeschi Penny Flohr Susana Miranda Daniel Nava Rodrigues Ruth Riisnaes Ines Figueiredo Mateus Crespo University of Trento, Italy Francesca Demichelis Alessandro Romanel Nicola Casiraghi Davide Prandi IRST, Meldola, Italy Ugo De Giorgi Samanta Salvi Dino Amadori Giorgia Gurioli Valentina Casadio RM/ICR prostate targeted theraphy group Johann de Bono Diletta Bianchini Zafeiris Zafeiriou Joaquin Mateo Pasquale Rescigno David Lorent Spyridon Sideris Nina Tunariu Raquel Perez-Lopez Emilda Thompson Jo Hunt Dee Moloney Bindu Baikady Ajit Sarvadikar Ada Balasopoulou Acknowledgements
- 28. in partnership with Acknowledgements Our patients and their families