Prospective Evaluation of Clinical Outcomes Using a Multiplex Liquid Biopsy Targeting Diverse Resistance Mechanisms in Metastatic Prostate Cancer

Abstract

Purpose: Nearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy.

Materials and methods: A novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board-approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings.

Results: Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]).

Conclusion: We demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.

Conflict of interest statement

Hamid EmamekhooConsulting or Advisory Role: Exelixis, Bayer, Bristol Myers Squibb, Cardinal Health, Seattle GeneticsResearch Funding: Bristol Myers Squibb, Exelixis, Replimune, Merck, Calithera Biosciences, Roche/GenentechTravel, Accommodations, Expenses: DAVA Pharmaceuticals Rana R. McKayConsulting or Advisory Role: Janssen, Novartis, Tempus, Exelixis, Pfizer, Bristol Myers Squibb, Astellas Medivation, Dendreon, Bayer, Sanofi, Merck, Vividion Therapeutics, Calithera Biosciences, AstraZenecaResearch Funding: Pfizer, Bayer, Tempus Xiao X. WeiHonoraria: OncLiveConsulting or Advisory Role: NovartisResearch Funding: Bristol Myers SquibbTravel, Accommodations, Expenses: Corvus Pharmaceuticals Rebecca SilverResearch Funding: Bayer Michael J. MorrisConsulting or Advisory Role: Bayer, Endocyte, Advanced Accelerator Applications, ORIC Pharmaceuticals, Johnson & Johnson, Curium Pharma, AthenexResearch Funding: Bayer, Sanofi, Endocyte, Progenics, Corcept Therapeutics, Roche/Genentech, JanssenTravel, Accommodations, Expenses: Endocyte, Fujifilm Felix Y. FengConsulting or Advisory Role: Astellas, Bayer, BlueEarth Diagnostics, Celgene, EMD Serono, Genentech, Janssen, Myovant, Ryovant, Bristol Myers Squibb, Exact Sciences, Varian, Bluestar Genomics, SerimmunResearch Funding: Zenith Epigenetics Howard I. ScherConsulting or Advisory Role: Janssen, Amgen, Janssen Research & Development, Menarini Silicon Biosystems, WIRB-Copernicus Group, ESSA, Ambry Genetics/Konica Minolta, Pfizer, Bayer, Sun PharmaResearch Funding: Janssen, Illumina, Epic Sciences, Menarini Silicon Biosystems, Thermofisher Scientific BiomarkersPatents, Royalties, Other Intellectual Property: BioNTech—Intellectual Property Rights, Elucida Oncology—Intellectual Property Rights, MabVAX—Intellectual Property Rights, Y-mAbs Therapeutics Inc—Intellectual Property RightsTravel, Accommodations, Expenses: Asterias Biotherapeutics, Menarini Silicon Biosystems, Amgen, WIRB-Copernicus Group, Konica Minolta, ESSA, Prostate Cancer Foundation, Sanofi, Bayer, Phosplatin Therapeutics Dana RathkopfConsulting or Advisory Role: Janssen, Genentech, AstraZeneca, Bayer, Myovant SciencesResearch Funding: Janssen Oncology, Medivation, Celgene, Takeda, Millennium, Ferring, Novartis, Taiho Pharmaceutical, AstraZeneca, Genentech/Roche, TRACON Pharma, Bayer, Phosplatin Therapeutics Scott M. DehmConsulting or Advisory Role: Celgene, Oncternal Therapeutics, Janssen Research & DevelopmentResearch Funding: Janssen Research & Development, Medivation/AstellasPatents, Royalties, Other Intellectual Property: Royalties from licensing genome-engineered prostate cancer cell lines Toni K. ChoueiriEmployment: Dana Farber Cancer HospitalLeadership: Dana Farber Cancer Hospital, NCCN, KidneyCAN, ASCOStock and Other Ownership Interests: Pionyr, Tempest TherapeuticsHonoraria: NCCN, UpToDate, Michael J. Hennessy Associates, ASCO, Harborside Press, Analysis Group, AstraZeneca, Alexion Pharmaceuticals, Sanofi/Aventis, Bayer, Bristol Myers Squibb, Genentech/Roche, GlaxoSmithKline, Merck, Novartis, Peloton Therapeutics, Pfizer, Corvus Pharmaceuticals, Ipsen, Foundation Medicine, Eisai, PlatformQ Health, Clinical Care Options, Navinata Health, Kidney Cancer Association, Exelixis, Prometheus, Lpath, The New England Journal of Medicine, Lancet Oncology, Cerulean Pharma, Alligent, EMD Serono, HERON, Lilly, Janssen Oncology, IQvia, Aveo, NCI GU Steering CommitteeConsulting or Advisory Role: Pfizer, Bayer, Novartis, GlaxoSmithKline, Merck, Bristol Myers Squibb, Roche/Genentech, Eisai, Foundation Medicine, Cerulean Pharma, AstraZeneca, Exelixis, Prometheus, Alligent, Ipsen, Corvus Pharmaceuticals, Lpath, Alexion Pharmaceuticals, Sanofi/Aventis, Peloton Therapeutics, UpToDate, NCCN, Michael J. Hennessy Associates, Analysis Group, Kidney Cancer Association, Clinical Care Options, PlatformQ Health, Navinata Health, Harborside Press, ASCO, The New England Journal of Medicine, Lancet Oncology, EMD Serono, HERON, Lilly, ESMOResearch Funding: Pfizer, Novartis, Merck, Exelixis, TRACON Pharma, GlaxoSmithKline, Bristol Myers Squibb, AstraZeneca, Peloton Therapeutics, Roche/Genentech, Celldex, Agensys, Eisai, Takeda, Prometheus, Ipsen, Corvus Pharmaceuticals, Cerulean Pharma, Seattle Genetics/Astellas, Bayer, Foundation Medicine, Roche, Calithera Biosciences, Analysis Group, NCI, Gateway for Cancer Research, Congressionally Directed Medical Research Programs (DOD)Patents, Royalties, Other Intellectual Property: International Patent Application No. PCT/US2018/058430, titled “Biomarkers of Clinical Response and Benefit to Immune Checkpoint Inhibitor Therapy,” and International Patent Application No. PCT/US2018/12209, titled “PBRM1 Biomarkers Predictive of Anti-Immune Checkpoint Response”Travel, Accommodations, Expenses: Pfizer, Bayer, Novartis, GlaxoSmithKline, Merck, Bristol Myers Squibb, Roche/Genentech, Eisai, Foundation Medicine, Cerulean Pharma, AstraZeneca, Exelixis, Prometheus, Alligent, Ipsen, Corvus Pharmaceuticals, Lpath, Alexion Pharmaceuticals, Sanofi/Aventis, UpToDate, Peloton Therapeutics, NCCN, Michael J. Hennessy Associates, Analysis Group, Kidney Cancer Association, Clinical Care Options, PlatformQ Health, Harborside Press, Navinata Health, The New England Journal of Medicine, Lancet Oncology, EMD Serono, HERON, Lilly, ESMOOther Relationship: Medical writing and editorial assistance support might have been funded by Communications companies funded by pharmaceutical companies such as ClinicalThinking, Health Interactions, Envision Pharma Group, Fishawack Group of Companies, Parexel Susan HalabiEmployment: ASCO Andrew J. ArmstrongHonoraria: Astellas Scientific and Medical Affairs IncConsulting or Advisory Role: Bayer, Dendreon, Pfizer, Astellas Scientific and Medical Affairs Inc, Clovis Oncology, AstraZeneca, Merck, Bristol Myers SquibbResearch Funding: Dendreon, Bayer, Pfizer, Novartis, Janssen Oncology, Astellas Pharma, Gilead Sciences, Roche/Genentech, Bristol Myers Squibb, Constellation Pharmaceuticals, Merck, AstraZeneca, BeiGenePatents, Royalties, Other Intellectual Property: Circulating tumor cell novel capture technologyTravel, Accommodations, Expenses: Astellas Scientific and Medical Affairs Inc Alexander W. WyattHonoraria: Janssen, Astellas Pharma, AstraZeneca, Merck, AstraZeneca CanadaConsulting or Advisory Role: AstraZenecaResearch Funding: ESSA Mary-Ellen TaplinHonoraria: Janssen-Ortho, Clovis Oncology, Astellas Pharma, Incyte, UpToDate, Research to Practice, Pfizer, Bayer, Amgen, AstraZeneca, Progenics, Guidepoint Global, Celgene, Merck, GlaxoSmithKline, Myovant Sciences, Roivant, AbbVie, Arcus Biosciences, Constellation PharmaceuticalsConsulting or Advisory Role: Janssen-Ortho, Bayer, Guidepoint Global, Best Doctors Inc, UpToDate, Clovis Oncology, Research to Practice, Myovant Sciences, Incyte, Pfizer, AstraZeneca, Arcus VenturesResearch Funding: Janssen-Ortho, Medivation, Bayer, PfizerTravel, Accommodations, Expenses: Medivation, Janssen Oncology, Tokai Pharmaceuticals, Astellas Pharma, Incyte, Pfizer, Clovis Oncology, Bayer Shuang G. ZhaoEmployment: Exact Sciences (I)Patents, Royalties, Other Intellectual Property: Patent applications pending with Decipher Biosciences, Exact Sciences Joshua M. LangStock and Other Ownership Interests: Salus DiscoveryConsulting or Advisory Role: Sanofi, Immunomedics, Janssen, Pfizer/Astellas, 4D PharmaResearch Funding: Medivation, Agensys, GlaxoSmithKline, Immunomedics, Bristol Myers Squibb, JanssenPatents, Royalties, Other Intellectual Property: I am listed on the patent on a technology for rare cell capture and analysis. This technology has been licensed by Salus Discovery LLC although no commercial products are availableNo other potential conflicts of interest were reported.

Figures

FIG 1.

Two distinct molecular clusters in circulating tumor cells from patients with metastatic prostate cancer. Hierarchical clustering of gene expression data of 11 genes from 99 patients with prostate cancer identified two distinct clusters (cluster 1: C1 and cluster 2: C2). Serum PSA (ng/mL), prostate cancer type, treatment with ARSI or chemotherapy in CRPC setting, sites of metastases at time of blood draw, and serum PSA at time of blood draw are indicated. X indicates presence of metastases at indicted site. AR, androgen receptor; ARSI, androgen receptor signaling inhibitor; AR-V, androgen receptor splice variant; CRPC, castration-resistant prostate cancer; CSPC, castration-sensitive prostate cancer; LN, lymph node; NEPC, neuroendocrine prostate cancer; PSA, prostate-specific antigen.

FIG 2.

C2 is prognostic for OS independent of AR-V status. (A) Swimmer plot ordered by OS for C2 (orange, top) and C1 (cyan, bottom). Gene expression for AR-V and AR pathway genes is indicated. X indicates the patient death. (B) Kaplan-Meier plot of OS by C2 versus C1. (C) Kaplan-Meier plot of OS by AR-V status. AR, androgen receptor; AR-V, androgen receptor splice variant; C1, cluster 1; C2, cluster 2; CRPC, castration-resistant prostate cancer; CSPC, castration-sensitive prostate cancer; HR, hazard ratio; NEPC, neuroendocrine prostate cancer; OS, overall survival.

FIG 3.

Multivariate survival analysis indicates that the association of cluster and risk of OS remained after adjusting covariates. Association of AR-V status and OS was no longer significant. AR-V, androgen receptor splice variant; CRPC, castration-resistant prostate cancer; HR, hazard ratio; NEPC, neuroendocrine prostate cancer; OS, overall survival; PSA, prostate-specific antigen.

FIG 4.

C2 has worse PSA, rPFS, and OS in two multicenter phase II trials of androgen receptor signaling inhibitors. Kaplan-Meier plot of (A) OS, (B) PSA PFS, and (C) rPFS for patients in C2 compared with C1 in patient treated with enzalutamide or abiraterone acetate. C1, cluster 1; C2, cluster 2; HR, hazard ratio; NR, not reached; OS, overall survival; PFS, progression-free survival; PSA, prostate-specific antigen; rPFS, radiographic progression-free survival.

FIG 5.

Treatment-emergent NEPC can be detected before the onset of clinical progression. (A) Heatmap of gene expression data demonstrates the acquisition of genes commonly upregulated in NEPC as a patient is treated with androgen deprivation therapy (yellow) and carboplatin and etoposide (blue). (B) Top panel shows ctDNA fractions (as a proportion of total cell-free DNA) at three sampled timepoints. Bottom panel shows a violin plot of the copy number profile at month 17. Blue indicates genes that pass the thresholds for identifying a copy deletion, and red indicates copy gain. Note that the violin for TP53 is distorted because of very low coverage (because of a deep deletion). For PTEN and RB1, the violins have long tails, suggesting that there may be intragenic differences in copy profiles. (C) Copy number profiles showing RB1 and PTEN exon-level data. All three patient timepoints are shown. Differences between the timepoint are a function of ctDNA fraction. Note that the high ctDNA fraction in the month 17 timepoint enables identification of an exon 1 deep deletion in PTEN and a 3′ deep deletion within RB1. AR, androgen receptor; AR-V, androgen receptor splice variant; ctDNA, circulating tumor DNA; NA, not available; NEPC, neuroendocrine prostate cancer.