'CK(-)PD-L1(+) Circulating Cells Associated with Malignancy (CCAM) at Diagnosis is a Prognostic Factor for Overall Survival in Lung Cancer Patients.'

Cox Proportional Hazards Model of Patients With Confirmed Lung Cancer.

Circulating Cells Associated with Malignancy (CCAM) Enumeration by Histology and Stage.

Assessment of PD-L1 Assay Sensitivity and Specificity.

Figure S1: Examples of a decision boundary that separate class A (red) from class B (blue) in 1 dimension (figure 1A) and 2 dimensions (figure 1B); Figure S2: Schematic of the supervised learning process (A) and leave one out cross validation (B); Figure S3: Single iteration of Leave-One-Out Cross-Validation performed at the blood sample level; Figure S4: An example of concordance of AURKA amplification in tumor and CTCs; Figure S5: (A) Kernel Density Estimate (KDE) curves of the classifier output are plotted for each patient sample colored by their diagnosis: NEPC (red) and CRPC (blue); Figure S6: Cell-level NEPC classifier (A) Receiver-Operating-Characteristic (ROC) curve generated on the classifier's single-cell output after LOOCV; Figure S7: To address whether the CTC classifier is simply stochastic, a reflection of an overall higher CTC count, linearity was assessed with a Pearson's coefficient showing a weak relationship between frequency of NEPC CTCs and total cell count; Table S1: A summary of cell-level features utilized to train Random Forest cell-level classifiers in both the LOOCV and for the classification of CTCs in the test cohort; Table S2: Patient characteristics (discovery cohort) including prior systemic therapies, serum markers including PSA (ng/ml), Chromogranin (ng/ml), NSE (ng/ml) and CTC counts; Table S3: Liver metastases in NEPC vs. CRPC; Table S4: The median concentration of CK-negative and AR-negative CTCs in CRPC, atypical CRPC, and NEPC patients; Table S5: Confusion matrix for the ability of CD56 staining to discriminate patients diagnosed with Small Cell Carcinoma NEPC vs CRPC; Table S6: Example of results from a single tube of blood.

Background Despite immune checkpoint inhibitor (ICI) monotherapy approvals in NSCLC, SOC predominately utilizes combinations of ICI with non-targeted chemotherapy or precision therapies targeting oncogenic drivers. Biomarkers guiding these clinical decisions rely on tumor genotyping to identify actionable mutations, tumor mutational burden (TMB) and on immunohistochemistry for PD-L1 expression. Currently, neither PD-L1 nor TMB perform adequately for ICI patient selection.1 Emerging evidence indicates a more complete profile of the tumor microenvironment (TME) may improve selection of patients likely to respond to ICI.2 The Xerna machine learning-based RNA sequencing biomarker assay classifies tumors into four TME subtypes; Immune Active (IA), Immune Suppressed (IS), Immune Desert (ID) and Angiogenic (A) . This classification identifies tumors likely to benefit from ICI (IA and IS) or anti-angiogenic agents (ID and A).3 We examined the distribution of actionable oncogenic driver mutations across Xerna TME subtypes to investigate the potential use for therapy selection. Methods Biomarker prevalence, and Xerna TME subtype classification, were determined for 104 metastatic lung cancer cases previously analyzed using the OncomapTM ExTra test, tumor-normal whole-exome and whole-transcriptome sequencing. DNA variants and high TMB (≥10 mut/Mb) were identified from DNA sequencing, and RNA expression levels were used to assign tumors to Xerna subtypes. Biomarker and associations were compared using Fisher's Exact Test. The study was approved by WCG IRB Ethics Board, approval number 20181863. Results In total, 53% of cases had high (IA+IS) vs. low (ID+A) Xerna immune subtypes and 60% harbored targetable oncogenic driver mutations (table 1). Actionable EGFR and KRAS mutations were detected in 31% and 20% of cases respectively, while high TMB was detected in 27% of cases. High TMB was significantly higher in IA (62%) vs. IS (14%) or A (13%) categories (p<0.05). Although no significant associations between Xerna subtype and oncogenic drivers were observed, EGFR mutations were least frequent in IA tumors (15%) while 33% of the IS subtype contained KRAS mutations (10% G12C). Conclusions The Xerna TME panel identified a high prevalence of patients who may benefit from ICI (IA+IS) and harbored actionable oncogenic drivers. Within this group, the prevalence of targetable oncogenic drivers within the IS phenotype, such as KRAS G12C, may represent the potential for novel ICI combination therapies.4 These findings further highlight the importance of adding TME analysis to comprehensive biomarker testing in NSCLC References Steuer CE, Ramalingam SS. Advances in Immunotherapy and Implications for Current Practice in Non-Small-Cell Lung Cancer. JCO Oncol Pract. 2021 Nov;17(11):662–668. doi: 10.1200/OP.21.00305. Epub 2021 Jun 25. Erratum in: JCO Oncol Pract. 2022 Mar;18(3):244. Horvath L, Thienpont B, Zhao L, Wolf D, Pircher A. Overcoming immunotherapy resistance in non-small cell lung cancer (NSCLC) – novel approaches and future outlook. Mol Cancer. 2020 Sep 11;19(1):141. Iyer, S., Ausec, L., Pointing, D., Zganec, M., Cvitkovic, R., Stajdohar, M., ... & Uhlik, M. T. (2022). Xerna? TME Panel: A pan-cancer RNA-based investigational assay designed to predict patient responses to angiogenic and immune targeted therapies.? Cancer Research,? 82(12_Supplement), 1232–1232. Mugarza E, van Maldegem F, Boumelha J, Moore C, Rana S, Llorian Sopena M, East P, Ambler R, Anastasiou P, Romero-Clavijo P, Valand K, Cole M, Molina-Arcas M, Downward J. Therapeutic KRASG12C?inhibition drives effective interferon-mediated antitumor immunity in immunogenic lung cancers. Sci Adv. 2022 Jul 22;8(29) Ethics Approval The study was approved by WCG IRB Ethics Board, approval number 20181863.

Circulating tumor cells (CTCs) have a great potential for noninvasive diagnosis and real-time monitoring of cancer. A comprehensive evaluation of four whole genome amplification (WGA)/next-generation sequencing workflows for genomic analysis of single CTCs, including PCR-based (GenomePlex and Ampli1), multiple displacement amplification (Repli-g), and hybrid PCR- and multiple displacement amplification-based [multiple annealing and loop-based amplification cycling (MALBAC)] is reported herein. To demonstrate clinical utilities, copy number variations (CNVs) in single CTCs isolated from four patients with squamous non-small-cell lung cancer were profiled. Results indicate that MALBAC and Repli-g WGA have significantly broader genomic coverage compared with GenomePlex and Ampli1. Furthermore, MALBAC coupled with low-pass whole genome sequencing has better coverage breadth, uniformity, and reproducibility and is superior to Repli-g for genome-wide CNV profiling and detecting focal oncogenic amplifications. For mutation analysis, none of the WGA methods were found to achieve sufficient sensitivity and specificity by whole exome sequencing. Finally, profiling of single CTCs from patients with non-small-cell lung cancer revealed potentially clinically relevant CNVs. In conclusion, MALBAC WGA coupled with low-pass whole genome sequencing is a robust workflow for genome-wide CNV profiling at single-cell level and has great potential to be applied in clinical investigations. Nevertheless, data suggest that none of the evaluated single-cell sequencing workflows can reach sufficient sensitivity or specificity for mutation detection required for clinical applications.

Chromosomal instability (CIN) increases a tumor cell's ability to acquire chromosomal alterations, a mechanism by which tumor cells evolve, adapt, and resist therapeutics. We sought to develop a biomarker of CIN in circulating tumor cells (CTC) that are more likely to reflect the genetic diversity of patient's disease than a single-site biopsy and be assessed rapidly so as to inform treatment management decisions in real time. Large-scale transitions (LST) are genomic alterations defined as chromosomal breakages that generate chromosomal gains or losses of greater than or equal to10 Mb. Here we studied the relationship between the number of LST in an individual CTC determined by direct sequencing and morphologic features of the cells. This relationship was then used to develop a computer vision algorithm that utilizes CTC image features to predict the presence of a high (9 or more) versus low (8 or fewer) LST number in a single cell. As LSTs are a primary functional component of homologous recombination deficient cellular phenotypes, the image-based algorithm was studied prospectively on 10,240 CTCs in 367 blood samples obtained from 294 patients with progressing metastatic castration-resistant prostate cancer taken prior to starting a standard-of-care approved therapy. The resultant computer vision-based biomarker of CIN in CTCs in a pretreatment sample strongly associated with poor overall survival times in patients treated with androgen receptor signaling inhibitors and taxanes. SIGNIFICANCE: A rapidly assessable biomarker of chromosomal instability in CTC is associated with poor outcomes when detected in men with progressing mCRPC.

Abstract Background: There is unmet need for biomarkers to guide the treatment selection in MBC. We described phenotypic CTC heterogeneity in metastatic castrate resistant prostate cancer(mCRPC), and showed that pts with CTC high heterogeneity have improved OS with chemotherapy, while with pts low CTC heterogeneity pts have longer OS with AR inhibitors (Scher et al. 2017 Cancer Research). Here, the same methodology was applied to evaluate the feasibility of CTC heterogeneity analysis in MBC pts. Material and methods: 92 blood samples from MBC pts were processed for HER2 CTC analysis utilizing the Epic Sciences platform. Following enumeration, multi-dimensional phenotypic characterization analysis was performed utilizing protein expression and digital pathology features. 20 features from each CTC were clustered using unsupervised approach (K-means) and the optimal number of clusters was determined using the elbow method with greater than 85% of variance taken into account. Shannon Index was used to score intra-patient CTC heterogeneity. Results: CTCs were detected in 77.2% (71/92) of the MBC pts. 1501 CTCs from 60 pts were clustered into phenotypic subtypes which are distinctively different in nuclear size, circularity, CK and HER2 intensity etc. HER2 expression was enriched in cell types that had higher CK intensity and was more circular morphologies often associated with replicative stress. A wide range of CTC phenotypic heterogeneity was observed across pts, Shannon Index scores from 0 (low heterogeneity) to 1.67 (high heterogeneity) with a median of 0.60. CTC heterogeneity was independent of CTC counts, some low count pts had heterogeneous CTC subtypes while some high count pts had homogeneous CTC subtypes. Conclusions: Diverse inter- and intra-patient phenotypic CTC heterogeneity were observed in this MBC cohort, consistent with that seen in mCRPC and other MBC cohorts (Beverly H et al, ASCO 2018). Studies linking degree and patterns of CTC heterogeneity to therapeutic outcomes are ongoing. Citation Format: Wang Y, Hom B, Ontiveros P, Landaverde C, Roth E, Levin MK, Hippely S, Landers M, Dittamore R, O'Shaughnessy JA. Diverse inter- and intra-patient circulating tumor cells (CTCs) phenotypic heterogeneity identified in metastatic breast cancer (MBC) cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-14.

225 Background: CIN is a mechanism by which tumor cells evolve, adapt, and evade therapeutic insults. We explored the relationship between the presence of LST as a biomarker of CIN through distinct CTC phenotypes, and sensitivity of tumors with the biomarker to standard of care (SOC) drugs. Methods: CTCs from 634 mCRPC blood samples were morphologically characterized by the Epic Sciences platform [Cytokeratin, CD45, DAPI, Androgen Receptor (AR) staining]. Training: 20 digital single cell image features were extracted from 608 CTC images from 26 patients (Pts) and single cell sequenced to determine the number of genomic LSTs. An image feature based algorithm to predict whether the number of LSTs was above the normal range defined by analysis of leukocytes was trained using this cohort (pLST) and parameters were locked. Analytical Validation (AV): the pLST algorithm was applied to a separate cohort of 570 CTCs from 54 Pts that were also sequenced to test single cell analytical performance. Clinical Validation: The pLST algorithm was then applied to CTC detected in 554 Pt samples obtained at prior to (BL, n= 324) and matched on-therapy (OnTx, n= 230) ~1 mo after starting SOC [including AR directed Tx and taxane]. A pre-defined patient scoring of > 3 pLST+ CTC/mL was related to PSA kinetics, time on drug, and overall survival (OS). Results: pLST identifies CTCs with a high number of genomic LSTs. Pts with pLST+ at BL or OnTx had poor survival (see table): 12/13 pts receiving ARSi’s and 17/18 pts receiving taxanes who were pLST+ On-Tx required a change of Tx in a median of 1.3 mo (0.9 to 2.1) or died within 6 mo. Conclusions: CTC morphology can be used to predict CIN as defined by high LSTs. CTC pLST is a strong marker of resistance to SOC Tx in mCRPC pts and treatment failure requiring a change in Tx earlier than standard criteria.[Table: see text]

e14033 Background: Immune checkpoint inhibitors (ICIs) are becoming standard treatment options in many indications. However, a substantial portion of patients will not respond. Single biomarkers such as PD-L1 are insufficiently accurate to predict patient benefit. We sought to expand the Epic Sciences non-invasive liquid biopsy platform to identify predictive, peripheral blood biomarkers for ICIs using both molecular analyses of CTCs and immune cell changes. Methods: Blood samples from prostate, kidney, and bladder cancer patients treated with ICIs were collected at baseline and on-therapy and sent to Epic Sciences. Nucleated cells were plated on glass slides and stained with CTC (pan-CK, CD45, PD-L1 and DAPI) and immune panels for activation (CD4, CD8, Ki-67, and DAPI) and exhaustion (CD8, Ki-67, PD-1, Lag-3, Tim-3, and DAPI). Changes in populations of immune cells and circulating tumor cells were assessed using high throughput digital pathology. Results: CTCs were detected in 73% (24/33) patients, of which 12% (4/33) had PD-L1+ CTCs detected. No PD-L1+ CTCs were detected in the nine on-therapy samples tested. Of 14 patients with matched samples, 57% (8/14) patients had an increase in activated CD4+ leukocytes and 36% (5/14) patients had an increase in activated CD8+ leukocytes in on-therapy samples compared to baseline. The exhaustion assay was performed on a subset (6 of 14) of matched samples. In baseline and on-therapy samples, one patient had higher levels of exhausted CD8+ leukocytes compared to healthy donor controls, and two patients had lower levels versus controls. No change in exhausted CD8+ leukocytes was observed from baseline to on-therapy. Conclusions: We developed a liquid biopsy-based platform that can simultaneously measure biomarkers in CTCs and leukocytes from a single peripheral blood sample. Changes in activated and exhausted immune cell populations with ICI treatment were detected and PD-L1 expression on CTCs was evaluated. Efforts to further stratify immune and rare cell populations are ongoing.

Background: Clinical benefit in response to chemotherapeutic agents that induce replication stress such as PARP inhibitors and cisplatin has been associated with mutations in DNA damage repair (DDR) genes. However, prediction of benefit is not solely explained by DDR mutations; therefore, additional biomarkers for prediction of clinical benefit of these agents are needed. SLFN11 is recruited to stalled replication forks where it sensitizes cells to DNA damage induced replication stress . Presence of SLFN11 protein expression in tumor cells is related to pre-clinical and clinical benefit of PARPi in small cell lung cancer. Previously, we reported high cellular heterogeneity in progressive mCRPC, and as such, we sought to examine whether SLFN11 protein expression was expressed in patients offered platinum chemotherapy to understand the subclonal heterogeneity of CTCs with and without SLFN11 protein expression.Material and methods: 30 blood samples from mCRPC patients prior to treatment with a platinum chemotherapy from MSKCC were sent to Epic Sciences for CTC enumeration and SLFN11 protein expression characterization. Nuclear localization of SLFN11 was reviewed by a trained technician. 70 CTCs from patients with and without SLFN11 CTCs were single cell sequenced and analyzed for copy number alterations (CNA) and clonality.Results: 83% (25/30) of patient samples had detectable CTCs, and 53% (16/30) samples had at least one SLFN11+ CTC. For patients with SLFN11(+) CTCs, 81% (13/16) had only nuclear localized SLFN11 expression; 6% (1/16) had a mix of CTCs with nuclear and non-nuclear localized SLFN11 expression; 13% (2/16) had no nuclear localized SLFN11 expression. In 13 patients with exclusively nuclear localized SLFN11 expression, the % of SLFN11(+) CTCs ranged from 2-100% with median of 11%. Single CTC NGS analysis of patients demonstrated genomic heterogeneity between CTCs. In patients with both SLFN11+ and SLFN11- CTCs, no discernable clonal or CNA difference was observed between SLFN11+ and SLFN11- CTCs.Conclusions: SLFN11 protein expression was observed subclonally in a majority fraction of patients with progressive mCRPC prior to platinum chemotherapy. SLFN11 expression heterogeneity was not consistent with a genetic mechanism. Studies examining whether SLFN11+ CTCs as a sensitivity marker to PARPi & cisplatin, but not ARSi or taxanes in mCRPC are ongoing.Citation Format: Luisa Fernandez, John Poirier, Angel Rodriguez, Melanie Hulling, Robin Richardson, Ramsay Sutton, Rhett Jiles, Joseph Schonhoft, Jerry Lee, Nadia Ebrahim, Emily Carbone, Ethan Barnett, Mark Landers, Yipeng Wang, Ryan Dittamore, Charles Rudin, Howard Scher. Characterization of SLFN11 protein expression in circulating tumor cells (CTCs) of patients with metastatic castration resistant prostate cancer (mCRPC) prior to platinum based chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1348.

e14531 Background: APE1/Ref-1 is a dual-function protein with a pleiotropic role in regulating transcription factors involved in cancer cell signaling via redox control, as well as responding to oxidative and base DNA damage. APX3330 is a highly selective inhibitor of the Ref-1 mediated redox function in tumors, while enhancing the neuronal protective function of APE1. APX3330 is undergoing clinical development as an anti-tumor agent that also protects and reverses oxidative damage to neurons. We now report on the CTC analysis conducted as part of the study (NCT0337508). Results of the clinical study are reported in a separate abstract. Methods: A total of 19 patients with 10 various solid tumors, including rectal, pancreatic, colon, endometrial, gall bladder, hepatocellular, prostate, melanoma, bladder, and ovarian cancers received APX3330 in escalating divided daily doses of 240, 360, 480, 600, and 720mg. Blood samples were collected from all patients prior to receiving APX3330 and after achieving steady-state serum concentrations of the drug. Samples were sent to Epic Sciences to analyze circulating tumor cells (CTCs) in peripheral blood via their CTC Platform. Results: Cumulatively, 37 samples from 19 patients were received. 35/37 (95%) samples passed technical quality control and were feasible for downstream analysis. 9/17 (53%) baseline samples (BL) had CTCs detected, while 9/18 (50%) on-treatment (OTx) draws had CTCs detected. 16 patients had BL and OTx samples that were further evaluated with the longitudinal analysis. Of these, 7/16 (44%), 6/16 (38%), and 3/16 (19%) patients showed a reduction trend, an increase trend, and no change in all CTC populations (delta greater than or equal to 0 CTC/ml), respectively. Patient follow-up is ongoing and the correlation of CTC biomarkers with clinical outcomes is pending. Conclusions: APX3330 is undergoing clinical evaluation as an anti-tumor agent that protects against and reverses CIPN. In this phase I study, 44% of evaluable blood samples showed a reduction in CTCs after initiation of treatment with APX3330. Additional studies are now being planned. Clinical trial information: NCT0337508.

TPS328 Background: Men with metastatic castration-resistant prostate cancer (mCRPC) who have a BRCA1/2 mutation ( BRCA1/2mut) or mutations in other HRD genes have a poor prognosis. The EPIC liquid biopsy test is a novel assay that can identify circulating tumor cells (CTC) with HRD associated phenotypes. Preliminary studies have shown that these men may respond to treatment with a PARP inhibitor. Pamiparib is an investigational PARP1/2 inhibitor that has shown brain penetration and potent PARP–DNA complex trapping in nonclinical studies. In early phase clinical studies (NCT02361723; NCT03333915), pamiparib was generally well tolerated and showed preliminary antitumor activity; 60 mg orally twice daily (BID) was established as the recommended investigational dose. Methods: This open-label, global, phase 2 study (NCT03712930) will evaluate antitumor activity and safety of pamiparib in mCRPC pts with CTC-HRD, assessed by the EPIC CTC-HRD assay, or deleterious germline/somatic BRCA1/2mut status. Patients must have progressed on/after ≥1 androgen receptor-targeted therapy, have received ≥1 taxane-based therapy, and have prostate-specific antigen (PSA) progression per PCWG3 criteria. Four cohorts of patients will receive pamiparib 60 mg BID in 28-day cycles. Cohort 1 will include ~50 pts with CTC-HRD+ +/- BRCA1/2mut mCRPC with measurable metastatic disease; Cohort 2 will include ~30 pts with CTC-HRD+ +/- BRCA1/2mut mCRPC with bone-only disease; Cohorts 3 & 4 will include ~20 pts with CTC-HRD-/unknown + BRCA1/2mut mCRPC with measurable metastatic disease (Cohort 3), or bone-only disease (Cohort 4). Disease status will be assessed every 8 wks for 24 wks, then every 12 wks; PSA levels will be tested every 4 wks. Co-primary endpoints are radiographic ORR assessed by IRC (pts with measurable disease) and confirmed PSA response rate per PCWG3 criteria (pts +/- measurable disease). Secondary endpoints include ORR, time to PSA response/progression, duration of PSA response, time to symptomatic skeletal event, radiographic progression-free survival, overall survival, and safety. Clinical trial information: NCT03712930.

Background: Upregulation of HER2 and AR protein expression, and increase in chromosomal instability, are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these biomarkers requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which precludes their utility to guide treatment in late stage metastatic pts. A CTC-based test could expand the clinical utility. Using the Epic Sciences platform, MBC blood samples were characterized for CTC prevalence, HER2 and AR expression on CTCs, and CTC chromosomal instability at time of disease progression. Material and methods: Blood samples were acquired from 127 total pts with HR+/HER2-, HR-/HER2+ and TNBC metastatic breast cancer pts. 114 samples were analyzed for HER2 and 110 were analyzed for AR using the Epic CTC Platform. Single-cell whole genome sequencing was performed on 157 CTCs from 19 pts to assess large scale transitions (a surrogate of chromosome instability) and gene copy number alterations. Results: 87/114 (76.3%) of pts had detectable CTCs, with a median of 1.1 CTC/mL. 17/114 (14.9%) had at least one HER2(+) CTC, 21/110 (19.1%) had at least one AR(+) CTC, and 10/110 (9%) had both AR(+) and HER2(+) CTCs detected in technical replicates. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. The majority of HER2+ and AR+ CTCs had high cytokeratin expression. Most pt samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. CTCs with larger nuclear area and higher N/C ratio had higher frequency of chromosomal instability. ERBB2, FGFR1 gain, PTEN, CDH1, BRCA1, BRCA2 loss were frequently observed and the prevalence were associated with CTC phenotypes. Conclusions: CTCs are detected in most MBC pts upon disease progression. CTC based biomarkers assessing HER2, AR and chromosomal instability status could guide therapy selection. The results warrant prospective evaluation of these biomarkers on MBC pts’ CTCs as a resistance marker for targeted therapies. Citation Format: Priscilla Ontiveros, Connie Landaverde, Maren K. Levin, Sarah Hippely, Mark Landers, Yipeng Wang, Ryan Dittamore, Joyce A. O'Shaughnessy. HER2, AR protein expression and chromosomal instability in circulating tumor cells (CTCs) of metastatic breast cancer (MBC) patients (pts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 457.

Background: PARPi have recently demonstrated clinical activity in a subset of mCRPC pts with defects in homologous recombination (HRD). Predicting PARPi response with HRD gene mutations have struggled to show clinical specificity. Previously, we described a novel circulating tumor cell (CTC) based method (CTC-HRD) that identifies a specific cell morphology associated with genomic scarring [e.g. large scale transitions (LST)] and demonstrated CTC-HRD's clinical utility in identifying pts likely to respond to PARPi and likely to resist to standard of care (SOC) therapies (Tx), including AR signaling inhibitors. We also observed some discordance between CTC-HRD and DNA damage response (DDR) mutational status from tissue sequencing. Here we compare the prevalence of CTCs, # LSTs/CTC and the % of CTCs with BRCA2 loss in mCRPC patient samples with matched tumor sequencing DDR status.Materials and methods: 302 CTCs from 26 mCRPC pts collected prior to Tx were collected from pts with matched tissue biopsy sequencing. CTCs were identified, assayed for CTC-HRD status, and low pass whole genome sequenced for LSTs and gene copy number alterations (CNA). We compared the prevalence of CTCs, HRD properties of each identified cell including BRCA2 copy loss, # of LSTs/CTC and their prevalence in cells derived from pts vs. CTC-HRD score and tissue DDR mutational status.Results:CTC-HRDDDR Tumor# PtsMedian CTC/mLMedian LST/CTCMedian % CTC BRCA2 Loss per Pt++913.83259.00%+-1010.42347.20%-+72.430.00%CTC-HRD positive pts, independent of tissue DDR status had higher CTC/mL, higher median # LST/CTC and higher percentages of BRCA2 loss in their CTCs vs. those pts who were CTC-HRD negative. Notably, we identified 10 pts that were negative for tissue DDR mutation but were CTC-HRD+ with a high frequency of CTC BRCA2 loss.Conclusions: These results highlight the concordance of the Epic CTC-HRD assay with known markers of HRD and demonstrate differences in CTC-HRD status from tissue DDR mutations which have struggled to achieve the required clinical specificity required for PARPi patient selection.Citation Format: Joseph Schonhoft, Adam Jendrisak, Jerry Lee, Angel Rodriguez, Ramsay Sutton, Yipeng Wang, Ryan Dittamore, Mark Landers. Comparison of the Epic CTC HRD assay vs. tumor DDR mutations in metastatic castration-resistant prostate cancer (mCRPC) patients (pts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2228.

5075 Background: Prediction of ARSi benefit in mCRPC is an unmet medical need. Recently, the Epic Sciences CTC based nl AR-V7 test validated as a predictive biomarker in two multi-center validation studies and has received Medicare coverage for use in mCRPC. While the nl AR-V7 biomarker is highly specific to resistance and predictive of improved response with taxane Rx, it is a measure of just one mechanism of resistance to ARSis. CTC Het measured by the Shannon Index and CTC chromosomal instability measured by predicted number of Large Scale Transitions (pLST) have both been associated with poor OS to ARSis in previous analysis. Here we investigate the relationship of Het and pLST to nlAR-V7 in order to assess multi-clonal resistance and determine if these biomarkers can provide added sensitivity in the nlAR-V7 negative patient population. Methods: 275 blood samples from 2nd+ line mCRPC patients prior to treatment with ARSi (n=148) or taxanes (n=137) were obtained between 2012 and 2017 from 3 clinical centers. Detectable CTCs in each blood sample were assayed for nlAR-V7, Het, and pLST using the Epic Sciences platform. Biomarkers were analyzed in context of each other and outcomes including clinical co-variates. Results: 94% of samples had detectable CTCs, 84% were evaluable for Het analysis (> 2 CTCs), and 76% were evaluable for pLST (> 3 CTCs). Conclusions: Addition of CTC Het (Shannon Index) and CTC chromosomal instability (pLST) biomarkers to nlAR-V7 identifies an additional 15% of mCRPC pts (38% of total) that are predicted to have poor survival to AR signaling inhibitors. [Table: see text][Table: see text]

3050 Background: MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), is a high throughput, targeted-DNA-sequencing panel for somatic mutations created by the Department of Pathology at Memorial Sloan Kettering Cancer Center (MSK). The MSK-IMPACT is FDA approved for tumor tissue profiling to guide treatment selection. Recognizing access to tumor tissue in many cancers is difficult and may harbor inter & intra lesional heterogeneity, we sought to evaluate concordance of sequencing single CTCs vs. paired biopsy analyzed by MSK-IMPACT, to assess CTC vs. tumor clonality, and their relationship to outcomes. Methods: 148 biopsy samples from 138 mCRPC pts were submitted for IMPACT analysis and a blood draw within 30-day prior to initiation of a new line of treatment. Blood samples were sent to Epic Sciences for CTC detection. The detected CTCs underwent single cell low pass whole genome sequencing for copy number variation (CNV). For each set of matched samples, DNA copy number profiles from IMPACT and CTC sequencing were compared for similarity. Results: Of the 114 successfully sequenced samples 58 were from lymph nodes, 23 from bone, 25 from liver or lung, and 8 from other soft tissue. Of the 111 patients with CTCs analyzed, a total of 1073 CTCs were sequenced (range 1-27, median = 4 per pt). Of patients with both IMPACT & CTC, >80% pts had multiple subclones with distinguishable CNV. Concordance of genomics clones was found in 63%, and discordant in 37% cases. The clonal concordance between tissue biopsy and CTC was higher when the biopsy was obtained from bone marrow or a visceral site (71% concordance) than from a lymph node (53% concordance). Conclusions: Single CTC sequencing is often concordant to metastatic tissue, but unique CTC clones and the presence of multi-clonal disease highlight the potential to be underrepresented through tissue biopsy, especially when taken from the lymph node.[Table: see text]

Introduction: Proxalutamide (code name: GT-0918) is a novel androgen receptor (AR) blocker and has shown efficacy in the treatment of castration resistant prostate cancer. Proxalutamide binds to AR and inhibits androgen-induced receptor activation. A Phase 1/2 clinical trial (ClinicalTrials.gov Identifier: NCT02826772) was conducted to identify dose-limiting toxicities (DLTs) and to assess safety, tolerability, and PK of GT0918 in pts with mCRPC who have progressed on standard of care and experimental therapies in the United States. To better understand patient responses and pharmacokinetics of GT0918, we sought to examine CTC enumeration, AR-N term expression, and AR-V7 nuclear expression using Epic CTC platform for patients treated with GT0918. Methods: A total of 18 metastatic castrate resistant prostate cancer (mCRPC) patients were recruited under either dose escalation or expansion studies and blood samples were collected at 3 time points: C1D1, C3D1, and C6D1/EOS. Patients with histologically confirmed mCRPC who had progressed after both hormonal therapy (abiraterone or enzalutamide) and chemotherapy (e.g., docetaxel) were eligible. Dosages of 300, 400, and 500 mg were evaluated in this study. CTCs were detected using the Epic Sciences platform and assessed for full length AR status using an AR N-terminus specific antibody and for nuclear localized AR-V7 truncation. AR-V7 nuclear localization was confirmed by trained technician. Results: 18/18 (100%) of patients had detectable CTCs, with 12/18 (67%) patients having AR-N term+ CTCs. 7/12 (58%) of AR-N term+ patients were AR-V7 positive. Reduction of CTC/mL, AR+ CTC/mL and % of AR+ CTC in follow up draws were observed in the whole cohort, with 500mg dose group showing more reduction than others. Sample accrual and patient follow up is ongoing and the correlation of CTC, AR, and AR-V7 status with the clinical outcomes will be investigated once the data becomes mature. Conclusions: In this clinical trial, we evaluated the pharmokinetic effects of GT0918 on CTC enumeration and Androgen Receptor CTC status. GT0918 might serve as a potential alternative for late stage mCRPC patients who have developed resistance to both hormone and chemotherapy treatments. The role of CTC as PK marker or patient selection device is ongoing. Citation Format: Lincy Chu, Phoebe Zhang, Amanda Anderson, Mark Landers, Yipeng Wang, Karl Zhou. Evaluation of pharmacokinetics of proxalutamide, a novel androgen receptor antagonist, in treatment for mCRPC patients via CTC enumeration and AR biomarker analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3885.

TPS5086 Background: Men with mCRPC who have a BRCA1/2 mutation ( BRCA1/2mut) or mutations in other genes resulting in HRD have a poor prognosis. A novel liquid biopsy test (EPIC Sciences) identifies CTCs with an HRD phenotype. Preliminary studies showed that these men may respond to treatment with a PARP inhibitor. Pamiparib, an investigational PARP1/2 inhibitor, has shown brain penetration and potent PARP–DNA complex trapping in nonclinical studies. In early phase clinical studies (NCT02361723; NCT03333915), pamiparib was generally well tolerated and showed preliminary antitumor activity; 60 mg orally twice daily (BID) was established as the recommended investigational dose. Methods: This open-label, global, phase 2 study (NCT03712930) evaluates the antitumor activity and safety/tolerability of pamiparib in mCRPC patients (pts) with CTC-HRD, assessed by the CTC-HRD assay, or deleterious germline/somatic mutations in BRCA1/2. Patients must have progressed on/after ≥1 androgen receptor-targeted therapy, received ≥1 taxane-based therapy, and have prostate-specific antigen (PSA) progression per PCWG3 criteria. Four cohorts of pts will receive pamiparib 60 mg BID in 28-day cycles. Cohort 1 will include ~50 pts with CTC-HRD+ +/- BRCA1/2mut mCRPC with measurable metastatic disease; Cohort 2 will include ~30 pts with CTC-HRD+ +/- BRCA1/2mut mCRPC with bone-only disease; Cohorts 3 & 4 will include ~20 pts with CTC-HRD-/unk + BRCA1/2mut mCRPC with measurable metastatic disease (Cohort 3), or bone-only disease (Cohort 4). Disease status will be assessed every 8 wks for 24 wks, then every 12 wks; PSA levels will be tested every 4 wks. Co-primary endpoints are radiographic ORR assessed by IRC (pts with measurable disease) and confirmed PSA response rate per PCWG3 criteria (pts +/- measurable disease). Secondary endpoints include ORR, time to PSA response/progression, duration of PSA response, time to symptomatic skeletal event, radiographic progression-free survival, overall survival, and safety. As of 05 December 2018, this study is actively enrolling. Clinical trial information: NCT03712930.