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BACKGROUND: A subset of TNBCs have homologous recombination deficiency with upregulation of compensatory DNA repair pathways. The combination of TAK-228 and TAK-117 (PIKTOR), investigational oral TORC1/2 and PI3Kα selective inhibitors, respectively, is hypothesized to increase genomic instability (GI) and to increase DDRD, leading to increased sensitivity to DNA damaging chemotherapy and to checkpoint inhibitors in metTNBC pts. METHODS: 10 metTNBC pts received 4 mg PO TAK-228 and 200 mg PO TAK-117 QDx3d QW until disease progression (PD), followed by cis 75 mg/m2 plus nab pac 220 mg/m2 for up to 6 cycles. Primary endpoints were objective response rate with cis/nab pac and safety. Blood samples and biopsies of metastatic lesions were collected prior to and at PD on PIKTOR. Blood CTC analyses included enumeration, cell morphology, phenotypic heterogeneity, and predicted genomic instability (pGI) derived from cell phenotypes. RESULTS: 10 pts received PIKTOR followed by cis/nab pac. Median age: 51 yrs; median number of prior chemotherapy regimens was 3 (range, 1-5); 7 pts had prior carboplatin; sites of metastases: lymph nodes (n=8); lung (n=6); chest wall (n=1); bone (n=1); brain (n=1). Median time on PIKTOR was 8 wks (range, 3-14). With cis/nab pac, 1 pt had PR, 2 had SD > 6 mos, 1 had SD and 6 had PD. 2 SD pts (sites LNs and bone) and 1 PD pt (sites LNs), all carboplatin-pretreated, whose pre-PIKTOR BCs were PDL1-negative (2 pts) or unknown (1 pt) have durable SD on pembrolizumab post-cis/nab pac for 1+ yrs. PIKTOR related AEs ≥30% included: fatigue (90%); nausea (80%); diarrhea (60%); vomiting (40%); stomatitis (40%); hyperglycemia (30%); rash (30%); cough (30%); chest pain (30%). Incidence and grade of cis/nab pac-related AEs were not greater than expected with this regimen. At PD on PIKTOR, higher CTC burden and pGI+ CTCs were observed in a subset of pts suggesting that some CTCs may have developed greater GI with PIKTOR treatment. Next generation sequencing (NGS) and reverse phase protein array (RPPA) analyses of biomarkers of DNA repair pathways pre-and post-PIKTOR tissues are underway and will be presented at the meeting. CONCLUSIONS: Priming pts’ metTNBC with PIKTOR did not lead to durable responses with cis/nab pac in most pts in this pretreated population. However, 3/10 pts who had carboplatin-pretreated disease in LNs +/- bone, have highly durable SD on pembrolizumab following PIKTOR therapy. Citation Format: Joyce O'Shaughnessy, Esther San Roman Rodriguez, Priscilla Ontiveros, Rick Wenstrup, Tiziano Pramparo, Jessica Lang, Victoria Zismann, Natalia Briones, William Hendricks, Virginia Espina, Claudius Mueller, Maren K Levin. Pilot trial of priming with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency (DDRD) followed by cisplatin (cis) and nab paclitaxel (nab pac) in chemotherapy-pretreated metastatic triple negative breast cancer (metTNBC) pts [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-25.

Background: The choice between hormonal therapies and chemotherapy is a frequent decision in the standard of care of metastatic breast cancer (mBCa) patients. We previously developed quantitative measures of phenotypic CTC heterogeneity in metastatic prostate cancer and found that higher heterogeneity was associated with better survival on chemotherapy vs. targeted hormonal therapies in a risk adjusted analysis (Scher et al. 2017 Cancer Research). Here we sought to test the feasibility of measuring CTC heterogeneity in mBCa patients. Methods: 295 blood samples from 165 pts (84 HR+, 19 Her2+, 8 HR+/Her2+, 54 TNBC) from mBCa patients were processed for CTC analysis utilizing the Epic Sciences Functional Cell Profiling platform. Following enumeration, multi-dimensional phenotypic characterization analysis was performed utilizing protein expression and digital pathology features. Features from each CTC (3994 CTCs from 165 pts, were compared by unsupervised clustering to assess intra-patient CTC heterogeneity among mBCa CTC phenotypes. Results: CTCs were detected in 76.9% (227/295) of mBCa patients (med=2.4 CTC/mL, Range 0 - 747). Phenotypic profiling identified distinct CTC subtypes characterized by morphological and protein expression features that correlated with mBCa subtypes. Prevalent CTC features that associated with mBCa were either high cytokeratin (CK) expression or lower nuclear to cytoplasmic (N/C) ratio with lower CK expression. Analysis of mBCa subtypes showed that TNBC pts were characterized by CTCs with a large nuclear area and high CK expression. We observed marked differences in CTC heterogeneity across patients, with some patients having more uniform CTC profiles, and others having higher phenotypic diversity. mBCa subtypes had similar intra-patient heterogeneity level. Conclusions: Distinct CTC phenotypes can be visualized and quantified across patients and associated with specific mBCa subtypes. Our analysis revealed significant heterogeneity between individual CTCs both between and within patients. Identification of patients with high heterogeneity may help to identify patients unlikely to respond to targeted therapies. Studies to link heterogeneity to efficacy of specific therapies and patient outcome are ongoing. Citation Format: Tiziano Pramparo, Adam Jendrisak, Priscilla Ontiveros, Megan Kearney, Lincy Chu, Nadia Ebrahim, Joseph Schonhoft, Rick Wenstrup, Yipeng Wang. Phenotypic profiling of circulating tumor cells (CTCs) in patients with metastatic breast cancer reveals clinically-relevant heterogeneity in CTC morphology and marker expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-09.

Purpose: Activated immune cell subpopulations and circulating tumor cells in patients receiving checkpoint inhibitor therapy were characterized with the aim of developing more sensitive diagnostic tools for prediction of immunotherapy response or resistance. Methods: Blood samples from bladder, kidney, and prostate cancer patients undergoing checkpoint inhibitor therapy were collected at baseline and on-therapy at convenience and sent to Epic Sciences for processing. Red blood cells were lysed and nucleated cells were plated onto glass slides and stained with CTC (pan-CK, CD45, PD-L1 and DAPI) and immune panels (CD4, CD8, Ki-67, and DAPI). 2-3 million cells per patient were imaged using high throughput digital pathology pipelines and changes in immune cell sub-populations and changes in circulating tumor cell (CTC) burden with PD1/PD-L1 immunotherapy were assessed. Results: A method to profile activated (CD8+Ki-67+ and CD4+Ki-67+) leukocyte subpopulations and PD-L1+ CTCs in a single blood sample was developed and feasibility was demonstrated in cell lines, healthy donor (HD), and patient blood. CTCs were detected in 73% (24/33) of patients tested. Of the 33 baseline samples tested, 12% (4/33) had PD-L1+ CTCs detected. No PD-L1+ CTCs were detected in on-therapy samples. 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. A decrease in activated CD4+ leukocytes was observed in 21% (3/14) patients and a decrease in activated CD8+ leukocytes was also observed in 7% (1/14) patients with therapy. Conclusions: We developed a liquid biopsy-based platform that can simultaneously measure immune related biomarkers in CTCs and leukocytes at single cell resolution from ~1 mL of blood. The platform has extreme sensitivity over conventional methodologies and immune cell populations existing in less than 1% of the total are readily quantified. Efforts to identify morphological sub-populations within each canonical leukocyte category and comparison of leukocyte and CTC biomarker panels to patient outcomes are ongoing. Citation Format: Rachel Krupa, Robin Richardson, Priscilla Ontiveros, Joseph Schonhoft, Jiyun Byun, David Lu, Sean Nisperos, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous quantification of activated immune cells and PD-L1 expressing circulating tumor cells (CTCs) in peripheral blood of cancer patients receiving checkpoint inhibitor therapy [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 4039.

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: 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.

Background: Upregulation of HER2 and AR are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these proteins and their localization requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which limits their utility to guide treatment in late stage metastatic patients. A CTC-based test could expand the clinical utility of these biomarkers. Here we utilized the Epic Sciences CTC platform for CTC detection and characterization. MBC blood samples were characterized for CTC prevalence, HER2 and AR expression at time of disease progression. Material and methods: HER2 and AR expression levels were determined based on model cell lines. A total of 72 blood samples were acquired from ER(+/-)/HER2(-) patients (by standard tissue pathology) at disease progression. 72 samples were analyzed for HER2 and 64 were analyzed for AR using the Epic Platform. Single-cell whole genome sequencing was performed to assess clonality and inter-patient heterogeneity of CTCs detected. Results: 55/72 (76.4%) of patients had CTCs detected across two slides. 13/72 (18.1%) had at least one HER2(+) CTC, 14/64 (21.9%) had at least one AR(+) CTC, and 7/64 (10.9%) had at both AR(+) and HER2(+) CTCs detected on replicate slides. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. Most patient samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. Subsequently, these cells will be individually sequenced to better determine the clonality of resistance. Conclusions: CTCs are detected in most MBC pts upon disease progression, with expression of known endocrine therapy resistance markers, HER2 and AR, observed that CTCs could guide subsequent therapy selection. Prospective evaluation of HER2 and AR on MBC pts' CTCs as predictive biomarkers of benefit from inhibitors of these proteins is needed. Citation Format: Ontiveros P, Landaverde C, Graf R, Levin MK, Hippely S, Wang Y, Landers M, Dittamore R, O'Shaughnessy JA. Enabling HER2 and androgen receptor (AR) protein expression and localization in circulating tumor cells (CTCs) of ER(+/-)HER2(-) metastatic breast cancer (MBC) patients (pts) [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 P3-01-07.

Background: The NOTCH pathway has been identified as a key therapeutic pathway in SCLC. Tarextumab (TRXT, anti-Notch2/3, OMP-59R5) is a fully human monoclonal antibody that targets the Notch2 and Notch3 receptors. PINNACLE is a Phase 1b/2 trial of TRXT in combination with etoposide and platinum therapy (EP) in patients with untreated extensive stage small cell lung cancer (ES-SCLC). Baseline CTCs and post treatment changes in CTCs have previously been shown to predict the response to chemotherapy in SCLC. CTCs may also serve as pharmacodynamic biomarkers. Here we describe a study of baseline and longitudinal CTCs in ES-SCLC patients from the PINNACLE phase 1b trial (clinicaltrials.gov:NCT01859741). Materials and methods: CTCs, CTC clusters, apoptotic CTCs and N-Cadherin+ CTCs were identified and enumerated from patient blood samples using Epic Sciences CTC technology. Baseline CTCs from 26 patients were correlated with clinical outcome: progression-free survival (PFS), overall survival (OS) and best overall response, as well as metastatic status. A mixed effects model was used to investigate the post treatment changes in CTCs among the dose groups. Association of CTCs with PFS/OS and best overall response, including CTCs at each time point, as well as temporal changes of CTC status, were studied. Multivariate analysis was performed to identify CTC numbers in a subset of time points to correlate with response to treatment. Results: CTCs were present in 81% of the patients (21/26). CTC clusters and apoptotic CTCs were detected in 38% and 77% of the patients, respectively. At baseline, CTC counts ≥ 5/mL were significantly associated with poor OS (p=0.04). There was a trend that the presence of CTC clusters was associated with worse OS. With a cut-off of 3.4/mL, apoptotic CTCs showed a trend in association with overall survival. CTC numbers in patients with liver metastasis were significantly higher than in patients without liver metastasis. CTCs were also found to be correlated significantly with the number of metastatic sites. When measuring at Day 7 post dosing, CTC numbers were significantly decreased. Conclusions: Our findings suggest that CTCs are frequently detectable in patients and are a prognostic factor in ES-SCLC. CTCs decrease with TRXT and platinum-based chemotherapy. Updated results will be presented. CTCs will be further evaluated in the phase 2 portion of the PINNACLE trial. Citation Format: Chun Zhang, Ryon Graf, Adam Jendrisak, Amanda K. Anderson, Priscilla Ontiveros, Sarah Orr, Anne Chiang, David Spigel, Charles Rudin, Eric Holmgren, Jakob Dupont, Gretchen Argast, Leonardo Faoro, Lei Zhou, John Lewicki, Ann M. Kapoun. Circulating Tumor Cells (CTCs) in patients with extensive stage small cell lung cancer and their association with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1727. doi:10.1158/1538-7445.AM2017-1727