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Purpose:Metastasis requires malignant cell circulation from the primary to a distant tissue. Elevated levels of circulating tumor cells (CTC) portend a poor prognosis in breast and other cancers. Recent studies have suggested that CTC clusters may be a factor in the metastatic process. We conducted a prospective retrospective study of the SWOG0500 clinical trial to test whether CTC clusters are associated with poorer prognosis.Experimental Design:CTC CellSearch galleries from SWOG0500 trial were reread using prespecified criteria for CTC clusters, doublets, and enumeration. Survival analysis methods include Kaplan–Meier plots and log-rank tests.Results:Patients were classified into three prognostic subgroups based on baseline CTC/7.5 mL whole blood (WB): Arm A: P < 0.0001). Furthermore, doublets or clusters were significantly more common in patients who were ultimately assigned to Arm C versus B (54% vs. 25%; P < 0.0001). In Arm C, doublets and clusters were associated with worse overall survival than only doublets, clusters, or no doublets nor clusters at baseline (P = 0.008) and first follow-up (P = 0.010). When compared with enumeration alone, doublets, clusters, or both were not prognostic in patients who had 5–19 or ≥20 CTC/7.5 mL WB.Conclusions:In patients with metastatic breast cancer starting first-line chemotherapy, mortality is independent of the presence of CTC clusters, but rather depends on the number of CTC/7.5 mL WB.

Supplementary Figures: Supplementary Figure 1. Summary of duration on AZD9496 therapy, previous therapies, and circulating biomarkers status at baseline for each patient ranked by ascending dose; Supplementary Figure 2. Tumor pharmacodynamics. Supplementary Tables: Supplementary Table 1. Study Plan showing time-points of collection of biosamples for exploratory research; Supplementary Table 2. ddPCR primers and probes;Supplementary Table 3. Antibodies and conditions for IHC analysis Antibodies for IHC analysis; Supplementary Table 4. Patients with {greater than or equal to}1 CTC/7.5 mL WB within 120 days of fulvestrant wash-out period prior C1D1.

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients.Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110–22. ©2017 AACR.

Purpose:Common resistance mechanisms to endocrine therapy (ET) in estrogen receptor (ER)–positive metastatic breast cancers include, among others, ER loss and acquired activating mutations in the ligand-binding domain of the ER gene (ESR1LBDm). ESR1 mutational mediated resistance may be overcome by selective ER degraders (SERD). During the first-in-human study of oral SERD AZD9496, early changes in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) were explored as potential noninvasive tools, alongside paired tumor biopsies, to assess pharmacodynamics and early efficacy.Experimental Design:CTC were enumerated/phenotyped for ER and Ki67 using CellSearch in serial blood draws. ctDNA was assessed for the most common ESR1LBDm by droplet digital PCR (BioRad).Results:Before starting AZD9496, 11 of 43 (25%) patients had ≥5 CTC/7.5 mL whole blood (WB), none of whom underwent reduction to +, two of whom had CTC-ER+ reduction. CTC-Ki67 status did not change appreciably. Patients with ≥5 CTC/7.5 mL WB before treatment had worse progression-free survival (PFS) than patients with P = 0.0003). Fourteen of 45 (31%) patients had ESR1LBDm+ ctDNA at baseline, five of whom had ≥2 unique mutations. Baseline ESR1LBDm status was not prognostic. Patients with persistently elevated CTC and/or ESR1LBDm+ ctDNA at C1D15 had worse PFS than patients who did not (P = 0.0007).Conclusions:Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER+ and ESR1LBDm+ ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker.

Supplementary Figure S1. Clinical timelines. Clinical timelines and treatment from first diagnosis until enrollment into MI-CTC-ONCOSEQ for the 11 metastatic breast cancer patients; Supplementary Figure S2. Sanger sequencing of WGA CTC DNA and Western blot confirming the presence of A569S mutation; Supplementary Figure S3. MCF-7 (ESR1 A569S) are not estrogen independent over 5 days in hormone-free conditions; Supplementary Figure S4. The tamoxifen metabolites 4-hydroxytamoxifen and endoxifen have no increased agonistic effect in MCF-7 cells over-expressing ER-A569S; Supplementary Figure S5. Inhibition of estradiol-stimulated MCF-7 parental or A569S- overexpressing cells. Supplementary Table S1. Oncomine Comprehensive Panel (OCP) target genes by presence of the MseI restriction site; Table S2. NGS of BT-474 cells spiked into blood and purified identifies expectedTP53 mutation; Supplementary Table S3. Results of CTC purification by DEPArray; Supplementary Table S4: Mean CTC Next Generation Sequencing parameters; Supplementary Table S5. NGS-identified prioritized mutations in individual and pooled CTC samples; Supplementary Table S6. 2 x 2 contingency tables of concordance for alterations in CTC vs tissue; Supplementary Table S7. Patient #2 analysis of primary, clinical metastatic tissue, research biopsy, pt-DNA by ddPCR; Supplementary Table S8. Primer/probe sequences used in Sanger sequencing and ddPCR.

Purpose: The vast majority of advanced ER POS breast cancers eventually cease responding to endocrine (ET) and other therapies leading to evolution of lethal disease. However, timely monitoring of the molecular events associated with response/progression in tissue biopsies is logistically difficult. The use of liquid biopsies, such as CTC and ctDNA, in this context has been of recent interest. Patients and Methods: Individual CTC and ctDNA were obtained at different time points from patients with advanced ER POS/HER2 NEG breast cancer enrolled in a Phase I trial of AZD9496, an oral selective estrogen receptor degrader (SERD) ET. The CTC, purified using tandem CellSearch®/DepArray™ technologies, were genomically profiled by DNA single cell next generation sequencing (scNGS). Plasma ctDNA was isolated from blood collected in Streck BCT tubes. Genomic profiling was performed by targeted gene panel scNGS for CTC and ddPCR for ERα gene (ESR1) mutations in ctDNA. Results: 123 high-quality CTCs from 12 patients profiled by scNGS showed 100% concordance with ctDNA in detection of driver ESR1 somatic mutations. CTC scNGS additionally revealed extensive intra-patient heterogeneity of driver alterations, that would have been unresolvable by bulk ctDNA profiling, including separate subclonal CTC populations emerging within the same patient. ScNGS revealed potential opportunities for targeted therapies in 73% of patients, directed at alterations in PIK3CA, FGFR2, KIT and BRAF, at times present as 2 or more targets in the same or different cell populations. In one patient, an emergent, distinct, BRAF p.V600E targetable alteration was detected in a subpopulation of CTCs collected at the progression time point but not at baseline. Conclusion: Serial monitoring of CTC and ctDNA genomic alterations is feasible and should enable real-time tracking of response/progression, tumor evolution and opportunities for precision medicine interventions. Citation Format: Andi K. Cani, Emily M. Dolce, Elizabeth P. Darga, Kevin Hu, Martha Brown, Chia-Jen Liu, Jackie Pierce, Kieran Bradbury, Kimberly Aung, Gaia Schiavon, Danielle Carroll, T. H. Carr, Teresa Klinowska, Justin Lindemann, Gayle Marshall, Vicky Rowlands, Elizabeth A. Harrington, J. Barrett, Anne Armstrong, Richard Baird, Erika Hamilton, Seock-Ah Im, Komal Jhaveri, Manish R. Patel, Caroline Dive, Scott A. Tomlins, Aaron M. Udager, Daniel F. Hayes, Costanza Paoletti. Monitoring circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) genomic alterations in ER positive (POS)/HER2 negative (NEG) advanced breast cancer during endocrine therapy: correlative study of AZD9496 oral SERD phase I trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3143.

Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). The CTC-endocrine therapy index (CTC-ETI), consisting of CTC-ER (estrogen receptor), BCL2, human epidermal growth factor receptor (HER2), and Ki67 expression, might predict resistance to endocrine therapy (ET) in patients with ER-positive MBC. One hundred twenty-one patients with ER-positive/HER2-negative MBC initiating a new ET after ≥1 lines of ET were enrolled in a prospective, multi-institutional clinical trial. CTC-ETI and clinical/imaging follow-up were performed at baseline and serial time points. Progression-free survival (PFS) and rapid progression (RP; determined at the 3-month time point) were primary endpoints. Associations with clinical outcomes used logrank and Fisher’s exact tests. At baseline, 36% (38/107) of patients had ≥5 CTC/7.5 ml whole blood (WB). Patients with ≥5 vs. P = 0.03). Elevated CTC at 1 month was associated with even worse PFS (1.9 vs. 5.0 months from the 1-month sample, P P = 0.008). Patients with high vs. low CTC and CTC-ETI more frequently experienced RP (CTC: 66% vs. 41%; P = 0.03; CTC-ETI: 79% vs. 40%; P = 0.002). In conclusion, CTC enumeration and the CTC-ETI assay are prognostic at baseline and follow-up in patients with ER-positive/HER2-negative MBC starting new ET. CTC at first follow-up might identify a group of patients with ER-positive MBC that could forego ET, but CTC-ETI did not contribute further.

Clinical decisions for precision and immuno-oncology therapies are based on predictive biomarkers commonly obtained from a single metastatic biopsy, or from archived primary tumor material. Circulating genomic biomarkers present a minimally invasive way to monitor the intra-patient tumor heterogeneity and its fluctuations in order to provide a real-time evaluation of the changing clonal architecture with potential therapeutic implications. Single-cell DNA next generation sequencing (scNGS) of circulating tumor cells (CTC) is a particularly well-suited method of unraveling and monitoring that heterogeneity to complement biomarker information obtained from tissue and cell-free circulating tumor DNA (ctDNA). In this proof-of-concept study we analyzed 123 CTC, 15 white blood cells (WBC), and ctDNA from 15 CTC-positive lobular breast cancer patients, five of whom had CTC available at both metastatic baseline and after progression on a variety of therapies chosen at their physician’s discretion. CTC were enriched with the CellSearch® system and isolated as single cells with the DEPArray™ system. Whole genome amplified CTC DNA underwent scNGS with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space to detect mutations, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). 99.1% of cells were informative, with a mean sequencing depth of 664x. Using our previously developed, CTC-based precision medicine reporting platform, MI-CTCSeq, multiple CTC in seven of 15 patients (47%) had mutations that were actionable by FDA-approved targeted therapies including in the oncogenes PIK3CA (alpelisib) and FGFR2 (erdafitinib). 13 patients (87%) displayed intra-patient, inter-CTC genomic heterogeneity of putative driver mutations. Two of five (40%) patients with CTC at both baseline and progression displayed fluctuations in their CTC subclonal makeup between timepoints. One of the two harbored a baseline ESR1 (estrogen receptor α) p.D538G activating mutation that largely disappeared at progression and was replaced by a CTC subclone with a different ESR1 activating mutation, p.Y537S. Intriguingly, this patient’s CTC also harbored an FGFR2 p.K659M mutation in an actionable “hotspot” at progression, which was absent at baseline, suggesting potential utility of serial monitoring by CTC scNGS. TMB scores and MSI status in CTC were highly concordant with those measured in clinical tissue biopsies. Taken together, these data suggest the non-invasive interrogation of the CTC genomic landscape and its serial monitoring to inform precision and immuno-oncology treatments in real time. Citation Format: Andi K. Cani, Emily M. Dolce, Elizabeth P. Darga, Kevin Hu, Chia-Jen Liu, James M. Rae, Daffyd G. Thomas, Scott A. Tomlins, Arul M. Chinnaiyan, Aaron M. Udager, Costanza Paoletti, Erin F. Cobain, Daniel F. Hayes. Serial monitoring of single-cell circulating tumor cell genomics in metastatic lobular breast cancer to identify precision and immuno-oncology biomarkers with therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1700.

The success of immune checkpoint inhibitors rests on biomarkers such as tumor mutation burden (TMB) and microsatellite instability (MSI), both FDA-approved predictors of anti PD-1/L1 therapy benefit. Tissue biopsies often collected once in the metastatic setting through an invasive procedure, or archived primary tumor tissue often collected much prior to treatment consideration, are the specimen types of choice for biomarker identification. The tissue sample originates from a limited region of one disease site, which may limit its usefulness given intra-patient tumor heterogeneity. TMB and MSI measurement by liquid biopsy, including proteins, circulating tumor cells (CTC), and cell-free circulating tumor DNA (ctDNA), is an attractive, minimally-invasive way to obtain a real-time picture of the entire disease. While TMB and MSI assessment from ctDNA have been reported, their measurement can be limited by low ctDNA tumor fraction. Single-cell next generation sequencing of CTC, on the other hand, is a particularly well-suited, but largely unexplored method of measuring TMB and MSI to complement tissue and ctDNA for better overall specificity of detection. In this proof-of-concept study, we show the ability to detect single-cell TMB and MSI. We analyzed 14 CTC and 4 ctDNA samples from 6 metastatic breast cancer patients, as well as 3 single cells and 1 cell pellet sample each from HCT-116 (MSI-High) and WiDr (MSI-Low) cell lines. CTC and cell line cells were enriched with the CellSearch® system and/or isolated with the DEPArray™ system. Whole genome amplified single-cell DNA was sequenced with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space. TMB and MSI scores obtained in CTC and ctDNA were compared to those measured in matched clinical tissue biopsies. Single-cell TMB scores and MSI status were assessable in all CTC tested. CTC TMB scores were highly concordant with the matched tissue samples (r=1.00), as were ctDNA TMB scores (r=0.98) in patients with assessable TMB scores in both biospecimen types compared. Importantly, TMB was detectable in CTC from one patient whose tissue sample was inadequate for clinical sequencing, and from another patient with inadequate, low tumor fraction ctDNA. Intriguingly, one patient harbored 3 TMB-high and 2 TMB-low CTC, potentially indicating intra-patient TMB heterogeneity. The known MSI-low status from clinical tumor tissue sequencing was correctly detected in CTC and ctDNA from all patients. MSI status and scores from single cells of HCT-116 and WiDr cell lines purified with the DEPArray™ system (mimicking CTC isolation), perfectly matched that of the corresponding cell pellet samples (r=1.00). Taken together, these data suggest the potential validity and continued interrogation of potential utility of CTC TMB and MSI detection to complement tissue and ctDNA in guiding checkpoint inhibitor immunotherapy. Citation Format: Andi K. Cani, Emily M. Dolce, Chia-Jen Liu, Brittany Rupp, Elizabeth P. Darga, Costanza Paoletti, Dafydd G. Thomas, Yi-Mi Wu, Dan R. Robinson, Sunitha Nagrath, Arul M. Chinnaiyan, Scott A. Tomlins, Aaron M. Udager, John M. Carethers, Erin F. Cobain, Daniel F. Hayes. Assessment of tumor mutation burden and microsatellite instability by single-cell circulating tumor cell genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 611.

Background Immune checkpoint inhibition is effective in several cancers. Expression of programmed death-ligand 1 (PD-L1) on circulating tumor or immune effector cells could provide insights into selection of patients for immune checkpoint inhibition. Methods Whole blood was collected at serial timepoints from metastatic breast cancer patients and healthy donors for circulating tumor cell (CTC) and platelet PD-L1 analysis with a phycoerythrin-labeled anti-human PD-L1 monoclonal antibody (Biolegend clone 29E.2A3) using the CellSearch® assay. CTC PD-L1 was considered positive if detected on at least 1% of the cells; platelet PD-L1 was considered positive if ≥100 platelets per CellSearch frame expressed PD-L1. Results A total of 207 specimens from 124 metastatic breast cancer patients were collected. 52/124 (42%) samples at timepoint-1 (at or close to time of progressive disease) had ≥5 CTC/7.5ml whole blood. Of those, 21 (40%) had positive CTC PD-L1. In addition, platelet PD-L1 expression was observed in 35/124 (28%) at timepoint-1. Platelet PD-L1 was not detected in more than 70 specimens from 12 healthy donors. Platelet PD-L1 was associated with ≥5 CTC/7.5ml whole blood (p = 0.0002), less likely in patients with higher red blood cell counts (OR = 0.72, p Conclusion PD-L1 expression was found in metastatic breast cancer patients on both CTC and platelets in an independent fashion. Inter-patient platelet PD-L1 expression was highly heterogeneous suggesting that it is a biological event associated with cancer in some but not all patients. Taken together, our data suggest that CTC and platelet PD-L1 expression could play a role in predicting which patients should receive immune checkpoint inhibition and as a pharmacodynamics biomarker during treatment.

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients. Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110–22. ©2017 AACR.

Purpose: Metastasis requires malignant cell circulation from the primary to a distant tissue. Elevated levels of circulating tumor cells (CTC) portend a poor prognosis in breast and other cancers. Recent studies have suggested that CTC clusters may be a factor in the metastatic process. We conducted a prospective retrospective study of the SWOG0500 clinical trial to test whether CTC clusters are associated with poorer prognosis. Experimental Design: CTC CellSearch galleries from SWOG0500 trial were reread using prespecified criteria for CTC clusters, doublets, and enumeration. Survival analysis methods include Kaplan–Meier plots and log-rank tests. Results: Patients were classified into three prognostic subgroups based on baseline CTC/7.5 mL whole blood (WB): Arm A: Conclusions: In patients with metastatic breast cancer starting first-line chemotherapy, mortality is independent of the presence of CTC clusters, but rather depends on the number of CTC/7.5 mL WB.

Purpose:Common resistance mechanisms to endocrine therapy (ET) in estrogen receptor (ER)–positive metastatic breast cancers include, among others, ER loss and acquired activating mutations in the ligand-binding domain of the ER gene (ESR1LBDm). ESR1 mutational mediated resistance may be overcome by selective ER degraders (SERD). During the first-in-human study of oral SERD AZD9496, early changes in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) were explored as potential noninvasive tools, alongside paired tumor biopsies, to assess pharmacodynamics and early efficacy.Experimental Design:CTC were enumerated/phenotyped for ER and Ki67 using CellSearch in serial blood draws. ctDNA was assessed for the most common ESR1LBDm by droplet digital PCR (BioRad).Results:Before starting AZD9496, 11 of 43 (25%) patients had ≥5 CTC/7.5 mL whole blood (WB), none of whom underwent reduction to Conclusions:Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER+ and ESR1LBDm+ ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker.

Introduction: Fifty percent of ER positive MBC patients do not benefit from ET. Potential mechanisms of resistance to ET in this patient population include absence of ER expression by deletion or suppression, alteration in ER signaling pathway genes, or upregulation of multiple growth factor receptor pathways. We hypothesized that genotyping and phenotyping of CTC combined with genomic analysis of ptDNA will provide important insights into the multiple mechanisms of ET resistance and that a set of blood tests might serve as a "liquid biopsy" abrogating the need for tissue specimens. Methods: Twenty-four patients providing informed consent were enrolled into the Mi CTC-ONCOSEQ study, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program). Seven of these patients (5 with ER immunohistochemistry (IHC) positive and 2 with ER negative cancers) who had available archived primary and metastatic cancer tissue, a research metastatic biopsy for genomic analysis, and who had ≥5CTC/7.5 ml whole blood (WB) characterized for ER protein (CTC-ER) are the focus of this report. All the patients were ET refractory. None of them was progressing on fulvestrant at the time of study entry. CTC enumeration and phenotyping was performed with CellSearch©. Circulating ptDNA was analyzed by droplet digital polymerase chain reaction (ddPCR). ER status from archived tissue was obtained from chart review. ER mRNA expression was determined in the research biopsy of metastatic tissue by using quantitative RNA sequencing. Mutational status of ER gene, ESR1, was determined by Next-gen Sequencing using the Illumina HiSeq 2500 platform. Results: The 2 control patients with triple negative breast cancer had negative CTC-ER. Discordance between CTC-ER and tissue ER by IHC was observed (Table). Two of the 5 ER positive patients retained CTC-ER positivity (39% and 19% of the CTC). One of them (#7) harbored an ESR1 mutation in the research biopsy tissue and in ptDNA, whereas the other (#14) had wild type (WT) ESR1. CTC-ER protein levels in patients #12, 17 and 24 were negative. All had WT ESR1 in the research biopsy tissue. Of note, patient #12 had WT ESR1 in the research biopsy, but an ESR1 mutation was detected in her ptDNA. Pt#CTC-ER Tissue-ER ESR1 status in research biopsyESR1 status in ptDNA N[deg]CTC/7.5ml WB% CTC-ER +Primary by IHCMet by IHCMet research biopsy by mRNA 71839%+++Y537SY537S141619%+NA+WTWT12130%+++WTD538G17160%++weakly+WTWT242750%+weakly+weakly+WTWT Conclusions: These exploratory data suggest heterogeneous mechanisms of resistance to ET in patients with previously determined ER-positive MBC, including ESR1 mutations in ER positive cases (seen in 2 patients) and loss of ER expression (seen in CTC of 3 patients). In contrast, other cancers continue to express WT ESR1, and therefore must have developed alternative mechanisms of resistance. At least 2 of these mechanisms can be detected and monitored with complementary circulating assays: CTC and ptDNA. Further investigations are needed to understand the heterogeneous mechanisms of resistance to ET. Citation Format: Paoletti C, Aung K, Cannell EM, Darga EP, Chu D, Kidwell KM, Thomas DG, Tokudome N, Brown ME, McNutt LM, Gersch C, Schott AF, Park BH, Robinson DR, Chinnaiyan AM, Rae JM, Hayes DF. Molecular analysis of cancer tissue, circulating tumor cells (CTC) and cell-free plasma tumor DNA (ptDNA) suggests variable mechanisms of resistance to endocrine therapy (ET) in estrogen receptor (ER) positive metastatic breast cancer (MBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-01.

Fulvestrant is a dose dependent selective estrogen receptor (ER) down-regulator (SERD) used in ER-positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch ® system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch ® CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene ( ESR1 ). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies.

Introduction: Only half of hormone receptor positive (HR+) metastatic breast cancer (MBC) patients (pts) benefit from endocrine therapy (ET). Circulating tumor cells (CTC) are prognostic in pts with MBC using CellSearch® technology. The CTC-endocrine therapy index (CTC-ETI) provides semi-quantitative analyses of CTC-ER (estrogen receptor), BCL2, HER2, and Ki67 expression. We hypothesized that CTC-ETI high (elevated CTC number and/or low expression of ER and BCL2, and high expression of HER2 and Ki-67) might predict resistance to ET in a prospective, multi-institutional clinical trial: COMETI-P2-2012.0 (NCT01701050). Methods: 121 pts with ER+, HER2 negative (-), and progressive MBC after one or more lines of ET or within 12 months (mos) of completing adjuvant ET, who were initiating a new ET, were enrolled after informed consent. CTC and CTC-ETI were determined as previously reported (Paoletti C et al, CCR 2015) at baseline (BL), 1, 2, 3, and 12 mos, and/or at the time of progression. Imaging was performed every 3 mos. Association of CTC levels and CTC-ETI with patient outcomes (progression free survival (PFS); rapid progression (RP) defined as progression within 3 mos) was assessed using logrank and Fisher's exact tests. Trial design estimated 85 PFS and 51 RP events, providing >90% power (2-sided a=0.05); pts with unsuccessful BL CTC-ETI or ineligible were unevaluable. Only baseline (BL) data are reported in this abstract. Results: 32% of enrolled pts had progression within 12 mos of completing adjuvant ET, whereas 40%, 20%, and 8% had 1, 2, ≥3 lines of ET for MBC. CTC-ETI was successfully determined in 93% of pts (90% CI, 88% to 97%). CTC were ≥5 CTC/7.5 ml whole blood in 37/108 (34%) pts evaluable for clinical validity. Elevated CTC was associated with worse PFS (median (m) PFS: 3.3 vs. 5.9 mos; P Conclusions: In this multi-institutional, prospective study, CTC-ETI was accurately determined, confirming the previously established analytical validity of the assay, meeting the primary objective of the trial. Elevated CTC and CTC-ETI high compared to low were associated with poor outcomes to ET. CTC-ETI distribution resulted in a small number of patients assigned to the intermediate group, restricting our ability to associate this group with outcomes. These results suggest that CTC-biomarker phenotype and enumeration have clinical validity. CTC-ETI may identify ER+ HER2– MBC pts who are unlikely to benefit from ET and might be better treated with ET in combination with other therapies or proceed to chemotherapy. Further analyses including CTC-ETI at serial time points during ET are planned. Citation Format: Paoletti C, Regan MM, Liu MC, Marcom PK, Hart LL, Smith II JW, Tedesco KL, Amir E, Krop IE, DeMichele AM, Goodwin PJ, Block M, Aung K, Cannell EM, Darga EP, Baratta PJ, Brown ME, McCormack RT, Hayes DF. Circulating tumor cell number and CTC-endocrine therapy index predict clinical outcomes in ER positive metastatic breast cancer patients: Results of the COMETI Phase 2 trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-01.

Introduction: Somatic mutations, including those in TP53, PIK3CA, and estrogen receptor alpha (ESR1), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity. Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch© (CXC kit). CTC and WBC were then purified using DEPArrayTM. DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1TM WGA). Genetic analysis was performed on individual CTC, pooled CTC and pooled WBC DNA by multiplexed PCR based targeted next generation sequencing (NGS) using the Oncomine Comprehensive Panel (targeting ∼130 onco- and tumor suppressor genes) and the Ion Torrent Proton. All patients had exome sequencing performed on research biopsies of metastases using an Illumina HiSeq 2500 platform. Results: This pilot study was conducted using high quality DNA from two patients assessed to date. Both patients had lobular carcinoma and as expected harbored somatic, deleterious CDH1 (E-cadherin) mutations (frameshift and non-sense) in both research biopsy and CTC-DNA. These data supported our approach. Patient #1 was TP53 wild type in her research biopsy, but multiple CTC harbored somatic TP53 frame-shift mutations (Table). Patient #2 harbored an ESR1 Y537S mutation in her research biopsy. However, only 4 of 7 CTC also harbored this somatic, heterozygous mutation. Prioritized mutations in CTCPt#Cell Type (CTC vs WBC), numberGeneMutationVariant fraction (expected 1=homozygous; 0.5=heterozygous)Found in research biopsy?1CTC_A2CDH1p.I584fs1YES CTC_A4 1 CTC_A7 0.54 CTC_pool* 0.74 WBC_pool 0 CTC_A2TP53p.152_156del1NO CTC_A4 1 CTC_A7 0.51 CTC_pool* 0.88 WBC_pool 0 2CTC_A9ESR1p.Y537S0.52YES CTC_D1 0.34 CTC_D2 0.46 CTC_D6 0.65 CTC_pool* 0.35 WBC_pool 0 CTC_A12 0 CTC_D3 0 CTC_D7 0 CTC_A12CDH1p.Q641X1YES CTC_A9 1 CTC_D1 1 CTC_D3 1 CTC_D6 1 CTC_pool* 1 WBC_pool 0 * pool of all CTC Conclusions: We demonstrate the ability to purify CTC, isolate, and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Both known and novel alterations were identified in comparison to research biopsy specimens. This approach allows single cell analysis demonstrating heterogeneity of mutational status in different single cells. Studies of CTC-ESR1 and other genetic abnormalities in patients with known tissue mutations who participated in Mi CTC-ONCOSEQ are now underway. Citation Format: Paoletti C, Cani AK, Aung K, Darga EP, Cannell EM, Hovelson DH, Yazdani M, Blevins AR, Tokudome N, Larios JM, Thomas DG, Brown ME, Gersch C, Schott AF, Robinson DR, Chinnaiyan AM, Bischoff F, Hayes DF, Rae JM, Tomlins SA. Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-19.