Supplementary Figure Legend: Supplementary Figure 1.: S1A.; S1B; Supplementary Figure 2.; Supplementary Figure 3.: S3A.; S3B.; S3C.; S3D.; Supplementary Figure 4.: S4A.; S4B.; S4C.; Supplementary Figure 5.: S5A.; S5B.; S5C.; Supplementary Figure 6.: S6A.; S6B.; S6C.; Supplementary Figure 7: S7A.; S7B; S7C. Tables: Supplementary Table S1.; Supplementary Table S2.: S2A; S2B; S2C; Supplementary Table S3.: S3A; S3B; S3C; Supplementary Table S4. Original Concept

AbstractPurpose: 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: <5CTC; Arm B/C: ≥5CTC and then B (<5CTC) and C (≥5CTC)/7.5 mL WB at first follow-up. At baseline, 19% of patients had CTC doublets or clusters, which were more likely in Arm B/C versus Arm A (38% vs. 1.4%; 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.

We have developed an automated assay to enumerate and characterize circulating multiple myeloma cells (CMMC) from peripheral blood of patients with plasma cell disorders. CMMC show expression of genes characteristic of myeloma and fluorescence in situ hybridisation results on CMMC correlated well with bone marrow results. We enumerated CMMC from over 1000 patient samples including separate cohorts of newly diagnosed multiple myeloma and high/intermediate risk smouldering multiple myeloma (SMM) with clinical follow-up data. In newly diagnosed myeloma patient samples, CMMC counts correlated with other clinical measures of disease burden, including the percentage of bone marrow plasma cells, serum M protein, and International Staging System stage. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (P < 0·001). Patients with CMMC counts ≥100 at remission showed reduced survival relative to patients with CMMC counts <100. Patients with undetectable CMMC in remission showed further overall survival benefits. In the SMM cohort, there was a trend toward higher CMMC in patients with higher-risk myeloma precursor states. Significantly higher CMMC counts were observed between intermediate/high risk SMM patients that progressed versus those without progression (P = 0·031). CMMC allow a non-invasive means of monitoring tumour biology and may have use as a prognostic test for patients with plasma cell disorders.

SummaryBackground Circulating tumor cells (CTCs) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in CTCs and tumor tissue were evaluated as prognostic or predictive markers of CXCR4 peptide antagonist LY2510924 plus carboplatin-etoposide (CE) versus CE in extensive-stage disease small cell lung cancer (ED-SCLC). Methods This exploratory analysis of a phase II study evaluated CXCR4 expression in baseline tumor tissue and peripheral blood CTCs and in post-treatment CTCs. Optimum cutoff values were determined for CTC counts and CXCR4 expression in tumors and CTCs as predictors of survival outcome. Kaplan-Meier estimates and hazard ratios were used to determine biomarker prognostic and predictive values. Results There was weak positive correlation at baseline between CXCR4 expression in tumor tissue and CTCs. Optimum cutoff values were H-score ≥ 210 for CXCR4+ tumor, ≥7% CTCs with CXCR4 expression (CXCR4+ CTCs), and ≥6 CTCs/7.5 mL blood. Baseline H-score for CXCR4+ tumor was not prognostic of progression-free survival (PFS) or overall survival (OS). Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTCs ≥6 at baseline and cycle 2, day 1 were prognostic of shorter PFS and OS. None of the biomarkers at their respective optimum cutoffs was predictive of treatment response of LY2510924 plus CE versus CE. Conclusions In patients with ED-SCLC, baseline CXCR4 expression in tumor tissue was not prognostic of survival or predictive of LY2510924 treatment response. Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTC count ≥6 at baseline and after 1 cycle of treatment were prognostic of shorter PFS and OS.

Abstract There is an increasing interest in the ability to dynamically track disease burden and perform molecular subtyping of patients with plasma cell disorders without invasive bone marrow sampling. Circulating multiple myeloma cells (CMMC) have been detected in elevated numbers in the peripheral blood of patients with plasma cell disorders using flow cytometry or circulating cell enrichment platforms. We developed an automated CELLSEARCH® assay to enrich, enumerate, and perform a triplex FISH assay for t(4;14), t(14;16), and del 17p on CMMC (CD138+CD38+, CD45-CD19-) isolated from a 4 mL peripheral blood sample (Gross, et.al. Blood 2011; 118(21):1825). Here we present the enumeration and cytogenetic profiling of CMMC from separate cohorts of patients across the spectrum of plasma cell disorders. The first cohort consisted of newly diagnosed multiple myeloma patients enrolled in the CoMMpass study (ClinicalTrials.gov Identifier: NCT01454297). One or more CMMC per four ml blood were detected in 684/698 (98%) of newly diagnosed myeloma patients with median CMMC count of 413 per 4 mL of blood. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (p<0.001). CMMC counts <100 at remission were associated with improved PFS and OS compared to those patients whose CMMC counts were > 100 at remission. CMMC FISH results (n = 57) showed overall agreement of 85%, 91% and 80% with bone marrow FISH results and 81%, 91%, and 95% agreement with bone marrow CNV/RNAseq results for the t(4;14), t(14;16), and del 17p assays, respectively. The second cohort of patients consisted of intermediate/high risk smoldering myeloma patients enrolled in a Phase 2 study of Siltuximab (ClinicalTrials.gov Identifier: NCT01484275). One or more CMMC per 4 mL blood was detected at baseline in 74/79 (94%) of intermediate/high risk smoldering myeloma patients with median CMMC count of 100 per 4 mL of blood. Significantly higher CMMC counts were observed between patients in the placebo arm that progressed versus those without progression (n = 34, p = 0.031). This is in contrast to standard metrics of percentage of bone marrow plasma cells and serum M protein levels where statistically significant differences were not seen between progressors and non-progressors in the placebo arm (p = 0.068 and p = 0.070, respectively). CMMCs were collected from a third cohort of 35 patients across the plasma cell disease spectrum with an emphasis on MGUS and SMM. CMMC counts were associated with the disease burden of patients within this cohort. CMMC may be a useful non-invasive tool for disease monitoring and characterization across the plasma cell disorder spectrum. In myeloma, CMMC may be a useful prognostic marker at remission to delineate those patients at risk for relapse. In SMM, CMMC may be useful for predictive patients at risk of progression to MM. Citation Format: Brad Foulk, Mike Schaffer, Steve Gross, Chandra Rao, Denis Smirnov, Shalini Chaturvedi, Manjula Reddy, Madeline Repollet, Claudia Rojas, Daniel Auclair, Mary DeRome, The MMRF CoMMpass Network, Brendan Weiss, A. Kate Sasser. Peripheral blood circulating multiple myeloma cells (CMMCs) correlate with disease burden and can be used to characterize high-risk cytogenetics in newly diagnosed and smoldering myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3163.

There is increasing evidence that breast cancer evolves over time under the selection pressure of systemic treatment. Today, treatment decisions in early breast cancer are based on primary tumour characteristics without considering the disease evolution. Chemoresistant micrometastatic disease is poorly characterised and thus it is not used in current clinical practice as a tool to personalise treatment approaches. The detection of chemoresistant circulating tumour cells (CTCs) has been shown to be associated with worse prognosis in early breast cancer. The ongoing Treat CTC trial is the first international, liquid biopsy-based trial evaluating the concept of targeting chemoresistant minimal residual disease: detection of CTCs following adjuvant chemotherapy (adjuvant cohort) or neoadjuvant chemotherapy in patients who did not achieve pathological complete response (neoadjuvant cohort). This article presents the rational and design of this trial and the results of the pilot phase after 350 patients have been screened and provides insights that might provide information for future trials using the liquid biopsy approach as a tool towards precision medicine (NCT01548677).

e18558 Background: CXCR4, highly expressed in SCLC, has been implicated in inducing tumor cell migration/invasion and shown to be prognostic of outcome. We evaluated CXCR4 expression in pretreatment CTCs and tumor tissue and post-carboplatin/etoposide (SOC) treatment CTCs in a phase 2 ED-SCLC study of a CXCR4 inhibitor (NCT 01439568). Methods: 89 pts were treated with 42 receiving SOC. CTCs were analyzed using CELLSEARCH CXC kit combined with a PE labelled anti-CXCR4 Ab. CXCR4 expression on CTCs was scored visually by an operator and with specialized software. Baseline tumor CXCR4 expression was quantified (H-score) by immunohistochemistry. Optimum cutoff values for CTC counts and CXCR4 expression on CTCs and tumor were determined from ROC curves. Kaplan Meier curves were used to assess cutoffs as PFS prognostic variables for SOC pts. Results: See Table. For all pts, baseline % CXCR4+CTCs correlated positively with tumor CXCR4 expression (r=0.423; p=0.001). The agreement between operator and software scoring of CXCR4 expression in CTCs was 83%. For the SOC pts, the presence of <6.9% CXCR4+CTCs at baseline was prognostic of better PFS (HR=0.34 [0.13-0.88]; log rank p=0.075), tumor tissue CXCR4 expression was not significant (log rank p=0.675). At C2D1 (Cycle 2 Day 1), <2 CTCs was also prognostic of better PFS (HR=0.19 [0.07-0.54), log rank p=0.004]. In the SOC pts, 83% (30/36) had ≥2 CTCs and 76% (25/33) had ≥6.9% CXCR4+ CTCs at baseline; 41% (11/27) had ≥2 CTCs and 25% (6/24) had ≥6.9% CXCR4+CTCs at C2D1. The mean percent decrease from baseline to C2D1 for CTCs (83%, p<0.001) and %CXCR4+ CTCs (52%, p=0.017) was significant. Conclusions: In ED-SCLC pts, while CXCR4 positivity in tumor was not prognostic in this small study, having either <6.9% CXCR4+ CTCs at baseline or <2 CTCs after 1 cycle of SOC treatment was prognostic of better survival. Clinical trial information: NCT01439568. Tumor CXCR4+ H-score Baseline* CTCs Baseline* %CXCR4+ CTCs C2D1* CTCs C2D1* %CXCR4+ CTCs 89 pts N^ 69 78 70 mean±SD 203±82 656±1662 24±26 median 200 51 18 (range) (5-300) (0-9948) (0 –100) 42 SOC pts N^ 33 36 33 27 24 mean±SD 200±85 702±1927 27±25 25 ± 80 11 ± 20 median 200 99 21 0 0 (range) (5-300) (0-9948) (0–100) (0 - 406) (0 – 67) * /7.5ml ^ pts with evaluable results.

Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement.

Background Enumeration of circulating tumor cells (CTC) from whole blood permits monitoring of patients with breast carcinoma. Analysis of apoptosis & Bcl‐2 expression in CTC might add additional prognostic and predictive information. We estimated the degree of these markers in CTC from patients being treated for metastatic breast cancer. Methods Eighty‐three evaluable patients initiating a new therapy for metastatic breast cancer were enrolled. Whole blood was collected at baseline, at one of three short term time windows (24, 48, or 72 h) after initiating treatment, and at first follow‐up (3–5 weeks). CTC were isolated, enumerated, and expression of M30 and Bcl2 was determined using the CellSearch® System. Results At baseline, window, and 3–5 weeks post‐treatment, 41/80 (51%), 40/80 (50%) and 21/75 (28%) patients had ≥5 CTC, respectively. At baseline, the proportion of CTC‐apoptosis (M30) was inversely correlated with CTC number, and modestly inversely correlated with CTC‐Bcl‐2. As expected, higher CTC levels at baseline or first follow‐up were associated with worse prognosis. Surprisingly, in patients with elevated CTC, higher levels of CTC‐apoptosis were associated with worse prognosis, while higher CTC‐Bcl‐2 levels correlated with better outcomes. Conclusions CTC apoptosis and expression of Bcl‐2 can be analytically determined in patients with metastatic breast cancer and may have biological and clinical implications. Characterization of CTC for these and other markers could further increase the utility of CTC monitoring patients in clinical investigations of new anti‐neoplastic agents.

Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelial cancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy.

11016 Background: Induction of apoptosis is a common effect of many new and existing drugs, and Bcl-2 expression is associated with resistance to apoptosis. CTCs are a minimally invasive blood test and are a source of tumor cells that can be obtained serially during therapy. Monitoring Bcl-2, apoptosis, and other markers in CTCs may be useful endpoints in trials of new and targeted agents. The goal of this pilot study is to estimate Bcl-2 expression and apoptosis in CTCs from pts being treated for metastatic breast cancer (MBC). Methods: Whole blood was collected from pts initiating therapy for MBC at baseline, post-treatment (24, 48, or 72 hours), and 3–4 weeks. Samples were split into 3 × 7.5ml fractions. CTCs were isolated and enumerated using the CellTracks and CellSpotter systems. CTCs were defined as DAPI+, cytokeratin+, CD45-, with a cellular morphology. Apoptosis defined by positive staining with MAb M30. Bcl-2 status defined by staining with MAb Bcl-2–100. Results: 39/81 patients (49%) had ≥5 CTCs at baseline. 25% had ≥5 CTCs at 3–4 weeks. 35% of pts with CTCs had positive Bcl-2 staining in 91–100% of their CTCs (highest decile). Other pts were equally distributed across the remaining Bcl-2 deciles. At baseline, apoptosis correlates with CTC number. In samples with 1–4 CTCs, 80% of cells were apoptotic. For 5–49 CTCs, 48% were apoptotic; for 50–99, 34% were apoptotic; for 100–500, 28% were apoptotic; and for >500, 22% were apoptotic. Bcl-2 staining was inversely correlated with apoptosis staining. Pts were risk stratified by changes in CTC number after one cycle of therapy, and apoptosis increased with improving prognostic groups. One pt had markedly elevated CTCs of >20,000. The cells were remarkably 100% Bcl-2 positive and M30 negative (1% staining). Conclusions: These data confirm the prevalence of CTCs seen in the pivotal trial of CTCs in MBC (Cristofanilli NEJM 2004) and provide novel observations for Bcl-2 expression and apoptosis in CTCs. Higher Bcl-2 expression appears to correlate with decreased apoptosis. High fractions of apoptotic cells correlate with lower numbers of CTCs, which is associated with a better prognosis. Analysis of the post-treatment (24, 48, or 72 hour) samples and correlation of markers with progression free survival are ongoing and will be presented. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Immunicon Immunicon Immunicon Immunicon

3693 Clinical studies have shown that circulating tumor cells (CTC) accurately predict progression free and overall survival in patients with metastatic breast, colon, or prostate cancer. The CellTracks™ system is an FDA-cleared, automated system to enumerate CTC based on immunomagnetic separation and immunofluorescent characterization of recovered cells. To enable pre-clinical studies of CTC in metastasis and response to therapy, CellTracks™ was modified to permit serial detection of CTC in whole blood of mice bearing xenografts of human breast cancer cell lines or primary tumor biopsy specimens. Initial studies involved xenografts of rapidly growing human breast cancer cell lines (SKBr3 and Sum159), which prohibited long-term analysis of CTC due to animal death. This report presents data from serial CTC analyses in mice bearing xenografts from a slower-growing cell line, allowing more detailed temporal correlations between CTC kinetics and tumor size. Materials and methods: Mice: SCID/NOD mice were injected bilaterally in the 4th mammary fat pad with 106 MDA MB-231 cells. Tumor size was monitored weekly. 100 µl of blood was collected weekly via cardiac puncture for CTC analysis using CellTracks™ (Immunicon Corporation). CTC were considered present if ≥2 events were recorded on CellTracks™. Results: Palpable tumors were detected between 21 and 35 days in 10 of 14 mice injected with MDA MB-231 cells. CTC (range, 2 - 45 CTC/100µl) were detected at some time point in 13 of 14 mice. In 7 of 10 mice that developed a palpable tumor (volume ≥ 400mm3), CTC were observed to rise > 5/100µl and then become undetectable at least 7-14 days before the xenograft reached 400 mm3. A second detectable peak of CTC occurred in 8 of these mice between 30 and 45 days later. Conclusions: These data further validate that CellTracks™ can be used in preclinical studies to serially measure CTC in mice inoculated with a third, more indolent type of human breast cancer cell line. Furthermore, mice bearing xenografts from this cell line showed an unexpected, but reproducible spike in CTC levels that preceded appearance of a palpable tumor. We speculate that this pattern may be a consequence of pulsatile invasion and metastatic processes that result in intermittent shedding of CTC into the circulation.

Purpose: To detect insulin-like growth factor-IR (IGF-IR) on circulating tumor cells (CTC) as a biomarker in the clinical development of a monoclonal human antibody, CP-751,871, targeting IGF-IR.Experimental Design: An automated sample preparation and analysis system for enumerating CTCs (CellTracks) was adapted for detecting IGF-IR-positive CTCs with a diagnostic antibody targeting a different IGF-IR epitope to CP-751,871. This assay was used in three phase I trials of CP-751,871 as a single agent or with chemotherapy and was validated using cell lines and blood samples from healthy volunteers and patients with metastatic carcinoma.Results: There was no interference between the analytic and therapeutic antibodies. Eighty patients were enrolled on phase I studies of CP-751,871, with 47 (59%) patients having CTCs detected during the study. Before treatment, 26 patients (33%) had CTCs, with 23 having detectable IGF-IR-positive CTCs. CP-751,871 alone, and CP-751,871 with cytotoxic chemotherapy, decreased CTCs and IGF-IR-positive CTCs; these increased toward the end of the 21-day cycle in some patients, falling again with retreatment. CTCs were commonest in advanced hormone refractory prostate cancer (11 of 20). Detectable IGF-IR expression on CTCs before treatment with CP-751,871 and docetaxel was associated with a higher frequency of prostate-specific antigen decline by >50% (6 of 10 versus 2 of 8 patients). A relationship was observed between sustained decreases in CTC counts and prostate-specific antigen declines by >50%.Conclusions: IGF-IR expression is detectable by immunofluorescence on CTCs. These data support the further evaluation of CTCs in pharmacodynamic studies and patient selection, particularly in advanced prostate cancer.