Table S1. Antibodies used for CTC and tissue studies. Table S2. Stage 2 stopping rules for the cumulative number of successfully assayed patients. Table S3. A. CTC-Enumeration Points based on CTC Enumeration B. CTC-Bio-Pointsa Footnotes:aOnly aliquots with CTC {greater than or equal to}5/7.5 ml WB evaluated bLow or negative ER expression is weighed more than BCL-2, HER2, and Ki67 due to its fundamental role in endocrine responsiveness. C. Calculation of CTC-ETI Scores. D. CTC-ETI Categories. Footnote: Abbreviations: CTC= circulating tumor cells; ER = estrogen receptor; ET= Endocrine Therapy; MBC = metastatic breast cancer; ET= endocrine therapy. Table S4. CTC-ETI determination in cultured human breast cancer cell lines spiked into normal human blood and evaluated by the CellSearch® system. Table S5. Examples of low, intermediate, and high CTC-ETI from patient samples. A. LOW CTC-ETI Patient # 5 (Primary Tissue: ER 90% positive, HER2 negative by FISH; Metastatic tissue: ER positive, HER2 negative by FISH)* B. INTERMEDIATE CTC-ETI Patient #4 (Primary Tissue: ER {greater than or equal to}95% positive, HER2 0 by IHC; Metastatic tissue: insufficient tissue to evaluate ER and HER2)* C. HIGH CTC-ETI Patient # 3 (Primary Tissue: ER 60% positive, HER2 1+/2+ by IHC; Metastatic tissue: ER 75% positive; HER2 1+ by IHC)* Footnote: *ER and HER2 as per initial clinical laboratory using the laboratory's criteria for positivity or negativity. Table S6.< CTC-ETI for each patient. Footnote: aAF =Analytical Failure (CTC<5 in 1 of 4 aliquots, while the other 3 aliquots had {greater than or equal to}5 CTC/7.5mL and the average was {greater than or equal to}5 CTC/7.5mL); bTF=Technical Failure (Machine failure in at least one of 4 aliquots). Table S7. Concordance between biomarkers in primary and metastatic tissues and CTC. aKappa score for concordance between CTC and tissue biomarker for respective biomarker.

Supplementary Figures. Figure S1. Clinical trial study design. Figure S2. Examples of biomarker staining of cultured human breast cancer cell lines after spike and capture from normal human blood using the CellSearch® system. A semi-quantitative scale was established based on relative expression of each biomarker: negative (0), weak (1+), intermediate (2+), and high (3+). Footnote: *hormone depleted condition. Figure S3. Demonstration of appropriate antibody staining of cultured human breast cancer cells with known positive and negative biomarker expression after spike and capture from normal human blood using the CellSearch® System. A: ER staining for MCF-7 and Sk-Br-3. B: BCL- 2 staining for MCF-7 and Sk-Br-3. C: HER2 staining for Sk-Br-3 and MCF-7. D: Ki-67 staining for MCF-7. Figure S4. Composite "CTC-Bio-Score" for different cell lines. A: Hormone depleted MCF-7 cells (Luminal-type). B: MDA-MB-231 (Basal-like type). C: Sk-Br-3 (HER2 positive). Figure S5. Heterogeneity of ER expression within the cultured MCF-7 human breast cancer cell line. Cells were cultured in hormone depleted media for 48 hours, harvested, and spiked into 7.5 ml of normal human blood. The blood was then processed using CellSearch® and stained for DAPI, CK, CD45, and ER. Examples of strong (2+, 3+), weak (1+), and no staining (0) are displayed. Apparent staining in the bottom panel is due to high gain analysis by the automated fluorescent microscopy, as indicated by very high background. Figure S6. Coefficient of variation of CTC-enumeration by mean CTC count. Figure S7. Heterogeneity between primary tissue and CTC for patient #4. The CTC galleries displayed were selected from over 1,000 images to illustrate CTC-ER heterogeneity.

Abstract Metastases from primary tumors are responsible for most cancer deaths. It has been shown that circulating tumor cells (CTCs) can be detected in the peripheral blood of patients with a variety of metastatic cancers and that the presence of these cells is associated with poor clinical outcomes. Characterization of CTCs in metastatic cancer patients could provide additional information to augment management of the disease. Here, we describe a novel approach for the identification of molecular markers to detect and characterize CTCs in peripheral blood. Using an integrated platform to immunomagnetically isolate and immunofluorescently detect CTCs, we obtained blood containing ≥100 CTCs from one metastatic colorectal, one metastatic prostate, and one metastatic breast cancer patient. Using the RNA extracted from the CTC-enriched portion of the sample and comparing it with the RNA extracted from the corresponding CTC-depleted portion, for the first time, global gene expression profiles from CTCs were generated and a list of cancer-specific, CTC-specific genes was obtained. Subsequently, samples immunomagnetically enriched for CTCs from 74 metastatic cancer patients and 50 normal donors were used to confirm by quantitative real-time reverse transcription-PCR CTC-specific expression of selected genes and to show that gene expression profiles for CTCs may be used to distinguish normal donors from advanced cancer patients as well as to differentiate among the three different metastatic cancers. Genes such as AGR2, S100A14, S100A16, FABP1, and others were found useful for detection of CTCs in peripheral blood of advanced cancer patients.

Supplementary Table 1 from Global Gene Expression Profiling of Circulating Endothelial Cells in Patients with Metastatic Carcinomas

Immunofluorescence characterization of EpCAM and c-MET in cell lines. Immunofluorescence of SNU5, HeLa and SIHA cell lines, with (clockwise from top left of each panel) external c-MET, EpCAM, DAPI from EpCAM slide, and DAPI from c-MET slide.

Supplementary Figure S1 from Global Gene Expression Profiling of Circulating Tumor Cells

Supplementary Table 2 from Global Gene Expression Profiling of Circulating Endothelial Cells in Patients with Metastatic Carcinomas

Supplementary Table S2 from Global Gene Expression Profiling of Circulating Tumor Cells

CD45+/CK+ cells captured with c-MET separated by disease site (2A). Boxplot of all samples from cancer patients versus healthy controls (2B). Representative sample of dual positive cells (2C).

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.

Background There are no biomarkers for assessment of disease burden or activity of therapy in SCLC. Patients and methods We conducted a prospective study enumerating serial CTCs in patients with newly diagnosed limited disease (LD) and extensive stage (ED) SCLC. CTCs demonstrating DNA damage and apoptosis based on γH2AX and M30 staining were also assessed. We correlated CTC number with disease stage, survival outcomes and tumor burden by RECIST. Results Between 03/2011-10/2013, 50 evaluable patients were enrolled (20 LD). Baseline CTC number was higher for ED (median CTC 71 vs. 1.5 for LD; p 0.0004). Patients with <5 CTC had longer PFS but not OS (11 vs. 6.7 months, p 0.0259 and 15.5 vs. 12.9 months, p 0.4357). A higher cutoff (CTC<50 or CTC≥50) was significantly correlated with both OS (20.2 vs. 11.8 months, p 0.0116) and PFS (10 vs. 4.8 months, p 0.0002). Patients with <5 CTC on day 1 of cycle 2 had longer PFS (10 vs. 3.17 months, p<0.001) and OS (18 vs. 9 months, p 0.0001). Patients with an increase in γ2HAX-positive CTCs after chemotherapy had longer OS compared to patients without an increase (25.3 vs. 9 months, p 0.15). Conclusions This study demonstrates that CTCs at baseline and Cycle 2 of chemotherapy correlate with disease stage and survival in patients with SCLC, suggesting that CTCs may be used as a surrogate biomarker for clinical response. Confirmatory prospective clinical trials are needed before we can incorporate routine evaluation of CTCs into clinical practice.

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.

Chest pain is a leading reason patients seek medical evaluation. While assays to detect myocyte death are used to diagnose a heart attack (acute myocardial infarction, AMI), there is no biomarker to indicate an impending cardiac event. Transcriptional patterns present in circulating endothelial cells (CEC) may provide a window into the plaque rupture process and identify a proximal biomarker for AMI. Thus, we aimed to identify a transcriptomic signature of AMI present in whole blood, but derived from CECs. Candidate genes indicative of AMI were nominated from microarray of enriched CEC samples, and then verified for detectability and predictive potential via qPCR in whole blood. This signature was validated in an independent cohort. Our findings suggest that a whole blood CEC-derived molecular signature identifies patients with AMI and sets the framework to potentially identify the earlier stages of an impending cardiac event when used in concert with clinical history and other diagnostics where conventional biomarkers indicative of myonecrosis remain undetected.

Abstract Amplification of the MET oncogene is associated with poor prognosis, metastatic dissemination, and drug resistance in many malignancies. We developed a method to capture and characterize circulating tumor cells (CTC) expressing c-MET using a ferromagnetic antibody. Immunofluorescence was used to characterize cells for c-MET, DAPI, and pan-CK, excluding CD45+ leukocytes. The assay was validated using appropriate cell line controls spiked into peripheral blood collected from healthy volunteers (HV). In addition, peripheral blood was analyzed from patients with metastatic gastric, pancreatic, colorectal, bladder, renal, or prostate cancers. CTCs captured by c-MET were enumerated, and DNA FISH for MET amplification was performed. The approach was highly sensitive (80%) for MET-amplified cells, sensitive (40%–80%) for c-MET–overexpressed cells, and specific (100%) for both c-MET–negative cells and in 20 HVs. Of 52 patients with metastatic carcinomas tested, c-MET CTCs were captured in replicate samples from 3 patients [gastric, colorectal, and renal cell carcinoma (RCC)] with 6% prevalence. CTC FISH demonstrated that MET amplification in both gastric and colorectal cancer patients and trisomy 7 with gain of MET gene copies in the RCC patient. The c-MET CTC assay is a rapid, noninvasive, sensitive, and specific method for detecting MET-amplified tumor cells. CTCs with MET amplification can be detected in patients with gastric, colorectal, and renal cancers. Implications: This study developed a novel c-MET CTC assay for detecting c-MET CTCs in patients with MET amplification and warrants further investigation to determine its clinical applicability. Mol Cancer Res; 14(6); 539–47. ©2016 AACR.

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.

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.

84 Background: Multiple GI malignancies including gastric, pancreatic, and colorectal cancers harbor genetic alterations in the MET oncogene. MET amplification is associated with poor prognosis, and c-MET targeted therapies are in development. We aimed to capture and identify c-MET CTCs using a non-invasive, rapid test. Methods: We modified the CELLSEARCH platform by using nanomagnetic particles conjugated to antibodies against the extracellular domain of c-MET to capture c-MET-expressing CTCs. The method was validated by spiking MET-amplified gastric cancer cells, c-MET-overexpressing, nonamplified cancer cells, and MET-negative cancer cells into peripheral blood from healthy volunteers. Peripheral blood samples were obtained from patients (pts) with refractory metastatic gastric, pancreatic, and colorectal cancers, prepared in duplicate, and characterized by immunofluorescence staining for intracellular c-MET-PE, DAPI, and pan-CK-FITC. CD45 + cells were excluded. CTC enumeration with c-MET capture was compared to EpCAM capture. After CTC isolation, MET DNA FISH was performed. Results: The novel c-MET CTC assay was found to be 80% sensitive for MET-amplified cells, 40-80% sensitive for c-MET-overexpressed cells, and 100% specific for c-MET negative cells and in 10 healthy volunteers. In 7/7 pts with metastatic pancreatic and 8/8 pts with metastatic colorectal cancers, we did not capture c-MET CTCs. Of 3 gastric cancer pts tested thus far, one pt had a significant number of c-MET + CTCs, with 52 CTCs and 90 CTCs in each of the duplicate samples, compared to 20 CTCs with EpCAM capture. CTC FISH demonstrated polysomy 7 and METamplification. This pt had HER2 amplification in primary tumor tissue and had progressed on FOLFOX and trastuzumab at the time of enrollment. Conclusions: We have developed a sensitive and specific c-MET CTC assay. c-MET-expressing CTCs can be detected in gastric cancer but not controls or other GI cancers to date. While not detectable in many pts, high levels in one pt support the importance of MET amplification as a resistance mechanism to HER2 therapy, and suggest that this approach may be useful to identify and follow patients who may be candidates for c-MET directed therapies.

AbstractBackground: Endocrine therapy (ET) fails to induce a response in one half of patients with hormone receptor (HR)–positive metastatic breast cancer (MBC), and almost all will eventually become refractory to ET. Circulating tumor cells (CTC) are associated with worse prognosis in patients with MBC, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR-positive MBC.Methods: The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC.Results: The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in patients with MBC. CTC-ETI was successfully determined in 44 of 50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Interobserver concordance of CTC-ETI determination was from 94% to 95% (Kappa statistic, 0.90–0.91). Inter- and cell-to-cell intrapatient heterogeneity of expression of each of the CTC markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissues.Conclusions: CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC biomarker expression, are being evaluated in an ongoing prospective trial. Clin Cancer Res; 21(11); 2487–98. ©2014 AACR.See related commentary by Mathew et al., p. 2421