83 Background: Absolute PSA change is an imperfect surrogate of clinical benefit; CTCs and PSA doubling time (DT) may also be used as a surrogate of progression (PD). Hormone therapy (HT) often leads to a rapid decline in PSA, however non-HT may be effective without causing a decline in absolute PSA or testosterone (T). SM-88, a non-HT novel combination Warburg effect therapy (amino acid analogue, CYP3a4 inducer, mTOR inhibitor and catalyst) does not affect T. We report outcomes leading to subsequent therapies of an ongoing PSA recurrent, non-metastatic PC trial. Methods: Prospective Phase II of rising PSA (per PCWG3), detectable CTCs, and no baseline lesions. Results: Since September 2016, 31 subjects enrolled with 17 on study for > 12 weeks. Mean age 68.9; BMI 28.7; 38% black and 62% post RT. Mean T increased 61 mg/dl from baseline 319 mg/dl (p=0.19). 82% (14/17) subjects experienced an improvement in PSA DT and 67% (10/15) experienced a decline in CTCs of >30%. Overall median PSA DT time on enrollment was 5.3 (1.4 – 37.6) and improved to 6.5 (Wilcoxon p=0.02) (see table). 3/4 subjects with PD failed to maintain a CTC drop >60% vs 2/17 without PD (p=.03); median time to nadir CTC was 3 cycles (1-7). Subjects avoiding subsequent therapy averaged a 50% CTC decline (15-100%). CTC and PSA DT effects were not correlated to T level. AEs possibly related to drug/unrelated were: No grade (G) 4 or 5; 0/1 G3; 1/7 G2; 13/19 G1. As reported elsewhere, typical HT-related side effects were not observed. Including all patients with >1 month of data, from initial diagnosis of PSA rise (median 9 months; 3-18), 96% (22/23) have remained metastases-free and 78% (18/23) remained free of additional HT (p<.05). Clinical trial information: NCT02796898. Conclusions: SM-88 may be a useful either before or as an additive to current PC treatments where normal T may be preferred. SM-88 might not worsen QOL parameters related to T level. An effect on PSA DT and CTCs were demonstrated even in an aggressive doubling time subgroup. Prospective trials are planned to confirm its utility.[Table: see text]

Abstract Circulating tumor cells (CTCs) are important clinical indicators of metastatic progression and treatment efficacy. However, because of their low number and heterogeneity, reliable patient‐derived CTC models are not readily available. We report here the isolation and characterization of the invasive population of CTCs, iCTCs, from blood of 10 patients with epithelial ovarian cancer (EOC) and one pancreatic cancer patient based on the avidity of tumor cells toward an artificial collagen‐based adhesion matrix (CAM), in comparison with tumor progenitor (TP) cells isolated from tumor cell lines, tumors and ascites from EOC patients. CAM‐avid cells identified to be iCTCs were indistinguishable with TP cells using either functional CAM uptake or surface markers (seprase and CD44). In addition, iCTCs were characterized using peritoneal and spontaneous metastasis models in vivo to evaluate their metastatic propensity and therapeutic response. TP cells and iCTCs had a doubling time of about 34‐42 hours. TP cells were rare (<3.5%) in most patient‐derived specimens, however, iCTCs emigrated into blood, at a high frequency, 64.2% (n = 49). Approximately 500 patient‐derived iCTCs recapitulated formation of iCTCs in mouse blood and formed micrometastases in the liver and/or lung, a degree of metastatic spread equivalent to the inoculation of 5 × 105 bulk tumor cells isolated from ascites and tumors. iCTCs were shown to be novel therapeutic targets for blocking metastasis using the reduced formation of iCTCs and micrometastases by RNAi, peptides, and monoclonal antibodies against seprase.

Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality. The goals of this study are to evaluate the association between levels of invasive circulating tumor cells (iCTCs) with CRC outcomes and to explore the molecular characteristics of iCTCs. Peripheral blood from 93 patients with Stage I–IV CRC was obtained and assessed for the detection and characterization of iCTCs using a functional collagen-based adhesion matrix (CAM) invasion assay. Patients were followed and assessed for overall survival. Tumor cells isolated by CAM were characterized using cell culture and microarray analyses. Of 93 patients, 88 (95%) had detectable iCTCs, ranging over 0–470 iCTCs/mL. Patients with Stage I–IV disease exhibited median counts of 0.0 iCTCs/mL (n = 6), 13.0 iCTCs/mL (n = 12), 41.0 iCTCs/mL (n = 12), and 133.0 iCTCs/mL (n = 58), respectively (p < 0.001). Kaplan–Meier curve analysis demonstrated a significant survival benefit in patients with low iCTC counts compared with in patients with high iCTC counts (log-rank p < 0.001). Multivariable Cox model analysis revealed that iCTC count was an independent prognostic factor of overall survival (p = 0.009). Disease stage (p = 0.01, hazard ratio 1.66; 95% confidence interval: 1.12–2.47) and surgical intervention (p = 0.03, HR 0.37; 95% CI: 0.15–0.92) were also independent prognostic factors. Gene expression analysis demonstrated the expression of both endothelial and tumor progenitor cell biomarkers in iCTCs. CAM-based invasion assay shows a high detection sensitivity of iCTCs that inversely correlated with overall survival in CRC patients. Functional and gene expression analyses showed the phenotypic mosaics of iCTCs, mimicking the survival capability of circulating endothelial cells in the blood stream.

e24072 Background: SM-88 is a relatively non-toxic novel combination of dysfunctional tyrosine and repurposed agents (CYP3a4 inducer, mTOR, oxidative stress catalyst) with activity in PC. PSA, mPFS and iCTCs were assessed in a Phase Ib/II trial. Methods: Blood from men treated with SM-88 for biochemically recurrent non-metastatic PC (nmPC) was prospectively examined for iCTCs and PSA at each cycle. A cell adhesion matrix (CAM)-based platform, Vita-Assay™ plate (two-step) and Vita-Cap™ tube (single-step), were used to enrich metastasis-initiating cells from blood. Isolated cells were identified and enumerated by flow cytometry using tumor progenitor and epithelial markers plus negative hematopoietic lineage markers to identify iCTCs. Serial changes in iCTCs and PSA were measured in 124 blood and 305 serum samples, respectively, from 13 PC patients to assess their concordance and relationship with radiographic or metastasis progression free survival (mPFS). Results: Among 13 subjects with Vita-Assay™ iCTC results, there were 11/13 with reduction within the 1st cycle (28 days). Thereafter all 11 had continued reduction of iCTCs with median of 6 wks to nadir, and 5 having undetectable levels a median of 7 wks on treatment. Two of 124 iCTC assays had high background red cell due to microclots in transit. Correlation of the iCTC assays enriched by Vita-Assay™ vs Vita-Cap™ was r2= 0.78 (P < 0.0001). 83% (10/12 subjects with ≥ 3 cycles of treatment) had an improvement in PSA velocity and at least one decrease in PSA. There were 2 biochemical progressions (PCWG3 criteria), one of which also had radiographic progression. iCTC results matched PSA PD and local PD was confirmed by CT 12 wks later. The other progression was predicted 8 wks earlier by iCTCs than PSA (iCTC sensitivity and specificity for PD = 100%). Other biomarkers (LDH, uNTX, neutrophil:lymphocyte, bsAlkP) did not predict mPFS or PSA rise. Conclusions: Treatment with SM-88 in patients with nmPC in a phase Ib/II cohort was associated with iCTC reduction, and PSA stability. iCTCs prospectively correlate with prolonged mPFS despite equivocal PSA results, and predicted both biochemical and radiographic progression. Clinical trial information: NCT02796898.

Goals. Management of epithelial ovarian cancer (EOC) could use serial measurements of invasive circulating tumor cells (iCTCs) for monitoring therapeutic response and early detection of disease progression/recurrence. Goals of this study are to develop an iCTC drug resistance (CDR) assay and to evaluate clinical significance of patient-derived, cultured iCTCs in selecting available therapies. Methods. The CDR assay using Taxol-Carboplatin and eight other EOC drugs at the concentration used for patients was performed. Blood was donated by six patients before primary Taxol-Carboplatin chemotherapy, one recurrent patient, six patients during and after their course of chemotherapy, and two patients with benign disease for procedure control. CDR score above and below 100 indicates sensitivity and resistance, respectively, to that drug. Results. Five of six pre-therapy samples had >20 iCTCs/>111 CDR for Taxol-Carboplatin sensitivity, and one had 40 iCTCs/23 CDR for resistance. The recurrent sample had 58 iCTCs/5 CDR for resistance. Four of six post-therapy patients had iCTCs decreased to 0/>153 CDR indicating sensitivity, while two patients had >45 iCTCs/<85 CDR indicating resistance. The patients' treatment history and follow-up confirmed that patients were in response or remission when iCTCs were sensitive to Taxol-Carboplatin, and patients were in relapse or recurrence when iCTCs were resistant to Taxol-Carboplatin. Conclusion. Further investigation on the CDR assay is warranted to examine its use in selecting drugs before treating a patient. (C) 2017 The Authors. Published by Elsevier Inc.

e16567 Background: SM88 is a novel combination of substituted amino acid ( AA) and repurposed agents with non-toxic activity in a variety of cancers including prostate (PC). Non-metastatic biochemically recurrent PC (nmPC) is ideal for such a well-tolerated, non-androgen based treatment. We present biomarker activity including CTCs (circulating tumor cells) after completion of the Ib dose escalating pharmacokinetic portion of the SM88 development plan. Methods: Planned analysis of an ongoing prospective Phase Ib/II open label study of nmPC (PCWG3 definition). Subject 1 received the 1stdose level of AA, and others received 2x dose. All subjects also received a combination of low dose re-purposed CYP3a4 inducer, oxidative stress catalyst, and mTOR inhibitor. Results: Four subjects completed at least 1 cycle (28d) with all PK sampling as prespecified. Median age was 73(70-80); all had prior ADT that was discontinued 3-6 yrs earlier, and either had curative intent prostatectomy (n = 2) or RT (n = 2); Testosterone level was castrate in one after prior RT. CTCs fell (p < 0.01) to undetectable (n = 1) or by > 25% (n = 3) (see table) while PSA remained stable (PCWG3 criteria). Preliminary LDH, neutrophil/lymphocyte ratio (N:L), urinary NTx, bone specific AlkPhos (bAP) trends are in the table below. There were no drug related serious adverse events (2 grade: 1- pigmentation, 1- vasomotor). EORTC-QLQ30 and PR25 scores were either improved or stable. Conclusions: Treatment with SM88 in patients with nmPC in the completed phase Ib cohort was associated with CTC reduction, and stable biomarker trends including PSA and LDH. There was no significant toxicity or adverse patient reported outcomes. Based on these results, the dose escalation was stopped at the second level and a cohort expansion phase II initiated. A phase III RCT is planned for confirmation of these results. Clinical trial information: NCT02796898. [Table: see text]

The ability to capture, enrich, and propagate circulating cancer cells/circulating tumor cells (CTCs) for downstream analyses such as ex vivo drug-sensitivity testing of short-term cultures of CTCs, single cell sorting of CTCs by fluorescence activated cell sorting (FACS), animal injection tumor and/or metastasis formation studies, next generation sequencing (NGS), gene expression profiling, gene copy number determination, and epigenomic analyses is of high priority and of immense importance to both the basic research and translational/clinical research communities. Vitatex Inc.'s functional cell separation technology, constructed as Vita-Assay™ (AG6W, AN6W, AR6W) culture plates, is based on the preferential adhesion of invasive rare blood cells of tissue origin to a tissue or tumor microenvironment mimic-the so-called cell adhesion matrix (CAM), which has a demonstrated ability to enrich viable CTCs from blood up to one-million fold.The CAM-scaffold allows for the functional capture and identification of invasive CTCs (iCTCs) including invasive tumor progenitor (TP) cells from cancer-patients' blood. CAM-captured CTCs are capable of ingesting the CAM (CAM+) itself. Green and red fluorescent versions of Vita-Assay™ (AG6W and AR6W) allow for direct visualization of CAM-uptake by cancer cells. Vita-Assay™ CAM-enrichment has allowed for sensitive multiplex flow cytometric and microscopic detection of iCTCs from patients with cancers of the breast, ovary, prostate, pancreas, colorectum, and lung; it has also been successfully utilized for ex vivo drug-sensitivity testing of ovarian-cancer patient CTCs. The CAM enrichment method is equally suitable for the separation of iCTCs and TP cells in ascites and pleural fluid.

Goals Contemporary management of epithelial ovarian cancer (EOC) uses biomarkers to monitor response to therapy. This study evaluates the role of invasive circulating tumor cells (iCTCs) in monitoring EOC treatment in comparison with serum cancer antigen 125 (CA125). Methods Molecular and microscopic analyses were used to identify seprase and CD44 as tumor progenitor (TP) markers. The iCTC flow cytometry assay was optimized using blood donated by 64 healthy donors, 49 patients with benign abdominal diseases and 123 EOC patients. Serial changes in iCTCs and CA125 were measured in 129 blood and 169 serum samples, respectively, from 31 EOC patients to assess their concordance during therapy and their relationship with risk of progressive disease (PD). Results The assay had 97% specificity and 83% sensitivity for detecting iCTCs in blood of EOC patients. iCTCs were detected in each monitoring patient (31/31, 100%) and in 110 of the 129 blood samples (85.3%). The concordance between changes in iCTCs/CA125 levels and changes in the intervals associated with no evidence of disease (NED) were markedly stronger (specificity: CA125 93.8%; iCTCs 90.6%), whereas increases in iCTCs (79.5%) were more sensitive than increases in CA125 (67.6%) to predict PD or relapse. Among the six patients who had greater than 6 measurements, iCTCs but not CA125 antedated changes in clinical status from PD to NED during and after chemotherapy and predated relapse. Conclusion Serial measurements of iCTCs could predict therapeutic responsiveness in 31 EOC patients who underwent standard taxol/carboplatin therapy.

The tumor invasive phenotype driven by seprase expression/activity has been widely examined in an array of malignant tumor cell types; however, very little is known about the transcriptional regulation of this critical protease. Seprase (also named fibroblast activation protein-α, antiplasmin-cleaving enzyme, and dipeptidyl prolyl peptidase 5) is expressed at high levels by stromal fibroblast, endothelial, and tumor cells in a variety of invasive tumors but is undetectable in the majority of normal adult tissues. To examine the transcriptional regulation of the gene, we cloned the human seprase promoter and demonstrated that endogenous seprase expression and exogenous seprase promoter activity are high in invasive melanoma cells but not in non-invasive melanoma cells/primary melanocytes. In addition, we identified a crucial TGF-β-responsive cis-regulatory element in the proximal seprase promoter region that enabled robust transcriptional activation of the gene. Treatment of metastatic but not normal/non-invasive cells with TGF-β1 caused a rapid and profound up-regulation of endogenous seprase mRNA, which coincided with an abolishment of the negative regulator c-Ski, and an increase in binding of Smad3/4 to the seprase promoter in vivo. Blocking TGF-β signaling in invasive melanoma cells through overexpression of c-Ski, chemically using SB-431542, or with a neutralizing antibody against TGF-β significantly reduced seprase mRNA levels. Strikingly, RNAi of seprase in invasive cells greatly diminished their invasive potential in vitro as did blocking TGF-β signaling using SB-431542. Altogether, we found that seprase is transcriptionally up-regulated in invasive melanoma cells via the canonical TGF-β signaling pathway, supporting the roles of both TGF-β and seprase in tumor invasion and metastasis.

Abstract Purpose: Cancer antigen 125 (CA125) is recognized as the leading biomarker in monitoring Epithelial Ovarian Cancer (EOC). More recently, invasive circulating tumor cells (iCTCs) have been introduced as a biomarker for metastatic potential. The goal of this research is to compare this biomarker with CA125 to determine the response to surgery and chemotherapy. Experimental Design: We compared clinical parameters with serial assays for iCTCs and CA125 in 129 fresh blood and 169 serum samples, respectively, from 31 patients who were receiving surgery and chemotherapy. iCTCs were capable of ingesting matrices (Cell Adhesion Matrix CAM+) and identified using positive tumor markers, Epi (EPCAM and CA125 or seprase and CD44), and negative selection markers, which exclude Epi-positive hematopoietic lineage (HL) cells, and enumerated by flow cytometry. iCTCs and CA125 levels were measured at time point less than 3-days apart. Results: A significant correlation was identified between changes in iCTC counts and CA125 levels (r=0.67, P<0.001) in the 83 samples collected prior to and after surgery and therapy. CA125 levels, but not iCTCs, were significantly reduced after surgery. During therapy, a significantly stronger concordance with clinical status was noted for decreasing values (specificity, 93.8% CA128; 90.6% iCTCs) compared with increasing values (sensitivity, 61.3% CA125; 83.9% iCTCs). Notably, increases in iCTCs, not CA125, were associated with increased risks for progressive disease (P<0.001). In the 8 patients who had >4 serial samples during therapy, changes in iCTC counts provided earlier measure of therapy response than CA125. Conclusion: This analysis showed that iCTC assay compared favorably with CA125 to monitor the response of chemotherapy in EOC patients, whereas changes of CA125 correlated better with reduced tumor burden after surgery. Citation Format: Wen-Tien Chen, Huan Dong, Qiang Zhao, Jie Yang, QIAO ZHANG, Stanley Zucker, Michael L. Pearl. Prognostic analysis of invasive circulating tumor cells (iCTCs) to monitor epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-180. doi:10.1158/1538-7445.AM2014-LB-180

The Vitatex cell-adhesion matrix (CAM) platform allows for isolation of invasive circulating tumor cells (iCTCs). Here we sought to determine the utility of prostate-specific membrane antigen (PSMA) as a metastatic castration-resistant prostate cancer (mCRPC) iCTC biomarker, to identify solitary cells and clusters of iCTCs expressing either epithelial, mesenchymal, or stem cell markers, and to explore the feasibility of iCTC epigenomic analysis. CTCs were isolated and enumerated simultaneously using the Vitatex and CellSearch platforms in 23 men with mCRPC. CAM-avid iCTCs were identified as nucleated cells capable of CAM uptake, but without detectable expression of hematopoietic lineage (HL) markers including CD45. iCTCs were enumerated immunocytochemically (ICC) and by flow cytometry. Whole-genome methylation status was determined for iCTCs using the Illumina HumanMethylation27 BeadChip. Thirty-four samples were collected for iCTC analysis. A median of 27 (range 0-800) and 23 (range 2-390) iCTCs/mL were detected by ICC and flow, respectively. In a subset of 20 samples, a median of seven CTCs/mL (range 0-85) were detected by the CellSearch platform compared to 26 by the CAM platform. iCTC clusters were observed in 17% of samples. iCTCs expressing PSMA as well as markers of EMT and stemness were detectable. The iCTC methylation profile highly resembled mCRPC. More CTCs were recovered using the CAM platform than the CellSearch platform, and the CAM platform allowed for the detection of iCTC clusters, iCTCs expressing EMT and stem-cell markers, and characterization of the iCTC methylome. Correlation with clinical data in future studies may yield further insight into the functional significance of these findings.

Goals. Circulating tumor cells (CTCs) have been introduced as a biomarker in detecting advanced epithelial ovarian cancer (EOC). The goals are to examine the prevalence of the invasive subpopulation of CTCs (iCTCs) in patients at high risk of EOC and to compare this biomarker to serum CA125.Methods. We used a unique cell adhesion matrix (CAM)-based, functional cell enrichment and identification platform to isolate iCTCs from 129 preoperative patients. We confirmed the identity of iCTCs using positive epithelial (Epi+) markers and negative hematopoietic lineage (HL-) markers. Sensitivity and specificity of the assays were examined and iCTCs/CA125 were correlated with overall survival (OS), progression-free survival (PFS) and clinical parameters.Results. We found a 412% sensitivity, 95.1% specificity and 77.8% positive predictive value (PPV) of the iCTC assay in detecting patients with stage land II EOC malignancy, and a 83% sensitivity and 97.3% PPV in detecting all stages of EOC malignancy. However, a positive CA125 test provided weak evidence to detect stage I and II malignancy (61.6% PPV) and all EOC (92.1% PPV), because of its 76.2% specificity. A significantly stronger concordance in OS and PFS of clinical factors (tumor stage, debulking and platinum sensitivity) was noted for elevated iCTCs than for serum CA125.Conclusion. The CAM-initiated CTC enrichment/identification method enabled the detection of early stage EOC. iCTCs were better correlated with worse OS and PFS, more specific and better PPV than CA125 in detecting EOC malignancy in patients at high risk of EOC. (C) 2014 Elsevier Inc. All rights reserved.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: Circulating tumor cells (CTCs) might function as prognostic markers for monitoring and assessing therapy efficacy ex vivo . This study aimed to measure overall population of CTCs based on expression of epithelial lineage (Epi) markers and the subpopulation of CTCs with the invasive phenotype (iCTCs) in preoperative patients for prognosis of advanced Epithelial Ovarian Cancer (EOC). Experimental Design: CTCs and iCTCs enriched by the Cell Adhesion Matrix (CAM)-coated devices from 122 preoperative patients were identified as Epi+ nucleic acid (NA)+ hematopoietic lineage (HL)- cells and CAM uptake (CAM+) Epi+ NA+ HL- cells, respectively. CTCs and iCTCs were enumerated using multiplex flow cytometry, validated by microscopy, and correlated with overall survival (OS) and progression-free survival (PFS). Results: iCTCs were detected in 0/39 (0%) benign patients, 5/11 (45.5%) Stage I, 2/4 (50%) Stage II, 41/43 (95.3%) Stage III and 16/16 (100%) stage IV patients (P-value <0.001). However, healthy (n=48) and benign women had noticeable CTCs (mean=10 and 3 CTCs/1.0-mL blood, respectively), and large variation (IQR=24.5 and 30 CTCs/1.0-mL blood, respectively), although CTCs were significantly different across different stage groups. Kaplan-Meier estimates of OS at 5 years in the iCTC positive and negative groups were 15.4% (95% CI, 7.2% to 26.3%) and 94% (95% CI, 82.5% to 98%), respectively. The PFS in the iCTC positive group was also significantly lower than that in the negative group (P-value <0.001). In multivariable Cox models, iCTCs were independently predictive of OS (P-value <0.001) and provided predictive power for disease recurrence independent of diagnostic disease stages, age, debulking status and platinum sensitivity. Conclusion: CTCs show the expression of heterogeneous epithelial antigens with potential biologic relevance. Co-expression of epithelial antigens and invasiveness of iCTCs in preoperative patients provides prognosis of advanced EOC that could help tailor appropriate therapy. Citation Format: Wen-Tien Chen, Qiang Zhao, Huan Dong, Jie Yang, Qiao Zhang, Tina Fan, Marc G. Golightly, Whitney C. Dessio, Stefan Madajewicz, Melissa Henretta, Michael L. Pearl. Circulating tumor cells with the invasive phenotype predict progression free survival and overall survival in preoperative patients diagnosed with advanced epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-73. doi:10.1158/1538-7445.AM2013-LB-73

11048 Background: Isolation, enumeration, and genomic profiling of CRPC CTCs offers the potential to discover genetic changes that occur in advanced disease. The Vitatex VitaCap platform captures CTCs based on their ability to invade a collagenous matrix (CAM), allows for capture of invasive CTCs (iCTCs) independent of EpCAM status, and yields viable cells suitable for comprehensive genomic study. Here we sought to compare CTC yields between the CAM and CellSearch platforms, to determine the utility of prostate-specific membrane antigen (PSMA) as an iCTC biomarker, to identify iCTC clusters and iCTCs expressing stem-like markers, and to explore the feasibility of iCTC epigenomic analysis. Methods: CTCs were isolated and enumerated simultaneously using the CellSearch and CAM platforms in 23 men with mCRPC. CAM-isolated iCTCs were defined as EpCAM+PSMA+ and were enumerated immunocytochemically (ICC) and by flow cytometry. iCTC clusters were enumerated by ICC. The Illumina Infinium HumanMethylation27 BeadChip was used to determine whole genome methylation status for CAM isolated cells. Results: 35 samples were collected for CAM analysis. A median of 27 (range 0-800) and 23 (range 2-390) iCTCs/ml were detected by ICC and flow respectively. In a subset of 20 samples, a median of 7 CTCs/ml (range 0-85) were detected by the CellSearch platform. CTCs were detectable by either CAM or CellSearch in >95% of samples. iCTC clusters were observed in 23% of samples with a median 7 clusters/ml (range 1-200). iCTCs expressing stem-like markers CD44 and Seprase were detected in 70% and 97% of samples by ICC and flow respectively, with a median of 9/ml (range 1-264) by flow. The iCTC methylation profile highly resembled mCRPC. Conclusions: The CAM and CellSearch platforms yield comparable CTC counts. iCTC clusters and iCTCs expressing stem-like markers are detectable using the CAM platform, and the iCTC methylome closely resembles that of mCRPC. Correlation with clinical data may yield further insight into the functional significance of these findings.

Objective: Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid present in the plasma, is released from activated platelets. Our previous study dem- onstrated that Sph-1-P promoted the spreading on and migration of human umbilical vein endothelial cells (HUVEC) through the extracellular matrix (ECM), suggesting a possible induction of cell surface proteases in the Sph-1-P activated endothelial cells. In the present study, we examined whether seprase, a type II transmembrane serine protease (TTSP) usually absent in tissue cells, can be induced in endothelial cells acti- vated by Sph-1-P. Methods: HUVEC migration through ECM was examined by modifi ed Boyden chamber assay. Western blotting and immunoprecipitation, using anti-seprase monoclonal antibodies (mAbs), were used to confi rm the seprase expression of HUVEC. Results: We show that Sph-1-P enhanced expression of active form seprase in a time- and dose-dependent manner in HUVEC. The Sph-1-P inducible active form seprase could be blocked by pertussis toxin and by C3 transferase, which inactivate Gi-type heterotrimetric G proteins and Rho, respectively. Conclusion: These results show that Sph-1-P can regulate migration of endothelial cells by inducing active form seprase expression, which, in turn, is mediated through a Gi-coupled cell surface receptor and the Rho protein.

144 Background: Isolation, enumeration, and genomic profiling of CRPC CTCs offers the potential to discover genetic changes that occur in advanced disease. The Vitatex VitaCap platform captures CTCs based on their ability to invade collagenous matrices (CAM), allows for CTC and CTSC capture independent of EpCAM status, and yields viable cells suitable for comprehensive genomic analysis. In this study we sought to compare CTC yields between the VitaCap and the Veridex CellSearch platforms, to determine the utility of prostate-specific membrane antigen (PSMA) as a mCRPC CTC biomarker, to identify CTSCs and CTC clusters, and to explore the feasibility of CTC epigenomic analysis. Methods: CTCs were isolated and enumerated simultaneously using the CellSearch and VitaCap platforms in 23 men. CAM isolated CTCs and CTSCs were defined as EpCAM+PSMA+ and CD44+DPPA5+ respectively, and were enumerated immunocytochemically (ICC) and by flow cytometry. CTC clusters were enumerated by ICC. The Illumina HumanMethylation27 BeadChip was used to determine whole genome methylation status for CAM isolated cells. Results: 35 samples were collected for CAM analysis. A median of 27 (range 0-800) and 23 (range 2-390) CAM+ CTCs/ml were detected by ICC and flow respectively. In a subset of 20 samples, a median of 7 CTCs/ml (range 0-85) were detected by the CellSearch platform. CTCs were detectable by either CAM or CellSearch in >95% of samples. CAM isolated CTC clusters were observed in 23% of samples with a median 7 clusters/ml (range 1-200). CTSCs were detected in 70% and 97% of samples by ICC and flow respectively, with a median of 9 CTSCs/ml (range 1-264) by flow. The CTC methylation profile highly resembled mCRPC with hypermethylation occurring more frequently in pathways associated with apoptosis (p<0.006), angiogenesis (p<0.007), and VEGF signaling (p<0.003). Conclusions: CAM and CellSearch platforms yield comparable CTC counts. CTSCs and CTC clusters are detectable using the CAM platform, and the CTC methylome closely resembles that of mCRPC. Correlation with clinical data may yield further insight into the functional significance of these findings.

Seprase (fibroblast activation protein α) has been examined as an invasion biomarker for various types of solid tumors. We studied whether plasma levels of seprase and homologous protease, DPP4 in cancer might serve as tumor biomarkers. We developed sensitive and specific Enzyme-Linked Immunosorbent Assays (ELISAs) to measure these proteases. In 747 plasma samples (from 139 healthy volunteers and 561 cancer patients), mean seprase and DPP4 levels were 0.51 ± 0.30 and 4.65 ± 6.37 μg/mL, respectively, and they were correlated with each other (R 2 = 0.382). Plasma DPP4 and seprase levels were significantly lower in cancer patients compared with healthy subjects (4.38 versus 5.65 μg/mL, p < 0.001 for DPP4; 0.46 versus 0.66 μg/mL, p < 0.001 for seprase). Higher DPP4 was associated with better survival in all cancers combined ( n = 346) as well as in head and neck malignancies ( n = 38). Higher seprase was associated with better survival in all non-metastatic cancers combined ( n = 151) as well as head and neck malignancies, but worse survival in colorectal cancers ( n = 47). This study demonstrates that in contrast to the high expression in solid tumors, plasma concentrations of seprase and DPP4 are reduced and correlate inversely with survival in most types of cancer, suggesting that these circulating proteases represent useful tumor markers.

Recent research advances show that tumor cell intravasation (entry into the circulation) and metastasis occur very early in breast cancer progression. Clinical studies also illustrate the potential importance of detection of circulating tumor cells (CTCs) in outcomes of patients with metastatic breast cancer. Whether these cells exhibit the invasiveness and express tumor stem or progenitor markers, hallmark of the metastatic phenotype, is less well characterized. To detect CTCs with the invasive phenotype and to explore their molecular features, we applied a functional cell separation method, called collagen adhesion matrix (CAM) assay, as enrichment and identification steps. The CAM-coated device successfully recovered tumor cells spiked in 1 ml of blood with a 54% +/- 9% (n = 18) recovery rate and 0.5-35% purity, and detected invasive tumor cells in 10/10 blood samples (100% yield) from patients with metastatic breast cancer with a range of 18-256 CTCs/ml and average of 126 +/- 25 (mean +/- SD) CTCs/ml. CTCs were detected in blood samples of 28/54 (52%) Stage I-III breast cancer patients with a mean count of 61 CTCs/ml. Furthermore, the relative frequency of these cells correlated to the staging, lymph node-status and survival of patients with early stage breast cancer. CAM-captured cells were capable of propagation in culture. Gene expression and multiplex flow cytometric analyses on CAM-captured cells demonstrated the existence of distinct populations of CTCs including these of epithelial lineage and stem or progenitor cells. Thus, CAM-initiated CTC detection provides advantages for examining invasiveness and tumor progenitor phenotypes.