CTC Counts Research Articles

Association of Metabolic, Inflammatory, and Tumor Markers With Circulating Tumor Cells in Metastatic Breast Cancer.

BackgroundCirculating tumor cells (CTCs) are associated with worse prognosis in metastatic breast cancer (BC). We evaluated the association of metabolic, inflammatory, and tumor markers with CTCs in women with metastatic BC before commencing a new systemic therapy.MethodsNinety-six patients with newly diagnosed or progressing metastatic BC without current diabetes or use of anti-inflammatory agents were recruited from four Ontario hospitals. Women provided fasting blood for measurement of metabolic, inflammatory, and tumor markers and CTCs. CTCs were assayed within 72 hours of collection using CellSearch. Other blood was frozen at –80°C, and assays were performed in a single batch. Associations between CTC counts with study factors were evaluated using Spearman correlation, and the chi-square or Fisher exact test. All statistical tests were two-sided and P value ≤ .05 was considered statistically significant.ResultsThe median age was 60.5 years; 90.6% were postmenopausal. The cohort included hormone receptor–positive (87.5%), HER2–positive (15.6%), and triple-negative (10.4%) BCs. Patients were starting firstline (35.5%), second-line (26.0%), or third-or-later-line therapy (38.5%). CTC counts (per 7.5 mL of blood) ranged from 0 to 1238 (median 2); an elevated CTC count, defined as five or more CTCs, was detected in 42 (43.8%) patients. Those with liver metastases (vs not) more frequently had an elevated CTC count (59.0% vs 33.3%, P = .02). CTCs were significantly associated with C-reactive protein (R = .22, P = .02), interleukin (IL)-6 (R = .25, P = .01), IL-8 (R = .38, P = .0001), plasminogen activator inhibitor 1 (R = .31, P = .001), carcinoembryonic antigen (R = .31, P = .002), and cancer antigen 15-3 (R = .40, P = .0001) and inversely associated with body mass index (R = –.23, P = .02) and leptin (R = –.26, P = .01).ConclusionsCTC counts were positively associated with tumor and inflammatory markers and inversely associated with some metabolic markers, potentially reflecting tumor burden and cachexia.

Prognostic role of circulating tumor cells in patients with EGFR-mutated or ALK-rearranged non-small cell lung cancer.

BackgroundCirculating tumor cell (CTC) counts at baseline and follow‐up are an independent prognostic factor in patients receiving standard chemotherapy for non‐small cell lung cancer (NSCLC). This study further explored the role of CTCs in EGFR‐mutated and ALK‐rearranged NSCLC patients administered targeted therapies as first‐line treatment.MethodsCTCs were enumerated with a novel high‐efficiency detection method from the blood of 43 patients with EGFR‐mutated or ALK‐rearranged NSCLC at baseline and at disease‐progression. Patients were stratified into favorable and unfavorable groups with baseline CTC counts of < 8 or ≥ 8 CTCs/3.2 mL, respectively.ResultsA total of 76.7% of the patients were positive for ≥ 2 CTCs /3.2 ml blood at baseline. The median progression‐free survival (PFS) and overall survival (OS) rates of the favorable compared to the unfavorable group were longer (11.6 vs. 8.5 months, P = 0.004 for PFS; 21.00 vs. 17.7 months, P = 0.013 for OS). Multivariate analysis demonstrated that baseline CTC count was a strong predictor of PFS (hazard ratio 2.835; 95% confidence interval 1.240–6.483; P = 0.014) and OS (hazard ratio 3.317; 95% confidence interval 1.360–8.092; P = 0.008).ConclusionBaseline CTC count could be a predictive biomarker for EGFR‐mutated and ALK‐rearranged NSCLCs, which allows for better guidance and monitoring of patients over the course of molecular targeted therapies.

A phase 1/2a trial of docetaxel plus ribavirin for reprogramming efficacy in patients with progressive metastatic castration resistant prostate cancer who have previously received docetaxel alone: DRREEM trial.

329 Background: We previously reported a novel cell reprogramming approach, termed drug efficacy reprogramming, as a new model for identifying candidate antitumor drugs targeting the cancer stemness-related gene network, and identified ribavirin as a candidate drug for overcoming docetaxel-resistant castration-resistant prostate cancer (CRPC). We conducted this phase 1/2a trial of docetaxel plus ribavirin for reprogramming efficacy in patients with progressive metastatic castration resistant prostate cancer who have previously received docetaxel alone. (DRREEM trial) Methods: In this clinical study, patients received intravenous docetaxel at 60-75 mg/m2is intravenously administered on Cycle1-Day1 in combination with the investigational drug (ribavirin). Docetaxel is administered at 3-week intervals (1 cycle) (total: 3 cycles). During administration, the dose may be reduced based on the subject’s condition if necessary. The primary endpoint was safety. Accessory evaluation items included prostate-specific antigen (PSA) response, objective response rate, health-related quality of lifel. Exploratory items included changes in CTC count, cfDNA, and exosome. Patients with progressive CRPC based on PSA and/or radiographic criteria, performance status (PS) 0–1, and normal renal and hepatic function were eligible. Results: Six patients were enrolled in this study; average age was 71.7±4.2. Average serum PSA concentration was 100.1±128.0 ng/ml (range: 3.0-336.8). The median cycle of docetaxel received before the study was 6 cycles. Safety: Grade 3/4 adverse events requiring dose modification were not observed. Two patients showed PSA reduction. Three patients showed stable disease. Changes in the blood concentrations of ribavirin, docetaxel, and prednisolone were within normal range. Conclusions: This combination of ribavirin with docetaxel was well tolerated with a promising response rate that justifies further investigations in docetaxel-resistant CRPC. This clinical study provides a useful drug re-positioning model in the area of translational medicine. Clinical trial information: UMIN000021107.

Phase I dose-escalation study of 225Ac-J591 for progressive metastatic castration resistant prostate cancer (mCRPC).

TPS399 Background: PC is a radiosensitive disease. PSMA is overexpressed in advanced PC with upregulation by androgen receptor (AR) pathway dysregulation; limited expression exists in other organs. A series of sequential studies of beta-emitting radiolabeled anti-PSMA monoclonal antibody (mAb) J591 have demonstrated accurate targeting, efficacy with dose-response effect, and safety with predictable, reversible dose-limiting myelosuppression. Alpha emitters are significantly more potent with a shorter range than beta emitters. Though there is no direct tumor-targeting, the bone-targeting alpha emitter Ra223 is approved. Anecdotal reports of PSMA small molecule targeted alpha emitters have hinted at efficacy but are limited by xerostomia and in mouse models may lead to long-term renal damage. Intact mAb J591 has comparatively no to minimal distribution in salivary glands and kidneys. Preclinical studies demonstrated purity, immunoreactivity, and stability with efficacy in a xenograft model [AACR 2017]. Methods: Men with progressive mCRPC following at least 1 potent AR-targeted agent (e.g. abi/enza) and docetaxel (or unfit/refuse chemo) without limit of # prior therapies (excluding beta-emitting bone-targeted radioisotopes) provided adequate organ function will undergo imaging with 68Ga-PSMA-11 PET/CT followed by a single dose of 225Ac-J591. Single-subject cohorts will be enrolled until grade &gt; 1 attributable toxicity, then transition to 3+3 design. Cohort 1 = 13.3 KBq/kg with planned escalation up to 93.3 KBq/kg of 225Ac with fixed 20 mg J591. Dose-limiting toxicity (DLT) is defined as attributable grade 4 heme toxicity or grade 3/4 non-heme toxicity. Planned cohort expansion will occur at recommended phase 2 dose (RP2D) in a 2-stage design. The primary endpoint is determination of DLT and RP2D. Secondary endpoints include toxicity, PSA decline rate, RECIST response, PFS, rPFS, OS, and patient reported outcomes (FACT-P and BPI-SF). Correlatives include baseline/follow up PSMA imaging, CTC count (CellSearch) changes, tissue and circulating genomic assessment, and immune studies. Enrollment began in October, 2017. Clinical trial information: NCT03276572.

High-purity capture of CTCs based on micro-beads enhanced isolation by size of epithelial tumor cells (ISET) method

In this paper, we develop a low-cost size-based microfluidic chip using conventional polycarbonate membrane to isolate CTCs from blood, and propose a strategy to increase the capture efficiency before cell filtration by a size enlargement method utilizing modified microbeads specifically binding to CTCs. Up to 91% of target cells were isolated from whole blood samples using our microfluidic capture system at a flow rate of 1mL/min. Moreover, a WBC depletion process is introduced which greatly decreases the WBC retaining on the filter membrane. The tests of immunofluorescence analysis of cells captured on the membrane were performed, which demonstrates that the device could provide a dependable CTC identification and CTC count in whole blood samples. Finally, the device was further validated in the detection of CTCs from blood samples of cancer patients, and it indicates a promising capability to detect CTC response to treatment.

Molecular analysis of single circulating tumour cells following long‐term storage of clinical samples

The CellSearch® semiautomated CTC enrichment and staining system has been established as the ‘gold standard’ for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time‐consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch®‐enriched cells in glycerol at −20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long‐term glycerol storage for single‐cell molecular analysis, we isolated individual CellSearch®‐enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at −20 °C. All isolated cells were subjected to whole‐genome amplification (WGA), and the efficacy of single‐cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both ‘spike‐in’ controls and clinical samples showed no statistical difference between values obtained pre‐ and postglycerol storage and that there is no further loss in integrity when DEPArray™‐isolated cells are then stored at −80 °C for up to 2 years. In summary, we have established simple yet effective ‘stop‐off’ points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.

824P - ARV7 status and CTC count: A combined biomarker for the baseline therapeutic decision in each line of mCRPC treatment

824P - ARV7 status and CTC count: A combined biomarker for the baseline therapeutic decision in each line of mCRPC treatment

Concordance between circulating tumor cells and clinical status during follow-up in anaplastic lymphoma kinase (ALK) non-small-cell lung cancer patients.

BackgroundThe identification of anaplastic lymphoma kinase (ALK) rearrangements is found in approximately 5% of non-small-cell lung cancers (NSCLCs). However, the development of liquid biopsies as a diagnostic tool is less developed in these cases. This study investigates the use of CTCs during treatment, together with an extended follow-up to correlate with clinical evolution.Patients and MethodsA total of 13 patients out of a cohort of 212 patients with lung adenocarcinoma, presented ALK rearrangements (6%) confirmed by tumor biopsy. A total of 60 serial blood samples were collected from these patients who were prospectively enrolled in the study.ResultsAll patients had a positive CTC count at baseline (mean = 3). The median follow-up was 9 months (range 1-17 months). Three patients underwent surgery and their CTC counts decreased after the procedure but still remained detectable. After radiotherapy, 3 cases showed an average decrease of 5 CTCs. A total of 6 patients were treated with ALK inhibitors and a partial response was observed in 3 of them, who also presented decreased CTC counts. The other 3 patients presented primary resistance, and their CTC counts were higher than those obtained prior to progression.ConclusionWe believe that the use of CTCs for dynamic monitoring of NSCLC with ALK rearrangement and to detect disease persistence or recurrence may be a reliable technique. CTC counts may also have potential use to monitor the efficacy of ALK inhibitors, facilitating detection of resistance to treatment.

RECIST response and variation of circulating tumour cells in phase 1 trials: A prospective multicentric study

BackgroundCirculating tumour cell (CTC) counting could be a new biomarker for better evaluation of tumour response to molecules tested in phase I trials. Patients and methodsConsenting patients with advanced metastatic cancer referred to various phase I units were enrolled prospectively in this study. CTCs from 7.5 ml of whole blood drawn at baseline and after starting experimental therapy were counted using the CellSearch system, and tumour response was assessed using RECIST 1.1 criteria at baseline and 2 months after treatment initiation. ResultsBetween March 2010 and May 2013, a total of 326 patients were enrolled, among whom 214 were evaluable (49% male, median age = 56; main cancer types: lung [28], colon [53], ovarian [18], breast [28]). At baseline, we detected ≥1 CTC/7.5 ml in 113/214 patients (53%), and at day 30, we observed ≥1 CTC/7.5 ml in 103/214 patients (48%). Two months after treatment initiation, 11 (5%) of the 214 patients were classified as having a partial response, with no CTCs in 9 of them or a decrease in the CTC count after therapy. In contrast, among the 104 patients (49%) classified as having progressive disease, 38 patients had a higher CTC count. The remaining 99 patients (49%), 33 of whom (33%) had a lower CTC count, were classified as having stable disease. The sensitivity and specificity of CTC variation for predicting progressive disease were 41% (32–51%) and 80% (73–88%) respectively. ConclusionAn early CTC change following therapy does not correlate with RECIST response in patients with advanced cancer enrolled in phase I trials.

Abstract 1712: Detection of circulating tumor cells in stage IV non-small cell lung cancer

Abstract Non-Small Cell Lung Cancer (NSCLC) is the most common cause of cancer related deaths in both men and women. Some lung cancer patients have a unique challenge of a tissue access due to tumor location or baseline lung disorders such as emphysema and chronic obstructive pulmonary disease (COPD). Liquid biopsy technologies can overcome tissue assess difficulties. However, as of today there are no commercially approved assays for CTC enumeration in lung cancer. Multiple non-commercial assays exist and some show correlation of CTC enumeration and changes in CTC counts on therapy with prognosis and response to therapy. Outside of absolute number of CTC there is little published data regarding prognostic significance of CTC morphologic heterogeneity in late stage NSCLC and its ability to predict treatment outcomes. Here we investigate CTC counts in 81 patients with stage IV NSCLC using a fluid phase biopsy and high definition (HD) diagnostic pathology imaging of all nucleated cells. HD-CTCs were detected in 51 (63%) patients at initiation of therapy with a median of 2.20 (range 0-509.20) and a mean of 26.21 HD-CTCs/mL (±15.64). There was no correlation between the absolute number of HD-CTCs at the time of initiation of new therapy and patient outcomes. A subset of 25 patients was further analyzed to determine the significance of HD-CTC kinetics, which may follow three distinct patterns: an increase in HD-CTCs with therapy, unchanged HD-CTCs numbers (stable), and a decline in HD-CTCs numbers. Patients that experienced an increasing, stable, or decreasing HD-CTC profile had an overall change of 118.40, 0.54, and 81.40 HD-CTCs/mL respectively during the first 3 months. The overall survival (OS) for increasing profiles was 31.08 months (±13.08, median 32.46, range 15.11-43.50), for stable profiles OS was 8.82 months (±3.57, median 7.23, range 3.91-19.75), and for decreasing profiles it was 15.30 months (±6.39, median 14.06, range 3.81-28.06) months. CTCs are identifiable in patients with stage IV NSCLC, but correlation of absolute HD-CTC counts with disease progression was not statistically significant. However, change in CTC counts were predictive of OS in patients with metastatic lung cancer receiving chemotherapy. Citation Format: Stephanie N. Shishido, Lyudmila Bazhenova, Anders Carlsson, James Hicks, Peter Kuhn. Detection of circulating tumor cells in stage IV non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1712. doi:10.1158/1538-7445.AM2017-1712

Abstract 3929: Heterogeneity in circulating tumor cells in blood samples of metastatic castration-resistant prostate cancer patient: comparison of isolation technique

Abstract Introduction/ObjectivesMetastatic castration-resistant prostate cancer (mCRPC) is a heterogen disease. Since most prostate cancer patients have a biopsy performed only at the time of diagnosis, representative tumor tissue sample giving real time information about the disease status is generally missing. CTC enumeration is a biomarker associated with clinical outcomes in patients with mCRPC. However, capturing these rare cells from whole blood is still a major challenge because they are extreme rare. Futhermore CTCs may exhibit a feature of epithelial-mesenchymal transition. First we assessed the method is best suited for the isolation of CTCs in mCRPC patients. Additionally was to evaluate the association between CTCs counts and ERG rearrangement in tumor samples. Materials &amp; Methods A tumor sample from metastatic site, along with blood samples were collected from mCRPC patients. Tumor samples were characterized of ERG rearrangement by fluorescence in situ hybridization. CTCs were detected using two methods. One was SreenCells system, CTC isolation based on size exclusion. The second method was the CellCollector which CTC captured using monoclonal specific antibody to EpCAM. This system can be used for CTC isolation ex vivo and in vivo. We used it here ex vivo. Captured CTCs were identified based on histological cell architecture by immunofluorescence staining using cytokeratin (8, 18, 19) and nuclear Hoechst staining positive as well as CD45 negative as criteria to exclude EpCam positive leucocytes Results We obtained duplicate blood samples from 10 mCRPC patient using SreenCells system and CellCollector isolation methods. Bothe methods detected CTCs in 8 of 10 blood samples, detection rate is 80 %. ScreenCells isolated in average of 22.6 CTCs (range of 0-80 CTCs). The CellCollector captured CTCs ex vivo in average of 3 CTCs (range of 0-6 CTCs). In a direct comparison of the ScreenCell system and the Cellcollector is significant different (p 0.0156) in the CTC counts. The CTC number of both systems is moderate correlated (r 0.5). All mCRPC tissue samples harbored an ERG rearrangement in range of 89 until 98 %. The CTC number of the ScreenCell system is good correlate with PSA level (r 0.776) and also with the ERG- rearrangement (r 0.5) of tissue samples. Conclusion Our results demonstrate that CTC isolation based on the physical properties has a higher sensitivity as the CTC isolation technique based on the biological cell properties in mCRPC patients. The size exclusion technique coupled with characterization of specific staining morphologies might be used to identify a heterogeneous CTC population which is always present in advanced cancer. It should be decided in dependence of the advanced cancer stage which CTC isolation technique can be used. The cost of the material and the hours of work are equal in both systems. Citation Format: Gerit Theil, Christine Weiss, Kersten Fischer, Andre Schumann, Paoloa Fornara. Heterogeneity in circulating tumor cells in blood samples of metastatic castration-resistant prostate cancer patient: comparison of isolation technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2017-3929

Abstract 798: Label-free enrichment and detection of circulating tumor cells in metastatic breast cancer patients show 82% of cohorts have detectable targets

Abstract Circulating tumor cells (CTC) are believed to be the culprit of metastasis and studies have shown that their enumeration has prognostic value in wide range of solid tumors. Although much progress has been made in CTC technology, their rarity in blood and their inherent heterogeneity still provide much challenge towards maturity of the technology. This paper presents semi-automated enrichment of CTC via density-based approach in a novel microfluidic disk, followed by multi-step on-disk immunofluorescence staining (pan-CK, EpCAM, Hoechst and CD45), fluorescence microscopy for image capture, and software image analysis. To characterize the performance of the system, we spiked 1 to 300 cells (mean 105, median 125) from six cell lines (DLD-1, Huh-7, MCF7, PC3, MDA-MB-231 and PC-9) from five cancers into whole blood from healthy donors. The recovery rate of the system is 87.4%±3.7% (R2=0.958) regardless of EpCAM expression levels of the cell lines. To interrogate the limitation of the technology, this data set included ultra-low cell counts spiked of 1 to 13 cells (mean 5.7, median 5) which might be indicative of CTC from metastatic breast cancer (MBC) patients. The recovery rate for this low cell count is 90%±10%. Notably, one single cell was spiked into healthy whole blood, processed via the technology, and one target cell was detected. This test was repeated in triplicate with all three tests recovered one cell each. Furthermore, the microfluidic disk technology enables operation over a range of blood volume (from 2 to 7.5ml) with no statistically significant difference in recovery rate. Recovered MCF-7 cells spiked in blood were subsequently cultured for 6 days and showed good viability with cell proliferation. We tested the technology in MBC patients along with CA15-3 and CT imaging. A total 34 of blood samples were collected from 21 patients over the course of their systemic treatment. Results showed CTC were detected in 28 samples (82%). The target CTC detected ranges from 0 to 138 (mean 16, median 4 CTCs per 7.5mL). 15 out of 34 samples (44%) had CTC number ≥ 5/7.5mL). In one patient with triple negative diagnosis, CTC count, CA15-3 and CT imaging were monitored during the course of chemotherapy. The CTC count remained zero at the end of the first and second treatment course, elevated to 24 CTCs at the end of the third course, and continued its elevation to 31 by the end of the fourth treatment course. During this entire treatment course, CA15-3 did not vary significantly. However, CT image confirmed the metastasized liver tumor grew from 6.15cm at the beginning of the second chemo course to 8.09cm at the beginning of the fourth course. Taken together, our label-free CTC enrichment technology has high analytical sensitivity (87%) over a wide range of cancers, able to handle a flexible blood volume (2mL to 7.5mL), and amenable to detect a high percentage (82%) of CTC in MBC patients. Citation Format: Yu-Jen Chang, Chen-Lin Chen, Wei-Fan Hsu, Meng-Ze Li, Wei-Yuan Ma, Ken-Chao Chen, Guan-Syuan Huang, Wai-Sang Wong, Jhan-Yu Syu, Thomas, Yo-Yan Huang, Ching-Hung Lin, Andrew M. Wo, Chiun-Sheng Huang. Label-free enrichment and detection of circulating tumor cells in metastatic breast cancer patients show 82% of cohorts have detectable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 798. doi:10.1158/1538-7445.AM2017-798

Circulating tumour cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer.

5049 Background: A heterogeneous landscape of patients with metastatic castration-resistant prostate cancer (mCRPC) exists in current clinical practice. We investigated the prognostic value of CTC enumeration and dynamics, in the context of second-line endocrine therapies (i.e. abiraterone or enzalutamide). Methods: In a prospective, multicentre study blood samples were collected from patients with mCRPC at baseline (n = 147) and follow-up (n = 95/147(64.6%)). At baseline, patients were stratified in favourable ( &lt; 5 CTCs/7.5mL) and unfavourable (≥5 CTCs/7.5mL) groups, whereas at follow-up, in those demonstrating a stable, in- or decreasing CTC count. PFS and OS were compared between groups. PSA changes at 10-12 weeks were evaluable in 83 patients. Results: Patients with ≥5 CTCs/7.5 mL (n = 59) at baseline had a shorter PFS (3.9 vs. 11.3 months, p&lt; 0.0001) and OS (9.34 months vs. not reached, p&lt; 0.0001). Patients demonstrating increasing CTCs (n = 21) on therapy had a shorter PFS (4.03 vs. 10.36 vs. 13.08 months, p &lt; 0.0001) and OS (11.2 months vs. not reached, p &lt; 0.0001), compared to patients with decreasing (n = 41) and stable (n = 33) CTCs, respectively. Multivariate Cox regression showed that the number of CTCs (HR (95%CI): 1.0054 (1.0006–1.010), p= 0.0260) and an increasing follow-up CTC count (HR (95%CI): 2.8987 (1.2856–6.536), p= 0.0103) were independent predictors of PFS. CTC increase was the sole independent predictor for OS (HR (95%CI): 7.3512 (1.7953–30.101), p= 0.0055). At 10-12 weeks, a PSA response of ≥30% and ≥50% was achieved in 46/83 (55.4%) and 33/83 (39.8%) patients, respectively, which was statistically different between chemo-naive or -pretreated patients (≥30%: p= 0.0395), patients with increasing, stable or decreasing CTC counts (≥30%: p= 0.0019; ≥50%: p= 0.0032), and patients with increasing or stable/decreasing CTC counts (≥30%: p= 0.0006; ≥50%: p= 0.0014). Conclusions: CTC levels are associated with PFS and OS in mCRPC patients, starting a new line of endocrine therapy. Follow-up CTC enumeration is associated with PSA response and its dynamics is an independent predictor of PFS and OS, thereby demonstrating the pharmacodynamic properties of CTCs.

Changes in CTC burden and prevalence of specific CTC subtypes in mCRPC patients (pts) receiving alpharadin (Ra-223) as single agent or in combination with other therapuetics (Tx).

5076 Background: Ra-223 prolongs life in mCRPC pts with symptomatic osseous metastasis with inconsistent effects on PSA. Survival times are prolonged further when combined with Abi/Enza. Data from preclinical studies suggest that Ra-223 may sensitize tumors to DDR agents and/or biologic therapies. But predictive biomarkers of benefit to each or both combinationso are lacking. We studied CTC counts and the prevelance of specific CTC subtypes in patients before and following Ra-223 therapy, both as a single agent and in combination, to identify biomarkers of sensitivity and treatement efficacy, and effects of Ra-223 on tumor biology. Methods: Pre and ~4 week post RA-223 therapy blood samples were collected from 35 pts (2 samples each) given as a single agent (n = 20 pts) or in combination with other therapies (n = 15 pts, 9 w/ Enza, 5 w/ Abi, 1 w/ Taxane). Samples were processed and CTCs analyzed using the Epic Sciences platform. Total CTC count and the prevalence of specific CTC phenotypes present pre and post Rx were identified utilizing high content digital pathology and associated with therapy type and post-treatment change. Results: CTC declines were observed in 55% (11/20) and 60% (9/15) of pts treated with single agent and combination respectively. In Ra-223 alone pts, a novel CTC subtype (high N/C ratio, high nuclear area) was identified at baseline 11/20 samples (med = 33% of CTCs). Which was no longer detected in 10 (90%) of the pts treated. This contrasts with a second novel CTC subtype present at baseline in 4 pts (med CTC = 9%) that increased to 9 cases (med CTC = 18%) at follow-up. Conclusions: A subset of pts demonstrate post-therapy CTC declines following Ra-223 alone or in combination. A novel CTC subtype resolved by RA-223 in conjunction with total CTC kinetics may indicate pt benefit from Ra-223. A novel emergent CTC subtype has also been identified in pts already receiving Ra-223. Single CTC sequencing and protein analysys of these CTC subtypes are ongoing, and may help describe tumor evolution and sensitization to novel therapuetics.

Prognostic role of circulating tumor cells-CTCs in metastatic renal cell carcinoma.

4568 Background: CTCs can be isolated in peripheral blood of cancer pts and have demonstrated to have prognostic role in several metastatic tumors such as breast, colorectal and prostate cancer. Few data are available for Renal Cell Carcinoma-RCC. Methods: We designed a multicenter prospective observational trial aiming to assess the association between CTC counts and PFS of RCC pts treated with an antiangiogenic tyrosine-kinase inhibitors as a first-line regimen for metastatic disease. OS and response rate were secondary objectives. Both basal and sequential counts were enumerated by Cellsearch system at 4 time points: day 0 of treatment, +1 mo, +3 mo, at progression or 12 mo in the absence of progression. Ethics Committee approval was obtained. Results: Among 246 pts, 195 are eligible for the present analysis, 71.4% males, median age 69 yrs (range, 27 to 91), 81% with previous partial/total nephrectomy. Treatment was sunitinib (77.5%), pazopanib (21%) or sorafenib (1.5%). According to Heng criteria there were 24.6% good, 62.6% intermediate and 24.6% poor prognosis pts. After a median follow-up of 31.5 mo, median PFS is 13.6 mo (23% censored), 49.2% of pts are still alive. Investigator-assessed best response was 3.8% complete, 37.3% partial response, 33% stable, 25.9% progression. At baseline 91 pts had 1 or more CTCs, median 2, range 1 to 263. Pts with at least 1 CTC had a significantly shorter PFS compared to negative pts (8.8 vs 16.6 mo, p = 0.03), HR = 1.41 (95%CI 1.02-1.9). Thirty pts had &gt; = 3 CTCs, with a median PFS of 5.8 vs 15 mo in the remaining pts (p = 0.002), HR = 1.99 (CI 1.28-3.03). Percentage of pts with &gt; = 3 CTCs increased from 6.6% of good, 18.4% intermediate and 38.9% poor Heng score pts (p = 0.042). Pts with &gt; = 3 CTCs had a shorter estimated OS of 13.8 mo vs 52.8 mo (p = 0.003), HR = 1.99 (CI 1,17-3.2). Correlation between CTC positivity and response rate was not significant. Conclusions: In this robust multicenter prospective cohort of first-line metastatic RCC pts, the presence of 3 or more CTCs predicts a significantly shorter PFS and OS. Further analyses are ongoing on apoptotic markers of CTCs and concomitant counts of endothelial cells collected in the same cohort.

Phase I dose-escalation study of fractionated-dose 177Lu-PSMA-617 for progressive metastatic castration resistant prostate cancer (mCRPC).

TPS5093 Background: PC is a radiosensitive disease. PSMA is selectively overexpressed in advanced PC with upregulation by androgen receptor (AR) pathway dysregulation; limited expression exists in other organs. A series of sequential studies of radiolabeled anti-PSMA antibody J591 revealed 1) targeting and safety [Bander 2003]; 2) safety and prelim efficacy [Milowsky 2004, Bander 2005]; 3) efficacy and initial dose-response [Tagawa 2013]; 4) dose-fractionation allows higher doses, ability to combine with docetaxel, confirmation of dose-response (PSA and overall survival) [ASCO 2010, 2014, 2016]; 5) predictable, reversible myelosuppression is dose-limiting [Tagawa 2013]. Small molecule PSMA inhibitor ligands can be successfully radiolabeled and are widely used for imaging and treatment in Europe. 177Lu-PSMA-617 is the most commonly used, but experience is mostly anecdotal/retrospective and no formal dose-escalation studies have been performed. Methods: Men with progressive mCRPC following at least 1 potent AR-targeted agent (e.g. abi/enza) and docetaxel (or unfit/refuse chemo) without limit of # prior therapies provided adequate organ function will undergo imaging with 68Ga-PSMA-HBED-CC PET/CT followed by escalating fractionated doses of 177Lu-PSMA-617. Cohort 1 = 3.7 GBq x2 two weeks apart up to 11.1 GBq x2 in a 3+3 dose-escalation study. Dose-limiting toxicity (DLT) is defined as attributable grade 4 heme toxicity or grade 3/4 non-heme toxicity. Planned cohort expansion will occur at recommended phase 2 dose (RP2D) in a 2-stage design. The primary endpoint is determination of DLT and RP2D. Secondary endpoints include toxicity, PSA decline rate, RECIST response, PFS, rPFS, OS. Correlatives include baseline/follow up PSMA imaging, whole body distribution of 177Lu-PSMA-617, CTC count (CellSearch) changes, tissue and circulating genomic assessment of DNA repair pathways, patient reported outcomes (FACT-P and BPI-SF). Clinical trial information: NCT03042468.

Circulating tumor cell subsets and macrophage polarization to predict efficacy of cabozantinib in advanced prostate cancer with visceral metastases.

5031 Background: The presence of VM in metastatic, castration-resistant prostate cancer (mCRPC) predicts poor survival. Cabozantinib (cabo) is a multi-kinase inhibitor that has clinical activity that did not improve survival in an unselected mCRPC population. Subgroup analyses suggested that the benefit may exist for patients (pts) with mCRPC-VM. The effect of cabo includes the tumor microenvironment, monocytes in particular, which in turn can alter tumor behavior. Methods: We conducted a single-arm study of cabo in men with mCRPC-VM. Pts received cabo 60 mg daily. Radiographs were used to assess response. Correlative blood samples were collected for the enumeration and characterization of circulating tumor cells using the NanoVelcro Assay and analysis of circulating monocytes by FACS. Results: A total of 17 pts enrolled with 16 evaluable for response. At 12 weeks, 19% experienced partial responses (PR), 44% stable disease (SD), and 38% progressive disease. The clinical benefit rate (PR+SD) at 12 weeks was 63%. Safety profile was consistent with previous reports. CTCs were detected in 80% of pts. NanoVelcro CTC counts showed reduction by week 8 in both PR+SD (88%) and PD (71%) groups with re-emergence at progression. Among pts with liver metastases, very-small-nuclear CTCs ( &lt; 8.5 μm) were seen in 29% of pts with clinical benefit compared to 60% in non-benefiters. Analysis of monocyte polarization after initiation of therapy showed that reduction of M1 polarization was associated with improvement in bone pain and/or bone scan. Conclusions: In heavily-pretreated mCRPC-VM, cabo provided clinical benefit with acceptable toxicity. Circulating biomarkers related to both tumor and microenvironment may be useful in identifying patients who benefit from this type of therapeutic approach. Clinical trial information: NCT01834651.

Circulating tumor cells and genomic profiling of patients with ERBB2 mutant, HER2 non-amplified metastatic breast cancer treated with neratinib.

e12554 Background: ERBB2 mutations in the absence of gene amplification are rare, with an incidence of 2-4%. Neratinib is a HER2/EGFR tyrosine kinase inhibitor being evaluated for use in ERBB2 mutated breast cancer. Neratinib has been found to have clinical activity on heavily pre-treated ERBB2 mutant breast cancer patients. We are evaluating the response and genomic profiles of 3 postmenopausal patients with metastatic ERBB2 mutant/non-amplified breast cancer receiving neratinib and fulvestrant NCT01953926, NCT01670877. Methods: Samples were collected at different points during treatment and CTCs were identified. Other representative cells were tracked but not classified as CTCs. CD45-CK+ cells with cytoplasmic and/or nuclear apoptosis were defined as CTC-Apoptotic. CD45- cells expressing little to no CK but otherwise meeting morphological criteria for CTCs are CTC-LowCK. CD45-CK+ cells with small nuclear size are CTC-SmallCK. Single CTCs will be analyzed using whole genome copy number variation to determine chromosomal alterations. Results: The patients had an average of 4.7 lines of therapy for metastatic disease before neratinib. Two had stable disease and one had progression. The patient who progressed had a rise in the number of CTCs from 37 to 52 cells/ml and drop in apoptotic cells from 5 to 0 cells/ml. Conclusions: The high CTC count of the patient who progressed may suggest more aggressive disease. The drop in the apoptotic cell count may correlate with a failure to respond to therapy. Samples have been sent for copy number variation profiling. The goal is to identify any genomic amplifications or deletions associated with clinical response and progression after targeted therapy. We hope to demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the use of fluid biopsies to monitor disease progression. [Table: see text]

A New Size-based Platform for Circulating Tumor Cell Detection in Colorectal Cancer Patients

BackgroundCirculating tumor cells (CTCs) might play a significant role in cancer progression and metastasis. However, the ability to detect CTCs is limited, especially in cells undergoing epithelial-mesenchymal transition. In this study, we evaluated a new size-based CTC detection platform and its clinical efficacy in colorectal cancer. Patients and MethodsBlood samples were obtained from 76 patients with colorectal cancer and 20 healthy control subjects for CTC analysis. CTCs were enriched using a high-density microporous chip filter and were detected using a 4-color staining protocol including 4′,6-diamidino-2-phenylindole (DAPI) for nucleated cells, CD45 monoclonal antibody (mAb) as a leukocyte marker, and epithelial cell adhesion molecule (EpCAM) mAb or cytokeratin (CK) mAb as an epithelial cell marker. CTC positivity was defined as DAPI-positive (DAPI+)/CD45−/EpCAM+ or CK+ cells and clinical outcomes of patients were analyzed according to CTC counts. ResultsCTCs were detected in 50 patients using this size-based filtration platform. CTC+ patients were more frequently identified with a high level of carcinoembryonic antigen and advanced stage cancer (P = .038 and P = .017, respectively). CTC counts for patients with stage IV cancer (12.47 ± 24.00) were significantly higher than those for patients with cancers that were stage I to III (2.84 ± 5.29; P = .005) and healthy control subjects (0.25 ± 0.55; P < .001). In addition, progression-free survival tended to be lower in CTC+ patients compared with CTC− patients (P = .092). In patients with stage I to III cancer, recurrence occurred only in CTC+ patients. ConclusionCTC positivity was found to correlate with clinical features of colorectal cancer patients. Our results suggest that this new size-based platform has potential for determining prognosis and therapeutic response in colorectal cancer patients.

55P CTC count &amp; gene expression profiling among Filipino NSCLC

55P CTC count &amp; gene expression profiling among Filipino NSCLC