Elevated Circulating Tumor Cells Research Articles

Abstract CT306: Radiolabeled anti-PSMA antibody J591 immunotherapy is associated with favorable circulating tumor cell (CTC) count control in men with castration-resistant prostate cancer

Abstract Background: J591 is a monoclonal antibody that selectively binds to prostate-specific membrane antigen (PSMA). Prospective phase I and II trials with therapeutic radiolabeled 177Lu-J591 have shown favorable PSA decline rates. Additionally, our group has previously reported that non-invasive evaluation of PSMA expression by imaging with planar gamma camera imaging is associated with response and survival. Initial anecdotal experience showed declines in CTC counts which led to prospective study. Methods: Three clinical trials of 177Lu-J591 in men with metastatic CRPC have included prospective evaluation of CTC counts (CellSearch methodology) prior to and following therapy (single-dose 177Lu-J591, fractionated-dose 177Lu-J591, and fractionated-dose 177Lu-J591 + docetaxel). Based upon prior experience of J591-based therapy without an effector molecule as immunotherapy, and an anecdotal report of CTC count decline following a low (imaging) dose of J591 without an effector molecule, a cohort of patients who received J591 without 177Lu labeling was also retrospectively analyzed. Results: 44 pts unselected for PSMA expression received 177Lu-J591 therapy had prospectively measured CTC counts. 39 of 44 pts (88.6%) had imaging evidence of PSMA expression. 57% of pts had baseline unfavorable (≥ 5 cells/7.5 mL) CTC counts. Of overall evaluable pts, 72.4% had decline in CTC counts (7.5 to 100% decline), with 50% of those with unfavorable baseline counts converting to favorable counts at 4-6 weeks. 17.1% with undetectable CTC counts remained undetectable at follow up. 7 pts with mCRPC and elevated CTC counts who received low-dose (naked J591) prior to imaging were also identified, and 4 (57%) of these pts demonstrated CTC count decline. Conclusion: In addition to previously reported PSA declines, treatment with 177Lu-J591 is associated with favorable CTC count decline in men with metastatic CRPC. Infusion with unlabeled J591 may also lead to CTC count clearance. Based on this finding, we are planning a prospective clinical trial with unlabeled (naked) J591 in men with metastatic CRPC and unfavorable CTC counts. Citation Format: Pravin Date, Beerinder S. Karir, Jaspreet S. Batra, Yuliya Jhanwar, Himisha Beltran, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Radiolabeled anti-PSMA antibody J591 immunotherapy is associated with favorable circulating tumor cell (CTC) count control in men with castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT306. doi:10.1158/1538-7445.AM2015-CT306

Abstract P4-01-19: The combined detection of CTC and serum HER2 ECD predict PFS for HER2-positive advanced breast cancer patients

Abstract Background: Circulating tumor cell (CTC) and serum HER2 ECD can all reflect an aggressive tumor behavior. We performed this prospective, monocenter, double-blinded study to investigate the potential clinical significance of combined detection of CTC and serum HER2 ECD for advanced breast cancer patients with histological HER2-positivity. Methods: A total of 88 eligible patients were enrolled in the present study from April 2012 to October 2013. We used Cell search system and ADVIA Centaur System to detect CTC and serum HER2 ECD respectively. Patients received systemic treatment according to national and international guidelines. Results: Twenty nine (33%) patients had ≥5 CTC, seventy three (83%) patients had serum HER2 ECD values of at least 15ng/ml, twenty seven (30.7%) patients had both elevated CTC and ECD values and fourteen (15.9%) patients had both normal CTC and ECD values. Patients with both normal CTC and serum HER2 ECD values exhibited a significantly longer median PFS than patients with both elevated values (9.0 months versus 2.8 months, p=0.023) and exhibited a trend toward longer PFS compared with patients with elevated CTC or ECD values (9.0 months versus 4.2 months, p=0.065), patients with both or one elevated values showed similar median PFS (2.8 months versus 4.2 months, p=0.211) (Figure1). Conclusions: The combined detection of CTC and serum HER2 ECD showed prognostic significance for HER-2 positive advanced breast cancer patients, patients with both normal values exhibited longer median PFS than others. Citation Format: Zefei Jiang, Jinmei Zhou, Tao Wang, Yi Liu, Lei Li, Huiqiang Zhang, Shaohua Zhang, Li Bian, Santai Song. The combined detection of CTC and serum HER2 ECD predict PFS for HER2-positive advanced breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-19.

Reply to F.-C. Bidard et al

We thank Bidard and Pierga for their thoughtful comments regarding SWOG S0500. We agree that the results of S0500 demonstrate that patients with elevated circulating tumor cells (CTCs) who do not have a CTC response after one cycle of cytotoxic therapy likely have tumors that exhibit general resistance to cytotoxic mechanisms. Unfortunately, this observation meant that our hypothesis—that switching to an alternate cytotoxic early would improve outcomes— was incorrect. However, the results do demonstrate that CTC levels after one cycle of cytotoxic therapy provide statistically significant and potentially clinically useful prognostic information. We believe this knowledge could be used to encourage patients who fail to decrease their CTC below the threshold to participate in trials of novel (noncytotoxic) and perhaps genomically selected, targeted therapies Bidard and Pierga suggest that S0500 would have been better performed in a non–first-line setting by enriching for a population with a higher level of chemoresistance. This concept is not fully applicable to the hypothesis behind the S0500 design. It had been assumed that, by switching early in the metastatic disease course, the different chemotherapy regimens would not be cross-resistant. Therefore, benefit from switching to an alternative class of cytotoxic therapy early, based on CTC level, would most likely be seen in the first-line setting before generalized resistance to all cytotoxic mechanisms could occur. In addition, secondary aims of the study were to confirm the good prognostic value of both low CTCs at both baseline and after one cycle of therapy. These end points were felt to be most appropriately tested in the first-line setting. Bidard and Pierga also question whether the CTC threshold of 5 CTC/7.5 mL was optimal for this study population. We believe that lowering the threshold would have risked confounding of the trial by the analytic variability of the assay ( 2-4 CTC/7.5 mL whole blood), making assessment of true CTC response more difficult. We completely support the ongoing clinical trials that continue to address the clinical utility of CTC in both the metastatic and adjuvant settings.

Circulating tumor cells in patients with metastatic castration resistant prostate cancer: exploratory findings at a tertiary referral hospital.

ObjectivesIn patients with metastatic castration-resistant prostate cancer (mCRPC), the finding of less than five circulating tumor cells (CTCs)/7.5 mL blood before start of cytotoxic treatment or shortly thereafter indicates prolonged survival. In this descriptive pilot study, we investigated whether this association depends on the sequence of the therapeutic attempts.Patients and methodsCTCs were determined in 41 mCRPC patients before and 2–3 months after starting first-line treatment with docetaxel (group 1) or second-line treatment with either radium-223 (group 2) or placebo/best supportive care (group 3). A “favorable” CTC count was defined as <5 CTC/7.5 mL blood. The results were related to overall survival.ResultsPretreatment, six of ten men in group 1, three of 19 in group 2, and three of 12 patients in group 3 had a favorable CTC count, leading to a significant difference between first- and second-line therapy (P=0.04). Decrease of pretreatment elevated CTCs to a favorable CTC count was significantly more often observed in patients on first-line therapy (three of four patients) than on second-line treatment (two of 26 men) (P=0.03). A favorable CTC count before or shortly after treatment start was observed in nine of ten patients on first-line and in eight of 31 men on second-line therapy (P=0.01). A favorable CTC count pretreatment or 2–3 months after therapy start was associated with beneficial overall survival in the three groups combined and in each group analyzed separately.ConclusionIn mCRPC, a favorable CTC count before or 2–3 months after start of therapy is associated with length of overall survival, though such favorable CTC counts are observed significantly less often in patients on second- than on first-line therapy.

Circulating tumor cell (CTC) enumeration in patients with metastatic neuroendocrine prostate cancer (NEPC) and castration-resistant prostate cancer (CRPC).

204 Background: Neuroendocrine prostate cancer (NEPC) is an aggressive variant of prostate cancer. Circulating tumor cell (CTC) counts as measured by CellSearch are prognostic for large groups with metastatic prostate cancer (PC), but are not well described in NEPC. Methods: With institutional review board approval, we retrospectively identified patients with metastatic PC and available CTC enumeration (CellSearch methodology) and compared counts/7.5 mL blood and overall survival (OS), measured from the first recorded CTC count until death or last follow up. Entry criteria for clinical trials were used to define NEPC, including histology (small cell/neuroendocrine carcinoma or adenocarcinoma with more than 50% NE staining), serum chromogranin greater than 5x ULN and/or neuron specific enolase greater than 2x ULN, and/or predominant liver/brain metastases with lack of prostate-specific antigen [Beltran ASCO 2013, clinicaltrials.gov NCT01799278 ]. Frequency of detectable and unfavorable counts was tabulated and OS was compared across groups. Results: Sixty one patients were identified: 21 NEPC with median age 73.7 and 40 patients with castration-resistant prostate cancer (CRPC) with median age 73.9 over contemporary time periods 2009 to 2012. Median OS for the entire group was 22.6 months (mo), with a trend for shorter OS in NEPC (20.7 mo) than CRPC (22.7 mo), p=0.11. 47.6% of NEPC and 55% of CRPC had detectable CTC counts (p=0.58); 38.1% of NEPC and 40.0% of CRPC had greater than or equal to five CTCs (p=0.89). CTC counts of 0 to 4 versus greater than or equal to five were prognostic for both groups: NEPC with 0 to 4 CTCs had median OS of 22.6 versus 6.6 mo for CTCs greater than or equal to 5 (p&lt;0.001) and CRPC with 0 to 4 CTCs median OS not reached (mean 40.6 mo) versus 11.2 mo for those with greater than or equal to five CTCs (p&lt;0.001). Conclusions: Patients with NEPC have similar frequency of detectable and elevated CTC counts by CellSearch methodology as compared to an overall CRPC population. CTC counts are prognostic for both groups.

Abstract S5-07: SWOG S0500 – A randomized phase III trial to test the strategy of changing therapy versus maintaining therapy for metastatic breast cancer patients who have elevated circulating tumor cell (CTC) levels at first follow-up assessment

Abstract Introduction: CTC are detectable in approximately 75% of patients with metastatic breast cancer (MBC), and are elevated (≥5CTC/7.5 ml whole blood (WB)) in about half. Elevated CTC at baseline are associated with a worse prognosis, and a decline in CTC levels suggests response to therapy. We initiated a prospective randomized clinical trial (SWOG S0500) to test whether a change in chemotherapy after failure to clear CTC after one cycle of first line chemotherapy would improve outcomes of patients starting first line cytotoxic chemotherapy for MBC. Methods: Patients initiating first-line chemotherapy for MBC were enrolled. All patients had measurable or evaluable disease that included bone metastases. Patients with elevated CTC at baseline and who continued to have elevated CTC after 21 days of therapy were randomly assigned to either continue initial therapy until progression or to change to a second line chemotherapy (physician choice) immediately at cycle 2. Patients with elevated CTC at baseline, which were not elevated at the 21-day follow-up were maintained on their initial therapy. Patients without elevated CTC at baseline were followed in an observation arm. The primary endpoint was overall survival, and progression-free survival was a secondary endpoint. It was expected that approximately 500-650 patients would have to be screened to enroll 120 patients in the randomized trial. Power was 81% with 2-sided α = 0.05 to detect a 70% increase in median overall survival for patients randomized to change therapy. Three interim analyses were planned during the course of the trial. Results: From 10/1/2006 until 3/15/2012, 624 patients were registered of whom 612 were eligible. A baseline CTC was obtained in 593 (97%) eligible patients of whom 317 (53%) had elevated CTC at baseline. Thirty-one patients (10%) with elevated baseline CTC did not complete the follow-up CTC due to death, progression, withdrawal, or assay failure. Of the 286 remaining patients, 123 patients (43%) continued to have elevated CTCs after the first cycle of chemotherapy and were randomly assigned to either maintain original chemotherapy (n = 64) or switched to new chemotherapy (n = 59). Final outcome results will be analyzed in October 2013 and will be reported if released by the SWOG Data Safety Monitoring Board. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S5-07.

Abstract P1-04-01: Significance of circulating tumor cells in metastatic triple negative breast cancer: Results of an open label, randomized, phase II trial of nanoparticle albumin-bound paclitaxel with or without the anti-death receptor 5 tigatuzumab (TBCRC 019)

Abstract Background: Circulating Tumor cells (CTCs) are prognostic at baseline and first follow-up in patients with metastatic breast cancer (MBC). Using the most commonly used assay (CellSearch®), we have previously reported the ability to detect apoptotic vs. non-apoptotic CTCs in patients with MBC. However, there has been concern regarding the performance of the CellSearch® assay in patients with triple negative (TN) MBC. We hypothesized that CellSearch® is an effective assay in patients with TN MBC, and that CTC-apoptosis might further separate prognosis. Therefore, we studied CTCs in patients with TN MBC who participated in a prospective randomized phase II trial testing for activity of tigatuzumab (TIG) in combination with nanoparticle albumin-bound paclitaxel (nab-PAC) conducted by the Translational Breast Cancer Research Consortium (overall results reported by Forero A., et al, ASCO 2013). Methods: Whole blood (WB) was drawn into a CellSave tube at baseline, day 15, and day 29 and CTC counts were determined using the CXC CellSearch® kit. Apoptosis was characterized by staining with a monoclonal antibody that detects a neo-epitope on fragmented cytokeratin (M-30) and independently by visual inspection (nucleic condensation and/or fragmentation, as well as granular cytokeratin). Association between levels of CTCs and CTC-apoptosis with the overall response rate (ORR) and progression free survival (PFS) at baseline, day 15, and day 29 was assessed using logistic regression, Kaplan Meier curves, and Cox proportional hazards modeling. Results: Of the 60 patients entered into the trial, 52 were evaluable for CTCs. Of these, 19/52 (36.5%), 14/52 (26.9%), and 13/49 (26.5%) had elevated CTCs (≥5CTC/7.5 ml WB) at baseline, day 15, and day 29, respectively. Patients with elevated CTCs at each time point had worse PFS than patients with low or no CTCs. Hazard rates (HR) at baseline, day 15, and day 29 were 2.38 (95% CI: 1.27-4.45, p = 0.007), 2.87 (95% CI: 1.46-5.66, p = 0.002), and 3.40 (95% CI: 1.68-6.89, p = 0.001), respectively. The odds of overall response for those who had elevated CTCs compared to those who did not at baseline, day 15, and day 29, were 0.25 (95% CI: 0.073-0.81, p = 0.024), 0.18 (95% CI: 0.04-0.67, p = 0.014), and 0.06 (95% CI: 0.01-0.28, p = 0.001), respectively. There was no apparent prognostic effect comparing the degree of CTC-apoptosis vs. non-apoptosis. Conclusions: Similar to observations in other intrinsic subgroups, CTCs were detected in a large fraction of TN MBC patients at baseline using CellSearch® assay, and reductions in CTC levels reflected response. In these homogenously prospectively enrolled TN MBC patients, regardless of treatment, CTCs are prognostic at baseline, day 15, and day 29. It does not appear that analysis of CTC-apoptosis is prognostic. Supported by Susan G. Komen for the Cure, Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP), Triple Negative Breast Cancer Foundation, The AVON Foundation, and The Breast Cancer Research Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-01.

Abstract OT1-3-01: Characterization of circulating tumor cells from subjects with metastatic breast cancer using the CTC-endocrine therapy index: The COMETI-P2-2012.0 trial

Abstract Introduction: Approximately 50% of patients with estrogen receptor (ER) positive metastatic breast cancer (MBC) benefit from endocrine therapy (ET). This percentage is even lower after first line ET. Other than absence of ER, there are no validated markers that predict lack of benefit from ET. Prior studies suggest that relative levels of four markers might predict lack of benefit from ET: low ER and BCL2 and high HER2, and Ki67. Elevated circulating tumor cell (CTC) levels are prognostic in patients with MBC. We have recently demonstrated the ability to quantify ER, BCL2, HER2 and Ki67 on CTC using the CellSearch® system, and we have developed an analytically validated CTC-Endocrine Therapy Index (CTC-ETI). We are now conducting a prospective, multi-institutional clinical trial to test whether pretreatment CTC-ETI predicts lack of benefit from ET, and whether serial CTC-ETI measurements can be used to monitor patients with ER positive MBC starting 2nd or later line of ET. Objectives: To demonstrate that CTC-ETI can be accurately determined in multiple centers across North America and whether high CTC-ETI is associated with rapid progression (within 3 months of starting ET), and finally to refine the current CTC-ETI algorithm. Secondary objectives: To determine if changes in CTC-ETI during ET are associated with rapid progression, to correlate the status of the biomarkers on baseline CTC with the status of the same biomarkers in primary and/or metastatic tissue collected from the subjects, and to store CTC positive cartridges for future molecular analyses. Methods: Women with ER positive, HER2 negative, progressive MBC after one or more lines of ET or during or within 12 months of completing adjuvant ET who are initiating a new ET are eligible. Subjects with brain metastases only or those who are progressing on fulvestrant are not eligible. All subjects must provide written informed consent. Patients may be treated with any approved ET for MBC, and may also receive everolimus. Patients may not be on any investigational therapy. Blood draws (approximately 40 ml) are collected at baseline, at 1, 2, and 3 months after starting therapy, and at 12 months or progression. CTC-ETI is performed as previously described. Statistical Plan: Enrollment of 200 subjects will provide a sufficient number of evaluable subjects and statistical power for evaluation of the primary and secondary objectives. A total of 120 subjects evaluable for clinical validity analyses will be required to obtain the minimum of 51 subjects with rapid progression for evaluation of the clinical validity of the CTC-ETI. Subjects will be considered evaluable for clinical validity if they have successful calculation of a baseline CTC-ETI and progression status within 3 months of starting ET can be ascertained. Current Status: The first patient was enrolled in May, 2013, and accrual is ongoing. Sponsored by Veridex, LLC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-3-01.

Abstract P2-11-16: Prospective comparison of prognostic factors in patients starting a third line of chemotherapy for metastatic breast cancer: An ancillary study to the CirCe01 trial

Abstract Background: Several prognostic factors and composite scores based on either patients general condition and/or usual blood analyses have been described in the setting of advanced metastatic cancers to estimate patients overall survival (OS). Over the past decade, circulating tumor cells (CTC) have been repeatedly reported as being a strong prognostic tool in metastatic breast cancer patients. We took the opportunity of the observational part of a prospective clinical trial (CirCe01, NCT01349842) that included 3rd line metastatic breast cancer patients to assess and compare several prognostic factors/scores and circulating tumor cell count. Patients and Methods: Metastatic breast cancer patients were included before the start of a 3rd line chemotherapy at the Institut Curie, Paris, France. The following adverse prognostic factors were assessed prospectively and compared to overall survival: altered performance status (PS≥2), lymphopenia (&amp;lt;1,000 lymphocyte/microl), elevated CTC count (CellSearch, ≥5CTC/7.5ml), low albumin (&amp;lt;35g/l), elevated CA 15.3 (&amp;gt;30UI/ml), elevated CEA (&amp;gt;5ng/ml), elevated LDH (&amp;gt;50UI/l). Two composite scores were also evaluated: the Barbot score (combining Karnofsky index, number of metastases, albumin and LDH; Barbot JCO 2008) and the Prognostic Inflammatory and Nutritional Index (PINI; combining albumin, prealbumin, orosomucoid and C-reactive protein). Metastatic sites (liver, lung, bone) were also analyzed. Survival was analyzed by Kaplan-Meier curves; multivariate analysis was done using a Cox model. Results: 56 patients have included prospectively and 36 of them (64%) died. CA 15.3 and CEA levels, lymphopenia and metastatic sites had no significant impact on OS in univariate analysis. The incidence of the significant prognostic markers (%), their correlations (p value) and impact (p value) on overall survival are shown. Correlation and impact on overall survival of prognostic markersFactorIncidenceCorrel. with PS (p value)Correl. with Alb (p value)Correl. with LDH (p value)Correl. with Barbot (p value)Correl. with PINI (p value)Univ. OS (p value)CTC &amp;gt; = 545%NSNS&amp;lt;0.001NSNS0.004PS &amp;gt; = 214%-0.0020.04&amp;lt;0.0010.003&amp;lt;0.001Alb &amp;lt;3516%--NS&amp;lt;0.001&amp;lt;0.001&amp;lt;0.001LDH&amp;gt;50065%---0.05NS0.032Barbot score &amp;gt;316%----0.005&amp;lt;0.001PINI &amp;gt;1013%-----&amp;lt;0.001 In multivariable analysis, the three independent prognostic markers on OS were: elevated CTC count (p = 0.003, HR = 3.4 95% CI [1.5-7.7]), poor PS (p = 0.005, HR = 4.1 95% IC[1.5-11.3]) and low albumin (p&amp;lt;0.0001, HR = 11.3 95% IC[3.9-32.5]). Discussion: CTC are an independent prognostic marker, even in advanced metastatic breast cancer. The disease aggressiveness being captured by the CTC count, usual clinical and biochemical tests appear to be sufficient to evaluate the patient prognosis, whereas more complex score (PINI, Barbot) appear to be of low interest. Financed by: Ligue contre le cancer and PHRC 2009. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-16.

Apoptotic circulating tumor cells (CTCs) in the peripheral blood of metastatic colorectal cancer patients are associated with liver metastasis but not CTCs

Enumeration of circulating tumor cells (CTCs) by the CellSearch system provides prognostic information in metastatic colorectal cancer, regardless of metastatic site. We found that CTCs generally represent <1% of observed events with CellSearch analysis and adapted scoring criteria to classify other peripheral blood events. Examination of twenty two metastatic colorectal cancer patients' blood revealed that patients with high CEA or liver metastases, but not lung or distant lymph node metastases, possessed significant numbers of apoptotic CTCs prior to treatment initiation by Fischer's exact test. Six out of eleven patients with liver metastasis possessed apoptotic CTCs whereas one of nine patients with other metastases had measurable apoptotic CTCs. An elevated CTC number was not necessarily associated with apoptotic CTCs or CTC debris by Spearman's correlation, suggesting the metastatic site rather than CTCs per se as contributing to the origin of these events.

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors.

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: (1) single cells and (2) multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor.

Monitoring apoptosis and Bcl-2 on circulating tumor cells in patients with metastatic breast cancer

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

Detection of circulating tumor cells in different stages of prostate cancer

To explore circulating tumor cell (CTCs) counts in different stages of prostate cancer (PC) in association with tumor burden, metastatic pattern and conventional serum biomarkers. Overall survival (OS) analyses were conducted with respect to optimized CTC cutoff levels. Circulating tumor cell counts were assessed in healthy controls (n = 15) as well as in locally advanced high risk (LAPC, n = 20), metastatic castration resistant (mCRPC, n = 40) and taxane-refractory (mTRPC, n = 15) PC patients. CTCs were detected using the CellSearch System. In metastatic PC (mPC), CTC counts were significantly increased compared to LAPC (p < 0.001). In LAPC, CTCs were at control level (p = 0.66). Patients with both bone and visceral lesions revealed the highest median CTC count (p = 0.004), whereas patients with sole soft tissue metastases displayed CTC counts comparable to controls (p = 0.16). No correlation was observed between CTC counts and osseous tumor burden assessed by bone lesion count (p = 0.54) or bone scan index (p = 0.81). CTC counts revealed a positive correlation with alkaline phosphatase (p < 0.001) and lactate dehydrogenase (p < 0.001) as well as a negative association with hemoglobin (p = 0.004) and PSA-doubling time (p = 0.01). Kaplan-Meier analyses demonstrated a cohort adjusted cutoff level of 3 CTCs with a shorter OS in case of ≥3 CTCs compared to <3 CTCs (p = 0.001), a cutoff level applicable in mCRPC (p = 0.003) but not in mTRPC patients (p = 0.054). Circulating tumor cell counts are applicable as a prognostic molecular marker, especially in mCRPC patients harboring bone metastases with or without visceral metastases. For clinical practice, mPC patients with elevated CTC counts in combination with short PSA-DT, high alkaline phosphatase and lactate dehydrogenase levels as well as low hemoglobin levels are at high risk of disease progression and limited OS.

Circulating tumor cells in melanoma patients.

Circulating tumor cells (CTCs) are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X) from blood of melanoma patients using a simple centrifugation device (OncoQuick), and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF) were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively) compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001). There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001), and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50%) stained for both pan-cytokeratin (KRT) markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14). Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA) may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs). The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids, and their role in metastatic progression.

High independent prognostic and predictive value of circulating tumor cells compared with serum tumor markers in a large prospective trial in first-line chemotherapy for metastatic breast cancer patients

Circulating tumor cells (CTCs) are a prognostic marker in metastatic breast cancer, but comparisons with serum tumor markers (CA 15-3, carcinoembryonic antigen and lactate dehydrogenase) variations are needed. CTCs were counted with CellSearch® at baseline, before cycle 2 (C2) and cycle 3 or 4 (C3/4) in 267 metastatic breast cancer patients on first-line chemotherapy with/without targeted therapy. Baseline CTC detection rate was 65% with ≥1 CTC/7.5 ml threshold and 44% with ≥5 CTC/7.5 ml and was independent of subtypes (luminal, triple negative, human epithelial growth factor receptor 2 (HER2)+). CTCs were associated with tumor markers, bone/liver involvement, tumor burden and performance status. CTC detection ≥1 CTC/7.5 ml was a strong prognostic factor for progression-free survival (PFS), P < 0.0001. Threshold of CTC ≥5 was statistically significant for PFS and overall survival (OS), P = 0.03 on multivariate analysis. Among patients with ≥5 CTC/7.5 ml at baseline, 50% had <5 CTC/7.5 ml at C2. Changes were correlated with both PFS and OS (P < 0.0001). All patients receiving anti-HER2 therapy had <5 CTC/7.5 ml after three cycles of treatment. This is the largest prospective series validating the prognostic value of CTC independently from serum tumor marker. Elevated CTCs before C2 are an early predictive marker of poor PFS and OS, which could be used to monitor treatment benefit. CTC decrease under treatment seems stronger with targeted therapy.

Circulating tumor cells predict progression free survival and overall survival in patients with relapsed/recurrent advanced ovarian cancer

Objective Serial circulating tumor cell (CTC) counts have demonstrated predictive and prognostic value in patients with metastatic breast, colorectal, and prostate cancer. In a phase III study of pegylated liposomal doxorubicin (PLD) with trabectedin vs. PLD for relapsed ovarian cancer, we evaluated the correlation, if any, between numbers of CTCs and progression free survival, (PFS) and overall survival (OS). Methods CTCs were isolated from peripheral blood (10 mL) using the CellSearch system and reagents (Veridex). A CTC is defined as EpCAM+, cytokeratin+, CD45−, and is positive for the nuclear stain DAPI. The normal reference range for CellSearch is < 2 CTC/7.5 mL of blood. Hazard ratios adjusted for known prognostic factors were estimated by Cox regression. Results Two-hundred sixteen patients had baseline CTC measurements of which 111 (51.4%) were randomized to the trabectedin + PLD arm; 143/216 patients (66.2%) were platinum-sensitive. Thirty-one of 216 patients (14.4%) had 2 or more CTCs detected prior to the start of therapy (range 2–566). Univariate Cox regression analyses indicated that patients with ≥ 2 CTCs prior to therapy had 1.89- ( p = 0.003) and 2.06-fold ( p = 0.003) higher risk for progression and death respectively. Multivariate analyses that include baseline CA-125, platinum sensitivity status, largest diameter lesion, number of tumor lesions, ECOG PS, and tumor grade show that patients with elevated baseline CTC had 1.58- ( p = 0.058) and 1.54-fold ( p = 0.096) higher risk for progression and death respectively. Conclusions Results from this study indicate that elevated numbers of CTCs impart an unfavorable prognosis for ovarian cancer patients.

Clinical validity of circulating tumor cell (CTC) enumeration in 841 subjects with metastatic breast cancer (MBC).

10592 Background: CTCs are associated with poor clinical outcomes in MBC. Multiple technologies have been evaluated for CTC capture, enumeration, and characterization, but only one (CellSearch; Veridex) has been evaluated independently by different investigators with serial assessments and longitudinal follow-up in a defined prospective clinical trial. We combined these datasets to further validate the prognostic and predictive significance of this biomarker in MBC. Methods: Six studies were identified in the peer-reviewed published literature; all used the same CTC decision point (>=5 CTC/7.5 mL blood) and clinical endpoints (PFS, OS) in patients with progressive MBC. CTC enumeration was performed with the FDA cleared immunomagnetic/immunofluorescence assay (CellSearch). Study teams contributed blinded data to create a dataset of 841 subjects with CTC results linked to clinicopathologic variables and survival. Analyses were defined prior to data collection and conducted in subsets where pertinent data were available. Results: >=5 CTCs predicted for worse PFS at baseline (HR 1.32; CI 1.13, 1.55) and 2-5 (HR 2.16; CI 1.67, 2.78), 6-8 (HR 2.75; CI 2.05, 3.70), 9-12 (HR 2.71; CI 2.08, 3.52), 13-20 (HR 2.64; CI 2.03, 3.42), and >=21 wks (HR 1.88; CI 1.45, 2.42) from initiating new therapy (tx). OS results were also significant. The predictive value of CTC level was not affected by type (chemotx, endocrine tx, biologic tx) or line (first, second, >=third) of tx, presence/absence of bone metastases, or presence/absence of disease limited to bone. Multivariate Cox regression analysis was performed with the baseline CTC level adjusting for clinicopathologic variables to predict PFS and OS; some factors maintained an association, but CTC level was the strongest predictor of outcomes. Conclusions: This analysis substantiates the prognostic and predictive utility of CTCs assessed before and during tx for MBC. Persistently elevated CTC levels are highly associated with treatment failure irrespective of clinicopathologic variables, disease subtype, and type or line or tx. Serial CTC assessments should be used as a surrogate marker for patient outcomes to assist in selecting appropriate treatment regimens in MBC.

Circulating tumor cells as emerging tumor biomarkers in breast cancer

Circulating tumor cells (CTCs) provide unique information for the management of breast cancer patients, since their detection and monitoring is useful for prognosis, prediction of response to therapy, or monitoring clinical course in patients with localized or metastatic disease. Currently, the most practical application of CTCs is monitoring of patients with metastatic disease. Elevated CTC levels prior to initiation of a new systemic therapy are associated with a worse prognosis while persistently elevated CTC levels strongly suggest that the therapeutic regimen with which the patient is being treated is not working. New areas of research are directed toward developing novel sensitive assays for CTC molecular characterization. Molecular characterization of CTCs is very important for the future use of CTCs as targets of novel therapies. This review has focused on the presentation of recent data showing that CTCs are emerging as novel tumor biomarkers for prognostic and predictive purposes in breast cancer.

Abstract P3-02-06: Correlation of BCL-2 and Apoptosis on Circulating Tumor Cells and Breast Cancer Tissue

Abstract Background: Circulating Tumor Cell (CTC) levels are prognostic markers in metastatic breast cancer (MBC). Further phenotypic characterization of CTC may provide an opportunity for non-invasive evaluation of predictive and prognostic markers. Apoptosis is a common form of chemotherapy-induced cell death. Monoclonal antibody (MAb) M30 recognizes a neo-epitope on fragmented cytokeratin and is a marker of apoptosis. BCL-2 is an anti-apoptotic marker and may predict for resistance to anti-neoplastic therapy. We have previously reported the results of a pilot clinical trial to estimate M30 and BCL-2 expression in CTC from patients with MBC. The current study was performed to correlate the expression of both BCL-2 and M30 in CTC with breast cancer tissue samples. Methods: Of the 85 patients in the original pilot study, 52 (61%) had evaluable tissue available (39 primary and 12 metastatic lesions; one unknown) and were included in this analysis. CTC were collected at baseline and were isolated, enumerated, and phenotypically characterized for M30 and BCL-2 using the CellSearch® System. CTC phenotype is reported as percentage of cells with positive staining. Tissue Microarrays (TMA) were constructed and immunohistochemically stained for M30 (anti-Cytodeath™ M30, Roche) and Bcl-2 (M-0887, DAKO) and scored using the Allred method. Results: Tissue staining for M30 and Bcl-2 were inversely correlated. Of the 52 patients with TMA available for investigation, 22 (42%) had &amp;gt; 5 CTC/7.5 ml whole blood. A positive, but non-significant, correlation was observed between increasing numbers of CTC levels and tissue BCL-2 Allred Score (Spearman r=0.36; p=0.1310). M30 staining was detected in ≥10% of CTC in 20 of the 22 (91%) patients with elevated CTC, while BCL-2 staining was detected in ≥10% of CTC in 19 of the 22 (86%) patients. Little if any correlation was observed between CTC M30 and tissue M30 expression, however CTC BCL-2 was positively correlated with tissue Bcl-2 expression (Spearman r=0.47; p=0.0440). Conclusions: BCL-2 and M30 can be characterized in both CTC and breast cancer tissue. The likelihood of having elevated CTC may be associated with higher BCL-2 expression, and CTC BCL-2 was associated with tissue BCL-2 expression. Although the clinical implications of these findings are unknown, they offer the opportunity for future trials to investigate whether CTC BCL-2 and M30 might be useful to identify patients with cancers that are resistant to standard therapies. Furthermore, CTC BCL-2 and M30 might be monitored in clinical trials of novel, apoptosis-inducing therapeutic agents. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-06.

Abstract P3-02-03: Prognostic Value of Circulating Tumor Cells (CTC) in HER-2 Positive Metastatic Breast Cancer (MBC) Patients (pts) Treated with Anti-HER2-Based Therapy

Abstract Background. Detection of ≥5 CTC per 7.5 mL of blood before starting systemic treatment is a strong and independent predictor of worse progression-free survival (PFS) and overall survival (OS) in MBC. Trastuzumab (Herceptin®) was shown to be highly effective in reducing CTC number in all HER-2 amplified MBC cases after the first dose (De Giorgi et al., SABCS 2009). Furthermore, trastuzumab can target chemotherapy-resistant CTC in pts with breast cancer (Bozionellou et al. 2004). To date no significant analysis exploring the effect of another anti-HER2 therapy, lapatinib (Tykerb®) on CTC has been published.We hypothesized that anti-HER2-based therapy may modify the detection rate and prognostic value of CTC in pts with HER-2 amplified MBC. Patients and Methods. We retrospectively evaluated 91 HER-2 positive MBC pts, treated at the M. D. Anderson Cancer Center with trastuzumab-based (n=59), or lapatinib-based therapy (n=32). In all pts, CTC were enumerated before starting systemic treatments, using CellSearch™. Follow-up CTC counts were available for 41 (69%) pts treated with trastuzumab and 25 (78%) pts treated with lapatinib. PFS and OS were estimated by Kaplan-Meier product limit, and compared between groups according to baseline CTC count (&amp;lt;5 vs ≥5) by log-rank test. Results. Median follow-up was 27 months (range, 8-65) and 16 months (range, 3-33) for pts receiving trastuzumab and lapatinib, respectively. Treatments administered and CTC counts are reported in table 1. Two (5%) pts receiving trastuzumab and 4 (16%) pts treated with lapatinib had CTC ≥5 at the follow-up assessment. Estimated median PFS and OS according to baseline CTC value, for both treatments are shown in table 2. An elevated CTC count was associated with poor prognosis in pts treated with lapatinib (median OS: 25.8 vs 13.6 months in pts with CTC &amp;lt;5 and ≥5, respectively, P=.072). Surprisingly, in the trastuzumab group, PFS showed a non-statistically significant trend in favor of pts with CTC ≥5 compared to those with &amp;lt;5. Conclusions. According to previously published data, our study confirms that trastuzumab-based therapies can drastically reduce the number of CTC and neutralize their negative prognostic value. Furthermore, we found that lapatinib had a limited effect on the detection rate and prognostic value of CTC, although the low number of pts and the high percentage of pre-treated women do not allow us to draw definitive conclusions. Table 1 Table 2 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-03.