Value Of Circulating Tumor Cells Research Articles

Effective capture of circulating tumor cells from a transgenic mouse lung cancer model using dendrimer surfaces immobilized with anti-EGFR.

The lack of an effective detection method for lung circulating tumor cells (CTCs) presents a substantial challenge to elucidate the value of CTCs as a diagnostic or prognostic indicator in lung cancer, particularly in nonsmall cell lung cancer (NSCLC). In this study, we prepared a capture surface exploiting strong multivalent binding mediated by poly(amidoamine) (PAMAM) dendrimers to capture CTCs originating from lung cancers. Given that 85% of the tumor cells from NSCLC patients overexpress epidermal growth factor receptor (EGFR), anti-EGFR was chosen as a capture agent. Following in vitro confirmation using the murine lung cancer cell lines (ED-1 and ED1-SC), cyclin E-overexpressing (CEO) transgenic mice were employed as an in vivo lung tumor model to assess specificity and sensitivity of the capture surface. The numbers of CTCs in blood from the CEO transgenic mice were significantly higher than those from the healthy controls (on average 75.3 ± 14.9 vs 4.4 ± 1.2 CTCs/100 μL of blood, p < 0.005), indicating the high sensitivity and specificity of our surface. Furthermore, we found that the capture surface also offers a simple, effective method for monitoring treatment responses, as observed by the significant decrease in the CTC numbers from the CEO mice upon a treatment using a novel anti-miR-31 locked nucleic acid (LNA), compared to a vehicle treatment and a control-LNA treatment (p < 0.05). This in vivo evaluation study confirms that our capture surface is highly efficient in detecting in vivo CTCs and thus has translational potential as a diagnostic and prognostic tool for lung cancer.

Survivin mRNA-circulating tumor cells are associated with prostate cancer metastasis.

Detection of circulating tumor cells (CTCs) has been made to develop reliable assays for early diagnosis of various cancers. Overexpression of survivin in cancer cells is strongly associated with tumor progression. Although upregulation of survivin is observed in various tumors, its expression profile in the peripheral blood of prostate cancer (PCa) patients has not yet been investigated. In this study, we validated the application of survivin as the tumor marker to detect CTC and assessed its utility for diagnosis of PCa distant metastasis. Immunohistochemistry and quantitative real-time PCR (QRT-PCR) were performed to confirm the levels of surviving expression in PCa tissues. In addition, CTC values in 3mL of peripheral blood from PCa patients, benign prostate hyperplasia (BPH) patients, and normal controls were also measured by the survivin-targeted PCR. Our results showed that surviving was overexpressed in PCa tissues. The median levels of blood surviving mRNA of PCa patients, BPH patients, and normal controls were 5.67 (range from 0 to 12.46), 2.24 (range from 0 to 6.55), and 1.85 (range from 0 to 3.82), respectively. The levels of survivin are positively associated with PCa distant metastasis. Our results concluded that quantitation of CTCs through survivin-PCR could be a promising marker for diagnosis of PCa metastasis.

The significance of circulating tumor cells in prostate cancer patients undergoing adjuvant or salvage radiation therapy.

Background:Following radical prostatectomy, success of adjuvant and salvage radiation therapy (RT) is dependent on the absence of micrometastatic disease. However, reliable prognostic/predictive factors for determining this are lacking. Therefore, novel biomarkers are needed to assist with clinical decision-making in this setting. Enumeration of circulating tumor cells (CTCs) using the regulatory-approved CellSearch System (CSS) is prognostic in metastatic prostate cancer. We hypothesize that CTCs may also be prognostic in the post-prostatectomy setting.Methods:Patient blood samples (n=55) were processed on the CSS to enumerate CTCs at 0, 6, 12 and 24 months after completion of RT. CTC values were correlated with predictive/prognostic factors and progression-free survival.Results:CTC status (presence/absence) correlated significantly with positive margins (increased likelihood of CTCneg disease; P=0.032), and trended toward significance with the presence of seminal vesicle invasion (CTCpos; P=0.113) and extracapsular extension (CTCneg; P=0.116). Although there was a trend toward a decreased time to biochemical failure (BCF) in baseline CTC-positive patients (n=9), this trend was not significant (hazard ratio (HR)=0.3505; P=0.166). However, CTC-positive status at any point (n=16) predicted for time to BCF (HR=0.2868; P=0.0437).Conclusions:One caveat of this study is the small sample size utilized (n=55) and the low number of patients with CTC-positive disease (n=16). However, our results suggest that CTCs may be indicative of disseminated disease and assessment of CTCs during RT may be helpful in clinical decision-making to determine, which patients may benefit from RT versus those who may benefit more from systemic treatments.

Abstract 1590: CD133-expressing circulating tumor cells might predict poor prognosis in patients with head and neck cancer

Abstract PURPOSE: The prognostic value of circulating tumor cells (CTCs) number in patients with head and neck carcinoma (HNSCC) has been reported to be positive. The hypothesis of the additional markers on CTCs might predict clinical outcomes have been proposed. This study aimed to elucidate the prognostic/predictive value of CD133-expressing CTCs in HNSCC patients. PATIENTS AND METHODS: In a prospective single-center study, CTCs were enumerated in the peripheral blood of 24 subjects, including 14 patients with HNSCC and 10 healthy donors, at baseline and 2 weeks after starting first dose of chemotherapy. CTCs were measured using an immunomagnetic separation technique by combined negative and positive selection strategies. Immunofluorescence staining methods were applied for surface marker identification. Tumor response at 12 weeks after chemotherapy of the whole cohort was performed by computed tomograpgy or magnetic resonance imaging scans following RECIST criteria 1.1. RESULTS: In the first step of efficacy evaluation phase, an average recovery rate of 85.5% and a R2 value of 0.9932 were found when compared between spiked and recovered blood samples. A CTCs detection rate of 100.0% (14/14) was found by this protocol and the number could successfully differ HNSCC patients from healthy donors (P = 0.001). The mean and median (range) of baseline CTC number were 105.0 and 87.2 (34.5-358.1) cells/mL of blood. The higher baseline CTCs number was associated with higher tumor stage (AJCC 7th edition, P = 0.003). The higher absolute number (&amp;gt;5cells/mL of blood) of baseline CD133-expressing CTCs could predict poor survivals (Kaplan-Meier survival curves, P = 0.013). In all assessable patients, a high correlation rate of 92.9% was found between changes of CTCs (decline or elevation) and CT responses (progressive disease [PD] and partial response [PR]/Stable disease [SD]) with a P value of 0.005 (Fisher exact test). CONCLUSION: The number of CD133-expressing CTCs might be a useful prognostic for survivals and predictors for imaging response evaluation at 12 weeks in patients with HNSCC. Further investigation in large-scale prospective trials is warranted on the basis of the findings in this study. Citation Format: Jason Chia-Hsun Hsieh, Harber Hung-Bo Shiao, Bill Yuan-Jhih Chao, Ming-Jung Lin, John Wen-Cheng Chang, Hung-Ming Wang, Tyler Min-Hsien Wu. CD133-expressing circulating tumor cells might predict poor prognosis in patients with head and neck 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 1590. doi:10.1158/1538-7445.AM2015-1590

Abstract 1583: Circulating tumor cells isolated from non-small cell lung cancer patients using the in vivo CellCollector Technology

Abstract Access to tumor tissue is often limiting and archival samples may not be reflective of the current disease. Alternative approaches using liquid biopsies are being explored to support personalized treatment of cancer patients. Circulating tumor cells (CTCs) detached from primary tumors or metastases into the blood may reflect the current molecular tumor profile providing the opportunity to monitor molecular changes in the tumor. As CTCs are rare cells in the bloodstream and most in vitro approaches for capturing CTCs are based on small samples volumes (5 - 10 ml), accurate and reliable quantification of CTCs is difficult to achieve. We report on a comprehensive clinical study using a novel CellCollectorTM for in vivo isolation of CTCs. Eighty four stage I - IIIB non-small cell lung cancer (NSCLC) patients and 7 healthy individuals were screened for CTCs using a CellCollectorTM (Gilupi, Germany) device. Molecular analysis of a patient with known mutation in the KRAS gene (G12D) was performed to confirm the predictive value of CTCs as “liquid biopsy”. CTCs were detected in 65 out of 84 NSCLC patients (77.4%) (median: 13 CTCs; range from 0 - 300). For comparison, using the U.S. Food and Drug Administration-cleared CellSearch® system only fifteen samples (17.8%) in this cohort were positive (median: 0 CTCs; range from 0-300). In all paired samples, the CellCollectorTM detected the same or higher numbers of CTCs. Healthy individuals were devoid of any cells classified as CTC. Investigating the KRAS status on CTCs captured by the CellCollectorTM, we were able to substantiate the G12D mutation that has earlier on been identified in the tissue of the primary lung tumor.Conclusions: The CellCollectorTM is a promising novel device for the in vivo isolation of CTCs and the device effectively isolated CTCs from NSCLC patients of all stages including the non-metastatic setting. The implementation of the CellColletorTM into clinical practice has the potential for early disease detection. Furthermore, this approach allows the molecular characterization of CTCs for possible therapeutic targets and early response to treatment which might be used in the setting of personalized cancer therapies and clinical development. Citation Format: Thomas Krahn, Lukasz Gasiorowski, Wojciech Dyszkiewicz, Grzegorz Dworacki, Maciej Zabel, Dave S. Hoon, Stefanie Herold, Bjoern Nowack, Johannes Tucholsky, Christian Schumann, Klaus Lücke. Circulating tumor cells isolated from non-small cell lung cancer patients using the in vivo CellCollector Technology. [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 1583. doi:10.1158/1538-7445.AM2015-1583

Formation mechanism of circulating tumor cells and progress of its application in I-III stage colorectal carcinoma

It has been a problem in clinical practice that high recurrence and poor survival of patients with colorectal cancer undergoing surgery. As an easy-accessible, repetitive, non-invasive "liquid biopsy" technology, circulating tumor cells(CTC) can assist us in many fields, for instance, real-time monitoring tumor status and providing information about recurrence and prognosis of colorectal cancer. In this review, generation mechanism and relevant major regulation molecules of CTC will be elucidated, and principles, superiorities and inferiorities among different detection technologies were compared. Besides, clinical application and prognostic value of CTC in stage I-III colorectal caner were discussed, and the dilemma of low detection rate of CTC will be tried to solve as well.

Prognostic Value of Circulating Tumor Cells in Ovarian Cancer: A Meta-Analysis.

BackgroundThe prognostic value of circulating tumor cells (CTCs) in ovarian cancer has been investigated in previous studies, but the results are controversial. Therefore we performed a meta-analysis to systematically review these data and evaluate the value of CTCs in ovarian cancer.Materials and MethodsA literary search for relevant studies was performed on Embase, Medline and Web of Science databases. Then pooled hazard ratios (HRs) for survival with 95% confidence intervals (CIs), subgroup analyses, sensitivity analyses, meta-regression analyses and publication bias were conducted.ResultsThis meta-analysis is based on 11 publications and comprises a total of 1129 patients. The prognostic value of the CTC status was significant in overall survival (OS) (HR, 1.61;95% CI,1.22–2.13) and progression-free survival (PFS)/disease-free survival (DFS) (HR, 1.44; 95%CI, 1.18–1.75). Furthermore, subgroup analysis revealed that the value of CTC status in OS was significant in "RT-PCR" subgroup (HR, 2.02; 95% CI, 1.34–3.03), whereas it was not significant in "CellSearch" subgroup (HR, 1.15; 95% CI 0.45–2.92) and "other ICC" subgroup (HR, 1.09; 95% CI 0.62–1.90). The presence of CTC was also associated with an increased CA-125 (OR, 4.07; 95%CI, 1.87–8.85).ConclusionOur study demonstrates that CTC status is associated with OS and PFS/DFS in ovarian cancer.

Circulating tumor cells and evaluation of targeted therapy effect in EGFR mutation/ALK translocation metastatic non-small cell lung cancer.

e19084 Background: Targeted therapies have considerably improved the prognosis of patients with non-small cell lung cancer (NSCLC) ,RESIST criteria was the most often used response assessment method to reflecting the clinical benefits. We propose a non-invasive, folate receptor (FR)–based circulating tumor cell (CTC) detection approach to interpret treatment response of targeted therapy between baseline and follow-up CTC values in EGFR mutation/ALK translocation advanced NSCLC. Methods: CTCs were collected from 4 ml of blood based on negative enrichment by immunomagnetic beads in blood from 51 advanced NSCLC patients before the start of targeted therapy and after one month, three months of therapy. The change of CTCs value, radiological response and patient reported outcome(PRO) were compared. Sequential analyses were conducted to monitor CTC signals during therapy and correlate radiological effects with treatment outcome. Results: CTCs were detected in pretreatment and protreatment blood samples from all...

A prospective evaluation of cell free DNA (cfDNA) genotyping and circulating tumor cells (CTC) in EGFR mutant NSCLC patients (pts) treated with erlotinib.

11068 Background: Genotype directed therapy is now standard of care for advanced NSCLC pts. However, adequate tumor tissue for comprehensive genotyping remains a challenge. Recent research suggests that CTC capture or cfDNA analysis allows for non-invasive diagnosis and monitoring of treatment. This prospective trial was designed to quantify the predictive value of CTC and cfDNA analyses of EGFR-mutant NSCLC pts treated with first-line erlotinib. Methods: TKI naive EGFR mutant NSCLC pts were enrolled in a phase II trial of erlotinib treatment. Paired blood for cfDNA and CTC analysis was collected at baseline prior to therapy and every 2 months during follow ups. Plasma genotyping was performed by ddPCR for EGFR19del, L858R, T790M while CTCs were isolated by CellSearch and analyzed by IF and MET-FISH. Repeat biopsies at progression were performed when feasible. Results: Between 2/10 and 1/15, 60 EGFRmutant pts (L858R:17, 19del:38, other:5) were enrolled. As of 1/15, 44 patients have discontinued therapy (3...

Effect of circulating tumor cells (CTCs) on metastatic progression in colorectal cancer.

e22026 Background: The purpose of the study was to analyze the content and to assess prognostic value of CTCs in peripheral blood of colorectal cancer patients with liver metastases. Methods: 75 co...

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.

Abstract P4-01-07: Different prognostic value of circulating and disseminated tumor cells in primary breast cancer. Influence of bisphosphonate intake?

Abstract Background: Disseminated tumor cells (DTCs) in the bone marrow and circulating tumor cells (CTCs) in blood of breast cancer patients (pts) were shown to provide independent prognostic information and can be regarded as an early indicator of therapy failure. Here we demonstrate a different prognostic value of DTCs and CTCs and explain these findings by early bisphosphonate intake. Patients and Methods: 10 ml blood and two bone marrow aspirates of 488 pts with first diagnosis of breast cancer between Aug 2006 and Dec 2010 were studied for DTCs and CTCs. CTCs were detected using the AdnaTest BreastCancer (AdnaGen AG, Hannover, Germany) for the detection of EpCAM, MUC-1 and HER2 transcripts. DTCs were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. In addition to chemo-, radio- and anti-hormonal therapy, bisphosphonates were given to DTC-pos pts [clodronate (2×520 mg per day) for at least two years] and 69 DTC-neg, postmenopausal pts [zoledronic acid (4 mg, twice a year) for three years]. Results: After a median follow-up time of 48 months (range: 5 to 87 months), the overall survival rate was 93% (456/488 pts) and relapses occurred in 11% (56/488 pts) of cases. CTCs were detected in 109/488 pts (22%) and significantly correlated with reduced disease free survival (DFS; p=0.02). Negative prognostic relevance was predominantly related to the lobular subtype (p=0.0047), the ER-pos subtype, (pER+/Her2=0.01; pER+/Her2+=0.25; pER-/Her2+=0.5) and pts who had received chemotherapy (p=0.015) or radiation therapy (p=0.0083) but not to pts with anti-hormonal regimens (p=0.06). DTCs were detected in 162/488 pts (33%). In contrast to CTCs, no prognostic significance could be shown with regard to DFS (p=0.49), all clinical parameters as well as treatment regimens. In contrast, only in CTC-pos pts, the presence of DTCs significantly correlated with DFS (p=0.04). We further investigated whether this lack in prognostic significance was due to bisphosphonate intake in case of DTC-positivity. Predominantly, in the CTC-neg group, bisphosphonate treatment significantly influenced DFS in both histological subtypes (pCTC-/ductal=0.04; pCTC-/lobular=0.0021). However, in the CTC-pos group of pts, no such effect was observed (pCTC+/ductal=0.76; pCTC+/lobular=0.51).Conclusion: Here we show, that CTCs, rather than DTCs were significantly prognostic for early relapse. The ER-pos subtype was mostly affected which is in concordance with our previous studies, demonstrating that CTCs show EMT and tumor stem cell characteristics and, thus, down regulate ER which probably makes anti-hormonal treatment less effective. In addition, the presence of these CTC-subtypes might also explain the negative prognostic impact of CTCs in pts receiving chemo- or radiation therapy which we could verify in a subgroup of our pts (n=238) showing a significant correlation of CTCs and EMT/stem cell like CTCs (p=0.017; data not shown). The lack of prognostic significance of DTCs can be related to bisphosphonate intake in all DTC-pos pts, a subgroup of DTC-neg pts as well as in DTC-pos/CTC-neg pts, underlining our assumption that CTCs might be a high risk indicator for already ongoing metastasis not limited to bone metastasis. Citation Format: Sabine Kasimir-Bauer, Bahriye Aktas, Siegfried Hauch, Rainer Kimmig, Oliver Hoffmann. Different prognostic value of circulating and disseminated tumor cells in primary breast cancer. Influence of bisphosphonate intake? [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 P4-01-07.

Abstract P4-01-04: Circulating tumor cells (CTC) are associated with defects in innate and adaptive immunity in inflammatory breast cancer (IBC) patients

Abstract Background: CTCs play a crucial role in tumor dissemination and are prognostic factor in primary and metastatic breast cancer patients. Immune cells in peripheral blood (PB) contribute to an unfavorable microenvironment for the CTCs survival. As such, effective host innate and adaptive immune surveillance systems could adversely influence tumor dissemination whereas dysfunctional immune systems could provide a favorable microenvironment for the dissemination of CTCs and cancer progression. This study aimed to correlate CTCs with the functions of innate [natural killer (NK) cells] and adaptive (T-cells) immune effector cells in PB of IBC patients. Methods: This prospective study included 65 IBC (21 non-metastatic, 14 de novo metastatic and 30 recurrent metastatic) patients treated between October 2008 and April 2012 at the MD Anderson Cancer Center. CTCs were enumerated before patients started a new line of chemotherapy using the CellSearch® system, 33 (50.8%) of patients were treatment naïve at the time of blood collection. The phenotype of T cells, their ability to secrete cytokines following activation through the T-cell receptor (TCR) and the NK cell subsets were analyzed by multiparameter flow cytometry and the results were correlated with CTCs and clinical outcome. For survival analysis immune cell counts were dichotomized to low or high category using the median count. Results: At least 1 CTC (≥ 1) or ≥ 5 CTCs per 7.5 mL of PB was detected in 40 (61.5%) or 21 (32.3%) of patients, respectively. Patients with at least 1 CTC or ≥ 5 CTCs had a significantly inferior overall survival (OS) [HR=2.48, p=0.003 and HR=1.85, p=0.045] than patients with no CTCs or with &amp;lt;5 CTCs, respectively. There was no correlation between CTCs count and total lymphocytes; however, patients with at least 1 CTC or ≥5 CTCs had significantly lower percentages of CD3+ and CD4+ T-cells compared with patients with no CTCs or &amp;lt; 5 CTCs, respectively. Patients with ≥ 1 CTC, had a lower percentage of TCR-activated CD8+ T-cells producing TNF-α (p=0.03) and IFN-γ (p=0.08), and a higher percentage of T regulatory lymphocytes (p=0.05) compared to patients with undetectable CTCs. Moreover, CTCs ≥5 was inversely associated with the percentage of the following NK cells subsets: non-ADCC NK (Spearman rho' = -0.30, p=0.02), ADCC NK (rho' = -0.15, p=0.20) and exhausted NK (rho = -0.24, p=0.04). We also observed increased prognostic value of CTCs in the context of adaptive immune cells, with worse OS for patients with ≥ 5 CTCs and low count of TCR-activated CD8+ T cells producing TNF-α (HR=6.72, p=0.0007) compared with patients with &amp;lt; 5 CTCs and high count of TCR-activated CD8+ that produced TNF-α. Conclusions: IBC patients with CTCs in PB had abnormalities in both innate and adaptive immunity as evidenced by low percentages of NK cell subsets, and low percentage of TCR-activated CD8+ T cells producing TNF-α, respectively. These data illustrate an inverse relationship between CTCs and both innate and adaptive immune cells in the PB microenvironment that could potentially impact tumor cell dissemination and initiation of the metastatic cascade. Moreover, immune cell profiling could add further prognostic value to CTCs in IBC patients. Citation Format: Michal Mego, Hui Gao, Evan N Cohen, Simone Anfossi, Antonio Giordano, Sanda Tin, Tamer M Fouad, Wendy A Woodward, Ricardo H Alvarez, Vicente Valero, Naoto T Ueno, Gabriel N Hortobagyi, Massimo Cristofanilli, James M Reuben. Circulating tumor cells (CTC) are associated with defects in innate and adaptive immunity in inflammatory breast cancer (IBC) 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 P4-01-04.

Abstract P4-11-21: Prevalence of circulating tumor cells (CTCs) after five years of zoledronate treatment in the adjuvant SUCCESS-A study

Abstract Aim: The prognostic value of CTCs at primary diagnosis has recently been confirmed by the SUCCESS A Study (Rack et al. JNCI 2014). Key questions on the role of adjuvant bisphosphonate treatment, including patient populations deriving benefit and optimal timing/scheduling of therapy are still controversial. Aim of this study was therefore to evaluate CTCs in the context of two different zoledronate regimens. Methods: The SUCCESS A trial is a large, randomized, open-label, 2x2 factorial design Phase III study in patients with high risk breast cancer (stage N1 or T2–T4 or grade 3 or age ≤ 35 or hormone-receptor negative). Patients were first randomized to adjuvant chemotherapy treatment with 3 cycles of Epirubicin-Fluorouracil-Cyclophosphamide (FEC) followed by either 3 cycles of Docetaxel or 3 cycles of Gemcitabine-Docetaxel. In addition, patients were randomized to 2 years vs. 5 years of zoledronate treatment. CTC status 5 years after primary diagnosis was assessed using the FDA-approved CellSearch System (Veridex, USA), and CTC positivity was defined as ≥ 1 CTC. We studied the influence of zoledronate treatment duration on CTC prevalence at 5 years in addition to well-known patient and tumour characteristics using a multiple logistic regression analysis on the ITT population. Results: Data on CTC status at 5 years after primary diagnosis were available for 728 (19.4%) out of 3754 randomized patients. 310 patients had been randomized to 2 years of zoledronate treatment and 418 patients had been randomized to 5 years of zoledronate treatment. In these patients a difference in CTC positivity after 5 years could not be shown between patients randomized to 2 or 5 years of zoledronate (p = 0.13, Wald test). The adjusted odds ratio for 2 years vs 5 years was 0.65 (95%CI: 0.37 to 1.13). Conclusions: The final survival analysis of the SUCCESS A trial will give further insight, whether CTCs can be used as early predictive marker for the efficacy of adjuvant zoledronate treatment. Citation Format: Brigitte Rack, Peter A Fasching, Lothar Häberle, Thomas Friedl, Mahdi Rezai, Jörn Hilfrich, Hans Tesch, Georg Heinrich, Helmut Forstbauer, Julia Neugebauer, Elisabeth Trapp, Susanne Albrecht, Bernadette Jäger, Tanja Fehm, Volkmar Müller, Andreas Schneeweiss, Klaus Friese, Werner Lichtenegger, Matthias W Beckmann, Wolfgang Janni. Prevalence of circulating tumor cells (CTCs) after five years of zoledronate treatment in the adjuvant SUCCESS-A study [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 P4-11-21.

Abstract P4-01-17: Expression of circulating tumor cells (CTC) and CK-19 mRNA (CK19) as prognostic factors in heavily pretreated metastatic breast cancer

Abstract Introduction: The expression of CTC and CK19 holds prognostic value for patients with breast cancer but their clinical significance remains still controversial. Methods: This clinical observational study included 58 preteated metastatic breast cancer patients who started a standard new treatment line. CTC and CK19 was measured with CellSearch® and RT-PCR respectively at inclusion time. Progression-free survival (PFS) was defined as the time elapsed between the initiation of the treatment and either the date of clinical or radiological progression or death or the last follow-up. Cox proportional hazards regression model was used to assess the univariate prognose value of CTC and CK19 on PFS, and Kaplan-Meier estimates. A multivariate Cox model was also performed to additionaly account for ER and visceral disease. CTC and CK19 positivity was considered when a value of 1 or more was observed. Results: Mean age was 59.68 (range 35-86), the average number of previous treatments was 2.98 (range 1-10), 38 patients (65.52%) were ER+ and 22 (37.93%) had visceral disease. Median PFS was 7 months (CI 95% 4-9). Univariate analyses showed a significant effect of the positivity of CK19 (HR=2.22, CI 95% 1.15-4.29, p=0.01) but did not reach statistical significance for CTC (HR=1.86, CI 95% 0.93-3.72, p=0.07). The estimate disease-free survival rate at 6 and 12 months were 63.6% and 43.4% for patients with CK19&amp;lt;1 and 47.1% and 10.6% for patients with CK19&amp;gt;=1, respectively. The estimate disease-free survival rate at 6 and 12 months were 77.8% and 28.8% for patients with CTC=0 and 43.6% and 21.5% for patients with CTC&amp;gt;=1, respectively. In the multivariate analysis the effect of CK19 and CTC were similar (HR=2.12 and 1.86, p=0.03 and 0.08 respectively), ER was statistically significant (HR=0.48, p=0.05, + vs -) but visceral diseases appeared not significant (HR=1.57, p=0.17, yes vs no). Conclusions: The expression of CK19 appeared clinically meaningful in pretreated metastatic breast cancer patients, even when adjusting by ER and visceral diseases. CTC showed a similar but minor effect and not statistically significant. These results support CK19 as an interesting biomarker for predicting clinical response in metastatic breast cancer. Citation Format: Serafin Morales, Ana Velasco, Jose Vidal, Anna Serrate, Joan Valls, Juan Carlos Samame, Rafael Gisbert, Desamparados Moral, Antonio Llombart, Antonieta Salud, Xavier Matias-Guiu. Expression of circulating tumor cells (CTC) and CK-19 mRNA (CK19) as prognostic factors in heavily pretreated metastatic breast cancer [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 P4-01-17.

Prognostic value of circulating tumor cells in patients with squamous cell carcinoma of the head and neck: a systematic review and meta-analysis.

The prognostic value of circulating tumor cells (CTCs) in patients with squamous cell carcinoma of the head and neck (SCCHN) is controversial. The objective of this study was to evaluate the prognostic value of CTCs in patients with SCCHN by conducting a meta-analysis. We systematically searched scientific literature published before June 10, 2014, using the PubMed, ScienceDirect, Cochrane Library, and EMBASE databases. Studies evaluating the correlation of CTC status with tumor-node-metastasis (TNM) disease stage, nodal involvement, and disease progression (recurrence or metastasis) in patients with SCCHN were selected for the analysis. Pooled odds ratio (OR) and 95 % confidence interval (CI) were calculated using the fixed-effects model or the random-effects model in the presence of heterogeneity. Our analysis consisted of eight studies, enrolling a total of 433 patients. The disease progression (recurrence/metastasis) rate in the CTC-positive patients was significantly higher (OR 3.44; 95 % CI 1.87-6.33; p < 0.01) compared with the patients without disease progression. However, there was no significant difference between TNM disease stage III-IV and stage I-II in the presence of CTCs (OR 1.54; 95 % CI 0.87-2.72; p > 0.05). CTC status did not correlate with nodal involvement (OR 1.20; 95 % CI 0.67-1.90; p > 0.05). This meta-analysis indicates that detection of CTCs has a predictive value in patients with SCCHN, particularly those with tumor progression. The presence of CTCs in patients with SCCHN has a poor prognosis compared with the patients without CTCs. Detection of CTCs might be served as a prognosticator in patients with SCCHN. Further studies based on homogeneous populations are warranted to confirm these findings.

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.

Detection of Circulating Tumor Cells at Surgery and at Follow-Up Assessment to Predict Survival After Two-Stage Liver Resection of Colorectal Liver Metastases.

The presence of circulating tumor cells (CTCs) is negatively associated with survival after resection of colorectal liver metastases (CLM). The current study aimed to determine the prognostic value of CTCs and disseminated tumor cells (DTCs) at the time of surgery and the prognostic value of CTCs at follow-up assessment, for patients scheduled to undergo two-stage hepatectomy with portal vein embolization (PVE) for CLM. Samples were collected at surgery (blood and bone marrow) and at follow-up assessment (blood) for the period 2008 through 2011. In this study, CTCs were detected with the CellSearch system, and DTCs were detected using standard immunocytochemical analysis. Of 24 patients, 18 completed both stages, and no patients were lost to follow-up. The median overall survival (OS) was 37 months, and the median recurrence-free survival (RFS) was 7 months. At surgery, CTCs were found in nine patients (38 %), and their presence was associated with reduced OS (p < 0.001) and RFS (p = 0.006). Follow-up CTC status was available for 11 patients. All eight patients with positive CTC status experienced recurrence. Two of three patients with negative CTC status remained recurrence free. In seven patients (32 %), DTCs were detected but were not associated with OS or RFS. The presence of CTCs at surgery is associated with worse OS and RFS for patients undergoing two-stage hepatectomy with PVE for CLM. Analysis of CTCs should be explored further for their potential to assist in treatment decisions and monitoring for CLM patients.

Role of circulating tumor cells as prognostic marker in resected stage III colorectal cancer

The prognostic role of circulating tumor cells (CTC) in early colorectal cancer (CRC) has not been determined yet. We evaluated the potential prognostic value of CTC in stage III CRC patients. Prospective multicenter study of 519 patients with stage III CRC recruited between January 2009 and June 2010. CTC were enumerated with the CellSearch System after primary tumor resection and before the start of adjuvant therapy. A total of 472 patients were included in the analysis. CTC ≥1, ≥2, ≥3 and ≥5 were detected in 166 (35%), 93 (20%), 57 (12%) and 34 (7%) patients, respectively. Median follow-up was 40 months. In the overall population, CTC ≥1 (disease-free survival (DFS): HR 0.97, P = 0.85; overall survival (OS): HR 1.03, P = 0.89), ≥2 (DFS: HR 1.07, P = 0.76; OS: HR 1.02, P = 0.95), ≥3 (DFS: HR 0.96, P = 0.87; OS: HR 0.74, P = 0.41) and ≥5 (DFS: HR 0.72, P = 0.39; OS: HR 0.48, P = 0.21) were not associated with worse DFS and OS. No clinicopathological characteristics were significantly associated with the presence of CTC. In patients with disease relapse, the proportion with CTC ≥1 was not significantly different between those with single versus multiple metastatic locations (37.9% versus 31.4%, P = 0.761). In the multivariate analysis, CTC ≥1 was not an independent prognostic factor for DFS (HR 0.97, P = 0.87) and OS (HR 0.96, P = 0.89). CTC detection was not associated with worse DFS and OS in patients with stage III CRC. Given the scarcity of CTC in these patients, it is likely that CTC determined by CellSearch system does not have a prognostic role in this setting. However, a longer follow-up is needed.

Are circulating tumor cells (CTCs) a feasible tool for predicting disease recurrence and survival in nonmetastatic (M0) colorectal cancer (CRC)?

650 Background: Based on the correlation between CTCs and clinical outcome of metastatic breast, prostatic and CRC cancer, CTCs might be an ideal model to investigate the individual risk to develop metastasis. On the other way, data on their significance in M0 CRC are still limited. In a previous study we investigated CRC cell death in the circulating compartment, finding high value of apoptotic CTCs (M30-positive) independently from the disease stage. Our aim was to evaluate the prognostic role of total and M30-positive CTCs in M0 CRC patients (pts). Methods: From January 2009 to December 2011, we prospectively enrolled 192 stage 0-III CRC pts (stage 0 identified rectal cancer pts with Tis or complete pathological response after neoadjuvant therapy). We used CellSearch assay to quantify total and M30-positive CTCs in peripheral blood of CRC pts candidate to curative resection at diagnosis, time of surgery and four months later. Multivariate survival analyses were conducted using a Cox-regression. Results: At baseline 81 out of 192 pts (43.3%) had at least 1 CTC/7.5 mL of peripheral blood, 40 pts (22.2%) had &gt;2 cells and 20 pts (11.6%) had &gt;3 cells. We found a weak association between OS and total CTCs number (p=0.069), a significant association with M30-positive CTCs (p=0.022) and no association between RFS and CTC status. Limiting the analysis to stage I-III (n=151), we found a strong association between OS and both CTCs number (p=0.021) and M30-positive cells (p=0.007). The multivariate analyses demonstrated a significant improvement of survival prediction adding baseline CTCs count to clinical and pathological characteristics (HR=1.51, 95% CI 1.16-1.96). Conclusions: We first demonstrated a significant association between OS and CTCs status in stage I-III CRC pts. This result is consistent with previous report in metastatic CRC. It highlights a potential advantage of CTCs count in stratifying CRC pts and identifying a subset of pts with a high metastatic risk that could benefit from an adjuvant therapy. Further studies are needed to explore this aspect, which might be particularly significant in stage II CRC where the indication for adjuvant therapy is still under debate.