Presence Of Circulating Tumor Cells In Patients Research Articles

Identification and Morphological Characterization of Features of Circulating Cancer-Associated Macrophage-like Cells (CAMLs) in Endometrial Cancers.

Simple SummaryCirculating cells in the blood of cancer patients with solid tumors are known to provide clinically relevant information about treatment response, tumor progression, metastasis, and the development of drug resistance. We identify for the first time the distinctive morphological characterization of endometrial giant macrophage-like cells, called CAMLs, in the context of the presence of circulating tumor cells in patients. Two distinct size-based subtypes of CAMLs, <20 µm (tiny) and >20 µm (giant), were demonstrated. The giant CAMLs have distinctive polymorphic morphologies with mononuclear or fused polynuclear structures, including (1) apoptotic CAMLs; (2) CAML–WBC doublets; (3) conjoined CAMLs; (4) CAML–WBC clusters; and (5) circulating tumor cell–CAML–WBC clusters. In enumerating CAMLs and circulating tumor cells simultaneously, we observed that all circulating tumor cell-positive patients are also positive for CAMLs, in contrast to 55% of patients out of all CAML-positive patients that were found positive for circulating tumor cells.The blood of patients with solid tumors contains circulating tumor-associated cells, including epithelial cells originating from the tumor mass, such as circulating tumor cells (CTCs), or phagocytic myeloid cells (differentiated monocytes), such as circulating cancer-associated macrophage-like cells (CAMLs). We report for the first time the identification and in-depth morphologic characterization of CAMLs in patients with endometrial cancers. We isolated CAMLs by size-based filtration on lithographically fabricated membranes followed by immunofluorescence, using a CD45+/CK 8,18,19+/EpCAM+/CD31+/macrophage-like nuclear morphology, from > 70 patients. Irrespective of the histological and pathological parameters, 98% of patients were positive for CAMLs. Two size-based subtypes of CAMLs, <20 µm (tiny) and >20 µm (giant) CAMLs, of distinctive polymorphic morphologies with mononuclear or fused polynuclear structures in several morphological states were observed, including apoptotic CAMLs, CAML–WBC doublets, conjoined CAMLs, CAML–WBC clusters, and CTC–CAML–WBC clusters. In contrast, CAMLs were absent in patients with non-neoplastic/benign tumors, healthy donors, and leucopaks. Enumerating CTCs simultaneously from the same patient, we observed that CTC-positive patients are positive for CAMLs, while 55% out of all CAML-positive patients were found positive for CTCs. Our study demonstrated for the first time the distinctive morphological characteristics of endometrial CAMLs in the context of the presence of CTCs in patients.

Circulating tumor cells detected in follow-up predict survival outcomes in tri-modality management of advanced non-metastatic esophageal cancer: a secondary analysis of the QUINTETT randomized trial

BackgroundOur aim was to establish if presence of circulating tumor cells (CTCs) predicted worse outcome in patients with non-metastatic esophageal cancer undergoing tri-modality therapy.MethodsWe prospectively collected CTC data from patients with operable non-metastatic esophageal cancer from April 2009 to November 2016 enrolled in our QUINTETT esophageal cancer randomized trial (NCT00907543). Patients were randomized to receive either neoadjuvant cisplatin and 5-fluorouracil (5-FU) plus radiotherapy followed by surgical resection (Neoadjuvant) or adjuvant cisplatin, 5-FU, and epirubicin chemotherapy with concurrent extended volume radiotherapy following surgical resection (Adjuvant). CTCs were identified with the CellSearch® system before the initiation of any treatment (surgery or chemoradiotherapy) as well as at 6-, 12-, and 24-months post-treatment. The threshold for CTC positivity was one and the findings were correlated with patient prognosis.ResultsCTC data were available for 74 of 96 patients and identified in 27 patients (36.5%) at a median follow-up of 13.1months (interquartile range:6.8-24.1 months). Detection of CTCs at any follow-up visit was significantly predictive of worse disease-free survival (DFS;hazard ratio [HR]: 2.44; 95% confidence interval [CI]: 1.41-4.24; p=0.002), regional control (HR: 6.18; 95% CI: 1.18-32.35; p=0.031), distant control (HR: 2.93; 95% CI: 1.52-5.65;p=0.001) and overall survival (OS;HR: 2.02; 95% CI: 1.16-3.51; p=0.013). After adjusting for receiving neoadjuvant vs. adjuvant chemoradiotherapy, the presence of CTCs at any follow-up visit remained significantly predictive of worse OS ([HR]:2.02;95% [Cl]:1.16-3.51; p=0.013) and DFS (HR: 2.49;95% Cl: 1.43-4.33; p=0.001). Similarly, any observed increase in CTCs was significantly predictive of worse OS (HR: 3.14; 95% CI: 1.56-6.34; p=0.001) and DFS (HR: 3.34; 95% CI: 1.67-6.69; p<0.001).ConclusionThe presence of CTCs in patients during follow-up after tri-modality therapy was associated with significantly poorer DFS and OS regardless of timing of chemoradiotherapy.

Factors of innate immunity in patients with colon cancer with different levels of circulating tumor cells.

e15501 Background: The purpose of this study was to demonstrate the importance of circulating tumor cells (CTCs) in the formation of immunosuppression in colon cancer. Methods: 200 patients with stage II-IV colon cancer underwent surgery as the first stage of treatment. Blood levels of CTCs were determined before surgery by flow cytometry using the CellSearch method (the sample was considered positive with CTCs &gt; 3), as well as some indicators of innate immunity (NK cells, neutrophilic and monocytic phagocytosis). Results: Regardless of the stage, patients with CTCs had statistically significantly lower levels of NK lymphocytes (16.8±1.9 versus 22.3±1.8%, p≤0.05) compared with the absence of CTCs; on the contrary, levels of NKT cells in this group were higher than in the absence of CTCs (8.8±0.9 versus 5.0±1.0%, p≤0.05). The presence of CTCs was accompanied by higher levels of CD16dim56bright cells (13.1±3.2 versus 5.9±1.0% p≤0.05) with a lower content of CD16+56dim (82.5±3.6 versus 90.4±0.8%), and levels of perforin and granzyme were similar. The presence of CTCs was characterized by a higher percentage of phagocytic monocytes in the Phagotest test, as well as a higher percentage of monocytes and neutrophils capable of developing oxidative burst upon fMLF stimulation in the Phagoburst test (10.8±3.3 versus 3.2±1.5% and 5.1 ±0.8 vs. 2.0±1.1%, respectively; p≤0.05). A separate analysis of phagocytosis indicators by disease stages revealed that the presence of CTCs in patients was accompanied by stimulation of phagocytosis in local tumors and inhibition in advanced disease, however, in the latter case, the ability of granulocytes and monocytes to generate reactive oxygen species (ROS) was preserved and even increased. Conclusions: The presence of CTCs in patients with colon cancer is characterized by a redistribution of natural killer subpopulations with an increase in the blood levels of NKT cells, some of which have immunosuppressive activity, due to a decrease in NK cells, and an increase in CD16dim56bright and a decrease in CD16+56dim, apparently, indicates disturbance of their maturation. The established changes in the presence of CTCs indicate the inhibition of antitumor properties of the NK cell and phagocytic components of innate immunity in tumor cell circulation. The N-formylpeptide fMLF is known as a powerful neutrophil chemoattractant, stimulator of their migration and cytokine production, which plays a role in the development of inflammatory bowel diseases. The growth-promoting activity described for the similar antimicrobial protein LL-37 can be mediated through neutrophil migration and ROS production. Thus, the presence of CTCs has a negative impact on the parameters of NK cell and phagocytic components of innate cellular immunity in patients with colon cancer, which can serve as one of the strategies for survival and dissemination of the tumor.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

SummaryCancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin−CD90−ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin−CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFβ signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFβ signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFβ/TGFBR1 inhibitors.

Abstract P3-01-21: Circulating tumor cells of breast cancer origin identified by fluorescence in situ hybridization and may be an early predictor of therapy failure in early breast cancer

Abstract Background: Most modern methods of detecting circulating tumor cells (CTCs) involve identifying cells with epithelial markers. This approach presents challenges, as not all epithelial cells found in circulation originate from the tumor and not all CTCs express epithelial markers. We propose using a size-exclusion filtration system to enrich for CTCs in peripheral blood followed by fluorescence in situ hybridization (FISH) of the filtered cells to identify cells of tumor origin in the early-stage breast cancer patients. We further hypothesize that the presence of CTCs may be indicators of therapy failure in early-stage breast cancer patients. Methods: Patients diagnosed with breast cancer (n = 9) were consented for CTC evaluation. Primary tumor DNA was analyzed by the Affymetrix OncoscanTM genome-wide microarray platform and investigated for somatic copy-number alterations (SCNAs). For each patient, two FISH probes were then identified for two regions of gain or a region of gain and a region of loss from the microarray results. Blood samples from patients were obtained before surgery, radiation therapy, endocrine therapy, and at 6-month or 1-year follow-up visits. Blood samples were filtered using ScreenCell® Cyto V2 devices, and FISH was performed. Cells were categorized as normal (diploid for all FISH probes), suspicious (single SCNA detected by FISH), or CTC (two SCNAs detected by FISH). Patients were identified as having CTCs present in their circulation when ≥2 CTCs were observed or when one CTC and &amp;gt;15 suspicious cells were observed. Results: The microarray data revealed that luminal A tumors ranged from 2-43 SCNAs; luminal B tumors ranged from 15-20 SCNAs; and ER+, PR+, HER2+ tumors ranged from 46-98 SCNAs. Although a correlation appears to exist between tumor genetic complexity and molecular subtype, the degree of complexity was highly varied within each subtype. We found that neither complexity of tumor profile, molecular subtype, nor stage could predict the presence of CTCs in patients. Molecular SubtypeSCNAsCTCsSuspicious Cells OnlyLuminal A2-433/52/5Luminal B15-200/21/2ER+, PR+, HER2+46-982/20/2 In pre-surgical blood samples, we detected CTCs in 63% of patients with stage 1 disease and in 60% of patients with luminal A tumors, 0% of patients with luminal B tumors, and 100% of patients with triple-positive tumors. StageCTCsSuspicious Cells OnlyIA/B5/82/8IIA0/11/1 Although limited in number, ongoing investigation revealed that one of our patients in early follow-up with a luminal A, stage IB tumor was identified to have persistent CTCs at 1-year after starting hormonal adjuvant therapy, suggesting residual tumor burden not detected by standard clinical modalities; this finding also suggests that this patient may be at highest risk for relapse and should be considered for additional therapies. Conclusion: Size-exclusion filtration followed by FISH analysis can accurately identify CTCs in early-stage breast cancer patients. Tumor complexity, molecular subtype, and stage did not predict the presence of CTCs in circulation. Our method for CTC detection may be able to serve as a diagnostic tool for treatment failure. Citation Format: Liu C, Althof PA, Maroni D, Stevens JM, Grabow CE, VanDyke AZ, Price JD, Sanmann JN, Thayer SP. Circulating tumor cells of breast cancer origin identified by fluorescence in situ hybridization and may be an early predictor of therapy failure in early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-21.

Abstract LB-B20: Characterization and Enumeration of Multiple Circulating Tumor Cell Phenotypes Using Two Distinct Platforms Establishes Presence of Epithelial-Mesenchymal Transition CTCs in Patients

Abstract We are employing a “liquid biopsy” approach for monitoring drug responses to new anti-cancer agents in early stage clinical trials by measuring drug effects in circulating tumor cells (CTCs), which requires the capability to capture and characterize CTCs from patients with diverse diseases including sarcomas, lymphomas and carcinomas. We have been applying the Apostream platform (DEP-FFF separation) with fluorescence imaging and have recently added the 5-channel CellSearch system (MAb coated ferrofluid) for specimen analysis. Use of both CD146 and EpCam capture per specimen, with addition of vimentin and the tumor markers, significantly increased the number of CTCs captured with the 5 channel CellSearch®. A “homebrew” CTC kit was developed that captures circulating cells that are either EpCAM or CD146 marker positive, and it was capable of identifying high numbers of EMT+ tumor marker+ double positive cells in patient specimens. There was statistical equivalency of events flagged as CTCs (CD45-/EpCam+/CK+) in patient specimens analyzed using Apostream or 5-Color CellSearch versus 4-channel CellSearch and 100% concordance between the CellSearch platforms. Reproducibility, as assessed by image re-analysis using Definiens software, was also high (&amp;gt;90%). Tumor marker specificity was established by analyzing normal donor blood, which varied from zero to 2 cells per ml blood depending on the marker set employed (only CD45 negative cells assessed). This approach solves a major limitation of using CTCs to monitor pharmacodynamic response by enumerating statistically significant cell numbers from patients with solid tumors of diverse histologies. Strikingly, both platforms demonstrated the presence of high numbers of CTCs with the Epithelial-Mesenchymal Transition (EMT) phenotype (10-100 cells per ml in Phase 1 trial populations), and widely used tumor markers such as MUC1, CEA, TLE1, or proteins generated by recombination events such as ASPL-TFE3. The presence of large numbers of CTCs expressing the EMT phenotype may be related to the intensive prior treatment (&amp;gt;3 prior therapies, median) of our phase 1 population. Funded by NCI contract # HHSN261200800001E. Citation Format: Lihua Wang, Francis Owusu, Sonny Khin, Ralph Parchment, Alice Chen, Shivaani Kummar, James H. Doroshow, Robert J. Kinders. Characterization and Enumeration of Multiple Circulating Tumor Cell Phenotypes Using Two Distinct Platforms Establishes Presence of Epithelial-Mesenchymal Transition CTCs in Patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B20.

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.

Importance of circulating tumor cells in newly diagnosed colorectal cancer.

Presence of circulating tumor cells (CTC) is associated with poor prognosis in patients with metastatic colorectal cancer (CRC). The present study was conducted to determine if the presence of CTC prior to surgery and during follow‑up in patients with newly diagnosed non-metastatic CRC can identify patients at risk for disease recurrence. In a prospective single center study 183 patients with newly diagnosed non-disseminated CRC, scheduled for surgery, were enrolled and followed-up for a median of 5.1 years. CTC were enumerated with the CellSearch system in 4 aliquots of 7.5 ml of blood before surgery and at several time-points during follow-up after surgery. The results showed that ≥1 CTC/30 ml of blood were detected in 44 (24%) patients before surgery. Patients with CTC before surgery had a significant decrease in recurrence-free survival (RFS, log-rank test p=0.014) and colon cancer related survival (CCRS, p=0.002). The 5-year RFS dropped from 75 to 61% and the 5-year CCRS from 83 to 69% for patients with CTC before surgery. The presence of CTC and positive lymph nodes remained significant factors in multivariate analysis for recurrence-free survival (RFS). Surprisingly, the presence of CTC weeks after surgery was not significantly associated with RFS and CCRD whereas CTC 2-3 years after surgery was again significantly associated with RFS and CCRD. The presence of CTC in patients with stage I-III CRC before surgery is associated with a significant reduction in RFS and CCRS. These findings suggest a role of CTC detection to assess which patients need adjuvant treatment.

Abstract 3064: Importance of circulating tumor cells in newly diagnosed colorectal cancer

Abstract Background: The presence of circulating tumor cells (CTC) is associated with poor prognosis in patients with metastatic colorectal cancer (CRC). This study was conducted to determine if the presence of CTC prior to surgery and during follow-up in patients with newly diagnosed non-metastatic CRC identifies patients who are at risk for disease recurrence. Methods: In a prospective single center study 183 patients with newly diagnosed non-disseminated CRC scheduled for surgery were enrolled from 2003 till 2008 and followed up for a median of 5.1 years. CTC were enumerated with the CellSearch System in 4 aliquots of 7.5 ml of peripheral blood before and after surgery (1-26 weeks), after adjuvant therapy and 1, 2, 3 and 4 years after surgery. Findings: ≥1 CTC/ 30ml of blood were detected in 44 (24%) patients before surgery. CTC frequency did not change significantly at the time points after surgery. Patients with CTC before surgery had a significant decrease in Recurrence Free Survival (RFS, logrank test p=0.014) and Colon Cancer Related Survival (CCRS, p=0.002). Five year RFS dropped from 75% to 61% and five year CCRS from 83% to 69% for patients with CTC before surgery. In a multivariate analysis of CTC, T-Stage and N-stage, the presence of CTC and N-stage remained as significant factors for RFS and CCRS. Surprisingly the presence of CTC after surgery was not significantly associated with RFS and CCRS whereas CTC 2-3 years after surgery was again significantly associated with RFS and CCRS. Interpretation: The presence of CTC in patients with stage I-III CRC before surgery is associated with a significant reduction RFS and CCRS. Although similar amounts of CTC were detected within 3 months after surgery they were not associated with RFS or CCRS. In contrast CTC were again highly significant for RFS and CCRS 2-3 years after surgery. These findings suggest a role of CTC detection, to assess which patients need adjuvant treatment. To implement CTC detection in the non-metastatic setting a validated CTC detection technology is needed with increased sensitivity and specificity. Citation Format: Guus van Dalum, Marco R. de Groot, Gerrit-Jan Stam, Loes F.A. Scholten, Walter J.B. Mastboom, Istvan Vermes, Arjan G.J. Tibbe, Leon W.M.M. Terstappen. Importance of circulating tumor cells in newly diagnosed colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2014-3064

Circulating tumor cells as a possible marker for micrometastatic disease in patients with localized pancreatic cancer.

175 Background: Circulating tumor cells (CTCs) are cells shed into circulation from tumors. They are increasingly recognized to be important biomarkers of disease burden in patients with solid tumors. Studies in pancreatic ductal adenocarcinoma (PDAC) have been limited due in part to low sensitivity of existing assays with extremely low numbers detected (0-15 per 7.5-15ml of blood). The development of newer microfluidic platforms has resulted in the ability to detect substantially greater numbers of CTCs. Methods: 15 patients with PDAC were enrolled in an IRB-approved study. Blood was collected in EDTA tubes and processed within 3 hours. CTCs were selected from 2-3ml of whole blood using monoclonal antibodies against epithelial cell adhesion molecule (EpCAM) and enumerated using a novel microfluidic platform. CTCs were confirmed by DAPI, CK8/18/19 and CD45 staining. Results: Patients with metastatic disease (n=12) had a mean of 29.7, median of 22 (3.5-107) CTCs per 1ml of blood. Patients with localized disease (n=3) had a mean of 3.8, median of 2.9 (2.3-6.2) CTCs per 1ml of blood. 0.5-1 CTCs per 1ml were detected in normal controls (n=2). The number of CTCs was significantly different between localized, metastatic and normal patients (p=0.01). 1 patient, initially thought to have localized disease by standard imaging but found to be metastatic at time of operation, had a mean of 45.9 CTCs per 1ml of blood compared to a mean of 3.8 CTCs per 1ml in patients who underwent a curative resection (p=0.009). Conclusions: Studies of CTCs in PDAC have been very limited. Our ability to detect large numbers of CTCs with good dynamic range suggests that further investigation into CTCs as a prognostic marker in PDAC is warranted. This is the first study that we are aware of to find CTCs in patients with localized disease. The presence of CTCs in patients with localized PDAC is surprising and may be associated with findings of unexpected metastatic disease at surgery. Further follow-up will be needed to determine if the presence of CTCs in these patients is a harbinger of shorter progression-free survival and overall survival after curative operations.

Abstract P2-10-26: Association between circulating tumor cells and molecular breast cancer subtypes

Abstract Background and Objective: Circulating tumor cells (CTCs) provide crucial information for the management of breast cancer patients. CTCs analysis can be used as a liquid biopsy approach for prognostic and predictive purposes in breast and other cancers. Several contradictory results presented about the breast cancer subtypes of CTCs recently. The aim of this study was therefore to analysis the association between the CTCs and molecular breast cancer subtypes. Methods: Blood samples were obtained from 143 breast cancer patients admitted to Breast Surgery Department, Affiliated Hospital of Medical College, Qingdao University. CTCs were detected by using immunomagnetic enrichment and a commercial kit, based on RT-PCR, was used to characterize CTCs. The expression of ER, PR, HER2 was detected by immunohistochemistry. The molecular subtypes were defined as: luminal A (ER+ and/or PR+, HER2−), luminal B (ER+ and/or PR+, HER2+), TNBC (ER−, PR−, HER2−), and HER2+, (ER−, PR−, and HER2+). The relations between the positive rate of CTCs and clinicopathological characteristics of primary tumor and molecular subtypes of breast cancer patients were analyzed. Results: Overall positive rate of CTCs was 39.8% (57/143). The positive rate of CTCs in patients with Luminal A, Luminal B, HER2+ and TNBC subtype was 33.8% (26/77), 47.1% (8/17),50.0% (6/12) and 45.9%(17/37). There was significant difference in positive rate of CTCs by different molecular subtypes (P = 0.0415), especially at different AJCC stages (P = 0.004). The positive rate of CTCs in patients with Luminal A subtype was significantly lower than patients with the others breast cancer subtypes (p = 0.0387). Conclusion: Our study suggests that presence of CTCs was more frequently found in patients with HER2+ and Luminal B subtypes. The presence of CTCs in patients with TNBC subtype was not as high as we wished. Theoretically, the positive rate of CTCs in TNBC subtype should be higher than that of others subtypes for being more aggressive histologically features and having increased potential to be invasive. One possible explanation for this might because of lacking epithelial markers expression and epithelial-mesenchyme transition (EMT) characteristics, CTCs of TNBC subtype are missed when using the standard EpCAM-based method. Shortage of patient's number was another possible reason. Further research on the Association between CTCs and molecular breast cancer subtypes will contribute to a better understanding of the biology of metastatic development in cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-26.

Prognostic value of circulating tumor cells in nonmuscle invasive bladder cancer: a CellSearch analysis

Circulating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC). Forty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration. CTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P < 0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category. The detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.

Abstract 4547: Clinical relevance of circulating tumor cells in esophageal cancer

Abstract Background: Current modalities to predict tumor recurrence and survival in esophageal cancer are insufficient. Locoregional and distant relapse is even common in lymph node-negative patients and more precise staging methods are therefore needed. Little is known about the utility of circulating tumor cells (CTC) as staging tool in esophageal cancer. Methods: In a prospective study we identified CTC in peripheral blood of esophageal cancer patients by two separate methods. We enrolled 85 consecutive esophageal cancer patients (03/2010-10/2011) who were surgically resected (R0) and patients received neither neoadjuvant nor adjuvant therapy. Peripheral blood was taken at the time of primary surgery and CTC were detected by either manual MACS enrichment (anti-cytokeratin plus anti-EpCam) with subsequent anti-cytokeratin staining or the automated CellSearch system (cleared by the FDA for CTC detection in metastatic breast, colon and prostate cancer) in parallel. CTC detection rates and correlation with clinicopathologic parameters were evaluated. The median clinical follow-up time is 11 months ranging from 1 to 19 months. Results: So far, 67 patients have been analysed of which 21 (31.3%) had CTCs using MACS, and 10 (14.9%) using CellSearch. CTCs detected with MACS significantly correlated with tumor size (p=0.040) and aggressive tumor biology as indicated by presence of regional lymph node (p=0.040) and distant metastases (p=0.019). CTCs detected with CellSearch correlated significantly with lymph node (p=0.044) and distant metastases (p=0.045), while no correlation was observed for tumor size or grading. CTC-positive patients had a worse overall survival than CTC-negative patients when tested with the CellSearch system (p=0.009). Conclusion: The presence of CTCs in patients with resectable esophageal cancer correlates with clinicopathologic risk factors.and predicts an unfavorable prognosis. Thus, our pilot study suggests that CTC detection may contribute to an improved staging in esophageal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4547. doi:1538-7445.AM2012-4547

P4-07-20: Apoptosis in Circulating Tumor Cells (CTCs) of Early and Metastatic Breast Cancer Patients.

Abstract Background: Metastasis has been associated with the presence of circulating (CTCs) and disseminated (DTCs) tumor cells in peripheral blood and bone marrow of breast cancer patients, respectively. Recent studies have confirmed that the detection of CTCs and DTCs represents a strong and independent prognostic factor for decreased disease-free and overall survival. However, it is not clear so far whether all CTCs are capable to generate metastasis or some of them are destined to die. The aim of the present study was to analyze the presence of apoptotic CTCs in patients with early and metastatic breast cancer. Patients and methods: Double staining immunofluorescent (IF) expreriments were performed in peripheral blood mononuclear cells (PBMC) cytospins of patients with early (n=23) and metastatic breast cancer (n=29), prior to the initiation of adjuvant and first-line chemotherapy, respectively. Pancytokeratin A45-B/B3 antibody (as a marker of CK-positive cells) was coupled with either M30 (marker of apoptotic cells) or Ki67 (marker of proliferating cells) antibodies. Apoptotic CTCs were also evaluated using a polycaspase detection kit. Results: Significantly lower proportions of apoptotic CTCs were detected in metastatic compared to early breast cancer patients using the polycaspase kit (3% vs 49%, p=0.0001). These results were confirmed with M30 staining (32% vs 67%, in metastatic and early disease, respectively, p=0.023). The median percentage of apoptotic CTCs per patient was also lower in advanced compared to early disease patients (0% vs 100% with the polycaspase kit and 15% vs 70% with M30 staining). Ki67 positive CTCs were identified in 56% of early and metastatic patients, while the ratio of Ki67-positive/Total CTCs was 15% and 36% respectively. Conclusions: Apoptotic CTCs are more commonly observed in early compared to metastatic breast cancer, whereas, Ki-67 positive CTCs are more frequently encountered in metastatic disease. The differential incidence of apoptotic and proliferative CTCs in early and metastatic disease is probably related to disease progression. The evaluation of these markers in CTCs from patients with breast cancer may provide useful prognostic information and could be used to monitor the effectiveness of therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-20.