Circulating Tumor Cells Levels Research Articles

Abstract P1-01-01: Circulating tumor cell number and CTC-endocrine therapy index predict clinical outcomes in ER positive metastatic breast cancer patients: Results of the COMETI Phase 2 trial

Abstract Introduction: Only half of hormone receptor positive (HR+) metastatic breast cancer (MBC) patients (pts) benefit from endocrine therapy (ET). Circulating tumor cells (CTC) are prognostic in pts with MBC using CellSearch® technology. The CTC-endocrine therapy index (CTC-ETI) provides semi-quantitative analyses of CTC-ER (estrogen receptor), BCL2, HER2, and Ki67 expression. We hypothesized that CTC-ETI high (elevated CTC number and/or low expression of ER and BCL2, and high expression of HER2 and Ki-67) might predict resistance to ET in a prospective, multi-institutional clinical trial: COMETI-P2-2012.0 (NCT01701050). Methods: 121 pts with ER+, HER2 negative (-), and progressive MBC after one or more lines of ET or within 12 months (mos) of completing adjuvant ET, who were initiating a new ET, were enrolled after informed consent. CTC and CTC-ETI were determined as previously reported (Paoletti C et al, CCR 2015) at baseline (BL), 1, 2, 3, and 12 mos, and/or at the time of progression. Imaging was performed every 3 mos. Association of CTC levels and CTC-ETI with patient outcomes (progression free survival (PFS); rapid progression (RP) defined as progression within 3 mos) was assessed using logrank and Fisher's exact tests. Trial design estimated 85 PFS and 51 RP events, providing >90% power (2-sided a=0.05); pts with unsuccessful BL CTC-ETI or ineligible were unevaluable. Only baseline (BL) data are reported in this abstract. Results: 32% of enrolled pts had progression within 12 mos of completing adjuvant ET, whereas 40%, 20%, and 8% had 1, 2, ≥3 lines of ET for MBC. CTC-ETI was successfully determined in 93% of pts (90% CI, 88% to 97%). CTC were ≥5 CTC/7.5 ml whole blood in 37/108 (34%) pts evaluable for clinical validity. Elevated CTC was associated with worse PFS (median (m) PFS: 3.3 vs. 5.9 mos; P<0.01). Low, intermediate, and high CTC-ETI were observed in 75 (69%), 6 (6%), and 27 (25%) pts, respectively. CTC-ETI was associated with PFS (logrank P<0.01): pts with low, intermediate, and high CTC-ETI had mPFS of 5.7, 8.5, and 2.8 mos, respectively. In the 96 pts eligible for determination, elevated CTC was associated with RP, (65.6% vs. 42.2%; P=0.05) as was CTC-ETI (P=0.003): 79.2% (95% CI, 57.8% to 92.9%) of pts with high CTC-ETI had RP versus 41.2% (95% CI, 29.4% to 53.8%) with low CTC-ETI; in the small group with intermediate CTC-ETI 1 of 4 pts (25%) had RP. Conclusions: In this multi-institutional, prospective study, CTC-ETI was accurately determined, confirming the previously established analytical validity of the assay, meeting the primary objective of the trial. Elevated CTC and CTC-ETI high compared to low were associated with poor outcomes to ET. CTC-ETI distribution resulted in a small number of patients assigned to the intermediate group, restricting our ability to associate this group with outcomes. These results suggest that CTC-biomarker phenotype and enumeration have clinical validity. CTC-ETI may identify ER+ HER2– MBC pts who are unlikely to benefit from ET and might be better treated with ET in combination with other therapies or proceed to chemotherapy. Further analyses including CTC-ETI at serial time points during ET are planned. Citation Format: Paoletti C, Regan MM, Liu MC, Marcom PK, Hart LL, Smith II JW, Tedesco KL, Amir E, Krop IE, DeMichele AM, Goodwin PJ, Block M, Aung K, Cannell EM, Darga EP, Baratta PJ, Brown ME, McCormack RT, Hayes DF. Circulating tumor cell number and CTC-endocrine therapy index predict clinical outcomes in ER positive metastatic breast cancer patients: Results of the COMETI Phase 2 trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-01.

Next-Generation CDK2/9 Inhibitors and Anaphase Catastrophe in Lung Cancer

The first generation CDK2/7/9 inhibitor seliciclib (CYC202) causes multipolar anaphase and apoptosis in lung cancer cells with supernumerary centrosomes (known as anaphase catastrophe). We investigated a new and potent CDK2/9 inhibitor, CCT68127 (Cyclacel). CCT68127 was studied in lung cancer cells (three murine and five human) and control murine pulmonary epithelial and human immortalized bronchial epithelial cells. Robotic CCT68127 cell-based proliferation screens were used. Cells undergoing multipolar anaphase and inhibited centrosome clustering were scored. Reverse phase protein arrays (RPPAs) assessed CCT68127 effects on signaling pathways. The function of PEA15, a growth regulator highlighted by RPPAs, was analyzed. Syngeneic murine lung cancer xenografts (n = 4/group) determined CCT68127 effects on tumorigenicity and circulating tumor cell levels. All statistical tests were two-sided. CCT68127 inhibited growth up to 88.5% (SD = 6.4%, P < .003) at 1 μM, induced apoptosis up to 42.6% (SD = 5.5%, P < .001) at 2 μM, and caused G1 or G2/M arrest in lung cancer cells with minimal effects on control cells (growth inhibition at 1 μM: 10.6%, SD = 3.6%, P = .32; apoptosis at 2 μM: 8.2%, SD = 1.0%, P = .22). A robotic screen found that lung cancer cells with KRAS mutation were particularly sensitive to CCT68127 ( P = .02 for IC 50 ). CCT68127 inhibited supernumerary centrosome clustering and caused anaphase catastrophe by 14.1% (SD = 3.6%, P < .009 at 1 μM). CCT68127 reduced PEA15 phosphorylation by 70% (SD = 3.0%, P = .003). The gain of PEA15 expression antagonized and its loss enhanced CCT68127-mediated growth inhibition. CCT68127 reduced lung cancer growth in vivo ( P < .001) and circulating tumor cells ( P = .004). Findings were confirmed with another CDK2/9 inhibitor, CYC065. Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.

EpCAM-based Flow Cytometric Detection of Circulating Tumor Cells in Gallbladder Carcinoma Cases

Purpose:Liquid biopsy has entered the arena of cancer diagnostics in the past decade and detection of circulating tumor cells (CTC) is one diagnostic component. CTCs in gallbladder cancer (GBC) have hitherto not been comprehensively analysed.Methods and Results:The current study focused on the diagnostic role of CTCs in 27 cases of treatment-naive GBC and 6 normal controls as well as 6 cases of cholecystitis. An EasySep kit featuring negative immunomagnetic bead separation and flow cytometric detection of EpCAM positive and CD45 negative cells revealed CTCs in 25 of the 27 cases. At a cut-off point of ≥1, the CTC count discriminated GBC from controls with a sensitivity, specificity and diagnostic accuracy of 92.6%, 91.7% and 92.3%, respectively. CTC levels in turn correlated significantly with clinico-pathological parameters of cases in terms of known prognostic indicators, with significant diagnostic potential at a cut-off point of >4, to discriminate disease stage I and II vs. III and IV GBC. With a cut-off of >3, the CTC count discriminated tumor stages I and II vs. III and IV and at >6 CTCs could discriminate metastatic vs. non metastatic GBCs with a sensitivity, specificity and diagnostic accuracy of 55. 6%, 100.0% and 85.2, respectively. A review of CTC in pancreatico-biliary malignancies is included.Conclusion:Detection and quantification of CTCs may serve as a non-invasive biomarker for GBC diagnosis in correlation with radiological studies.

Circulating tumour cells and pathological complete response: independent prognostic factors in inflammatory breast cancer in a pooled analysis of two multicentre phase II trials (BEVERLY-1 and -2) of neoadjuvant chemotherapy combined with bevacizumab

We present a pooled analysis of predictive and prognostic values of circulating tumour cells (CTC) and circulating endothelial cells (CEC) in two prospective trials of patients with inflammatory breast cancer (IBC) treated with neoadjuvant chemotherapy combined with neoadjuvant and adjuvant bevacizumab. Nonmetastatic T4d patients were enrolled in two phase II multicentre trials, evaluating bevacizumab in combination with sequential neoadjuvant chemotherapy of four cycles of FEC followed by four cycles of docetaxel in HER2-negative tumour (BEVERLY-1) or docetaxel and trastuzumab in HER2-positive tumour (BEVERLY-2). CTC and CEC were detected in 7.5 and 4 ml of blood, respectively, with the CellSearch System. From October 2008 to September 2010, 152 patients were included and 137 were evaluable for CTC and CEC. At baseline, 55 patients had detectable CTC (39%). After four cycles of chemotherapy, a dramatic drop in CTC to a rate of 9% was observed (P < 0.01). Pathological complete response (pCR) rate was 40%. No correlation was found between CTC or CEC levels and pCR rate. Median follow-up was 43 months. CTC detection (≥1 CTC/7.5 ml) at baseline was associated with shorter 3-year disease-free survival (39% versus 70% for patients without CTC, P < 0.01, HR 2.80) and shorter 3-year overall survival (OS) (P < 0.01). In multivariate analysis, independent prognostic parameters for shorter survival were absence of hormonal receptors, no pCR and CTC detection at baseline. CEC level at baseline or variations during treatment had no prognostic value. In this pooled analysis of two prospective trials in nonmetastatic IBC, detection rate of CTC was 39% with a strong and independent prognostic value for survival. Combination of pCR after neoadjuvant treatment with no CTC detection at baseline isolated a subgroup of IBC with excellent OS (94% 3-year OS), suggesting that CTC count could be part of IBC stratification in prospective trials.

Screening for Circulating Tumour Cells Allows Early Detection of Cancer and Monitoring of Treatment Effectiveness: An Observational Study

Background:Circulating-Tumour-Cells (CTC) provide a blood biomarker for early carcinogenesis, cancer progression and treatment effectiveness. An increase in CTCs is associated with cancer progression, a CTC decrease with cancer containment or remission. Several technologies have been developed to identify CTC, including the validated Isolation-by-Size-of-Epithelial-Tumour (ISET, Rarecells) technology, combining blood filtration and microscopy using standard histo-pathological criteria.Methods:This observational study compared CTC count to cancer status and cancer risk, by monitoring treatment effectiveness in cancer patients and by screening for CTC in asymptomatic patients with risk factors, including family history of cancer.Results:Between Sept-2014 and Dec-2016 we undertook 600 CTC tests (542 patients), including 50% screening requests of patients without cancer diagnosis but with risk factors. CTC were detected in all cancer patients (n=277, 100%), and in half of the asymptomatic patients screened (50%, 132 out-of 265 patients). Follow-up tests including scans, scheduled within 1-10 months of positive CTC tests, found early cancerous lesions in 20% of screened patients. In 50% of male patients with CTC and normal PSA (prostate-specific-antigen) levels, PSMA-PET scans revealed increased uptake in the prostate, indicative of early prostate cancer. Other types of cancers detected by CTC screening and subsequent scans included early breast, ovarian, lung, or renal cancer. Patients with CTC were advised on integrative approaches including immune-stimulating and anti-carcinogenic nutritional therapies. CTC repeat tests were available in 10% of patients with detected CTC (40 out-of 409 patients, n=98 CTC tests) to assess treatment effectiveness, suggesting nutritional therapies to be beneficial in reducing CTC count.Conclusions:CTC screening provided a highly sensitive biomarker for the early detection of cancer, with higher CTC counts being associated with higher risk of malignancy. CTC monitoring over time indicated treatment effectiveness. Nutrients with anti-carcinogenic properties could reduce CTC count, and included curcumin, garlic, green tea, grape seed, modified citrus pectin, and medicinal mushroom extract.

Effect of small and radical surgical injury on the level of different populations of circulating tumor cells in the blood of breast cancer patients.

Circulating tumor cells (CTCs) constitute a heterogeneous population. Some tumor cells are cancer stem cells (CSCs), while others are in the process of the epithelial-mesenchymal transition (EMT); however, most CTCs are neither stem cells nor in the EMT. This prospective study of 22 patients with nonspecific-type invasive carcinoma of the breast identified different populations of CTCs by flow cytometry in the blood of patients before biopsy, after biopsy and after surgical tumor removal without neoadjuvant chemotherapy. The results showed that minor surgical injury (biopsy) was accompanied by a significant increase in the blood levels of CTCs without signs of the EMT or stemness (Epcam+CD45-CD44-CD24-Ncadh-) and CTCs with signs of stemness and without signs of the EMT (Epcam+CD45-CD44+CD24-Ncadh-). Our results suggest that minor surgical injury to a tumor contributes to the release of CTCs into the bloodstream, including a population of stem cells.

P1.07-031 Clinical Evaluation of Folate Receptor-Positive Circulating Tumor Cells Detection in Patients with Small Cell Lung Cancer

Small cell lung cancer (SCLC) is distinguished by extremely high numbers of circulating tumor cells (CTCs) in comparison to other malignancies; however, the role of CTCs in evaluating chemotherapy effect of SCLC is to be further clarified. The purpose of this study was to investigate the predictive and prognostic role of folate receptor–positive CTCs in unresectable SCLC. In this single-center prospective study, blood samples for folate receptor–positive CTCs analysis were obtained from 80 patients with chemotherapy-naive unresectable SCLC at baseline, after two cycles of chemotherapy, and on disease progression. All patients received chemotherapy with EC or EP regimen for at least two cycles. CTCs number at baseline, after chemotherapy and changes with chemotherapy were evaluated as predictive factors for chemotherapy effect, along with clinical characteristics. Of all 80 patients, CTCs was detected at baseline as positive (CTCs>8.7 FU/3mL) in 67 patients, with the percentage of 83.8%, which was not associated with age, sex, smoking status or disease stage. In 72 evaluable patients, the disease control rate was 83.9% (52/62) and 50% (5/10) in CTCs positive and negative patients respectively (P=0.004). In CTCs positive patients, those harboring low levels of folate receptor (8.7<CTCs<14, n=29) were associated with longer PFS (275 days, 95%CI:82-468) than those with high expression levels (CTCs ≥14, n=38)(PFS: 210days, 95% CI:159-261; P=0.0458). Reduction in CTCs number after two cycles of chemotherapy was associated with partial response (P=0.049) but not with stable disease (P=0.47). The results of a multivariate analysis showed that the baseline CTCs level was an independent prognostic factor for survival time (HR=2.19; P=0.024). CTCs number at baseline could be used as a useful prognostic biomarker for SCLC. Reduction in CTCs number with chemotherapy could predict better chemotherapy effect of SCLC.

P1.05-009 Clinical Value of Circulating Tumor Cell in the Differential Diagnosis of Solitary Pulmonary Nodule

The diagnosis of lung cancer suffers from the lack of accurate, noninvasive and early diagnostic tests. Low-dose helical computed tomography (LDCT) identifies millions of solitary pulmonary nodules (SPN) annually, many of which are undiagnosed as either malignant or benign. When removed surgically, 18%-25% of the nodules are benign, which leads to unnecessary treatment procedures for surgeons, stress and panic for patients and waste of medical resources for government. Therefore, an accurate noninvasive test that can discriminate benign SPN from malignant is urgently needed. Circulating tumor cells (CTCs) are cells shed from either primary or secondary tumors that migrate into the circulatory system and exist at the early stage of cancer. In recent years, CTC has become the research hotspot because of its great significance in the early diagnosis of cancer, disease monitoring, prognosis evaluation and guiding individualized treatment. In this study, we evaluated the application value of CTC in the differential diagnosis of SPN. Peripheral blood samples were collected from 134 patients with solitary pulmonary nodule in Shanghai Chest Hospital from September 2013 to January 2015, including 80 patients with malignant nodule and 54 with benign nodule. CTC levels of the above subjects were detected by LT-PCR (ligand-targeted polymerase chain reaction, LT-PCR) assay, and serum CEA and CYFRA21-1 were detected by flow fluorescence assay. The CTC levels of malignant SPN patients were significantly higher than that of benign SPN patients (P<0.001). The area under the Receiver Operating Characteristic (ROC) curves of CTC and CEA were 0.817(95% CI: 0.743∼0.891) and 0.613(95% CI: 0.508∼0.718) respectively, while the CYFRA21-1 had no significant meaning in the differential diagnosis of SPN. The positive and negative predictive value (PPV and NPV) in differential diagnosis of SPN for CTC were 89% and 74%. Then the patients were divided into three groups according to the nodule diameter to evaluate the diagnostic value of CTC in SPN with different size. For SPN with diameter less than 8 mm, the PPV and NPV of CTC were 86% and 57%; For SPN with diameter between 8 mm and 20 mm, the PPV and NPV of CTC were 88% and 81%; For SPN with diameter greater than 20 mm, the PPV and NPV of CTC were 92% and 68%. Compared with traditional tumor marker, CTC detection could provide more clinical value in differential diagnosis of solitary pulmonary nodule.

P2.03b-092 Predictive and Prognostic Effect of Circulating Tumor Cells in Non-Small Cell Lung Cancer Treated with Targeted Therapy: Topic: Biomarkers

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 and the feasibility whether CTCs as a prognostic factor in advanced NSCLC with EGFR mutation/ALK translocation. This was a prospective, unopen-labeled, single-center trial. From July 25, 2014 to March 11,2016, 308 advanced NSCLC patients were enrolled to quantified CTC level by negative enrichment using immunomagnetic beads in combination with folate receptor-directed polymerase chain reaction(PCR) that allows secondary amplification of tiny amounts of CTCs in 3mL peripheral blood before the start of targeted therapy and after one month, every two months hereafter of treatment. Among whom, 272 NSCLC patients with EGFR mutation (exon 19Del mutation: n=135, L858R mutation: n=137), 39 ALK translocation or undefined lung cancer patients. Sequential analyses were conducted to monitor CTC values during therapy and correlate radiological effects with treatment outcome. There was no significant difference between CTC values and patients’ characteristics including stage (P= 0.1015), EGFR mutation status (19 del:14.5 CTCs, L858R:12.6 CTCs, P=0.1868), age (≤60 versus >60 years), gender, smoking status. Of 272 eligible and evaluable patients received EGFR-TKI, we found that patients harboring lower CTC levels (<20.5) were associated with longer PFS (432 days, 95%CI:332.7-541.3) than those with higher CTC levels (≥20.5) (PFS: 308days, 95% CI: 245.3-370.7; P=0.017). Patients with CTC <20.5 had a DCR of 77.11% compared with 58.49% in CTC >20.5 groups (P=0.008), patients with CTC <20.5 had a ORR of 51.20% compared with 33.96% in CTC >20.5 groups (P=0.029). Decreased CTC values correlated well with partial response after one month or three months’ treatment of EGFR-TKI (P=0.0082 and P=0.0023), but not with stable disease (P=0.1843 and P=0.8606). Multivariate analysis showed that CTC level was an independent prognostic factor for PFS (CTC level<20.5 vs ≥20.5, hazard ratio, 0.497; P=0.014). The change of CTCs correlated significantly with radiological response. This strategy may enable non-invasive, predictive and prognostic, specific biomarker in patients undergoing targeted therapies.

Diagnostic Value of Folate Receptor-positive Circulating Tumor Cell in Lung Cancer: A Pilot Study

背景与目的评价一种通过叶酸受体(folate receptor, FR)检测循环肿瘤细胞(circulating tumor cell, CTC)的方法用于肺癌临床诊断的实用性和可行性及进一步探究CTC在肺癌术后复发的预测价值。方法通过免疫磁珠负向富集方法从3 mL外周血中捕获循环肿瘤细胞，再用肿瘤特异性叶酸配体-寡核苷酸偶和物标记捕获的循环肿瘤细胞，洗去没有结合的偶和物后，洗脱下特异性结合的偶合物的寡核苷酸用于定量PCR扩增分析。结果97例肺癌患者的CTC水平高于肺部良性疾病患者(P < 0.001)。本检测方法以8.7 Folate Units/3 mL为cutoff值，结果显示靶向PCR法对肺癌的检测灵敏度为82.5%，特异性为72.2%，特别是在Ⅰ期肺癌灵敏度达到86.8%。与其他肿瘤标志物(NSE、CEA、CYFRA21-1)比较，CTC对肺癌及Ⅰ期肺癌具有较高的诊断准确性(0.859; 95%CI: 0.779-0.939)和(0.912; 95%CI: 0.829-0.994)。5例肺癌患者术后2周内CTC水平高于cutoff值。结论叶酸受体阳性循环肿瘤细胞可以应用于肺癌的临床诊断，即使是对早期非小细胞肺癌(non-small cell lung cancer, NSCLC)的诊断。

Point of care assessment of melanoma tumor signaling and metastatic burden from μNMR analysis of tumor fine needle aspirates and peripheral blood

This study evaluates μNMR technology for molecular profiling of tumor fine needle aspirates and peripheral blood of melanoma patients. In vitro assessment of melanocyte (MART-1, HMB45) and MAP kinase signaling (pERK, pS6K) molecule expression was performed in human cell lines, while clinical validation was performed in an IRB-approved study of melanoma patients undergoing biopsy and blood sampling. Tumor FNA and blood specimens were compared with BRAF genetic analysis and cross-sectional imaging. μNMR in vitro analysis showed increased expression of melanocyte markers in melanoma cells as well as increased expression of phosphorylated MAP kinase targets in BRAF-mutant melanoma cells. Melanoma patient FNA samples showed increased pERK and pS6K levels in BRAF mutant compared with BRAF WT melanomas, with μNMR blood circulating tumor cell level increased with higher metastatic burden visible on imaging. These results indicate that μNMR technology provides minimally invasive point-of-care evaluation of tumor signaling and metastatic burden in melanoma patients.

Clinical value of circulating tumor cell in the differential diagnosis of solitary pulmonary nodule

Objective To evaluate the application value of circulating tumor cell (CTC) in the differential diagnosis of solitary pulmonary nodule (SPN). Methods Peripheral blood samples were collected from 134 patients with solitary pulmonary nodule in Shanghai Chest Hospital from September 2013 to January 2015, including 80 patients with malignant nodule and 54 with benign nodule. CTC levels of the above subjects were detected by ligand-targeted polymerase chain reaction (LT-PCR) assay, and serum carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1) were detected by flow fluorescence assay. Results By Mann-Whitney U Test, the CTC levels of malignant SPN patients [11.06(8.77-14.41)units/3 ml] were significantly higher than those of benign SPN patients [6.65(4.49-7.84)units/3 ml] (Z=-6.217, P<0.001). The sensitivity and specificity of differential diagnosis of SPN for CTC were 80%(64/80)and 85%(46/54)respectively. According to the diameter of SPN, the patients were divided into three groups to evaluate the diagnostic value of CTC in SPN with different size. For SPN with diameter less than 8 mm, the sensitivity and specificity of CTC were 6/9 and 4/5 respectively. For SPN with diameter between 8 mm and 20 mm, the sensitivity and specificity of CTC were 83%(35/42) and 85%(29/34). For SPN with diameter greater than 20 mm, the sensitivity and specificity of CTC were 79% (23/29)and 13/15. Conclusion Comparing with the traditional tumor markers, CTC could provide more clinical value in the differential diagnosis of solitary pulmonary nodule. (Chin J Lab Med, 2016, 39: 941-945) Key words: Solitary pulmonary nodule; Neoplastic cells, circulating; Diagnosis, differential

Early detection of poor outcome in patients with metastatic colorectal cancer: tumor kinetics evaluated by circulating tumor cells.

BackgroundColorectal cancer (CRC) is the third most prevalent cancer worldwide. New prognostic markers are needed to identify patients with poorer prognosis, and circulating tumor cells (CTCs) seem to be promising to accomplish this.Patients and methodsA prospective study was conducted by blood collection from patients with metastatic CRC (mCRC), three times, every 2 months in conjunction with image examinations for evaluation of therapeutic response. CTC isolation and counting were performed by Isolation by Size of Epithelial Tumor Cells (ISET).ResultsA total of 54 patients with mCRC with a mean age of 57.3 years (31–82 years) were included. Among all patients, 60% (n=32) were carriers of wild-type KRAS (WT KRAS) tumors and 90% of them (n=29) were exposed to monoclonal antibodies along with systemic treatment. Evaluating CTC kinetics, when we compared the baseline (pretreatment) CTC level (CTC1) with the level at first follow-up (CTC2), we observed that CTC1-positive patients (CTCs above the median), who became negative (CTCs below the median) had a favorable evolution (n=14), with a median progression-free survival (PFS) of 14.7 months. This was higher than that for patients with an unfavorable evolution (CTC1− that became CTC2+; n=13, 6.9 months; P=0.06). Patients with WT KRAS with favorable kinetics had higher PFS (14.7 months) in comparison to those with WT KRAS with unfavorable kinetics (9.4 months; P=0.02). Moreover, patients whose imaging studies showed radiological progression had an increased quantification of CTCs at CTC2 compared to those without progression (P=0.04).ConclusionThis study made possible the presentation of ISET as a feasible tool for evaluating CTC kinetics in patients with mCRC, which can be promising in their clinical evaluation.

Correlation between epidermal growth factor receptor tyrosine kinase inhibitor efficacy and circulating tumor cell levels in patients with advanced non-small cell lung cancer.

ObjectiveThe aim of this study was to investigate the correlation between the efficacy of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and circulating tumor cell (CTC) levels in patients with advanced non-small cell lung cancer (NSCLC). The efficacy of EGFR-TKIs in reducing CTC counts in patients with advanced NSCLC was studied.Patients and methodsA total of 66 patients with advanced NSCLC were enrolled and divided into two groups (those with high CTC counts and those with low CTC counts) based on the patients’ median CTC counts. All the patients were treated with an EGFR-TKI, and the treatment efficacy and prognoses were compared.ResultsThe treatment efficacies were 53.3% (16/30) and 27.8% (10/36) for the low CTC group and high CTC group, respectively, and this difference was statistically significant (P<0.05). The median overall survival was 22.8 months (95% confidence interval [CI]: 18.9–26.8 months) for the low CTC group and 18.3 months (95% CI: 2.9–8.2 months) for the high CTC group. The median progression-free survival was 11.5 months (95% CI: 8.1–15 months) and 5.6 months (95% CI: 2.9–8.2 months) for the low and high CTC groups, respectively, and the difference was statistically significant (P<0.05).ConclusionThe CTC count can be used as an index for predicting the EGFR-TKI effect on patients with advanced NSCLC. Efficacy and prognosis of EGFR-TKI treatment and CTC count were considered important, and the CTC count could be used to predict the efficacy of EGFR-TKI treatment and prognosis of advanced NSCLC. The change in CTC expression levels can be used as an index for evaluating the prognosis of patients with advanced NSCLC.

Multiparameter Evaluation of the Heterogeneity of Circulating Tumor Cells Using Integrated RNA In Situ Hybridization and Immunocytochemical Analysis.

Circulating tumor cells (CTCs) are routinely identified as cytokeratin (CK)-positive, epithelial cell adhesion molecule (EpCAM)-positive, and CD45-negative and are enriched based on EpCAM. However, there are a number of methodological challenges regarding both isolation and characterization of these rare CTCs including downregulation or absence of EpCAM in a variety of solid tumors leading to the omission of subpopulations of CTCs, difficulties in analyzing RNA and protein targets in CTCs due to the rarity of these cells, and low levels of targets and technological limitations of visualizing the targets of interest on each individual cell. Building on our previous CTC research on CD45-based negative magnetic separation and four-color fluorescent immunocytochemical (ICC) staining, RNA in situ hybridization (ISH) was applied to fluorescently target mRNA sequences corresponding to tumor-related genes at the single CTC level. Multiple categories of markers are targeted including CK, human epidermal growth factor receptor family markers, Hedgehog pathway markers, human papillomavirus markers, and protein arginine methyltransferase 5. In addition, an integrated method of RNA ISH and fluorescent ICC staining was developed to visualize CTCs on both mRNA and protein levels. The robustness of the integrated co-ICC and RNA ISH staining was demonstrated by a series of tests on representative tumor markers of different categories. The integrated staining can incorporate the advantages of both RNA ISH and fluorescent ICC staining and provide more intense signals and more specific bindings. With this integrated staining methodology, distinct staining patterns were applied in this report to facilitate the searching and characterization of rare subgroups of CTCs. These results support the existence of diverse groups of CTCs at both protein and mRNA transcript levels and provide an analytical tool for the research on CTCs of rare subgroups.

Detection and Prognostic Significance of Circulating Tumor Cells in Patients With Metastatic Thyroid Cancer.

Individual patient prognostication for advanced thyroid cancer (TC) is challenging. Circulating tumor cells (CTCs) have been shown to be a valuable prognostic marker for other solid cancers. We hypothesized that CTCs are present in the blood of patients with advanced TC and their number can predict overall survival (OS). This is a prospective study at a tertiary cancer hospital. Patients, Interventions, and Main Outcome Measures: Initial studies were performed with TC cell lines to determine the feasibility of detection using the Veridex CellSearch. CTC enumeration was performed in blood samples from 18 patients with distantly metastatic medullary TC (metMTC), 14 with distantly metastatic differentiated TC (metDTC), and 10 controls with a history of TC but no evidence of disease. The prognostic value of CTC levels to predict OS in metMTC patients was assessed. CellSearch detected cells from MTC and DTC but not anaplastic TC cell lines. Six metMTC patients but no metDTC or control patients had more than or equal to 5 CTCs detected by the CellSearch assay. Median survival in metMTC patients with more than or equal to 5 CTCs was 13 months vs 51.5 months for those with less than 5 CTCs (P = .0116). The hazard ratio for mortality of patients with more than or equal to 5 CTCs compared with those with less than 5 CTCs was 3.95 (1.20-13.0, P = .0245). The presence of more than or equal to 5 CTCs in patients with metMTC is associated with worse OS. Larger cohorts are required to validate the prognostic value of CTC enumeration.

Diagnostic value of human circulating tumor cell detection for non-small cell lung cancer

Objective To estimate the diagnostic value of circulating tumor cell detection for non-small cell lung cancer. Methods A Non-intervention clinical study was conducted in this research. From October 2014 to April 2015, totally 162 NSCLC who presented at Thoracic Surgery Department, 119 benign pulmonary disease and 52 healthy individuals were collected from Shanghai Chest Hospital. Folate receptor (FR) based polymerase chain reaction (PCR) method was used to detect the circulating tumor cell (CTC) level, CEA and CYFRA21-1 was detected by the flowcytometry fluorescence luminance method, SCC was detected with Chemiluminescent microparticle immunoassay. The differences among groups were analyzed by the Kruskal-Wallis test(multi group comparison) and the Mann-Whitney U test(two group comparison), and the chi-square test was used in the positive rate comparison; the Receiver Operating Characteristics (ROC) curve was established. Results The median level of CTC in NSCLC patients was 11.90 Units/3 ml, which was significantly higher than those of benign pulmonary disease (6.72 CTC Units/3 ml) and healthy individuals (5.82 CTC Units/3 ml, χ2=125.990, P<0.01). Areas Under Curve (AUCs) of ROC curve for NSCLC was 0.853 2(95% CI: 0.809 5, 0.896 9). The cut-off value for discriminating NSCLC with benign pulmonary disease/healthy people was 8.74 CTC Units/3 ml with sensitivity being 77.16% and specificity being 90.06%.The positive rate of CTC in Stage I NSCLC patients was 68.7%, which was much higher than that of the combination of tumor markers(χ2=32.98, P<0.01). Conclusion With relatively high sensitivity and specificity, the detection of circulating tumor cell may has a clinical value of application and extension. (Chin J Lab Med, 2016, 39: 589-594) Key words: Carcinoma, non-small-cell lung; Neoplastic cells, circulating; Tumor markers, biological

Abstract 2252: Correlation of baseline value of folate receptor-positive circulating tumor cells and efficacy of pemetrexed and dynamic monitoring study in non-small cell lung cancer patients receiving first-line platinum-based chemotherapy

Abstract Background: The efficacy of pemetrexed associates with the expression of folate enzymes, however, few serological marker is available to predict its efficacy. Moreover, dynamic monitoring of circulating tumor cell (CTC) count can predict the outcome of tumor therapy, the progress of tumor and the overall survival of the patient. By quantitative determination of folate receptor-positive CTCs, this study investigated the correlation of its baseline values to the efficacy of pemetrexed and performed a dynamic monitoring study in non-small cell lung cancer (NSCLC) patients who underwent first-line platinum-based chemotherapy. Methods: This study enrolled 70 NSCLC patients who received first-line platinum-based chemotherapy. The CTCs were quantified by negative enrichment using immunomagnetic beads in combination with folate receptor-directed polymerase chain reaction(PCR) that allows secondary amplification of tiny amounts of CTCs in peripheral blood, thereby enhancing the sensitivity of detection. In this study, the CTC levels were examined by collecting 3mL of anti-ENTA whole blood samples before the treatment and after every chemotherapy, and followed up until progressive disease (PD) or completion of first-line chemotherapy. Based on the changes in CTC levels, we evaluated the correlation of the changes in CTC levels and the progress of the disease as well as the correlation of the CTC levels and the therapeutic outcomes. Results: The CTC level of seventy patients was 13.68 CU/3mL. Of twenty-two patients who received pemetrexed disodium/platinum combined therapy (AP/AC), the patients harboring high levels of folate receptor showed greater efficacy than those with low expression levels (8.7&amp;lt;CTC level&amp;lt;16, n = 7; PFS: 448 vs.94days, P = 0.0199; ORR: 75% vs.11%). In the dynamic monitoring study, the CTC level (AUC = 0.8026,P = 0.0033), the CTC ratio (AUC = 0.8422,P = 0.0003), the rate of CTC changes (OR = 102.005,P = 0.0012) after the second chemotherapy and the CTC level (AUC = 0.9487,P&amp;lt;0.0001), the CTC ratio (AUC = 0.8889,P&amp;lt;0.0001), the changing rate of CTCs (OR = 51.662,P = 0.0031) after the fourth chemotherapy positively correlated to the disease progression. Conclusions: The patients with high expression of folate receptor-positive CTCs appear to have superior response to pemetrexed than those with low expression. Additionally, the changes of CTC count can be used as a dynamic monitoring indicator in the treatment process to evaluate tumor burden and therapeutic outcomes. Citation Format: Xiaoxia Chen, Caicun Zhou, Xuefei Li. Correlation of baseline value of folate receptor-positive circulating tumor cells and efficacy of pemetrexed and dynamic monitoring study in non-small cell lung cancer patients receiving first-line platinum-based chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2252.

Abstract 2247: A multicenter clinical trial of lung cancer circulating tumor cell assay with the largest sample size (1210 cases) in China

Abstract Background: As a novel biomarker of primary tumors, circulating tumor cells (CTCs) are well known to play an important role in cancer diagnosis, recurrence and metastasis, and disease monitoring. This study investigated the application of CTCs in lung cancer therapy by quantitative determination of folate receptor-positive CTCs. Methods: This study enrolled 1210 subjects (including 560 lung cancer patients, 350 patients with benign lung diseases, 150 healthy subjects and 150 non-lung cancer malignant tumor patients) and quantified the tiny amounts of CTCs in peripheral blood by negative enrichment using immunomagnetic beads in combination with folate receptor-directed polymerase chain reaction (PCR). Results: Following ROC curve analyses of CTC levels in the patients with benign lung diseases or benign lung diseases and the healthy subjects, the cut-off of CTCs was 8.70 CTC Units/3mL with a specificity of 88.2% (441/500) and a sensitivity of 79.6% (446/560) as determined by the method mentioned above. The ROC area under curve of more than 0.8 (AUC = 0.8797, P&amp;lt;0.0001) suggested the excellence of ligand-directed PCR to diagnose lung cancer. For stage II-IV lung cancer patients, the sensitivities were 70.8%(34/48), 79.6% (125/157), and 90.2% (185/205) respectively, and the sensitivity in stage I patients can reach up to 68.0% (102/150) as well. CTC levels appeared to have greater accuracy to diagnose lung cancer (AUC: 0.8833, P&amp;lt;0.0001) than the combination of the other four lung cancer-specific serum tumor markers (CEA, NSE, CYFRA21-1 and SCC)(AUC: 0.8557, P&amp;lt;0.0001). CTCs were related to tumor stage and tumor size other than pathological type. By quantitative determination of folate receptor-positive cells in 150 different types of cancers, the CTC level in lung cancer was found to be higher than those in gastric, breast, colorectal, liver, and esophageal cancers. Conclusions: LT-PCR achieves quantitative determination of folate receptor-positive CTCs to diagnose lung cancer with high sensitivity and specificity, and significantly greater diagnosis accuracy than the model of combined lung cancer-specific serum tumor markers. Citation Format: Jiatao Lou, Caicun Zhou, Jing Wu, Lihua Qiao, Xiaohui Liang, Xiaoqian Wang, Xiaoxia Chen, Xuefei Li, Chao Zhao. A multicenter clinical trial of lung cancer circulating tumor cell assay with the largest sample size (1210 cases) in China. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2247.

Abstract 3954: Prospective evaluation of circulating tumor cells (CTCs) in head and neck cancer patients receiving definitive radiotherapy with a nanotechnology based system

Abstract Background: Circulating tumor cells (CTCs) are an important biomarker in cancer. There has been substantial interest in utilizing CTCs to enable personalized treatment. However, there is limited data on CTCs in patients with head and neck cancers (HNC) where there is growing data that curative treatment needs to be risk-based. The purpose of this prospective, correlative study is to investigate the clinical significance of CTCs, as measured by a highly sensitive CTC capture technology UiChip, in HNC patients undergoing definitive treatment. Methods: HNC patients (M0) undergoing definitive treatment (radiation+/- chemotherapy) were enrolled. Peripheral blood was collected prior to starting RT, at the first week, mid-point, final week of treatment, and every 4 to 12 weeks after completion. Quantification of CTCs was conducted using UiChip. The primary endpoint was change in CTCs pre- and post- RT. Results: 35 patients are included in this analysis (median age 58, 34% non-smokers, 69% HPV/p16 positive, and 17% node (-)) and 90% received chemotherapy. CTCs were detected in all patients pre-RT (100%). There was no association between pre-RT CTC level and tumor or nodal stage. Median CTCs significantly decreased from 71 CTCs/mL (range, 7-849) before RT to 32 (2-209) at completion of RT and 27 (2-78) at 4 to 12 weeks post-treatment. Ten patients had persistent disease and/or distant metastasis at a median follow-up of 10.6 months (range, 3.7-17). CTCs declined throughout treatment in patients with complete clinical and/or radiographic responses, in contrast to an elevation in CTCs at mid or post-RT in the 7 patients with pathologic residual disease/distant failures. Conclusions: We demonstrated that the novel UiChip can capture CTCs in a diverse population of head and neck cancer patients. Our pilot data suggest that individual patient CTC changes during and after treatment may be a predictive biomarker for radiotherapy response and clinical outcomes. Table: Median CTCs/mL Peripheral Blood (Median% of Pre-Treatment Baseline Count)Pre-RTMid-RTPost-RTNumber (%) of CTCs in Complete Responders to RT (N = 18)67 IQR: 39-11737 (38%) IQR: 22-79 (18-94%)21 (18%) IQR: 7-40 (13-42%)Number (%) of CTCs in Partial Responders to RT (N = 7)120 IQR: 36-30366 (123%) IQR: 20-134 (30-172%)51 (45%) IQR: 23-54 (17-63%) Citation Format: Joseph M. Caster, Michael J. Eblan, Kyle Wang, Ja Hye Myung, Bhishamjit Chera, Seungpyo Hong, Andrew Z. Wang. Prospective evaluation of circulating tumor cells (CTCs) in head and neck cancer patients receiving definitive radiotherapy with a nanotechnology based system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3954.