Low Circulating Tumor Cells Count Research Articles

Frequency and Prognostic Value of Circulating Tumor Cells in Cancer of Unknown Primary.

Cancer of unknown primary (CUP) is defined as a primary metastatic malignancy, in which the primary tumor remains elusive in spite of a comprehensive diagnostic workup. The frequency and prognostic value of circulating tumor cells (CTCs), which are considered to be the source of metastasis, has not yet been systematically evaluated in CUP. A total of 110 patients with a confirmed diagnosis of CUP according to the European Society for Medical Oncology (ESMO) guidelines, who presented to our clinic between July 2021 and May 2023, provided blood samples for CTC quantification using CellSearch methodology. CTC counts were correlated with demographic, clinical, and molecular data generated by comprehensive genomic profiling of tumor tissue. CTCs were detected in 26% of all patients at initial presentation to our department. The highest CTC frequency was observed among patients with unfavorable CUP (35.5%), while patients with single-site/oligometastatic CUP harbored the lowest CTC frequency (11.4%). No statistically significant association between CTC positivity and the number of affected organs (P = 0.478) or disease burden (P = 0.120) was found. High CTC levels (≥5 CTCs/7.5 mL; 12/95 analyzed patients) predicted for adverse overall survival compared to negative or low CTC counts (6-months overall survival rate 90% vs 32%, log-rank P < 0.001; HR 5.43; 95% CI 2.23-13.2). CTC dynamics were also prognostic for overall survival by landmark analysis (log-rank P < 0.001, HR 10.2, 95% CI 1.95-52.9). CTC frequency is a strong, independent predictor of survival in patients with CUP. CTC quantification provides a useful prognostic tool in the management of these patients.

A Novel Karyoplasmic Ratio-Based Automatic Recognition Method for Identifying Glioma Circulating Tumor Cells.

BackgroundDetection of circulating tumor cells (CTCs) is a promising technology in tumor management; however, the slow development of CTC identification methods hinders their clinical utility. Moreover, CTC detection is currently challenging owing to major issues such as isolation and correct identification. To improve the identification efficiency of glioma CTCs, we developed a karyoplasmic ratio (KR)-based identification method and constructed an automatic recognition algorithm. We also intended to determine the correlation between high-KR CTC and patients’ clinical characteristics.MethodsCTCs were isolated from the peripheral blood samples of 68 glioma patients and analyzed using DNA-seq and immunofluorescence staining. Subsequently, the clinical information of both glioma patients and matched individuals was collected for analyses. ROC curve was performed to evaluate the efficiency of the KR-based identification method. Finally, CTC images were captured and used for developing a CTC recognition algorithm.ResultsKR was a better parameter than cell size for identifying glioma CTCs. We demonstrated that low CTC counts were independently associated with isocitrate dehydrogenase (IDH) mutations (p = 0.024) and 1p19q co-deletion status (p = 0.05), highlighting its utility in predicting oligodendroglioma (area under the curve = 0.770). The accuracy, sensitivity, and specificity of our algorithm were 93.4%, 81.0%, and 97.4%, respectively, whereas the precision and F1 score were 90.9% and 85.7%, respectively.ConclusionOur findings remarkably increased the efficiency of detecting glioma CTCs and revealed a correlation between CTC counts and patients’ clinical characteristics. This will allow researchers to further investigate the clinical utility of CTCs. Moreover, our automatic recognition algorithm can maintain high precision in the CTC identification process, shorten the time and cost, and significantly reduce the burden on clinicians.

Neutrophil-Lymphocyte Ratio and Circulating Tumor Cells Counts Predict Prognosis in Gastrointestinal Cancer Patients.

PurposeThe purpose of this study is to explore the prognostic value of associating pre-treatment neutrophil–lymphocyte ratio (NLR) with circulating tumor cells counts (CTCs) in patients with gastrointestinal cancer.Materials and MethodsWe collected the related data of 72 patients with gastric cancer (GC) and colorectal cancer (CRC) who received different therapies from August 2016 to October 2020, including age, gender, primary tumor location, TNM stage, tumor-differentiation, NLR, CTCs, disease-free survival (DFS) and overall survival (OS). We chose the optimal cut-off value of NLR >3.21 or NLR ≤3.21 and CTC >1 or CTC ≤1 by obtaining receiver operating characteristic (ROC) curve. The Kaplan–Meier survival analysis and Cox regression analysis were used to analyze DFS and OS. To clarify the role of the combination of NLR and CTCs counts in predicting the prognosis, we analyzed the DFS and OS when associated NLR and CTCs counts.ResultsA high NLR (>3.21) was associated with shorter DFS (P <0.0001) and OS (P <0.0001). Patients with high CTCs level (>1) had shorter DFS (P = 0.001) and OS (P = 0.0007) than patients with low CTCs level. Furthermore, patients who had both higher NLR and higher CTCs counts had obvious shorter DFS (P <0.0001) and OS (P <0.0001).ConclusionsPatients with higher NLR and more CTCs respectively tended to have poor prognosis with shorter DFS and OS, which might be regarded as predictors of gastrointestinal cancer. In particular, associating NLR and CTCs counts might be a reliable predictor in patients with gastrointestinal cancer.

Circulating Tumor Cells Undergoing the Epithelial-Mesenchymal Transition: Influence on Prognosis in Cytokeratin 19-Positive Hepatocellular Carcinoma.

PurposeThe purpose of this study was to elucidate the relationship between cytokeratin 19 (CK19) expression and levels of circulating tumor cells (CTCs) in preoperative peripheral blood of patients with hepatocellular carcinoma (HCC), and the potential influence of that relationship on prognosis.Patients and MethodsCanPatrol™ CTC-enrichment technique and in situ hybridization (ISH) were used to enrich and classify CTCs undergoing the epithelial–mesenchymal transition (EMT) from blood samples of 105 HCC patients. CK19 immunohistochemistry staining was performed on HCC tissues and compared with demographic and clinical data.ResultsIn total, 27 of 105 (25.7%) HCC patients were CK19-positive. CK19-positive patients had significantly lower median tumor-free survival (TFS) than CK19-negative patients (5 vs 10 months, P = 0.047). In total, 98 (93.3%) patients showed pre-surgery peripheral blood CTCs (range: 0–76, median: 6), and 57 of 105 (54.3%) patients displayed CTC counts ≥6. Furthermore, CK19-positive patients with CTC count ≥6 showed significantly higher percentage than CK19-negative ones (77.8% vs 46.2%, P = 0.004). CK19-positive patients showed a significantly higher proportion of mesenchymal CTCs among CTCs undergoing EMT than CK19-negative patients (mean rank: 62.28 vs 49.79, P = 0.046). We also found that CK19-positive patients with high CTC count showed significantly shorter median tumor-free survival than CK19-negative patients with low CTC count (5 vs 16 months, P = 0.039).ConclusionHigh CTC count and high percentage of mesenchymal CTCs are closely related to the expression of CK19, which is associated with poor prognosis in HCC patients.

Tumor Evolution and Therapeutic Choice Seen through a Prism of Circulating Tumor Cell Genomic Instability.

Circulating tumor cells (CTCs) provide an accessible tool for investigating tumor heterogeneity and cell populations with metastatic potential. Although an in-depth molecular investigation is limited by the extremely low CTC count in circulation, significant progress has been made recently in single-cell analytical processes. Indeed, CTC monitoring through molecular and functional characterization may provide an understanding of genomic instability (GI) molecular mechanisms, which contribute to tumor evolution and emergence of resistant clones. In this review, we discuss the sources and consequences of GI seen through single-cell analysis of CTCs in different types of tumors. We present a detailed overview of chromosomal instability (CIN) in CTCs assessed by fluorescence in situ hybridization (FISH), and we reveal utility of CTC single-cell sequencing in identifying copy number alterations (CNA) oncogenic drivers. We highlight the role of CIN in CTC-driven metastatic progression and acquired resistance, and we comment on the technical obstacles and challenges encountered during single CTC analysis. We focus on the DNA damage response and depict DNA-repair-related dynamic biomarkers reported to date in CTCs and their role in predicting response to genotoxic treatment. In summary, the suggested relationship between genomic aberrations in CTCs and prognosis strongly supports the potential utility of GI monitoring in CTCs in clinical risk assessment and therapeutic choice.

CTC-Derived Models: A Window into the Seeding Capacity of Circulating Tumor Cells (CTCs).

Metastasis is the main cause of cancer-related death owing to the blood-borne dissemination of circulating tumor cells (CTCs) early in the process. A rare fraction of CTCs harboring a stem cell profile and tumor initiation capacities is thought to possess the clonogenic potential to seed new lesions. The highest plasticity has been generally attributed to CTCs with a partial epithelial-to-mesenchymal transition (EMT) phenotype, demonstrating a large heterogeneity among these cells. Therefore, detection and functional characterization of these subclones may offer insight into mechanisms underlying CTC tumorigenicity and inform on the complex biology behind metastatic spread. Although an in-depth mechanistic investigation is limited by the extremely low CTC count in circulation, significant progress has been made over the past few years to establish relevant systems from patient CTCs. CTC-derived xenograft (CDX) models and CTC-derived ex vivo cultures have emerged as tractable systems to explore tumor-initiating cells (TICs) and uncover new therapeutic targets. Here, we introduce basic knowledge of CTC biology, including CTC clusters and evidence for EMT/cancer stem cell (CSC) hybrid phenotypes. We report and evaluate the CTC-derived models generated to date in different types of cancer and shed a light on challenges and key findings associated with these novel assays.

Abstract GS3-07: Clinical utility of circulating tumor cell count as a tool to chose between first line hormone therapy and chemotherapy for ER+ HER2- metastatic breast cancer: Results of the phase III STIC CTC trial

Abstract Background: In ER+ HER2- metastatic breast cancer (MBC) patients, the clinical choice between 1st line hormone therapy (HT, the recommended option) or chemotherapy (CT) is based on the absence of “visceral crisis” or adverse prognostic factors, with no proven/objective criteria. In that context, STIC CTC (NCT01710605) was set up as a strategy trial to test whether circulating tumor cells (CTC) count could help customize the choice between 1st line HT or CT. Methods: For this multicenter phase 3 non-inferiority trial, the main inclusion criteria were: ER+ HER2- MBC with no prior therapy, PS≤2, no contra-indication to HT or CT and informed consent. The a priori treatment choice (HT or CT) and CTC count (CellSearch®) were obtained in all patients prior to randomization. Patients were randomized 1:1 between clinically-driven choice (CTC count not disclosed, HT or CT administered as decided a priori), or a CTC-driven choice (HT if &amp;lt;5 CTC/7.5ml, CT if ≥5 CTC/7.5ml). The primary objective was treatment efficacy (PFS hazard ratio), non-inferiority being established if the upper bound of the PFS HR 2-sided 90%CI is ≤1.25; secondary objectives included subgroup analyses (CTC status, patient characteristics) and OS. Results: 761 MBC patients were randomized between 02/2012 and 08/2016. Baseline characteristics: 7.8% of patients had a PS=2, 24.1% had a de novo metastatic disease; 63.3% received prior adjuvant HT and 49.9% prior adjuvant CT; 31.3% had ≥3 metastatic sites. A priori treatments (HT or CT) and CTC count (&amp;lt; or ≥5 CTC/7.5ml) were well balanced between the two arms. After randomization, in the clinically-driven arm, N=267 (72.4%) patients received HT and N=102 (27.6%) CT (as decided a priori). In the CTC-driven arm: (1) the a priori choice of HT was confirmed by a low CTC count in N=181 (67.5%) of patients, while N=87 (32.5%) were switched to CT due to a high CTC count; (2) the a priori choice of CT was confirmed by high CTC count in only N=48 (48%) patients, while N=52 (52%) were switched to HT. The primary endpoint was met, PFS being not inferior in the CTC-driven arm (HR=0.98, 90%CI=[0.84–1.13]). Analyses focusing on discordant subgroups showed that for patients with a priori choice of HT but with high CTC count (leading to a switch to CT in the CTC-arm), PFS was significantly longer in the CTC-driven arm than in the standard arm (HR=0.67, 95%CI=[0.49–0.92]; p=0.01), with a non-significant trend toward longer OS (HR=0.68, 95%CI=[0.44–1.07]; p=0.09). Inversely, for patients with a priori choice of CT but with low CTC count (i.e. de-escalation to HT in the CTC arm), PFS was not statistically different between the two arms. Conclusion: This trial demonstrates the clinical utility of CTC count as an objective decision tool when considering 1st line therapy in ER+ HER2- MBC. In most patients, CTC count did confirm the a priori clinical choice; however, trial results show that in discrepant cases, CTC count may be trusted for either escalating (i.e. considering CT in patients if high CTC count) or de-escalating (i.e. considering HT in patients if low CTC count) 1st line therapy. Funding: French National Cancer Institute; Menarini Silicon Biosystems. Citation Format: Bidard F-C, Jacot W, Dureau S, Brain E, Bachelot T, Bourgeois H, Goncalves A, Ladoire S, Naman H, Dalenc F, Gligorov J, Espie M, Levy C, Ferrero J-M, Loirat D, Cottu P, Dieras V, Simondi C, Berger F, Alix-Panabieres C, Pierga J-Y. Clinical utility of circulating tumor cell count as a tool to chose between first line hormone therapy and chemotherapy for ER+ HER2- metastatic breast cancer: Results of the phase III STIC CTC trial [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 GS3-07.

Abstract A051: Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution

Abstract Liquid biopsy components from blood, such as cell free DNA (cfDNA) and circulating tumor cells (CTCs), are prognostic for overall survival in advanced prostate cancer patients and allow the study of clonal evolution. cfDNA is easily obtained and has been widely used for molecular characterization and reflects pooled genomic profiles in a patient, but has limitations regarding gene copy number calls. CTC single-cell genomic studies generate precise gene copy number calls and elucidate intrapatient intercellular genomic heterogeneity. The main limitation of CTC analyses has been the low CTC count found in many cancer patients. We elected to study whether liquid biopsy by apheresis in advanced prostate cancer patients increases the yield of CTC to study tumor genomics, intrapatient heterogeneity, and ex vivo organotypic 3D models. Advanced metastatic prostate cancer patients being considered for clinical trials were invited to consent to apheresis. Apheresis CTC counts using CellSearchTM (Menarini) were acquired from 16 patients. The contents of the CellSearch cartridges were sorted into pure single cells by fluorescence-activated cell sorting and subsequently assessed by array comparative genomic hybridization (aCGH, Agilent Technology) for copy number aberrations (CNA). Exome and aCGH from tissue biopsies were compared to the single cell aCGH results. We generated patient-derived organoid (PDOs) cultures from apheresis products by preenrichment using density gradient (Lymphoprep) and subsequent CTC enrichment by EpCAM positive selection (EasySep StemCell Technologies). PDOs were characterized by immunofluorescence (IF) as DAPI+/CK+/EpCAM+ and CD45- cells and subsequently by aCGH for CNA. All sixteen patients (median age of 70 years; range 60-77 years) tolerated apheresis without any adverse effects. CTC counts from peripheral blood (PB) prior to apheresis ranged from 13 to 711 (median = 96), and did not significantly change post apheresis. The estimated CTC yield per apheresis ranged from 660-35473 per apheresis product (median = 3351). This constitutes an increase of 102-fold when compared to median CTC capture from 7.5mL of PB. A total of 170 single CTCs from 15 apheresis patients were genomically profiled and the copy number aberration profiles confirmed prostate cancer with multiple genomic hallmarks including CNAs such as AR amplification, chromosome 8q gain (MYC locus), and PTEN, RB1, BRCA2, TP53, CHD1 loss. CNA profiles of PDOs showed similar genomic aberrations to same patient CTCs and also reflected intrapatient heterogeneity detected by single CTC analysis. In conclusion, apheresis from advanced prostate cancer patients is a well-tolerated procedure and in our study increased the CTC yield by 102-fold when compared to PB. CTC and PDOs from apheresis products shared similar CNA profile compared with tissues biopsies and furthermore gave us an insight of the tumor heterogeneity and clonal evolution. Citation Format: Maryou B.K. Lambros, Veronica Gil, Mateus Crespo, Mariane S. Fontes, Alan Mackay, Gemma Folwer, Gemma Folwer, Berni Ebbs, Rui Neves, Penny Flohr, Susana Miranda, Semini Sumanasuriya, Daniel N. Rodrigues, Rita Pereira, Geroge Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Niven Mehra, Jane Goodall, Claudia Bertan, Suzanne Carreira, Nikolas H. Stoecklein, Leon W.M.M. Terstappen, Gunther Boysen, Joahnn S. De Bono. Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A051.

P3.01-008 Association between Icotinib Efficacy and Circulating Tumor Cell Levels in Advanced Non-Small Cell Lung Cancer

Advanced non-small cell lung cancer (NSCLC) studies indicated a potential association between chemotherapy efficacy and circulating tumor cells (CTC) counts in peripheral blood. The icotinib efficacy and circulating tumor cells (CTC) counts in non-small cell lung cancer remain unknown. The aim is to investigate association between the icotinib efficacy and CTC counts in advanced NSCLC patients. A total of 74 advanced NSCLC patients consented to provide 5ml of peripheral blood before systematic therapy, 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 icotinib, and the icotinib efficacy and prognoses were compared. The treatment efficacies were 46.88% (15/32) and 23.81% (10/42) for the low CTC group and the high CTC group, respectively. The median overall survival was 22.0 months (95%CI: 19.6-28.7 months) for the low CTC group and 18.3 months (95% CI: 15.3-25.4 months) for the high CTC group. The median progression-free survival was 11.6 months (95% CI: 8.7-15.6 months) and 7.2 months (95% CI: 3.4-8.7 months) for the low group and the high CTC group, respectively. The CTC counts can be used as a important biomarker for therapy monitoring the icotinib effect on patients with advanced NSCLC. Efficacy and prognosis of icotinib and CTC counts were considered important, and the CTC counts could be used to predict the efficacy of icotinib and prognosis of advanced NSCLC. The change in CTC count levels can be used as a biomarker for evaluating the prognosis of patients with advanced NSCLC.

Abstract 993: Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC)

Abstract Introduction: CTC count is an independent predictor of overall survival in mCRPC. Isolation of CTC from peripheral blood (PB) for genomic and functional analysis is challenging, especially in patients (pts) with low CTC count. It has been shown that DLA increases CTC yield. However, it has yet to be proven whether CTC isolation from DLA can be used in complementary studies such as molecular characterization and growth of organoid culture for drug sensitivity studies. Here we present preliminary data of an on-going study, which evaluates DLA in mCRPC pts, focusing on safety, CTC enrichment, molecular characterization and feasibility for organoid culture. Methods: mCRPC pts considered for clinical trials were selected according to performance status (ECOG 0-1) and number of CTC found in 7.5ml PB (&amp;gt;20 cells/7.5mL). DLA products (200x106 cells) were processed using the CellSearch CTC kit (Janssen Diagnostics, LLC) according to manufacturer procedures. The contents of CellSearch cartridges were sorted into single cell by fluorescence activated cell sorting (FACS) and subsequently assessed by array comparative genomic hybridization (aCGH) for copy number aberrations (CNA). Enrichment of CTC for organoid culture was performed by density gradient of mononuclear cells followed by positive selection using magnetic beads. Results: Overall 12 mCRPC patients underwent DLA without any complication or toxicity. The mean CTC count was 90 CTC/7.5 ml peripheral blood (median = 31) and ranged from 20 to 324. CellSearch CTC count in the DLA yielded a mean of 466 (median=203) and ranged from 60 to 2496 with an up to 40-fold increase (mean = 13, median = 6) in CTC count separation when comparing 1mL of PB to 1mL of DLA. Molecular analyses of FACS single CTC from the DLA by aCGH showed that these CTC genomic profiles had the typical hallmarks of mCRPC with CNAs including AR and MYC locus (8q) amplification, and PTEN, RB1, TP53, CHD1 loss. Additionally, ex vivo culture of CTC-derived organoids was successfully achieved. aCGH of these organoids matched the genomic profile that of the CTC from the same patient. Conclusion: DLA from mCRPC pts was well tolerated and yields higher CTC capture than PB and may provide an alternative to tissue biopsy and routine blood volumes. Our strategy allowed us to isolate genomic DNA with good quality for molecular characterization and viable CTC for organoid culture and functional studies. Citation Format: Maryou B. Lambros, Veronica S. Gil, Mateus Crespo, Mariane S. Fontes, Rui N. Neves, Niven Mahra, Gemma Fowler, Berni Ebbs, Penny Flohr, George Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Joost F. Swennenhuis, Kiki C. Andree, Semini Sumanasuriya, Matthew Clarke, Pasquale Rescigno, Zafeiris Zafeiriou, Joaquin Mateo, Diletta Bianchini, Nikolas H. Stoecklein, Leon W. Terstappen, Gunther Boysen, Johann S. De Bono. Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2017-993

Abstract 1732: Accuracy of extrapolation of circulating tumor cell count from small blood volumes: statistical estimation using the AccuCyte - CyteFinder system

Abstract Background: Baseline and post-treatment counts of circulating tumor cells (CTCs) are prognostic of patient outcome in cancer. The FDA-cleared CellSearch® system has defined CTC count as the number of cells per 7.5 mL of blood, with poor prognosis being 5 or more for breast and prostate cancers, and 3 or more for colon cancer. There are little reported data on whether it is reasonable to extrapolate CTC counts from smaller blood volumes, particularly when CTC count is low, given sampling variability. The AccuCyte - CyteFinder system (RareCyte) collects 7.5 mL of blood for analysis, isolates the buffy coat and smears it onto 8 slides, which are then stained with markers for CTCs that are identified by fluorescence microscopy. We investigated the statistical accuracy of extrapolation of total CTC count from fewer than 8 slides. Methods: 172 blood samples from various cancers were processed to microscope slides, stained, and analyzed by CyteFinder. Counts were made on a per slide basis, with total “true” CTC count being the sum of all 8 slides. Samples with no CTCs were excluded. To extrapolate the total CTC count from k slides, all possible combination of k slides were used to estimate the total count of 8 slides. This was done for k = 1-7 for all 172 samples and extrapolated counts were rounded to the nearest integer. The proportion of times that extrapolated counts fell within a specified percentage of the true CTC count was calculated across the entire sample set, as well as for 6 sub-categories defined by true CTC count range. Results: The 6 CTC count categories were: 1-4 (N=39), 5-10 (N=21), 11-25 (N=21), 26-50 (N=14), 51-100 (N=24), more than 100 (N=53). Cumulative proportions within 5, 10, 25 and 50% of the true total CTC count were determined for the entire sample set as well as by category. In the entire sample set, the proportion of extrapolated counts that fell within 25% of the true count ranged from 0.448 (1 slide) to 0.723 (4 slides) to 0.951 (7 slides). Generally, the lower the true total count, the lower the fraction of extrapolated counts that fell within the specified percentage of the true count. For example, for true count 5-10, the fraction that fell within 25% of the true count ranged from 0.208 (1 slide) to 0.513 (4 slides) to 0.951 (7 slides). In contrast, for true count more than 100, the fraction that fell within 25% of the true count ranged from 0.751 (1 slide) to 0.968 (4 slides) to 1.000 (7 slides). Conclusions: In this sample set, nearly three quarters of cases could be estimated within 25% of the true CTC count by extrapolating from 4 AccuCyte slides. Estimating each slide to represent ~1 mL of processed whole blood, our analysis suggests that a ~4 mL sample will have this level of accuracy relative to a CTC count from 7.5 mL. At low true CTC counts the accuracy of extrapolation decreases, indicating that a larger volume of blood is required for this level of accuracy. Citation Format: Jeffrey L. Werbin, Paulina Varshavskaya, Arturo B. Ramirez, Jackie L. Stilwell, Daniel E. Sabath, Ping-Yu Liu, Eric Kaldjian. Accuracy of extrapolation of circulating tumor cell count from small blood volumes: statistical estimation using the AccuCyte - CyteFinder system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1732. doi:10.1158/1538-7445.AM2017-1732

Prognostic value of circulating tumor cells in advanced gastric cancer patients receiving chemotherapy.

The identification of circulating tumor cells (CTCs) may provide important prognostic information in several types of solid tumors, including gastric cancer. The aim of this study was to investigate whether CTC count may be used to predict survival in patients with advanced gastric cancer treated with chemotherapy. The CELLection™ Epithelial Enrich kit was used to isolate and purify CTCs from samples of peripheral blood. Immunofluorescent staining was used for CTC counting. High CTC counts were associated with poor tumor differentiation and high serum CEA levels (P=0.021 and 0.005, respectively). After 3 months, 16 patients with decreasing CTC counts after the first cycle of chemotherapy obtained complete response, partial response or stable disease, while 13 patients with increasing CTC counts developed progressive disease. The patients with decreasing CTC counts also exhibited longer progression-free survival (PFS) (P≤0.001) and overall survival (OS) (P=0.002) compared with those with increasing CTC counts. Among all 59 patients, those with a CTC count of ≤2 cells/5 ml blood exhibited longer PFS (P≤0.001) and OS (P≤0.001) compared with those with a CTC count of >2 cells/5 ml blood. The multivariate analysis suggested that an increase of the CTC count after the first cycle of chemotherapy was only an independent prognostic marker of poor PFS (P=0.019). However, a baseline CTC count of >2 cells/5 ml blood was an independent poor prognostic marker for PFS (P=0.008) and OS (P=0.001) in all 59 patients. Our study suggested that patients with a low baseline CTC count or decrease of the CTC count after the first cycle of chemotherapy may benefit significantly from palliative chemotherapy. In conclusion, CTC count may be a good chemotherapy monitoring marker and an ideal prognostic marker for patients receiving palliative chemotherapy.

Personalized Treatment Through Detection and Monitoring of Genetic Aberrations in Single Circulating Tumor Cells.

Circulating tumor cells (CTCs) present a viable alternative to access tumor materials other than primary biopsies in cancer. This disease is among the most widespread in the world and is difficult to target because of its complex nature, challenges in getting quality samples and dynamic temporal changes in response to treatment. Conventional methods of detection and monitoring the disease profile do not suffice to be able to target the heterogeneity that exists at the cellular level. CTCs have been identified as a possible substitute for tumor tissue samples, and can be used to complement current disease management. Challenges in CTCs molecular analysis lie in the purity of the sample, which is masked by the presence of large quantities of white blood cells (WBCs) . In this chapter, we present a microfluidic biochip platform that performs secondary purification to isolate single CTCs efficiently. Studying single CTCs will allow for sensitive detection of critical mutations and addressing intercellular variances that will be otherwise missed easily due to low mutation frequencies when evaluating bulk cell retrieval. Using the biochip, we isolated single CTCs, and conducted personalized integrated EGFR mutational analysis using conventional polymerase chain reaction (PCR) and Sanger sequencing. We also demonstrated that high quality next generation sequencing (NGS) libraries can be readily generated from these samples. In our initial study, we revealed that the dominant EGFR mutations such as L858R and T790M could be detected in Non Small Cell Lung Cancer (NSCLC) patients with low CTC counts. We envision the biochip will enable efficient isolation of rare single cells from samples. This technology coupled with downstream molecular characterization of CTCs will aid in realizing the personalized medicine for cancer patients.

A High Circulating Tumor Cell Count in Portal Vein Predicts Liver Metastasis From Periampullary or Pancreatic Cancer: A High Portal Venous CTC Count Predicts Liver Metastases.

Circulating tumor cells (CTCs) released from a periampullary or pancreatic cancer can be more frequently detected in the portal than the systemic circulation and potentially can be used to identify patients with liver micrometastases. Aims of this study is to determine if CTCs count in portal venous blood of patients with nonmetastatic periampullary or pancreatic adenocarcinoma can be used as a predictor for subsequent liver metastases. CTCs were quantified in portal and peripheral venous blood samples collected simultaneously during pancreaticoduodenectomy in patients with presumed periampullary or pancreatic adenocarcinoma without image-discernible metastasis. Postoperatively patients were monitored for liver metastasis by abdominal magnetic resonance imaging or computed tomography every 3 months for 1 year. Sixty patients with a pathological diagnosis of periampullary or pancreatic adenocarcinoma were included in the study. Multivariate analysis indicated that portal CTC count was a significant predictor for liver metastases within 6 months after surgery. Eleven of 13 patients with a high portal CTCs count (defined as >112 CMx Platform estimated CTCs in 2 mL blood) developed liver metastases within 6 months after surgery. In contrast, only 6 of 47 patients with a low portal CTC count developed liver metastases (P < 0.0001). A value of 112 CMx Platform estimated CTCs had 64.7% sensitivity and 95.4% specificity to predict liver metastases within 6 months after surgery. We concluded that a high CTC count in portal venous blood collected during pancreaticoduodenectomy in patients with periampullary or pancreatic adenocarcinoma without metastases detected by currently available imaging tools is a significant predictor for liver metastases within 6 months after surgery.

Abstract P2-02-17: T-DM1 in HER2-negative metastatic breast cancer patients with HER2-amplified circulating tumor cells: Current status of the CirCe T-DM1 phase II trial

Abstract Background: Liquid biopsy can reassess key therapeutic targets in metastatic breast cancer. Several studies showed that a low albeit significant rate of metastatic breast cancer initially considered as HER2-negative can be reclassified as HER2-positive by systematic biopsy procedures. We report here the current status of the CirCe T-DM1 trial [NCT01975142] which aims to demonstrate the clinical utility of HER2 status reassessment on circulating tumor cells (CTCs). Methods: The first step of the trial consists in CTC count and HER2/CEP17 FISH on detected CTCs (CellSearch, Janssen Diagnostics) in patients (pts) with measurable disease progressing after the second line of chemotherapy. Pts with amplified CTCs (HER2/CEP17 ratio equal or higher than 2.2) are eligible to the treatment step of the study in two distinct cohorts: low CTC count (1 or 2 HER2-amplified CTCs) and high CTC count (3 and more HER2-amplified CTCs). In the treatment step has a Simon's two stage design, the anti-HER2 antibody-drug conjugate T-DM1 being administered until tumor progression. The primary objective of the trial is the confirmed response rate (RECIST). This trial is supported by Roche. Results: CirCe T-DM1 has been initiated in 10 centers in France. As of June 2015, 105 metastatic breast cancers pts considered as HER2-negative were screened. 29 pts (27%) had no CTC detected, 68 pts (65%) had at least 1 CTC detected with no HER2 amplification, and 8 pts (8%) exhibited HER2-amplified CTCs. Among the 8 pts, 1 pt had 5 HER2-amplified CTC, 2 pts had 2 HER2-amplified CTC and 5 pts had 1 HER2-amplified CTC. HER2/CEP17 ratios among HER2-amplified CTCs ranged from 2.5 to 7. Five of the 8 pts were treated by T-DM1. One objective confirmed partial tumor response has been observed (20%). Conclusion: The accrual is ongoing; the first efficacy assessment will occur after having treated 14 pts. This innovative trial highlights the promise and the complexity of liquid biopsy-based programs in the era of precision medicine: scarcity of the target, reliability and reproducibility of the target assessment, major efficacy when the target is matched to the appropriate drug. Citation Format: Bidard F-C, Romieu G, Jacot W, Cottu P, Dieras V, Lerebours F, Servent V, Luporsi E, Lortholary A, Tubiana-Mathieu N, Espie M, Bollet M, Bourgeois H, Renaud N, Pelissier S, Armanet S, Baeten K, Pierga J-Y. T-DM1 in HER2-negative metastatic breast cancer patients with HER2-amplified circulating tumor cells: Current status of the CirCe T-DM1 phase II trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-17.

Prognostic Importance of Circulating Tumor Cells in Nonsmall Cell Lung Cancer: A Prospective Study

Purpose: To investigate the prognostic value of circulating tumor cells (CTCs) and to predict the treatment response in a non-small cell lung cancer (NSCLC).Methodology: A single-center prospective study involving 93 patients with NSCLC was conducted. Blood samples were analyzed for CTC count before and after chemotherapy. Clinical relevance of CTCs with patient`s characteristics and treatment response were determined.Results: Higher levels of CTCs were associated with severe stage of NSCLC (p = 0.003), tumor histology (p = 0.014) and metastases (p = 0.013). Significant difference in CTC count was observed in favorable (CTCs &lt; 5) and unfavorable (CTCs ≥ 5) groups. Progression-free survival (PFS) was 5.8 months (range: 5.32 to 6.43) and 2.2 months (range: 1.85 to 3.01) in the favorable and unfavorable groups, respectively (HR: 3.88, 95% CI, p &lt; 0.001). Similarly, overall survival (OS) was 7.3 months (95% CI, 6.51 to 7.92) and 3.9 months (95% CI, 1.99 to 5.13), respectively (HR: 4.8, 95% CI, p &lt; 0.001). Multivariate regression analysis revealed CTCs as strong predictors of OS and PFS. Significant reduction (p &lt; 0.001) in CTC count was also observed after one cycle of chemotherapy.Conclusion: Patients with low CTC count live longer and remain progression-free for a longer period of time than those with high CTC count. High CTCs can be detected in severe forms of lung cancer and can be used as a valid prognostic marker. However, this assertion requires validation in larger prospective clinical cohorts.Keywords: Circulating tumor cells, Non-small cell lung cancer, Circulating tumor cell, Prognosis

Circulating Tumor Cell Enumeration in a Phase II Trial of a Four-Drug Regimen in Advanced Colorectal Cancer

BackgroundMultidrug regimens are active against advanced colorectal cancer (ACRC). However, the increased toxicity requires the use of biomarkers to select the patients who will derive the most benefit. We assessed circulating tumor cells (CTCs) as a prognostic biomarker in patients treated with a 4-drug regimen. Patients and MethodsA single-arm phase II trial (Erbitux Study of CPT11, Oxaliplatin, UFToral Targeted-therapy [eSCOUT]) was undertaken in patients with previously untreated KRAS wild-type ACRC using a regimen of irinotecan, oxaliplatin, and tegafur-uracil with leucovorin and cetuximab. Baseline CTCs were enumerated using CellSearch. The endpoints were an objective response rate (ORR) and overall survival (OS). We modeled our results and compared them with those modeled for the capecitabine, oxaliplatin, bevacizumab +/- cetuximab (CAIRO2) trial, stratifying patients a priori into low (< 3) and high (≥ 3) CTC groups. ResultsFor 48 eligible patients, the best ORR from the 4-drug regimen was 71%, with a disease control rate of 98%. The median OS for patients with a high and low CTC count was 18.7 and 22.3 months (log-rank test, P = .038), respectively. In our modeled data, for patients with a low CTC count, no differences were found between the median OS in the eSCOUT trial and that in the CAIRO2 trial (22.2 vs. 22.0 months). However, for the high CTC group, a clinically relevant improvement was seen in median OS (eSCOUT vs. CAIRO2, 18.7 vs. 13.7 months; P = .001). ConclusionThese data are hypothesis generating—for patients with ACRC, stratification by CTC count can identify those who might benefit the most from an intensive 4-drug regimen, avoiding high-toxicity regimens in low CTC groups. This hypothesis warrants validation in a phase III biomarker-driven trial.

Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients

BackgroundOncogenic mutations are powerful predictive biomarkers for molecularly targeted cancer therapies. For mutation detection patients have to undergo invasive tumor biopsies. Alternatively, archival samples are used which may no longer reflect the actual tumor status. Circulating tumor cells (CTC) could serve as an alternative platform to detect somatic mutations in cancer patients. We sought to develop a sensitive and specific assay to detect mutations in the EGFR gene in CTC from lung cancer patients.MethodsWe developed a novel assay based on real-time polymerase chain reaction (PCR) and melting curve analysis to detect activating EGFR mutations in blood cell fractions enriched in CTC. Non-small-cell lung cancer (NSCLC) was chosen as disease model with reportedly very low CTC counts. The assay was prospectively validated in samples from patients with EGFR-mutant and EGFR-wild type NSCLC treated within a randomized clinical trial. Sequential analyses were conducted to monitor CTC signals during therapy and correlate mutation detection in CTC with treatment outcome.ResultsAssay sensitivity was optimized to enable detection of a single EGFR-mutant CTC/mL peripheral blood. CTC were detected in pretreatment blood samples from all 8 EGFR-mutant lung cancer patients studied. Loss of EGFR-mutant CTC signals correlated with treatment response, and its reoccurrence preceded relapse.ConclusionsDespite low abundance of CTC in NSCLC oncogenic mutations can be reproducibly detected by applying an unbiased CTC enrichment strategy and highly sensitive PCR and melting curve analysis. This strategy may enable non-invasive, specific biomarker diagnostics and monitoring in patients undergoing targeted cancer therapies.

Three-arm randomized phase II study of cisplatin and etoposide (CE) versus CE with either vismodegib (V) or cixutumumab (Cx) for patients with extensive stage-small cell lung cancer (ES-SCLC) (ECOG 1508).

7508 Background: Targeted inhibition of the Hedgehog (HH) pathway by V &amp; Insulin-like Growth Factor type-1 Receptor (IGF-1R) by Cx enhances efficacy of chemotherapy, and also demonstrates activity against the tumor cell fraction responsible for disease recurrence in SCLC. Methods: Patients (Pts) with newly diagnosed ES-SCLC with measurable disease, ECOG PS 0-1 were randomized to receive (four 21-day cycles) CE alone [(C 75 mg/m2 D1 &amp; E 100 mg/m2 D1-3) Arm A] or in combination with either V [(150 mg/day PO) Arm B] or Cx [(6mg/kg weekly IV) Arm C]. Pts with responsive or stable disease on Arms B &amp; C were continued on V or Cx respectively until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS), stratified on gender. Circulating tumor cells (CTCs) were isolated/enumerated by Veridex Cell Search Platform at baseline, after 1or 2 cycles of chemotherapy, at completion of 4 cycles &amp; 3 months (m). thereafter for correlation with efficacy parameters. The study was designed to detect a PFS HR of 0.58 with 90% power &amp; an overall one-sided type I error rate of 0.10 for each of the comparisons of the V and Cx arms to CE alone. Results: 155 eligible pts were treated; 136 have died &amp; 149 have experienced a PFS event. Pt. demographics &amp; disease characteristics are well-balanced between the three arms except higher rate of PS 0 on Arm B (p=0.03). The median PFS on Arms A, B &amp; C are 4.7, 4.4 &amp; 4.6 m, the median OS 9.1, 9.8 &amp; 10.1 m, &amp; the RR are 43%, 52% &amp; 49% respectively. None of the comparisons of these outcomes are statistically significant. PFS HR for V+CE vs. CE: 1.32, p=0.21, and for Cx+CE vs. CE: 1.12, p=0.58. The median OS among those with low CTC count (≤100 per 7.5ml) at baseline is 10.7 m vs. 7.2 m for those with high CTC count [HR1.70, p=0.01]. Toxicities in all three arms are as expected with the combination of CE alone. Conclusions: There is no significant improvement in PFS or OS with the addition of either V or Cx to CE vs.CE alone in pts with ED-SCLC. Low baseline CTC count is associated with improved OS, suggesting its role as a prognostic biomarker in SCLC. Clinical trial information: NCT00887159.

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic™), real-time PCR (EntroGen™) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.