CTC Detection Research Articles

Prevalence of ARV7 in Asian metastatic castrate-resistant prostate cancer (mCRPC) patients using multiple detection platforms.

245 Background: The presence of AR-V7 in metastatic castrate resistant prostate cancer (mCRPC) men has been associated with worse outcome in men initiated on 2nd generation androgen receptor signalling inhibitors (ARSI) in the Caucasian population. A multinational study was conducted to investigate this in the Asian population. Methods: mCRPC patients were recruited prospectively across 5 countries. Blood samples were collected and processed from patients with progressive disease immediately before the initiation of a new treatment and at progression. AR-V7 detection were performed using 3 methods: CTC enrichment followed by automated immunofluorescent staining (Clearbridge [CB]), CTC enrichment followed by reverse-transcription PCR analysis (IBN), and the AdnaTest Prostate Cancer(Adna) platform for CTC analysis and detection. Only blood samples collected in Singapore underwent all 3 methods of detecting AR-V7. Comparison of AR-V7 prevalence using the 3 detection methods were done on patients with the AdnaTest platform as gold standard. We examined associations between AR-V7 status and PSA response rates, PSA progression free survival and overall survival(OS). Results: 102 patients were recruited. 72 patients had ARSi while 30 patients had chemotherapy. 66 patients were included for the comparison of AR-V7 detection methods. AR-V7 prevalence rate was 14.3% (95% CI 4.8-30.3), 21.6% (95% CI 12.9-32.7) and 33.7% (95% CI 24.6-43.8) based on Adna, CB and IBN respectively. Concordance between Adna and CB was 75% while Adna and IBN was 68%. AR-V7- patients had a trend towards higher PSA response, lower risk of PSA progression as compared to AR-V7+ patients. AR-V7- patients had a significantly lower risk of death as compared to AR-V7+ patients detected by Adna and IBN platforms but not the CB platform. The association between ARV7 status and outcomes did not vary when compared across treatment groups. Conclusions: AR-V7 positivity in Asian mCRPC patients is consistent with the data reported in Western populations with lower PSA response rates, PSA progression free survival and OS. This data suggest that ARV7 is more likely a prognostic than a predictive biomarker. Clinical trial information: 2015/2797.

Detection and prognostic role of heterogeneous populations of melanoma circulating tumour cells

BackgroundCirculating tumour cells (CTCs) can be assessed through a minimally invasive blood sample with potential utility as a predictive, prognostic and pharmacodynamic biomarker. The large heterogeneity of melanoma CTCs has hindered their detection and clinical application.MethodsHere we compared two microfluidic devices for the recovery of circulating melanoma cells. The presence of CTCs in 43 blood samples from patients with metastatic melanoma was evaluated using a combination of immunocytochemistry and transcript analyses of five genes by RT-PCR and 19 genes by droplet digital PCR (ddPCR), whereby a CTC score was calculated. Circulating tumour DNA (ctDNA) from the same patient blood sample, was assessed by ddPCR targeting tumour-specific mutations.ResultsOur analysis revealed an extraordinary heterogeneity amongst melanoma CTCs, with multiple non-overlapping subpopulations. CTC detection using our multimarker approach was associated with shorter overall and progression-free survival. Finally, we found that CTC scores correlated with plasma ctDNA concentrations and had similar pharmacodynamic changes upon treatment initiation.ConclusionsDespite the high phenotypic and molecular heterogeneity of melanoma CTCs, multimarker derived CTC scores could serve as viable tools for prognostication and treatment response monitoring in patients with metastatic melanoma.

1428P - Circulating tumour cell detection in epithelial ovarian cancer using dual-component antibodies targeting EpCAM and FRα

BackgroundCirculating tumor cell (CTC) detection methods based on epithelial cell adhesion molecule (EpCAM) have low detection rates in epithelial ovarian cancer (EOC). Meanwhile, folate receptor alpha (FRα) has high expression in EOC cells. We explored the feasibility of combining FRα and EpCAM as CTC capture targets in EOC. MethodsEpCAM and FRα antibodies were linked to magnetic nanospheres (MNs) using the principle of carbodiimide chemistry. Blood samples from healthy donor spiked with A2780 ovarian cancer cells were used for detecting the capture rate. Ninety-five blood samples from 30 patients with EOC were used for comparing the positive rate of detection when using anti-EpCAM-MNs alone with that when using combination of anti-EpCAM-MNs and anti-FRα-MNs. Samples from 28 patients initially diagnosed with EOC and who did not undergo any treatment and 20 patients with ovarian benign disease were used for evaluating the sensitivity and specificity of combination of anti-EpCAM-MNs and anti-FRα-MNs. ResultsRegression analysis between the number of recovered and that of spiked A2780 cells revealed yEpCAM = 0.535x (R2 = 0.99), yFRα = 0.901x (R2 = 0.99) and yEpCAM+FRα = 0.928x (R2 = 0.99). In mixtures of A2780 and MCF7 cells, the capture rate was 92% using the combination of anti-EpCAM-MNs and anti-FRα-MNs, exceeding the rate when using anti-EpCAM-MNs or anti-FRα-MNs alone by approximately 20% (P<0.01). The combination of anti-EpCAM-MNs and anti-FRα-MNs showed significantly increased positive rate compared with anti-EpCAM-MNs alone (χ2 = 14.45, P<0.001). Sensitivity values were 0.536 and 0.75 when using anti-EpCAM-MNs alone and when using the combination of anti-EpCAM-MNs and anti-FRα-MNs, respectively. Specificity values were 0.9 and 0.85, respectively. The combination of anti-EpCAM-MNs and anti-FRα-MNs improved the sensitivity of CTC detection in patients with newly diagnosed EOC (χ2 = 4.17; P=0.041). ConclusionsThe combination of FRα and EpCAM is feasible as a CTC capture target of CTC detection in patients with EOC. Legal entity responsible for the studyThe authors. FundingThe NNSFC (National Natural Science Foundation of China) (81802980, 81770169, 81670144) and the Health Committee Research Project Fund of Hubei Province (WJ2019M179). DisclosureAll authors have declared no conflicts of interest.

Circulating Tumor Cell and Circulating Tumor DNA Assays Reveal Complementary Information for Patients with Metastatic Urothelial Cancer

Despite considerable advances in the management of urothelial carcinoma (UC), better risk stratification and enhanced detection of minimal residual disease are still urgent priorities to prolong survival while avoiding the morbidity of overtreatment. Circulating tumor cells and DNA (CTCs, ctDNA) are two biologically distinct “liquid biopsies” that may potentially address this need, although they have been understudied in UC to date and their relative utility is unknown. To this end, matched CTC and ctDNA samples were collected for a head-to-head comparison in a pilot study of 16 patients with metastatic UC. CTCs were defined as cytokeratin- and/or EpCAM-positive using the RareCyte direct imaging platform. ctDNA was assayed using the PlasmaSelect64 probe-capture assay. 75% of patients had detectable CTCs, and 73% had detectable somatic mutations, with no correlation between CTC count and ctDNA. 91% of patients had tissue confirmation of at least one plasma mutation and, importantly, several clinically actionable mutations were detected in plasma that were not found in the matching tumor. A ctDNA fraction of >2% was significantly associated with worse overall survival (p=0.039) whereas CTC detection was not (p=0.46). Notably, using a predefined gene panel for ctDNA detection had a high but not complete detection rate in metastatic UC, similar to what has been described for a custom tissue-personalized assay approach. In sum, both liquid biopsies show promise in UC and deserve further investigation. Patient summaryNew “liquid biopsy” blood tests are emerging for urothelial cancer aimed at early detection and avoiding overtreatment. Our results suggest that two such tests provide complementary information: circulating tumor cells may be best for studying the biological features of a person’s cancer, whereas circulating tumor DNA may be better for early detection.

Abstract 1389: Single-cell sequencing to identify genetic variant accelerating shedding of CTCs into peripheral blood in gastric cancer patients

Abstract Circulating tumor cells (CTCs) are shed from primary or metastatic tumors into blood circulation, which may carry specific genetic characteristics related to hematogenous metastasis. Drawbacks of conventional EpCAM or cell size-dependent CTC detection and relevant single-cell sequencing technologies have limited discovery of specific genetic variants in CTCs. In the current study, a novel integrated SE-iFISH strategy, despite cell size or epithelial marker status, was applied to detect CTCs in advanced gastric cancer (GC) patients. Obtained CTCs were classified into diverse subtypes upon different chromosome ploidy. Fifty-three aneuploid CTCs identified by iFISH in 6 of naïve GC patients were isolated by a novel non-laser microscopic single cell manipulator (NMSCM), and subjected to modified Multiple Displacement Amplification (MDA) and subsequent single-cell sequencing. Compared with the conventional PCR, MDA generates 2 to 100-kb products with a lower error rate and achieves high fidelity whole genome amplification. Sequencing targeting 50 tumor genes of single CTC as well as paired primary tumor specimens was performed. Moreover, leukocytes genomic DNA as germline controls were included. Obtained results showed that high frequent SNV in genes such as TP53 were detected in both CTCs and the paired primary tumor mass. Furthermore, CTCs had considerable genetic heterogeneity compared to the paired primary tumor lesions. In particular, the CDKN2AA68V variant is frequently detected in CTCs but the paired primary GC tumor. We propose that the specific CDKN2AA68V variant carried by GC CTCs with high frequency, but low in the examined whole population of cancer cells in paired primary tumor lesions, may serve as the driver to promote CTCs shed into peripheral blood and to further facilitate hematogenous metastasis. Systematic in vitro and ex vivo studies are in progress to investigate roles of CDKN2A mutation in hematogenous dissemination in GC. Citation Format: Yilin Li, Xiaotian Zhang, Jifang Gong, Daisy Dandan Wang, Mingxia Qi, Yanyan Li, Dan Liu, Chunxiang Wang, Peter Ping Lin, Lin Shen. Single-cell sequencing to identify genetic variant accelerating shedding of CTCs into peripheral blood in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1389.

Abstract 401: Clinical impact of viable circulating tumor cells (v-CTC) detection and PD-L1 expression on v-CTC in the patients with resectable pancreatic cancer

Abstract [Introduction] The capture and analysis of CTCs as “liquid biopsy” provides the possibility to avoid invasive biopsies, obvious implications in cancer diagnosis and staging. We tested novel methods for viable CTCs (v-CTC) isolation in the patients (pts) with pancreatic cancer (PC), and investigated the clinical potential of v-CTCs in prognosis. We analyzed the PD-L1(L1) expression in both PC tumors and v-CTCs. [Pts and Methods] 7.5 ml of venous blood was collected prospectively from 39 PC pts, either surgery first (S group) or pre-treatment, consisted of Gem:800mg/m2; and S-1:80mg/m2 given concurrently with IMRT to 60Gy (NACRT:N group). To detect v-CTCs, we employed a telomerase-specific replication-selective adenovirous expressing GFP. For S group, samples were obtained before/after resection. For N group, samples were obtained before/after NACRT and after resection. To distinguish between leucocyte and cells with either epithelial or mesenchymal origin, cells were stained by anti-CD45, anti-Cytokeratin and anti-Vimentin Abs. GFP-positive and CD45-negative cells were counted as v-CTC. To assess L1 expression in PC tissues (IHC) and v-CTCs, L1 IHC kit (22C3, for tissues) and anti-human L1 mAb(MIH1,for CTCs) were employed. [Results]S group: 24 pts aged 53~85 years (male/female=12/12) were enrolled. 24 pts underwent curative resection. No v-CTCs were detected in 6 pts at both before and after resection, and 5 of 6 pts survived without recurrence. V-CTCs were identified in 18 of 24 pts, and 13 of 18 pts developed liver metastasis. Marked decrease of CTC counts were seen after resection in 10 of 18 pts, but 9 pts developed recurrence. N group: 15 PC pts aged 44~77 years (male/female=4/11) were enrolled. 15 pts underwent curative resection. No v-CTCs were detected in 5 pts, and 5 pts survived without recurrence. V-CTCs was identified in 10 of 15 pts, and only 3 out of 10 pts developed disease recurrence. Marked increase in CTC counts was observed after NACRT in 5 of 6 CTC-positive pts before NACRT, and 3 of 5 pts developed liver metastasis and died. NACRT may induce tumor cell dissemination into the blood circulation for CTC-positive pts. PD-L1 expression: L1 expression were assessed for 21 pts (S group:18, N group: 3). No patients with IHC-L1 high expression (≥50%) were observed (&amp;lt;1% [negative]:9, 10%:6, 20%:4, 40%:2). For S group, IHC-L1 low +/CTC-L1 + were detected in 12/18 pts (66%). IHC-L1-/CTC-L1+ were detected in 3/18 pts (17%). IHC-L1 - /no detectable CTCs were detected in 3/18 pts (17%). For N group, IHC-L1 - were detected in 3 pts, but CTCs-L1 + were detected in 2 pts (1 pt had no CTCs). The majority of detectable v-CTCs were positive for L1-expression (89% [92/103 CTCs]), despite L1-negative or -low expression in PC tissues. [Conclusions] Viable CTC detection appears as a promising prognostic marker. Immunotherapy with anti-PD-1/PD-L1 Abs may effectively target v-CTCs. Note: This abstract was not presented at the meeting. Citation Format: Masahiro Tanemura, Kenta Furukawa, Manabu Mikamori, Takurou Saito, Masahisa Otsuka, Yozo Suzuki, Kentaro Kishi, Hironao Yasuoka, Masahiko Tsujimoto, Yasuo Urata, Hiroki Akamatsu. Clinical impact of viable circulating tumor cells (v-CTC) detection and PD-L1 expression on v-CTC in the patients with resectable pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 401.

CTC versus biopsy tissue sequencing: A concordance analysis of genomic copy number profile from mCRPC patients (pts).

3050 Background: MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), is a high throughput, targeted-DNA-sequencing panel for somatic mutations created by the Department of Pathology at Memorial Sloan Kettering Cancer Center (MSK). The MSK-IMPACT is FDA approved for tumor tissue profiling to guide treatment selection. Recognizing access to tumor tissue in many cancers is difficult and may harbor inter &amp; intra lesional heterogeneity, we sought to evaluate concordance of sequencing single CTCs vs. paired biopsy analyzed by MSK-IMPACT, to assess CTC vs. tumor clonality, and their relationship to outcomes. Methods: 148 biopsy samples from 138 mCRPC pts were submitted for IMPACT analysis and a blood draw within 30-day prior to initiation of a new line of treatment. Blood samples were sent to Epic Sciences for CTC detection. The detected CTCs underwent single cell low pass whole genome sequencing for copy number variation (CNV). For each set of matched samples, DNA copy number profiles from IMPACT and CTC sequencing were compared for similarity. Results: Of the 114 successfully sequenced samples 58 were from lymph nodes, 23 from bone, 25 from liver or lung, and 8 from other soft tissue. Of the 111 patients with CTCs analyzed, a total of 1073 CTCs were sequenced (range 1-27, median = 4 per pt). Of patients with both IMPACT &amp; CTC, &gt;80% pts had multiple subclones with distinguishable CNV. Concordance of genomics clones was found in 63%, and discordant in 37% cases. The clonal concordance between tissue biopsy and CTC was higher when the biopsy was obtained from bone marrow or a visceral site (71% concordance) than from a lymph node (53% concordance). Conclusions: Single CTC sequencing is often concordant to metastatic tissue, but unique CTC clones and the presence of multi-clonal disease highlight the potential to be underrepresented through tissue biopsy, especially when taken from the lymph node.[Table: see text]

Dual-antibody Modified PLGA Nanofibers for Specific Capture of Epithelial and Mesenchymal CTCs

CTC detection has great potential to provide crucial clinical information for early cancer diagnosis, patient prognosis, personalized therapy, and cancer progress monitoring etc. It has been proved that the disease progress is associated with an increase in mesenchymal CTCs, while most mature techniques have been developed based on epithelial CTC detection. In present work, a dual-antibody nanointerface against EpCAM and N-Cadherin was developed to capture epithelial CTCs as well as mesenchymal CTCs from blood samples. A uniform poly-(lactic-co-glycolic acid) (PLGA) nanofiber substrate was fabricated by electrospinning to provide a platform for cell capture (i.e., MCF-7 and GIST882 cells), modified with BSA and dual antibodies. Our results showed the dual-antibody substrates exhibited an improved capture efficiency of target cells compared to the mono-antibody ones, revealing potential application of the dual-antibody PLGA nanofibers for efficient capture of epithelial and mesenchymal CTCs in clinic.

EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients

Although clinically relevant, the detection rates of EpCAM positive CTCs in non-small cell lung cancer (NSCLC) are surprisingly low. To find new clinically informative markers for CTC detection in NSCLC, the expression of EGFR and HER3 was first analyzed in NSCLC tissue (n = 148). A positive EGFR and HER3 staining was observed in 52.3% and 82.7% of the primary tumors, and in 62.7% and 91.2% of brain metastases, respectively. Only 3.0% of the brain metastases samples were negative for both HER3 and EGFR proteins, indicating that the majority of metastases express these ERBB proteins, which were therefore chosen for CTC enrichment using magnetic cell-separation. Enrichment based on either EGFR or HER3 detected CTCs in 37.8% of the patients, while the combination of EGFR/HER3 enrichment with the EpCAM-based CellSearch technique detected a significantly higher number of 66.7% CTC-positive patients (Cohen’s kappa = −0.280) which underlines the existence of different CTC subpopulations in NSCLC. The malignant origin of keratin-positive/CD45-negative CTC clusters and single CTCs detected after EGFR/HER3 based enrichment was documented by the detection of NSCLC-associated mutations. In conclusion, EGFR and HER3 expression in metastasized NSCLC patients have considerable value for CTC isolation plus multiple markers can provide a novel liquid biopsy approach.

Detection and Quantification of Circulating Tumor Cells in PBMCs or Blood Using Flow Cytometry

Abstract Circulating Tumor Cells (CTCs) are cells that have detached from primary tumor tissues or metastases and entered into peripheral blood circulation, led to cancer recurrence and distal metastasis. Evaluation of CTCs conduces to tumor diagnosis, treatment, monitoring, and prognosis. Tumor cell spiking assay was frequently used to evaluate the sensitivity, specificity, accuracy, and repeatability of the methods or systems for enumeration of rare CTCs. In this work, a novel flow cytometer NovoCyte® Quanteon™ was employed to measure and quantify spiked human tumor cells of colon carcinoma (SW620) in normal PBMCs or peripheral blood. First, immunofluorescence labeled (EpCAM-APC antibody) SW620 cells (0–256 cells) were serially diluted in 1×106 PBMCs and tested directly on Quanteon. The data indicate that CTC detection using Quanteon has a sensitivity of 0.0001% (one CTC per million cells) and an excellent linear relationship between the number of cells detected and the predicted number added. Furthermore, SW620 cells (0–2000 cells) were serially diluted in 2mL normal whole blood, following the traditional isolation of PBMCs, magnetic EpCAM-positive selection, immunofluorescence labeling (CD45 FITC, EpCAM APC) and washing. The results show that the average recovery of CTCs is more than 75%. Flow cytometry is a valuable tool for therapeutic monitoring and prognosis assessment. Detection of CTC is critical for a “liquid biopsy”. These experimental results demonstrate that the NovoCyte Quanteon flow cytometer can provide high specificity and accuracy of CTC detection through both direct identification and EpCAM affinity bead-based enrichment methods.

Circulating Tumor Cells in Early Breast Cancer.

Circulating tumor cells (CTCs) are particularly rare in non-metastatic breast cancer, and the clinical validity of CTC detection in that clinical setting was initially not well recognized. A cytological CTC detection device (CellSearch) fulfilling the CLIA requirements for analytical validity was subsequently developed and, in 2008, we reported the first study (REMAGUS02) showing that distant metastasis-free survival was shorter in early breast cancer patients with one or more CTCs. In the past 10 years, other clinical studies and meta-analyses have established CTC detection as a level-of-evidence 1 prognostic biomarker for local relapses, distant relapses, and overall survival. This review summarizes available data on CTC detection and the promises of this proliferation- and subtype-independent metastasis-associated biomarker in early breast cancer patients.

Feasibility of intraoperative CTC detection from portal vein during resection for pancreatic adenocarcinoma

Feasibility of intraoperative CTC detection from portal vein during resection for pancreatic adenocarcinoma

Circulating Tumor Cells for the Management of Renal Cell Carcinoma.

Renal cell carcinoma is a highly malignant cancer that would benefit from non-invasive innovative markers providing early diagnosis and recurrence detection. Circulating tumor cells are a particularly promising marker of tumor invasion that could be used to improve the management of patients with RCC. However, the extensive genetic and immunophenotypic heterogeneity of cells from RCC and their trend to transition to the mesenchymal phenotype when they circulate in blood constitute a challenge for their sensitive and specific detection. This review analyzes published studies targeting CTC in patients with RCC, in the context of the biological, pathological, and molecular complexity of this particular cancer. Although further analytical and clinical studies are needed to pinpoint the most suitable approach for highly sensitive CTC detection in RCC patients, it is clear that this field can bring a relevant guide to clinicians and help to RCC patients. Furthermore, as described, a particular subtype of RCC—the ccRCC—can be used as a model to study the relationship between cytomorphological and genetic cellular markers of malignancy, an important issue for the study of CTC from any type of solid cancer.

Abstract 5590: MiSelect R: A novel single-cell retrieval system for CTC from whole blood for biomarker analysis in cancer targeted and immunotherapy

Abstract Background: Circulating tumor cells' (CTCs) presence in patient's peripheral circulation has been recognized as a useful tool of liquid biopsy for cancer treatment. In addition to enumeration, analysis of associated protein and molecular alterations offers great potential for precision medicine. MiSelect R was invented for purification and characterization of rare cells including CTC. This study evaluates MiSelect R analytic performances and validates with clinical samples. Methods: Lung cancer cell line H2228 was spiked into 8 mL of whole blood (16 cells/ 8mL) for single-cell retrieval, subsequent RT-PCR and sequencing analysis on ALK-translocation. Breast cancer cell lines, SKBr3 (high EpCAM expressing) and MDA-MB-231 (low EpCAM expressing), were spiked into 8mL whole blood from healthy subjects at various concentrations (4-1024 cells/ 8mL) for recovery rate and linearity. Experiments with low cell spikes (32 cells/ 8mL) vs. high cell spikes (1024 cells/ 8mL) were carried out to assess the within-device precision. The assay specificity was examined in blood of healthy subjects. Further evaluation was performed in metastatic breast cancer (mBC) patients. Results: Spiked H2228 cells were retrieved with greater than 90% recovery rate and 70% purity from whole blood. Each retrieved single H2228 cell was confirmed to have ALK-EML4 translocation by RT-PCR and sequencing analysis. The detection ranges of 4 to 1024 SkBr3 and MDA-MB-231 cells per 8mL were confirmed to be linear with average recovery rate of 91% (R2 = 0.9949) and 70% (R2 = 0.9983), respectively. The within-device precision showed 9.23% and 7.82% CV for low cell spikes vs. high cell spikes. Great assay specificity was achieved with no CTC detected in healthy subjects (N = 42). 100% detection rate of CTC was achieved in untreated mBC patients and 47% for patients regardless of treatment history. Also, the HER2 score analyzed by MiSelect R in isolated CTCs was in agreement with that by IHC analysis. Nonetheless, the HER2 expression level was heterogeneous in the CTCs isolated from HER2-positive patients. Furthermore, the PD-L1 expression in CTCs was shown to be heterogeneous within and between cancer patients. Conclusion: The MiSelect R System showed excellent capability for single-cell retrieval of rare cells from whole blood. It also demonstrated outstanding analytic performances and clinical sensitivity for CTC detection as well as versatile capability for multiple-biomarker characterization. It potentially provides important cellular and molecular information at single-cell level from liquid biopsy for prognosis. Citation Format: Ju-Yu Tseng, Yen-Ru Chen, Chia-Ying Lee, Li-Fan Wu, Cheng-Hsu Wang, Shin-Hang Wang, Hui-Min Yu, Wei-Feng Fang, Mu-Yi Chen, Chwen-Cheng Chen. MiSelect R: A novel single-cell retrieval system for CTC from whole blood for biomarker analysis in cancer targeted and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5590.

Abstract 1561: Increased phenotypic depth for automated visual identification of biomarkers on circulating tumor cells by cocktailing epithelial markers EpCAM and cytokeratin

Abstract Background: Canonical methods for CTC identification (such as CellSearch®) have typically relied on co-expression of surface (EpCAM) and cytoplasmic (cytokeratin, CK) epithelial markers to separate them from white blood cells. RareCyte has developed AccuCyte®-CyteFinder®, a platform for automated visual identification and retrieval of rare cells in blood by immunofluorescence (IF). For CTC detection, EpCAM and CK are placed in individual fluorescence channels, allowing for identification of CTCs that may be EpCAM(-) /CK(+) as well as EpCAM(+)/CK(-). Recently we have developed technology that increases the number of markers from 4 to 6 to allow for biomarker phenotypic analysis in the additional two channels. Given that our platform does not require co-expression of EpCAM and CK, we hypothesized that we could gain a third biomarker channel by combining detection reagents for EpCAM and CK into a cocktail within a single channel. We tested feasibility of epithelial marker cocktailing with an assay to detect androgen receptor (AR) in prostate CTCs. Methods: Normal human whole blood samples were spiked with prostate cancer lines PC3, LNCaP, and 22RV1 as model CTCs (mCTCs). Blood samples from University of Washington patients with advanced cancer were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer using a CTC assay for detection of AR in which anti-cytokeratin and anti-EpCAM antibodies were combined in a reagent cocktail into a single channel; the CD45 WBC marker and a nuclear dye were included in the assay. After development using spike-in models, the assay was applied to a patient sample. Results: No CTCs were identified in non-spiked normal donor blood samples. In spike-in models, the epithelial cocktail assay performed as well as the non-cocktail assay in identifying CTCs. AR fluorescence signal was present in LNCaP and 22RV1 mCTCs (which express AR) and absent in PC3 mCTCs (which do not express AR), confirming specificity of AR staining. When the assay was applied to a blood sample from a prostate cancer patient with known high CTC count, 160 CTCs per mL were identified; of these, 114 were observed to be AR-positive (71%). Conclusions: Combining the epithelial markers cytokeratin and EpCAM into a single-wavelength cocktail is feasible for IF identification of CTCs, allowing an additional channel for phenotypic investigation. In a patient with advanced prostate cancer with high CTC count, there was high incidence (71%) of AR expression in identified CTCs. Citation Format: Yao Sun, Arturo Ramirez, Daniel Campton, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel Sabath, Eric Kaldjian. Increased phenotypic depth for automated visual identification of biomarkers on circulating tumor cells by cocktailing epithelial markers EpCAM and cytokeratin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1561.

Simultaneous detection of circulating and disseminated tumor cells in primary breast cancer patients following neoadjuvant chemotherapy.

Pathological complete response (pCR) is a common endpoint in neoadjuvant chemotherapy (NACT) of primary breast cancer patients (PBC), but does not address the systemic prevalence of minimal residual disease. In this study, we compared pCR with the detection of circulating (CTC) and disseminated tumor cells (DTC) following NACT, as well as their impact on survival. Patients with PBC receiving NACT and consecutive surgery were eligible for this study. CTCs were detected using the CellSearch® system and DTCs were determined using immunocytochemistry (cytokeratin staining with the A45-B/B3 antibody). pCR was defined as ypT0/ypTis and ypN0. 58 patients were included in the analysis with a median follow-up of 30months. Of these, 5 (9%) presented with CTCs and 36 (62%) with DTCs. 16 patients (28%) achieved a pCR. No significant correlation between CTCs, DTCs and pCR and no statistically significant impact on disease free (DFS) or overall survival (OS) was apparent. Both CTCs and DTCs are detectable after NACT. As we could not show a significant relationship between CTC detection, DTC detection and pCR, all three methods may provide independent information regarding treatment response. Since we were unable to show a significant impact on survival, larger prospective studies that include CTCs and DTCs are needed. These trials should include the molecular characterization of primary tumor tissue, CTCs and DTCs to determine whether these cells are independent subpopulations of malignant cell clones.

DNA‐Templated Magnetic Nanoparticle‐Quantum Dot Polymers for Ultrasensitive Capture and Detection of Circulating Tumor Cells

AbstractCirculating tumor cell (CTC) enumeration and analysis has emerged as an important platform for cancer diagnosis and prognosis. A great challenge, however, is to efficiently capture low abundant CTCs with high purity from blood samples in a rapid and high‐throughput manner for accurate and sensitive CTC detection. Herein, a new class of DNA‐templated magnetic nanoparticle‐quantum dot (QD)‐aptamer copolymers (MQAPs) is developed for rapid magnetic isolation of CTCs from human blood with high capture efficiency and purity approaching 80%. The phenotype of CTCs is simultaneously profiled with QD photoluminescence (PL) at single cell level. These MQAPs are constructed through hybridization chain reaction to achieve amplified magnetic response, extraordinary binding selectivity for target cells over background cells, and ultra bright ensemble QD PL for single cell detection. MQAPs are free from nonspecific binding that would otherwise compromise the capture purity of target cells. As a result, facile isolation and enumeration of rare CTCs in blood samples could be achieved in 20 min with high sensitivity and accuracy.

Abstract SY24-01: High-throughput CTC detection for noninvasive cancer monitoring

Abstract Circulating tumor cells (CTCs) provide a noninvasive source of patient-derived material to repeatedly sample cancer cells during the course of therapy, assessing changes in tumor burden, genetic composition, and transcriptional and RNA-based abnormalities. While some CTCs are viable and tumorigenic ex vivo, the majority of these cells die in the circulation, and some localized cancers with vascular invasion appear to shed CTCs long before the initiation of metastatic lesions. CTC detection therefore also offers an opportunity for early cancer detection. Two major technologic hurdles have limited the broad clinical application of CTC analyses: 1) the capture of these rare cells, estimated at approximately one tumor cell per billion normal blood cells in the circulation; and 2) the complex imaging criteria required for scoring rare and heterogeneous tumor cells using antibody-dependent staining. To address the primary challenge of capturing rare CTCs admixed with large numbers of normal blood cells, we have previously described a microfluidic device, the CTC-iChip, capable of high throughput depletion of normal hematopoietic cells, producing an output that is highly enriched for CTCs. This negative-depletion strategy enables isolation of CTCs across most types of cancer, independent of cell surface markers, and it ensures the highest quality of cellular RNA for downstream molecular analyses. To address the secondary challenge of imaging CTCs using accurate and high throughput platforms, we took advantage of the intact cancer cell–derived transcriptome within microfluidically enriched cancer cells to establish digital PCR platforms, capable of highly sensitive, reliable, and robust detection of CTCs from different cancer types. As proof of principle, we tested the application of such a digital CTC detection in patients with hepatocellular cancer, prostate cancer, and melanoma, demonstrating accurate measurement of tumor burden and associated RNA-based alterations and genotypes, as well as cancer cell–derived predictive markers of drug response. Taken together, the digital scoring of CTCs from microfluidically enriched blood cell populations provides a high throughput and highly quantitative platform for serial noninvasive monitoring of cancer. For future early cancer detection applications, the use of intact cancer cells in the circulation as a source of molecular markers may help distinguish invasive from indolent cancers. Citation Format: Shyamala Maheswaran, Mark Kalinich, Irun Bhan, David Miyamoto, Yu Zheng, Xin Hong, Tanya Todorova Kwan, Ravi Kapur, Lecia Sequist, Ryan Sullivan, Aditya Bardia, Richard Lee, David Ting, Mehmet Toner, Daniel A. Haber. High-throughput CTC detection for noninvasive cancer monitoring [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 SY24-01. doi:10.1158/1538-7445.AM2017-SY24-01

Abstract 1941: Cell surface vimentin is a novel marker for CTC detection in neuroblastoma

Abstract Among children in the United States, brain cancers now account for most number of cancer-related deaths. Neuroblastomas (NB) and sarcomas account for 13% of all childhood cancers in the United States. Despite significant progress yielding increased 5-year survival, 1250 deaths are expected this year from childhood cancers and cancer incidence has been steadily increasing. One of the key contributors to cancer mortality is tumor metastasis. It is generally believed that CTCs are shed into the circulation from primary tumors and contain unique driver mutations enabling their aggressive phenotype. However, detection of CTCs from NB is difficult as there is no direct method. Our lab has developed a monoclonal antibody targeting vimentin on the cells’ surface. Cell surface vimentin (CSV) is only observed in tumor cells; it remains intracellular in normal cells. We have published data demonstrating the superior specificity and sensitivity of our approach in breast, colon, and prostate cancer. Here, we report direct CTC detection in NB using our CSV mAb. As part of our collaborative effort analyzing NB blood samples from a multicenter Phase II trial, we present novel discoveries regarding treatment of NB with difluoromethylornithine (DFMO) and its effects on CTC release into the circulation in patients under remission. Among our observations, approximately ~1/3 patients have low or no CTCs while ~1/4 with high numbers of CTC are responding to DFMO treatment. Remaining patients show oscillating CTC numbers. Further, treatment with DFMO showed a statistically significant decline in CTCs after the beginning of therapy. Ongoing work including whole genome analysis can possibly reveal new insight into CTCs’ entering the circulation. These data demonstrate that our CTC capture using CSV is a novel and unique approach for detection in NB. Citation Format: Izhar S. Batth, Heming Li, Giselle Saulnier Sholler, Shulin Li. Cell surface vimentin is a novel marker for CTC detection in neuroblastoma [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 1941. doi:10.1158/1538-7445.AM2017-1941

Abstract 1931: The first circulating tumor cell detection technique from frozen PBMCs

Abstract Circulating tumor cells (CTCs) enter the vasculature or lymphatic system after shedding from the primary tumor. CTCs may serve as “seed” cells for tumor metastasis. The utility of CTCs in clinical application is not fully investigated, partly due to the necessity for fresh blood samples and the lack of CTC specific antibody. Of note, there is no CTC detection tool for pediatric tumors. To overcome these drawbacks, we developed a protocol for CTC capture from frozen peripheral blood mononuclear cells (PBMCs) based on the cell-surface vimentin (CSV) antibody 84-1. CSV is a unique marker compared to others such as EpCAM because it is only specific to tumor cells and has been demonstrated to be a viable target for isolation and enumeration of CTCs across tumor types. In this study, we report the first CTC isolation technology from frozen PBMCs of osteosarcoma patients using the CSV antibody 84-1. The CTCs captured using this new protocol were validated by single cell gene sequencing analysis and mesenchymal marker α-SMA staining. Moreover, spiking analysis was also performed to ensure the specificity and sensitivity of this unique CTC capture analysis. Linear regression analysis yielded a positive correlation between the number of detected sarcoma cells and the number of sarcoma cells spiked in both fresh and frozen PBMCs. In summary, our results demonstrate for the first time, a technology to specifically detect and isolate mesenchymal origin CTCs from frozen PBMCs. This technology can be easily expanded to other types of cancers including pediatric tumors because the antibody used is universal and CTC specific. Such a technology will boost the feasibility and utility of CTC-based diagnosis and therapeutic treatment monitoring in a centralized laboratory. Citation Format: Heming Li, Izhar Singh Batth, Xueqing Xia, Frank J. Hsu, Neeta Somaiah, Keila Enitt Torres, Ruoyu Wang, Shulin Li. The first circulating tumor cell detection technique from frozen PBMCs [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 1931. doi:10.1158/1538-7445.AM2017-1931