CellSearch Circulating Tumor Cells Research Articles

Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Abstract 7492: Automated identification and enumeration of CELLSEARCH Circulating Tumor Cells (CTC) with a deep learning algorithm

Abstract Introduction: CTC enumeration in blood samples with CELLSEARCH® is a prognostic biomarker in metastatic breast, prostate and colorectal cancer (full intended use documents.cellsearchctc.com). Currently, CTC identification is performed by visual assessment, which is time-consuming and potentially affected by subjective interpretations. Recently, we developed a Deep Learning (DL) algorithm for automated CTC identification in CELLSEARCH images (CellFind®, Menarini Silicon Biosystems, research use only). This study aims to assess the CTC identification performance of CellFind on 2 different datasets: the first for comparison with human reviewers, and the second for validation with much higher number of images. Methods: CellFind is composed of an image segmentation followed by a classification network, which was trained using 13067 CTC and 52890 non-CTC images from 215 breast, 123 prostate and 180 colorectal cancer samples. The performance of CellFind was tested on 2 separate datasets. 1) The first dataset was made of CELLSEARCH gallery images from 26 breast cancer, 68 prostate cancer, and 40 benign samples. 8 human reviewers, qualified for CTC image analysis, performed blind labeling. The Ground Truth (GT) was generated by the classification of the 3 most experienced reviewers by majority voting (1621 CTCs out of 17080 images). Accuracy and F1 metrics were used to rank the performance of DL and 5 reviewers against the GT. 2) The second dataset was made of CELLSEARCH gallery images from 63 breast, 66 prostate and 32 colorectal cancer samples. 3 experienced reviewers created the GT classification by majority voting, identifying 20052 CTCs out of 117447 images.Finally, each patient was assigned to the favorable or unfavorable group by applying the validated cutoff on CTC enumeration per sample (CTC≥5 for breast and prostate cancer, CTC≥3 for colorectal cancer). Results: 1) On 17080 gallery images from 134 samples, CellFind reached top human-level performance, as ranked both by accuracy (97.8% for DL vs. 96.8-98.0%) and F1 (88.6% for DL vs. 83.3-89.9% for reviewers). On the 94 cancer samples, DL accuracy on favorable/unfavorable prognosis was 98.9% vs. 95.8-97.9% for reviewers. 2) On the bigger dataset with 117447 gallery images from 161 cancer samples, CellFind obtained accuracy = 96.0% and F1 = 87.8% for CTC identification (TP = 17039, FP = 1735, FN = 3013, TN = 95660). For favorable/unfavorable prognosis the DL accuracy was 95.4%. Conclusion: Automated identification and enumeration of CTCs in CELLSEARCH images with CellFind can remove human subjectivity from the review process and maximize standardization among different research centers. CellFind performed better than most operators and reduced the data processing time required for each blood sample by the operator. Citation Format: Luca Biasiolli, Pietro Ansaloni, Nicolò Gentili, Daniele Giardiello, Francesco Montanari, Ramona Miserendino, Giulio Signorini, Gianni Medoro. Automated identification and enumeration of CELLSEARCH Circulating Tumor Cells (CTC) with a deep learning algorithm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7492.

Abstract 3098: The first circulating tumor cell-derived explant (CDX) model of a Merkel cell carcinoma

Abstract NeuroEndocrine Carcinomas (NECs) are rare yet lethal diseases; they are frequently metastatic and have limited treatment options. Whilst progress has been made in understanding the biology of Small Cell Lung Cancer (SCLC), the most studied NEC, research on NECs from other anatomical sites lags behind. In SCLC, circulating tumor cells (CTCs) can be used to generate patient-relevant mouse models; termed CTC-Derived eXplants (CDX) which are faithful in biology and chemotherapy response to donor patients1,2. A pilot prospective study is on-going to assess the potential utility of cell-free DNA (cfDNA) and/or CellSearch (CS) CTCs (EpCAM/cytokeratin positive) as liquid biopsies and feasibility of CDX generation from patients with advanced stage extrapulmonary NECs undergoing palliative chemotherapy. CS CTCs are enumerated before and during treatment and marker-independent RosetteSep-enriched CTCs from parallel blood samples are implanted in immunodeficient mice to attempt CDX generation. Twenty-seven patients were recruited from Jun-19 to Nov-21. CS-CTCs were present in 19/27; 70% pre-treatment samples (mean 57, median 2, range 0-685 CTCs per 7.5mL of blood). CDX generation was successful from a pre-treatment CTC sample from a patient originally diagnosed with a NEC of unknown origin who responded to Platinum/Etoposide. Whole-exome sequencing of this CDX and matched pre-treatment cfDNA from the donor revealed largely overlapping copy number changes and common mutations including an activating PIK3CA mutation. In addition, there was no evidence of TP53 or RB1 loss (hallmarks of SCLC) in neither the CDX nor the donor’s cfDNA. Immunohistochemical (IHC) analysis of this CDX showed a small cell NEC morphology, 90% Ki-67 positive cells, expression of EpCAM, pan-cytokeratins and neuroendocrine (NE) diagnostic markers, concordant with the donor’s biopsy. Further IHC and RNA sequencing revealed expression of the NE transcription factor atonal homolog 1 (ATOH1)2, CK20 and abundant Merkel cell polyomavirus (MCPyV) transcripts. These characteristics prompted reclassification of the original patient diagnosis as Merkel cell carcinoma (MCC). There was only 1 CS-CTC in the parallel pre-treatment blood sample implying that EpCAM-negative CTCs may have given rise to the CDX. The CDX showed in vivo sensitivity to Platinum/Etoposide, mirroring treatment response of the donor. Ex vivo cultures from CDX-derived cells grow as suspension clusters similar to ‘classic’ SCLC and MCPyV-positive MCC cell lines. In summary, this first Merkel Cell Carcinoma CDX recapitulates the biology of the donor’s tumor. As the donor remains alive 25 months from diagnosis of advanced disease, their CDX can serve as an avatar to guide future treatment. 1. Hodgkinson et al., Nature Med 2014, 20: p. 897-903. 2. Simpson et al., Nature Cancer 2020, 1: p. 437-451. Citation Format: Melissa Frizziero, Elaine Kilgour, Kathryn L. Simpson, Dominic Rothwell, Kristopher Frese, Melanie Galvin, Yitao Chen, Alastair Kerr, Sam Humphrey, Mukarram Hossain, Juan W. Valle, Mairéad G. McNamara, Caroline Dive. The first circulating tumor cell-derived explant (CDX) model of a Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3098.

Abstract P2-02-06: Her2-low ctcs in breast cancer: Pipeline for phenotypic driven, single-cell collection and molecular analysis

Abstract Background The presence of circulating tumor cells (CTCs) expressing HER2 at low score (HER2-low) in the peripheral blood of metastatic breast cancer (MBC) patients has been associated with resistance to treatment and more aggressive metastatic behavior. However, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges beyond the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs to perform single-cell molecular analysis. Methods 4 different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, SKBR3), that are known to express HER2 at different immunohistochemistry scores (respectively classified as 0, 1+, 2+, 3+), were spiked (around 500 cells each) in healthy donor blood tubes (7.5 ml). Samples were subsequently enriched through the CellSearch™ (Menarini Silicon Biosystems, Bologna, Italy), HER2 stained using the CellSearch CTC Kit and analyzed with the ACCEPT tool (Zeune et al., 2017). Enriched cells were additionally characterized by the DEPArray NxT™ Cell Browser and subsequently collected in pooled and single cells. The HER2 signal-intensity scores (fluorescein isothiocyanate, FITC mean), detected by both tools in each cell line, was compared using the nonparametric Mann-Whitney U test. The optimal cut-offs to distinguish HER2 1+ from HER2 0 and HER2 2+ cells were calculated performing Receiving Operator Curves (ROC). Results Detected mean intensities retrieved from CellSearch™ and analyzed with the ACCEPT tool were respectively 0.34 (MDA-MB-231, 0), 1.44 (T47D, 1+), 15.28 (MDA-MB-453, 2+) 166 (SKBR3, 3+), resulting in the possibility to discriminate both 2+ and 3+ cells from 1+ and 0 (P < 0.00001). No statistical significance has been observed between 1+ and 0 (P=0.19). Conversely, HER2 signal-intensity scores detected with the Cell Browser were: 3.69 (0), 4.38 (1+), 6.28 (2+) and 42.82 (3+). These results allow DEPArray Nxt to efficiently differentiate each single cell line, in particular HER2 1+ cells from both 0 and 2+ (P < 0.00001), enabling the collection of these specific cells. The area under the ROC was 0.7 and 0.72 (respectively 0 vs 1+ and 1+ vs 2+) and the optimal calculated cut-offs were 3.23 (lower) and 4.61 (higher). Conclusions HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. Downstream single-cell or pooled collection can be subsequently performed. Further molecular characterizations, as DNA genome sequencing, could highlight the underlying altered pathways responsible for resistance to treatment and molecular patterns accountable for worse prognosis. Citation Format: Paolo D'Amico, Carolina Reduzzi, Wenan Qiang, Qiang Zhang, Lorenzo Gerratana, Ami N Shah, Andrew A Davis, Anthony Kang, Meilynn Shi, Youbin Zhang, Saya Jacob, Amir Behdad, Giuseppe Curigliano, Massimo Cristofanilli. Her2-low ctcs in breast cancer: Pipeline for phenotypic driven, single-cell collection and molecular analysis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-06.

Correlation of baseline circulating tumor cells (CTC) and associated genomic profile with survival outcomes in patients (pts) with metastatic castration-sensitive prostate cancer (mCSPC) in a real-world cohort.

5077 Background: We recently published, in the context of SWOG1216 trial in pts with mCSPC, that higher baseline CTC level were associated with inferior survival outcomes (Goldkorn. Agarwal, CCR, 2021). Here in, we validate these findings in a real world population of mCSPC and interrogate tumor genomic profile with respect to the CTC level. Methods: Eligibility criteria: new mCSPC receiving ADT without or with intensification (docetaxel or novel hormonal therapy) and enumeration of baseline CTCs by FDA cleared Cell Search CTC assay. Gene alterations were determined by comprehensive genomic profiling (CGP) of tumor tissue (Foundation Medicine). CTC counts were categorized as 0, 1-4 and ≥5/7.5 ml. Relationships between CTC counts and number (no.) of genes altered and individual gene alterations were assessed via Kruskal-Wallis and chi-squared tests, respectively. Relationships between progression-free survival (PFS), overall survival (OS) and individual mutations were assessed via log-rank tests. Relationships between CTC counts, PFS and OS were assessed by Cox proportional hazards models, both unadjusted and adjusted for multiple variables (Table). Results: Overall 103 pts were eligible. Median age: 67 yrs, Gleason score: 9, PSA at ADT initiation: 41 ng/mL. 67 (65%) pts had de-novo metastatic disease and 44 (43%) pts underwent ADT intensification therapy. Pts with greater CTC counts tended to have greater no. of altered genes (p=0.017), greater no. of total alterations (p=0.017) and higher rate of TP53 mutations (p=0.036). In univariate analyses (UVA) and multivariable analyses (MVA), both CTC counts and no. of genes altered were strongly associated with both PFS and OS (Table). CGP of tumors with respect to CTC counts will be presented in meeting. Conclusions: Herein, we validate our previous findings from SWOG1216 trial of association of higher CTC level with inferior survival outcomes in a real world mCSPC cohort. The CTC enriched population is associated with a distinct tumor genomic landscape, which may guide further drug development in this pt population at the highest risk of progression and/or death.[Table: see text]

Association of circulating tumor cells (CTC) with survival outcomes in patients (pts) with metastatic castration-sensitive prostate cancer (mCSPC) in a real-world cohort.

59 Background: In mCSPC, baseline CTC counts have been shown to correlate with PSA responses and progression free survival (PFS) in small studies in the context of androgen deprivation therapy (ADT) without modern intensification with docetaxel or novel hormonal therapy. Similar correlation of CTC count with PSA responses and PFS was recently reported from an ongoing phase 3 trial in mCSPC setting (SWOG1216) without reporting the association in the context of ADT intensification. Furthermore, none of these studies correlated CTCs with overall survival (OS). Herein we evaluated whether CTCs were associated with outcomes including OS in a real world mCPSC population treated with intensified as well as non-intensified ADT. Methods: Eligibility criteria: new mCSPC receiving ADT with or without intensification and enumeration of baseline CTCs by FDA cleared Cell Search CTC assay. The relationship between CTC counts (categorized as: 0, 1-4, and ≥5/7.5 ml) and both PFS and OS was assessed in the context of Cox proportional hazards models, both unadjusted and adjusted for age, Gleason, PSA at ADT initiation, de novo vs. non-de novo status, and ADT intensification vs. non-intensification therapy. Results: Overall 99 pts were identified. Baseline characteristics are summarized in Table. In unadjusted analyses, CTC counts of ≥5 as compared to 0 were strongly associated with inferior PFS (hazard ratio [HR] 3.38, 95% CI 1.85-6.18; p < 0.001) and OS (HR 4.44 95% CI 1.63-12.10; p = 0.004). In multivariate analyses, CTC counts of ≥5 as compared to 0 continued to be associated with inferior PFS (HR 5.49, 95% CI 2.64-11.43; p < 0.001) and OS (HR 4.00, 95% CI 1.31-12.23; p = 0.015). Within the ADT intensification subgroup also, high CTC counts were associated with poor PFS and OS. For PFS, the univariate HR for CTC ≥5 vs. 0 was 4.87 (95% CI 1.66-14.30; p = 0.004) and multivariate HR for CTC ≥5 vs. 0 was 7.43 (95% CI 1.92-28.82; p = 0.004). For OS, the univariate HR for CTC ≥5 vs. 0 was 15.88 (95% CI 1.93-130.58; p = 0.010) and multivariate HR for CTC ≥5 vs. 0 was 24.86 (95% CI 2.03-304.45; p = 0.012). Conclusions: To best of our knowledge this is the first study to show that high baseline CTC counts are strongly associated with inferior PFS as well as OS in pts with newly diagnosed mCSPC, even in those who received intensified ADT therapy. Identifying these pts at highest risk of progression and death can help with counselling and prognostication in clinics as well as design and enrollment in future clinical trials. [Table: see text]

Development and validation of circulating tumor cell (Epic Sciences) enumeration as a prognostic biomarker in men with metastatic castration-resistant prostate cancer.

157 Background: We evaluated the prognostic significance of circulating tumor cell (CTC) number as determined on the Epic Sciences platform in men with metastatic castration resistant prostate cancer (mCRPC) treated with an androgen receptor signaling inhibitor (ARSI). Methods: A pre-treatment blood sample was collected from men with progressing mCRPC starting either abiraterone or enzalutamide as a 1st, 2nd or 3rd line systemic therapy at Memorial Sloan Kettering Cancer Center (Discovery cohort, N=175) or as a 1st and 2nd line therapy as part of the multi-center PROPHECY trial (NCT02269982) (Validation cohort, N=107). Enumeration was performed on the Epic Sciences platform and associated with overall survival (OS) in the Discovery cohort, and progression-free survival (PFS) and OS in the Validation cohort. Matched blood samples from the Validation cohort were to CTC counts measured on the CellSearch Circulating Tumor Cell kit. Results: In the MSKCC Discovery cohort, CTC count was a statistically significant prognostic factor of OS as a dichotomous (< 3 CTCs/mL versus ≥ 3 CTCs/mL; HR = 1.8, (1.3-3.0, 95% CI)) and as a continuous variable when adjusting for line of therapy, presence of visceral metastases, PSA, lactate-dehydrogenase, and alkaline-phosphatase. The findings were validated in an independent dataset (PROPHECY trial) - (HR (95% CI) = 1.8, (1.1-3.0) for OS and 1.7 (1.1-2.9), for PFS). A strong correlation was observed between CTC counts determined in matched samples on the CellSearch and Epic platforms (r = 0.84). Conclusions: The findings validate the prognostic significance of pretreatment CTC number determined on the Epic Sciences platform for predicting OS in men with progressing mCRPC starting an ARSI.

Prospective Multicenter Study of Circulating Tumor Cell AR-V7 and Taxane Versus Hormonal Treatment Outcomes in Metastatic Castration-Resistant Prostate Cancer.

PURPOSEAndrogen receptor splice variant 7 (AR-V7) detection in circulating tumor cells (CTCs) is associated with a low probability of response and short progression-free (PFS) and overall survival (OS) in men with metastatic castration-resistant prostate cancer (mCRPC) treated with enzalutamide or abiraterone. However, it is unclear whether such men benefit from taxane chemotherapy.PATIENTS AND METHODSPROPHECY is a multicenter prospective blinded study of patients with poor-risk mCRPC starting abiraterone or enzalutamide and observed through subsequent progression and taxane chemotherapy. We assessed AR-V7 status using the Johns Hopkins modified AdnaTest CTC AR-V7 messenger RNA assay and the Epic Sciences CTC nuclear-localized AR-V7 protein assay before treatment. The primary objective was to validate the independent prognostic value of CTC AR-V7 status based on radiographic/clinical PFS. OS, confirmed prostate-specific antigen (PSA), and objective radiologic responses were secondary end points.RESULTSWe enrolled 118 men with mCRPC treated with abiraterone or enzalutamide, 51 of whom received subsequent docetaxel or cabazitaxel. Pretreatment CTC AR-V7 status by the Johns Hopkins and Epic Sciences assays was independently associated with worse PFS (hazard ratio [HR], 1.7; 95% CI, 1.0 to 2.9 and HR, 2.1; 95% CI, 1.0 to 4.4, respectively) and OS (HR, 3.3; 95% CI, 1.7 to 6.3 and HR, 3.0; 95% CI, 1.4 to 6.3, respectively) and a low probability of confirmed PSA responses, ranging from 0% to 11%, during treatment with abiraterone or enzalutamide. At progression, subsequent CTC AR-V7 detection was not associated with an inferior PSA or radiographic response or worse PFS or OS with subsequent taxane chemotherapy after adjusting for CellSearch CTC enumeration and clinical prognostic factors.CONCLUSIONDetection of AR-V7 in CTCs by two different blood-based assays is independently associated with shorter PFS and OS with abiraterone or enzalutamide, but such men with AR-V7–positive disease still experience clinical benefits from taxane chemotherapy.

Abstract 5379: Non-invasive profiling of advanced prostate cancer via multi-parametric liquid biopsy and radiomics

Abstract Introduction: Biomarkers of treatment response in advanced prostate cancer (PC) are urgently needed. Liquid biopsy profiling and radiomic imaging enable non-invasive, repeatable tumor profiling, and combining these modalities could yield powerful new predictive tools. We tested the feasibility of concurrent, multi-parametric molecular analysis of circulating tumor cells (CTCs) and matched plasma cell-free DNA (cfDNA), combined with radiomic analysis of CT scans from the same patients. Methods: Under IRB-approved informed consent, blood was collected from 23 patients with metastatic castrate resistant PC (mCRPC). CTCs were enumerated using the RareCyte and CellSearch platforms, and individual single CTCs and white blood cells (WBCs) were retrieved using RareCyte's integrated robotic micropipette. DNA was extracted from single cells (scDNA), as well as from matched cfDNA and buffy coats (bcDNA), then sequenced using Ampliseq HD Pan-cancer panel. Concurrent radiomic analysis was performed on bone metastatic lesions identified on CT scans from the same patients, and manually segmented regions of interest were queried for a variety of texture metrics. Results: Of 19 patients with both CellSearch and RareCyte enumeration, CTCs were detected in 12 by CellSearch (63%; median: 3/7.5 mL; range 1-343/ 7.5 mL) and in 14 by RareCyte (74%; median: 1/7.5 mL; range 1-363/7.5 mL). Using matched bcDNA as a gold standard, sensitivity and positive predictive value of germline SNP detection were 74% and 93% for scDNA, and 98% and 99% for cfDNA. A total of 48 and 18 somatic alterations were identified across the cohort in CTCs and cfDNA, respectively, including mutations in AR, TP53, CCND3, FGFR1, ALK, and ROS1. Of 14 patients whose single CTCs were recovered by RareCyte, 12 (86%) had detectable somatic mutations in scDNA, and 7 (50%) had detectable somatic mutations in matched cfDNA. While some mutations were concordant between matched scDNA and cfDNA, most were distinct. Radiomic entropy in CT scans was associated with CellSearch CTC-counts (AUC of 0.74) using the FDA-cleared prognostic threshold of <5 vs. ≥5 CTCs/7.5ml. Conclusion: We successfully piloted an integrated, multi-parametric analysis of matched single CTCs, WBCs, plasma cfDNA, and buffy coats, along with matched radiomic analysis of CT scans in patients with mCRPC. Germline variants were detected more readily in cfDNA, whereas somatic mutations in commonly altered genes were detected more frequently in CTCs. CellSearch CTC count, an FDA cleared prognostic biomarker, correlated with radiomic entropy, the first such association to our knowledge between liquid biopsy and radiographic readouts. The results demonstrate the feasibility of this approach and its potential to generate comprehensive molecular data and new biomarker profiles. Prospective validation in a large PC cohort is underway. Citation Format: Gareth J. Morrison, Jonathan Buckley, D. Gigi Ostrow, Bino Varghese, Nolan Ericson, Steven Cen, Alexander Cunha, Yi-Tsung Lu, Tad George, Jeffrey Smith, David Quinn, Vinay Duddalwar, Timothy Triche, Amir Goldkorn. Non-invasive profiling of advanced prostate cancer via multi-parametric liquid biopsy and radiomics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5379.

Abstract IA19: Multiple uses of circulating tumor cells in lung cancer?

Abstract Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA. Citation Format: Caroline Dive. Multiple uses of circulating tumor cells in lung cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA19.

Baseline circulating tumor cell (CTC) count as a prognostic marker of PSA response and progression in metastatic castrate sensitive prostate cancer (mCSPC): Results from SWOG S1216, a phase III randomized trial of androgen deprivation plus orteronel (cyp17 inhibitor) or bicalutamide.

5506 Background: In mCSPC, androgen deprivation therapy (ADT) combined with chemotherapy or androgen receptor signaling inhibition (ARSI) is the new standard of care. Biomarkers that predict clinical outcomes with these therapies are needed. We hypothesized that CellSearch CTC count, an FDA-cleared biomarker in metastatic castrate resistant PC (mCRPC), may be a valuable biomarker in mCSPC. Methods: In S1216, peripheral blood was drawn with informed consent at registration (baseline), and CTCs were enumerated on the FDA-cleared CellSearch platform (Menarini) per standard manufacturer protocol. CTC counts were analyzed centrally for associations with 2 pre-specified trial intermediate endpoints: 7-month PSA (7mPSA) ≤ 0.2 ng/ml vs. 0.2–4.0 vs. > 4.0, (intermediate endpoint for overall survival, OS); and progression-free survival (PFS) < vs. > 2 years. Because OS data have not matured, analysis was pooled and equal numbers of samples were analyzed from each treatment arm and outcome measure (7mPSA and PFS) as stipulated by the Data Safety Monitoring Committee. Results: From 2014 to 2017, 523 baseline samples were collected. In the 7mPSA analysis (n = 264), CTCs were detected in 38% of men, with a median of 4 CTCs in those with detectable CTCs. In the PFS analysis (n = 336), CTCs were detected in 37% of men, with a median of 3 CTCs in those with detectable CTCs. Adjusting for disease burden (minimal vs. extensive) and ADT status (already initiated or not) at the time of CTC measurement, men with undetectable CTCs were 6.1-fold more likely to attain 7mPSA ≤ 0.2 (OR 6.1, 95% CI 2.1-17.2, p < 0.001) and 3.7-fold more likely to achieve > 2 years PFS (OR 3.7, 95% CI 1.7-8.1, p < 0.001) compared to men with baseline CTCs ≥ 5. Other cutpoints previously validated in mCRPC studies (CTC < 5 vs. ≥5 and CTCs 0 vs. ≥1) also strongly discriminated 7mPSA and PFS with statistical significance in this mCSPC cohort. Conclusions: CTC count at the start of treatment for mCSPC was highly prognostic of 7-month PSA response (intermediate endpoint for OS) and of PFS at 2 years. To our knowledge, this is the first such strong evidence from a prospective phase 3 trial of this magnitude. Additional analyses are planned when the trial is fully reported. Baseline CTC count may serve as a valuable prognostic marker to discriminate men likely to respond favorably to hormonal therapies from those who may benefit from early alternate interventions.

Clinical Utility of Circulating Tumour Cell Androgen Receptor Splice Variant-7 Status in Metastatic Castration-resistant Prostate Cancer

BackgroundDetection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumour cells (CTCs) is associated with worse outcome in metastatic castration-resistant prostate cancer (mCRPC). However, studies rarely report comparisons with CTC counts and biopsy AR-V7 protein expression. ObjectiveTo determine the reproducibility of AdnaTest CTC AR-V7 testing, and associations with clinical characteristics, CellSearch CTC counts, tumour biopsy AR-V7 protein expression and overall survival (OS). Design, setting, and participantsCTC AR-V7 status was determined for 227 peripheral blood samples, from 181 mCRPC patients with CTC counts (202 samples; 136 patients) and matched mCRPC biopsies (65 samples; 58 patients). Outcome measurements and statistical analysisCTC AR-V7 status was associated with clinical characteristics, CTC counts, and tissue biopsy AR-V7 protein expression. The association of CTC AR-V7 status and other baseline variables with OS was determined. Results and limitationsOf the samples, 35% were CTC+/AR-V7+. CTC+/AR-V7+ samples had higher CellSearch CTC counts (median CTC; interquartile range [IQR]: 60, 19–184 vs 9, 2–64; Mann-Whitney test p<0.001) and biopsy AR-V7 protein expression (median H-score, IQR: 100, 63–148 vs 15, 0–113; Mann-Whitney test p=0.004) than CTC+/AR-V7− samples. However, both CTC− (63%) and CTC+/AR-V7− (62%) patients had detectable AR-V7 protein in contemporaneous biopsies. After accounting for baseline characteristics, there was shorter OS in CTC+/AR-V7+ patients than in CTC− patients (hazard ratio [HR] 2.13; 95% confidence interval [CI] 1.23–3.71; p=0.02); surprisingly, there was no evidence that CTC+/AR-V7+ patients had worse OS than CTC+/AR-V7− patients (HR 1.26; 95% CI 0.73–2.17; p=0.4). A limitation of this study was the heterogeneity of treatment received. ConclusionsStudies reporting the prognostic relevance of CTC AR-V7 status must account for CTC counts. Discordant CTC AR-V7 results and AR-V7 protein expression in matched, same-patient biopsies are reported. Patient summaryLiquid biopsies that determine circulating tumour cell androgen receptor splice variant-7 status have the potential to impact treatment decisions in metastatic castration-resistant prostate cancer patients. Robust clinical qualification of these assays is required before their routine use.

21O - EPAC-Lung: Pooled analysis of circulating tumor cells in advanced non-small cell lung cancer

Background: We assessed the clinical validity of circulating tumor cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a European pooled analysis of individual patient data. This is the largest study of its kind and the first to examine between-centre heterogeneity of CTC identification in NSCLC. Methods: Nine European NSCLC CTC centers were asked to provide reported/unreported anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 - March 2017. We used Cox regression models, stratified by centre, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinico-pathological models using likelihood ratio (LR) statistics and c-indices. Results: Seven out of nine eligible centers provided data for 550 eligible patients, including 209 patients whose prognostic information was previously unpublished. CTC counts of ≥ 2 and ≥5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: HR 1.72, p < 0·001; ≥5 CTCs: HR 2.21, p < 0·001) and overall survival (≥2 CTCs: HR 2·18, p < 0·001; ≥5 CTCs: HR 2·75, p < 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinico-pathological models (log-transformed CTCs p < 0·0001; ≥2 CTCs p < 0·0001; ≥5 CTCs p < 0·0001), while more moderate improvements were observed with the use of c-index models. There was minor evidence of between-center heterogeneity in the effect on PFS, but not OS.No difference in CTC profile was observed between key NSCLC molecular subsets such as EGFR, ALK, and KRAS. Conclusions: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC. CTC count improves prognostication when added to full clinico-pathological predictive models. ≥2 CTCs is an appropriate cutoff to move towards establishing clinical utility. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: C.R. Lindsay: Institutional funding for an ongoing phase II trial for which I am PI; Supported by Roche as part of an ESMO translational fellowship awarded in 2014-2016. F.H. Blackhall: Grants: AstraZeneca, Novartis, Pfizer, Amgen, BMS; Consultancy fees: Cell Medica, MSD; Speaker bureau: BI; Advisory board work: Regeneron, Medivation, AbbVie, Takeda, Roche, Ibsen. M.G. Krebs: Advisory board: J&J. L. Terstappen: Inventor on a number of US patents related to CellSearch, rights of which assigned to Johnson&Johnson, CellSearch kits obtained from Johnson&Johnson through a collaborative agreement with the MCBP. J-C. Soria: Consultancy fees: AZ, Astex, Clovis, GSK, GamaMabs, Lilly, MSD, Mission Therapeutics, Merus, Pfizer, PharmaMar, Pierre Fabre, Roche/Genentech, Sanofi, Servier, Symphogen, Takeda; Full time employee: MedImmune; Shareholder: AZ, Gritstone. All other authors have declared no conflicts of interest.

Scanning Electron Microscopy of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles.

To explore morphological features of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs), we developed a protocol for scanning electron microscopy (SEM) of CTCs and tdEVs. CTCs and tdEVs were isolated by immunomagnetic enrichment based on their Epithelial Cell Adhesion Molecule (EpCAM) expression or by physical separation through 5 μm microsieves from 7.5 mL of blood from Castration-Resistant Prostate Cancer (CRPC) patients. Protocols were optimized using blood samples of healthy donors spiked with PC3 and LNCaP cell lines. CTCs and tdEVs were identified among the enriched cells by fluorescence microscopy. The positions of DNA+, CK+, CD45− CTCs and DNA−, CK+, CD45− tdEVs on the CellSearch cartridges and microsieves were recorded. After gradual dehydration and chemical drying, the regions of interest were imaged by SEM. CellSearch CTCs retained their morphology revealing various shapes, some of which were clearly associated with CTCs undergoing apoptosis. The ferrofluid was clearly distinguishable, shielding major portions of all isolated objects. CTCs and leukocytes on microsieves were clearly visible, but revealed physical damage attributed to the physical forces that cells exhibit while entering one or multiple pores. tdEVs could not be identified on the microsieves as they passed through the pores. Insights on the underlying mechanism of each isolation technique could be obtained. Complete detailed morphological characteristics of CTCs are, however, masked by both techniques.

Abstract 5601: Single-cell molecular profiling of circulating tumor cells (CTCs) within the TRACERx study reveals heterogeneous patterns in early non-small cell lung cancer (NSCLC)

Abstract Introduction: For early stage NSCLC, surgery with curative intent is the most common therapeutic option; however, tumour recurrence occurs in approximately 50% of cases and most commonly at distant sites. Analysis of CTCs represents a potential means of tracking cancer cells from the primary tumour and has the potential to provide insights into the metastatic process and mechanisms of resistance to therapy linking cell phenotype and genotype. Within the TRACERx study, we are carrying out whole genome analysis of single CTCs obtained from NSCLC patients undergoing surgery in order to examine the relationship between CTCs, the primary tumour and metastatic relapse. Methods: Blood from 163 patients was collected at surgery from the tumour draining pulmonary vein and from peripheral vein in longitudinal samples. CTCs were enriched based on their EpCAM expression using CellSearch® and then enumerated according to their positivity for epithelial markers (Cytokeratins) and lack of detectable blood markers (CD45). Samples containing at least five CellSearch® CTCs (CS-CTCs) were processed through DEPArray™ for single cell isolation followed by whole genome amplification (WGA) using the Ampli1™ WGA kit. Genome wide copy number alterations (CNA) were detected through low-depth whole genome sequencing (WGS) and mutational analysis of individual CTCs was carried out using whole exome sequencing (WES). CTC genomic profiles were compared to the corresponding spatially separated tumour sections obtained at surgery. Results: In a pilot study of 30 patients undergoing tumour resection, the presence of pulmonary vein CS-CTCs has been linked to poor clinical outcome (Crosbie et al, J Thorac Oncol, 2016). Here we extended the evaluation of pulmonary vein CS-CTCs to a further 122 patients and confirmed the initial observation that the presence of pulmonary vein CS-CTCs is a poor prognostic indicator. CNA analysis of 100 single CTCs from the first 12 patients revealed heterogeneous patterns between patients and within individual patients. Three types of pulmonary CS-CTC candidates were observed: Type 1, which share clear copy number changes with both tumour and other CS-CTCs; Type 2, where copy number changes were detected but were not shared with the tumour; Type 3, where no copy number changes were detected. Using WES of both CTCs and matching excised tumour regions, we identified common genetic alterations shared by CTCs and the tumour, as well as private genetic changes detected only in CTCs. We are currently evaluating metastatic biopsies from relapsing patients and comparing their molecular profiles to those seen in CS-CTCs obtained at surgery. Conclusion: The combined tumour and CTC data show that single CTC analysis provides an additional layer of complexity with a valuable new perspective on tumour heterogeneity and early dissemination in NSCLC. Citation Format: Francesca Chemi, Sakshi Gulati, Dominic G. Rothwell, Debbie Burt, Daniel Slan-Tan, Barbara Mesquita, Chris Wirth, Gareth Wilson, Jackie Pierce, Ged Brady, Charles Swanton, Caroline Dive. Single-cell molecular profiling of circulating tumor cells (CTCs) within the TRACERx study reveals heterogeneous patterns in early non-small cell lung cancer (NSCLC) [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 5601.

Abstract 3645: Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT

Abstract Introduction. Composition of cell suspensions enriched for Circulating Tumor Cells (CTC) by the CellSearch system has not yet been explored as the system only presents DAPI+/Cytokeratin+ objects to the operator to identify CTC. We used ACCEPT (http://github.com/LeonieZ/ACCEPT) to identify and characterize all DAPI+ nuclei in EpCAM enriched samples and explored avenues to increase the number of nuclei that can be assigned to a cell lineage. Addition of CD16, expressed on granulocytes and NK-cells, the detection of a cellular plasma membrane and the use of LED light, as opposed to the mercury arc lamp, to improve the excitation of CD45-APC were tested. Methods. In 127 controls and 300 samples from 192 metastatic lung cancer patients, CD16-PerCP was added to the CellSearch CTC identification antibody cocktail. A CellTracks Analyzer equipped with six filter cubes was used to acquire the images. For exploration, after the CellTracks image acquisition 5 cartridges were scanned again on a microscope that contains a LED light source to determine if this would improve the detection of CD45-APC. Also, wheat germ agglutinin (wga) conjugated to AlexaFluor488 stains the cellular plasma membrane and was added to the CellSearch antibody cocktail to discriminate between bare nuclei and unstained cells in 4 samples. The stack of fluorescence images was analyzed with the open source imaging program ACCEPT to define and identify various cell populations. Results. In 427 samples 26,285 ±26,590 [603-156,519] (mean, SD, [range]) nuclei were identified with ACCEPT in the CellSearch images of which only 14% ±15 were identified as leukocytes or CTC, remaining 86% ±15 cells unidentified. Adding CD16 increased the leukocyte population by 47% ±17, thereby reducing the unidentified population to 39% ±19 of the total number of nuclei. CD16-staining was found on 13% of the CTC. Using a LED-light source improved the identification of APC+ cells, and reduced the amount of unidentified nuclei in these samples from 29% (±5) to 14% (±10). Exploration with wga indicated that 43% (±11) (mean 987 cells, range 331-2,638 cells) of the unstained cells contained a cellular membrane. Conclusion. In 427 blood samples run on CellSearch, only 14% of the nuclei obtained after EpCAM enrichment could be assigned as a leukocyte or a CTC. By adding CD16 to the antibody cocktail, the unidentified population is reduced from 86% to 39%. Also, 13% of nuclei scored as CTC express CD16, suggesting these are granulocytes that are incorrectly designated as tumor cells. Using a LED light source improves the identification of dimly stained CD45+ cells. Exploration with wga indicates that 57% of the unidentified nuclei are simply bare nuclei and the origin of 43% still remains unknown. Further studies are needed to determine whether EpCAM+/Cytokeratin- CTC are present among the nucleated cells of which the origin is not yet known. Citation Format: Sanne De Wit, Leonie L. Zeune, T. Jeroen N. Hiltermann, Harry J. Groen, Leon W.M. Terstappen. Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT [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 3645.

Prognostic value of chromogranin-a (CGA) compared to circulating tumor cells (CTCs) in metastatic castration resistant prostate cancer (mCRPC).

249 Background: Serum CGA has been identified as a candidate prognostic biomarker for mCRPC. In a two cohort study, we compared the prognostic value of serum CGA with a validated CTC assay. Methods: In the discovery cohort (DC), blood samples were collected from 256 men with mCRPC. In an independent validation cohort (VC), 92 men with mCRPC were enrolled in a biospecimen collection study. In both cohorts, men receiving proton pump inhibitors and those with non-castrate levels of testosterone ( &gt; 50ng/dl) were excluded. Serum CGA was measured in a homogeneous automated immunofluorescent assay using time-resolved amplified cryptate emission. In the VC, CTC enumeration was performed using the FDA cleared CELLSEARCH CTC test prior to treatment with abiraterone acetate/ prednisone. Cox proportional hazard regression and Kaplan-Meier analysis were performed for associations with elevated CGA (above reference range), unfavorable (≥ 5) CTCs, and overall survival (OS). Results: In the DC, 200 men were eligible for analysis. The median age was 72 years (yrs), 81/200 pts had a Gleason score (GS) ≥ 8, 34/200 had an elevated CGA. At a median follow up of 2.2 yrs, 156/200 were deceased. In the subset of men with GS ≥ 8, elevated CGA was associated with shorter OS [hazard ratio (HR) 2.19, p = 0.017]. In the VC, 71 men were eligible for analysis. The median age was 71 yrs, 36/71 tumors were GS ≥ 8, 31/71 pts had an elevated CGA, 26/66 had unfavorable CTCs (≥ 5). At a median follow up of 1.8 yrs, 31/71 were deceased. Elevated CGA (HR 1.91, p = 0.043) and unfavorable CTC counts (HR 2.97, p = 0.0012) were adversely associated with OS. In the high GS group, both CTCs and CGA had the same area under the curve (AUC) of 0.72. Pts with elevated CTC and CGA had the poorest OS (HR 3.76, p = 0.008). Conclusions: Elevated serum CGA was negatively associated with OS in men with mCRPC. Serum CGA represents a prognostic biomarker that may complement CTC enumeration. Clinical trial information: NCT#01953640.

Genomic and phenotypic evidence for prostate cancer osteomimicry in circulating tumor cells from men with metastatic castration resistant prostate cancer (mCRPC) treated with radium-223.

160 Background: Radium-223 is a targeted alpha therapy that improves survival in men with mCRPC. The biologic basis for radium-223 efficacy is not completely understood. We hypothesized that PC osteomimicry, a form of epithelial plasticity leading to an osteoblastic phenotype, may contribute to the intralesional deposition of radium-223 and subsequent irradiation of the tumor microenvironment. Methods: We conducted a pharmacodynamic study of radium-223 in men with bone predominant mCRPC to investigate genomic and phenotypic alterations in circulating tumor cells (CTCs), ctDNA, and metastases. Prior to radium and 3 and 6 months after radium, liquid and metastatic biopsies were collected, including CTCs for phenotypic characterization and CTC/ctDNA genomic analysis. The primary objective was to describe the prevalence of CTC bone alkaline phosphatase (BAP) over time. We measured radium-223 decay products in tumor and surrounding normal bone during treatment. Results: We enrolled 20 men with heavily pre-treated symptomatic bone predominant mCRPC and treated with radium-223 over a median of 6 doses. 55% had elevated serum BAP. PFS was 5.5 mo; OS was 13.3 mo; 10% had unfavorable (≥5) to favorable ( &lt; 5) Cellsearch CTC conversion; 5% had ≥30% PSA decline. We found evidence of persistent CTC BAP expression in both EpCAM+ and EpCAM- CTCs in the majority of men over time during radium treatment. We identified genomic gain of key osteomimicry regions in CTC DNA, including loci for BAP, osteopontin, and OB-cadherin. Radium-223 uptake was observed in tumor to a greater degree than surrounding normal bone. CTC DNA and matched ctDNA and CTC cultures suggested persistence of CTCs with aggressive genomic alterations such as AR, FOXA1, and MYC gain and PTEN and RB1 loss. We established multiple CTC cultures and one CTC PDX; cells exhibited evidence of epithelial plasticity with BAP expression. Conclusions: Osteomimicry may contribute to the uptake of Radium-223 within bone metastases and may thereby enhance the therapeutic benefit of radium-223. We found genomic and phenotypic evidence of osteomimicry in CTCs and CTC cultures from men with mCRPC. Clinical trial information: NCT02204943.

Detection of circulating tumor cells using oHSV1-hTERT-GFP in lung cancer.

BackgroundThis study was conducted to evaluate the clinical utility of the oHSV1‐hTERT‐GFP circulating tumor cell (CTC) detection method in the peripheral blood of patients with lung cancer by comparing its sensitivity to the CellSearch CTC detection method.MethodsThe oHSV1‐hTERT‐GFP and CellSearch CTC detection methods were compared using peripheral blood samples of patients pathologically diagnosed with lung cancer.ResultsA total of 240 patients with lung cancer were recruited, including 89 patients who were newly diagnosed and 151 patients who had previously received treatment. Sixty‐six newly diagnosed patients were evaluated using both methods. The CTC detection rates were 71.2% and 33.3% using the oHSV1‐hTERT‐GFP and CellSearch methods, respectively; this difference was statistically significant (P = 0.000). Among the entire cohort (n = 240), the CTC detection rate using the oHSV1‐hTERT‐GFP method was 76.3%, with a CTC count of 0–81. The CTC detection rates were 76.7%, 68.9%, and 76.3% in patients with squamous cell carcinoma, adenocarcinoma, and small cell lung cancer, respectively. There was no statistically significant difference in the CTC detection rates between these different pathological subtypes (P = 0.738). The CTC detection rates of 79.8% and 74.4% in patients with stage I–III and IV lung cancer, respectively, were not significantly different (P = 0.427).ConclusionThe oHSV1‐hTERT‐GFP method is highly effective for detecting CTCs in patients with lung cancer, independent of pathological type and disease stage, and is ideal for large‐scale clinical applications.

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