Collection Of Circulating Tumor Cells Research Articles

A Micropillar Array Based Microfluidic Device for Rare Cell Detection and Single-Cell Proteomics.

Advancements in single-cell-related technologies have opened new possibilities for analyzing rare cells, such as circulating tumor cells (CTCs) and rare immune cells. Among these techniques, single-cell proteomics, particularly single-cell mass spectrometric analysis (scMS), has gained significant attention due to its ability to directly measure transcripts without the need for specific reagents. However, the success of single-cell proteomics relies heavily on efficient sample preparation, as protein loss in low-concentration samples can profoundly impact the analysis. To address this challenge, an effective handling system for rare cells is essential for single-cell proteomic analysis. Herein, we propose a microfluidics-based method that offers highly efficient isolation, detection, and collection of rare cells (e.g., CTCs). The detailed fabrication process of the micropillar array-based microfluidic device is presented, along with its application for CTC isolation, identification, and collection for subsequent proteomic analysis.

Presence of Circulating Tumor Cells Predates Imaging Detection of Relapse in Patients with Stage III Melanoma.

Stage III melanoma includes nodal metastasis or in-transit disease. Five-year survival rates vary between 32% and 93%. The identification of high-risk patients is important for clinical decision making. We demonstrated previously that ≥1 circulating tumor cells (CTCs) at baseline was associated with recurrence. In this study, we investigated how frequently CTCs were identified prior to radiologically detected recurrence. Stage III patients (n = 325) had imaging at baseline and q 3 months. Baseline and q 6-12 months blood draws (7.5 mL) were performed to identify CTCs up to 3.5 years from diagnosis. CTC assessment was performed using the immunomagnetic capture of CD146-positive cells and anti-MEL-PE. The presence of one or more CTCs was considered positive. We analyzed the cohort of patients with relapse confirmed by radiologic imaging. CTC collection dates were assessed to determine the lead time for CTC detection. CTC-negative patients were significantly less likely to relapse compared to patients positive for CTCs (p-value < 0.001). Within the 325-patient cohort, 143 patients (44%) had recurrence, with a median follow-up of 52 months from diagnosis. The cohort (n = 143) with positive imaging and CTC results revealed 76% of patients (108/143) had CTC+ results before the radiological identification of relapse. The median time between positive CTC and positive imaging was 9 months. CTCs were positive in >75% of patients prior to relapse at a median of 9 months before radiologic detection.

A phase II study of olaparib (AZD2281) in patients (Pts) with metastatic/advanced urothelial carcinoma and other genitourinary (GU) tumors with DNA-repair defects.

TPS4607 Background: Olaparib is a poly-ADP-ribose polymerase (PARP) inhibitor that induces synthetic lethality in tumor cells with dysfunctional DNA damage repair (DDR) from loss of function of BRCA or other DDR genes. It improves survival in advanced ovarian, pancreatic, prostate and breast carcinoma with DDR gene alterations and is FDA approved for these indications. PARP inhibitors still have an undefined role in non-prostate GU tumors. We hypothesize that PARP inhibitors will have activity in DDR deficient tumors, regardless of histology. We are conducting a phase 2 trial of olaparib in non-prostate GU cancers with DDR gene defects. Methods: Eligible pts must have metastatic/advanced urothelial carcinoma or other non-prostate GU cancers and have measurable disease by RECIST 1.1. Pts in Cohorts 1 and 2 must harbor tumor or liquid pathologic DDR alterations identified by FoundationOne CDx (F1CDx) panel and reviewed by a molecular genetics review panel: Cohort 1 includes BRCA1, BRCA2, ATM, BAP1, MSH2, PALB2, and BRIP1 alterations, and pts with high tumor mutational burden (TMB) (&gt;15 mutations/megabase); Cohort 2 (exploratory cohort) includes ABL1, ATR, ATRX, BARD1, BRD4, CCND1, CHEK1, CHEK2, DOTIL, FANCC, FANCE, FANCG, FANCL, IKBKE, MEN1, MLH1, MSH2, MSH6, MUTYH, NPM1, PMS2, POLD1, POLE, RAD51, SMARCB1, STEK11, and TP53 alterations. Cohort 3 (natural history) will include pts without pathologic alterations in any Cohort 1 or 2 genes. Pts with eligible gene alterations (Cohorts 1 and 2) will be given olaparib 300mg twice daily until progressive disease, unacceptable adverse events, withdrawal of consent or death. They will have circulating tumor cells (CTCs) collected at baseline and every 4 weeks, and CT restaging every 8 weeks. Pts in Cohort 3 will receive standard of care treatment and have baseline CTC collection and be followed for survival. The primary objective is to evaluate the objective response rates (ORR) in Cohorts 1 and 2. Secondary objectives include progression-free survival (PFS), overall survival (OS) and safety. In these cohorts, the trial will have a Simon optimal two-stage design. In Cohort 1, 25 evaluable pts will be required to provide 90% power with alpha = 0.1 to differentiate between a promising ORR of 30% versus an unacceptably low ORR of 10%. In Cohort 2, 35 evaluable pts will be required to provide 90% power with alpha = 0.05 to differentiate between a promising ORR of 30% versus an unacceptably low ORR of 10%. Exploratory objectives include estimating the distribution of DDR alterations in non-prostate GU malignancies, assessing the effectiveness of NGS in detecting DDR alterations in circulating tumor DNA (ctDNA), assessing levels of baseline CTCs and changes in CTC level with olaparib, and correlating with clinical outcomes. This is an ECTCN network clinical trial and is open to enrollment. Clinical trial information: NCT03375307 .

Abstract 3382: Detection of CTCs using a micro-channel combined fluid dynamics and electrophoresis in colorectal cancer

Abstract Background: Circulating tumor cells (CTCs) are tumor cells that can be isolated from peripheral blood. Investigation of CTCs might allow comprehensive studies at the DNA, RNA, and protein. However, fewer CTCs generally can be collected from patients with colorectal cancer, limiting the possibility of clinical applications of CTC analysis despite the development of many different methods. We have developed a micro-channel device that integrates hydrodynamic filtration (HDF) capable of size separation and dielectrophoresis (DEP) capable of electrical property separation, using peripheral blood from colorectal cancer patients. Method: A separation test was performed using a whole peripheral blood sample obtained from colorectal cancer patients with KRASmutation in the tumor tissue. The sample blood was diluted 2-fold with PBS, and red blood cells and plasma were removed using HDF. After that, Leukocytes and CTCs were separated in the same channel by DEP. DNA was extracted from CTCs (port 1) and leucocyte fraction (port 2), and KRAS mutations were detected using droplet digital PCR. In order to evaluate the separated fractions, microwell slide was used for evaluation by nuclear staining, CD45 and EpCAM antibody staining. Results: 52 colorectal cancer patients were participated. The median age of patients was 71 years, 38 (73%) were male. Twenty-eight (53.9%) had colon cancers. Four stage III and 48 stage IV patients were included. The quality of DNA extracted from all patients were suitable for molecular genetic analysis. The DNA concentration of port 1 was significantly lower than that of port 2 (371 ng/mL vs 36667 ng/mL, P&amp;lt;0.001). Using droplet digital PCR, KRAS mutations were detected in the port 1 of 8 (15.4%) patients and in port 2 of 15 (28.8%) patients. In 35.5% of patients, KRAS mutations were detected in either port 1 or port 2. Conclusion: This device does not require pretreatment and is easy to extract CTCs. Since CTCs are contaminated with the leukocyte fraction, it is necessary to improve the accuracy of CTCs collection by adjusting the conditions of DEP. Citation Format: Ryo Ohta, Takeshi Yamada, Nobuhiko Taniai, Hiroshi Yoshida. Detection of CTCs using a micro-channel combined fluid dynamics and electrophoresis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3382.

Success rates and clinicopathologic associations with experimental outcomes of a novel circulating tumor cell (CTC) technology in advanced colon cancer (CC) and pancreatic cancer (PC).

805 Background: Studying CTC cultures (cx) ex-vivo can provide valuable insights into cancer metastases (mets). However, current immunoaffinity- and size-based-methods used to isolate and expand these cells have low success rates for culturing CTCs (6-20%). Here, we report our experience using a novel technology developed at Georgetown for culturing CTCs from patients (pts) with metastatic (m) CC, mPC, and locally advanced PC (LAPC). Methods: 44 peripheral blood samples were prospectively collected from 35 pts with adenocarcinoma across 3 cohorts (18 samples from 15 mCC pts, 21 samples from 15 mPC pts, and 5 samples from 5 LAPC pts). Pts were previously treated, treatment-naïve, or actively undergoing treatment. FiColl-Paque-based separation of red blood cells was performed, plasma and buffy coat cells were resuspended in cx medium, and successfully grown CTC cxs were processed and injected subcutaneously (subQ) in the flanks of mice. A single predetermined sample from each pt was used in assessing the clinicopathologic associations with experiment outcomes and survival analyses. Results: CTC cxs were successfully grown from 81.8% (36/44) of all samples. Cxs grew from 72.2% (13/18) of mCC, 85.7% (18/21) of mPC, and 100% (5/5) of LAPC samples. At the time of analysis, 25 injected mice were evaluable for subQ tumor growth. 4 mice failed to grow tumor, 10 were still under monitoring, and 11 grew tumor, 9 of which also developed mets. These were mostly macro-mets and partially mirrored the met pattern seen in the matched pt. Across all cohorts, samples were predominantly collected from pts during systemic treatment (79.5%), during 2nd- (36.4%) and 1st-line treatment (20.5%), at a median of 15 (0-111) and 5 (1-20) months after diagnosis in mPC/mCC and LAPC pts, respectively, after scans with new/progressing disease (72.7%), and with a median of 3 (1-9) sites of disease on scans done within 2 months of collection . In all pts, no clinicopathologic or genomic factor predicted for cx growth. In the mCC subgroup, female sex (p=0.044), longer time from diagnosis to CTC collection (p=0.033), and presence of lung mets (p=0.022) were associated with cx growth. With cohorts combined, the median pt progression-free survival was 174 days (95% CI 97-220) and median overall survival was not reached. There was no statistically significant relationship between cx growth and pt survival. Conclusions: This novel platform demonstrated historically high rates of successful CTC cx and xenograft tumor growth using samples from advanced CC and PC pts. Studies to elucidate reasons for mice failing to grow tumor and others with matched tumor-CTC molecular analyses are ongoing to validate this promising technology to ultimately use to identify biomarkers for metastases, uncover novel therapeutic targets, and inform clinical decisions.

Abstract LB117: Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry

Abstract Introduction: CTCs play a critical role in breast cancer (BCa) metastases. Rapid identification of live CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic disease. However, CTCs are present at very low concentrations in the peripheral blood, and CTC enumeration is costly and technically challenging. We previously reported that cell-size based device utilizing a microfluidic cassette could be used to isolate live blood cells (Zhang et al. 2019 AACR). Here we describe a pilot study using novel microfluidic Parsortix workflow to identify live CTCs for patients with Stage IV BCa. Methods: Four whole blood samples (7.5ml/each) were collected from each of 10 patients with stage IV BCa patients who had continued clinical progression of their disease on systemic therapy, under a Northwestern University IRB-approved study (NU20B03). All samples were stored at 15-30 °C in EDTA tubes. Live CTCs enrichment was performed using a cell size and deformability based Parsortix system (Angle) which utilizes a microfluidic based disposable cassette containing a step separation structure. The four samples from each patient were loaded onto the device and the separation procedure was started by using 4.5μm, 6.5μm 8μm and 10μm separation cassettes, respectively. After separation and cleaning, the CTCs captured by the Parsortix system were harvested by inverting the flow direction and then flushing from the cassette in 200μL PBS. The harvested CTCs were multi-stained directly by fluorescence labeled antibodies for Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes) (Menarini). Flow cytometry (FCM) was used to identify the CTCs which were classified as CK+, DAPI+ and CD45-. White blood cells from patient samples and the human breast cancer cell line MDA-MB-231 were used as negative and positive controls, respectively. Results: A total of 500 and 200 MDA-MB-231 cells were processed as controls, which were collected completely by Parsortix and identified by FCM confirming that Parsortix system could recover the CTCs effectively. CTCs were classified based on morphology and phenotype as CK+, DAPI+ and CD45-. Live CTCs (≥1) were detected in 8 out of 10 (80%), 9 out of 10 (90%), 7 out of 10 (70%) and 5 out of 10 (50%) samples by using 4.5μm (Group 1), 6.5μm (Group 2), 8μm (Group 3) and 10μm (Group 4) separation cassettes. Maximum CTCs isolated from each group were 18, 32, 12 and 8 from Group 1, 2, 3 and 4, respectively. The corresponding average CTCs isolated from each sample were 3.5 CTCs, 5.6 CTCs, 2.5 CTCs and 1.7 CTCs from Group 1, 2, 3, and 4, respectively. The 6.5μm cassettes (Group 2) using for CTCs collection had significantly higher efficacy on collection of CTCs compared to other three groups. Conclusions: In this pilot study, our findings demonstrated that the cell-sized dependent Parsortix system using multiple staining and FCM is an effective and specific approach for rapid recovery and identification of CTCs from Stage IV BCa patients. With further optimization, this strategy can help to accurately evaluate CTCs and offers a potential technique for diagnosis and treatmentmonitoring of patients with metastatic breast cancer. Citation Format: Qiang Zhang, Marwa Manai, Andrew Davis, Carolina Reduzzi, Paolo D’Amico, Saya Liz Jacob, Jianhua Jiao, Weijun Qin, Lisa Flaum, Amir Behdad, Massimo Cristofanilli, Ami Shad, Leonidas Platanias, William Gradishar. Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry [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 LB117.

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 &amp;lt; 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 &amp;lt; 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.

Evaluation of the Safety and Feasibility of Apheresis in Dogs: For Application in Metastatic Cancer Research.

Simple SummaryIn cases of cancer metastasis, some tumor cells circulate in blood (CTCs) and function as a seed for metastasis. These cells are important for detailed study of the molecular basis of metastasis. However, their few numbers and difficult isolation hinder its analysis. Apheresis, a process used to isolate a specific component of blood, can efficiently separate peripheral blood monocyte (PBMC), whose density is closely like that of CTCs. In the present study we used the dogs as a precious model of human’s cancers to check the safety and feasibility of apheresis and to separate PBMC and infused MCF7 cells using Spectra Optia apheresis machine (Terumo). The process was performed safely to capture PBMC and MCF7. The captured MCF7 cells were regrown up in vitro and characterized. In conclusion, Spectra Optia apheresis machine (Terumo) can be used safely to isolate CTCs from dogs blood with precautions to keep hemodynamic stability.In patients with solid tumors, circulating tumor cells (CTCs) spread in their blood and function as a seed for metastases. However, the study of CTCs has been limited by their rarity, low frequency, and heterogeneity. The efficient collection of CTCs will contribute to further research of metastatic cancers. Apheresis is a process in which the whole blood of an individual is passed through a machine that isolates a particular constituent and returns the remainder to the circulation. In the present study, we investigated the safety and feasibility of apheresis to separate peripheral blood monocytes (PBMCs), whose density is closely similar to that of CTCs, and to capture intravenously administered human breast cancer cells, MCF7s, from the dogs. No life-threatening events were observed in dogs during the apheresis process. The changes in the hemogram were transient and recovered gradually within a few days after apheresis. During apheresis, 50 mL of PBMCs could be collected from each dog. Notably, a thrombus was formed along the circuit wall during apheresis, which decreased the blood collection pressure. MCF7 cells were successfully captured by the apheresis machine. The captured cells were regrown in vitro and characterized compared with the original cells. In conclusion, apheresis could be safely performed in dogs to isolate CTCs with precautions to maintain hemodynamic stability.

Detection Methods and Clinical Applications of Circulating Tumor Cells in Breast Cancer.

Circulating Tumor Cells (CTCs) are cancer cells that split away from the primary tumor and appear in the circulatory system as singular units or clusters, which was first reported by Dr. Thomas Ashworth in 1869. CTCs migrate and implantation occurs at a new site, in a process commonly known as tumor metastasis. In the case of breast cancer, the tumor cells often migrate into locations such as the lungs, brain, and bones, even during the early stages, and this is a notable characteristic of breast cancer. Survival rates have increased significantly over the past few decades because of progress made in radiology and tissue biopsy, making early detection and diagnosis of breast cancer possible. However, liquid biopsy, particularly that involving the collection of CTCs, is a non-invasive method to detect tumor cells in the circulatory system, which can be easily isolated from human plasma, serum, and other body fluids. Compared to traditional tissue biopsies, fluid sample collection has the advantages of being readily available and more acceptable to the patient. It can also detect tumor cells in blood earlier and in smaller numbers, possibly allowing for diagnosis prior to any tumor detection using imaging methods. Because of the scarcity of CTCs circulating in blood vessels (only a few CTCs among billions of erythrocytes and leukocytes), thorough but accurate detection methods are particularly important for further clinical applications.

Abstract PS2-14: Her2 expression in matched metastatic tumor and circulating tumor cells (ctcs) in breast cancer: Implications for profiling and monitoring of her2 status to help guide anti-her2 therapy

Abstract INTRODUCTION: Despite improvements in early detection, 1 in 8 women in the US (12%) will develop invasive breast cancer over the course of her lifetime. Approximately 20% of breast cancer is HER2 positive. During treatment and at disease progression, HER2 receptor conversion may occur. Once metastatic, it may be difficult to access multiple metastatic sites or perform serial biopsies. Therefore, accuracy of results may be sub-optimal as tissue biopsy is a single time point collection and limited by sampling (inter-tumoral and intra-tumoral heterogeneity). A liquid biopsy is a contemporaneous non-invasive and cost-effective method that allows for collection and analysis of tumor material and includes circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA). We compared prospectively the expression of HER2 in metastatic tumors to HER2 amplification in CTCs. METHODS: We enrolled patients with metastatic breast cancer in the Individualized Molecular Analyses Guide Efforts in Breast Cancer (IMAGE) II Study (NCT02965755). All patients regardless of subtype, had at least one line of therapy (chemotherapy, hormone therapy, or anti-HER2 therapy as appropriate). We analyzed HER2 status on tumor biopsies obtained 0-43 months (mean 7.3 months) prior to enrolling in IMAGE, and CTCs isolated from peripheral blood (PB) drawn ideally before starting a new treatment, 1-2 weeks after starting a new treatment and at the time of first restaging. CTCs were captured by Target Selector TM (Biocept) and analyzed for HER2 amplification by FISH. The biomarker expression profile on the metastatic tumor and CTCs were compared for each patient. Concordance of HER2 expression between CTCs and the metastatic tumor tissue was analyzed using McNemar’s test. RESULTS: For 36 evaluable patients, the specificity of HER2 on CTCs to tissue was 92.9% for PB samples collected within 5 weeks of the tumor biopsy and 100% at for PB samples collected between 5-10 weeks post biopsy, with overall concordance of 65% (independent of CTC collection time point), accuracy of 76.5% and specificity of 79.7%. A change in HER2 in amplification between the metastatic tumor and CTCs was noted in 36% (13/36) of patients with 7 patients HER2+ in tissue, HER2- on CTCs and 8 patients HER2- on tissue, HER2+ on CTCs. CONCLUSION: These data demonstrate high accuracy of HER2 amplification on CTCs at baseline and within 10 weeks of treatment and provide a sensitive and specific mechanism to monitor for changes in HER2 status that may be due to either tissue heterogeneity or receptor switch, a well-established phenomenon. This ability to effectively and contemporaneously monitor HER2 status on CTCs has the potential to identify patients who may benefit from the addition of anti-HER2 therapy and those are on anti-HER2 therapy who may not benefit optimally and for whom additional therapeutic options may warrant consideration. Citation Format: Vered Stearns, Jennifer Lehman, Christine Mitchell, Barbara Blouw, Lan Huynh, Veena Singh. Her2 expression in matched metastatic tumor and circulating tumor cells (ctcs) in breast cancer: Implications for profiling and monitoring of her2 status to help guide anti-her2 therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-14.

Phase II/III study of SM-88 in patients with metastatic pancreatic cancer.

437 Background: SM-88 (racemetyrosine, Tyme Inc) is a dysfunctional tyrosine derivative used with MPS (methoxsalen 10mg, phenytoin 50mg and sirolimus 0.5mg). SM-88 was well tolerated with improvement in survival among select heavily pretreated PDAC patients who achieved stable disease (HR 0.08, p = 0.02) (Noel et al. Annal Oncol 2019). Circulating tumor cells (CTCs) were prognostic in identifying a PDAC subgroup that may be more likely to benefit from SM-88. Preliminary radiomic analysis of the largest metastases at baseline correlated with baseline CTCs (Ocean et al, Annal Oncol 2019). Here we describe the subsequent randomized portion of the trial in third-line patients only, of SM-88 vs physician/patient choice chemotherapy, to evaluate the potential role of SM-88 in metastatic PDAC through analysis of CTCs and passively acquired biometrics data from a wearable device. Methods:Prospective open-label RCT (Tyme 88 Panc Part 2, NCT03512756) after 2 prior lines for metastatic PDAC. A cell adhesion matrix (CAM) was used to enrich solitary CTCs and cells in clusters floating in the medium after 24 hour culture. Isolated CTCs were collected each cycle on day 1, isolated, and enumerated by flow cytometry using the epithelial cell surface marker Epi+ and cellular uptake of green fluorescent labeled CAM (GCAM+). Results:As of Sept 15, 67 subjects were consented. Randomized and evaluable subjects (n=38) included: mean age 65y (45-86); BMI 24.6 (18.8-38.7); female 39.5%; White 76.3%. Of treated subjects 65.8% (25/38) had 166 AEs, with 25.7% (26/101) being at least possibly SM-88-related, with 1 Grade 3. Four CTC subpopulations defined by GCAM, Epi+ and cluster status, were enumerated and correlated to each other (r=0.03-0.71). At least one CTC subpopulation was detected at baseline (mean 33.8 cells/2mL) in all subjects (n=27). The longest metastatic lesion diameter at baseline correlated with baseline CTCs (r=0.55 for Epi+ cluster; r=0.52 for GCAM+ cluster). CTCs were successfully separated and enumerated at each cycle for correlation with survival, response and other parameters. The median baseline daily step count during the first two weeks on treatment was 3993.8 (IQR: 2745.6 - 5078) for those alive vs. 689.3 (IQR: 630.0-2083.6) among deaths in evaluable subjects (p = NS). Passively acquired mean heart rate during week 3 on trial was 89.3 (SD 10.5) among those who died vs. 78.0 (SD 9.2) among those living; medians are 87.0 for deaths vs. 79.2 for alive (p= NS). Conclusions: In a preliminary exploratory analysis, passively acquired biometrics from a wearable device can be collected for correlation with other clinical outcomes. CTC collection and enumeration is also feasible for correlation with traditional trial outcomes. Given that the longest lesion diameter is correlated with CTCs at baseline, additional radiologic feature analysis (eg radiomics) may be important predictor of CTCs. SM-88 was well tolerated with no treatment-related Grade 4 or 5 events. Clinical trial information: NCT03512756.

Abstract A12: Presence of circulating tumor cells is an adverse risk factor for early-stage uveal melanoma

Abstract Introduction: Uveal melanoma (UM) is a rare melanoma; only 5% of cases at initial presentation have distant metastasis. As approximately 50% of patients will develop metastatic disease, there is a need to detect minimal residual disease (MRD). Liquid biopsy using circulating tumor cells (CTCs) has been evaluated in multiple tumors; high CTC count predicts for increased risk of metastasis. We conducted a pilot study to check feasibility of CTCs in UM and evaluate if CTCs vary with risk status in early-stage UM. Methods: We enrolled 40 UM patients ≥18 years, after obtaining informed consent in an IRB-approved protocol from 12/1/2014 to 2/1/2018. Peripheral blood was obtained to detect CTCs using CellSearch Circulating Melanoma Cell Assay. Demographic information, mutational analysis, date of diagnosis of primary UM or metastasis, and dates of CTC collection were collected. Risk stratification was done by gene expression profiling (GEP) by DecisionDX-UM assay. Landmark overall survival (OS) was calculated using “Landest” R package comparing CTC detected vs. not detected in early-stage UM. Results: 39 patients were available for analysis. Median age of whole cohort was 52 years (20-83 years), 100% were non-Hispanic Caucasians, and 44% were male and 56% female. 20 patients had early-stage vs. 19 patients had metastatic disease at enrollment. 87% (13/15) of early-stage disease had class II disease. GNAQ &amp; GNA11 mutations were present in 36% (12/39) and 13% (5/29), respectively. At initial blood draw, 36% of the patients had detectable CTCs [30% (6/20) in early stage vs. 42% (8/19) in metastatic], which increased to 54% at median study follow-up of 16.4 months [40% (8/20) in early stage vs. 68% (13/19) in metastatic]. Mean CTCs were higher in metastatic (9) vs. early stage (1.83) (p&amp;gt;0.05). 5 patients in early-stage disease developed distant metastasis; 60% (3/5) had detectable CTCs before radiographic detection of metastasis. Out 5, 80% had class II disease and 20% had unknown GEP. Landmark overall survival (from study enrollment) at 24 months was statistically worse in CTC detected vs. not detected in early-stage UM (P&amp;lt;0.05). Conclusion: CTCs are more frequently detected in metastatic compared to early-stage UM. In early-stage UM, presence of CTCs predicts for increased risk of metastatic disease and is associated with worse outcome. Citation Format: Jason Roszik, Dan Gombos, Joshua Upshaw, Vanessa Sarli, Salyna Meas, Anthony Lucci, Carolyn Hall, Sapna Patel, Kartik Anand. Presence of circulating tumor cells is an adverse risk factor for early-stage uveal melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A12.

Abstract A089: Detection of circulating tumor cells (CTC) by microfluidic technology based on lateral magnetophoresis and CTC-based multigene profiling analysis in prostate cancer patient

Abstract Introduction By using the circulating tumor cells (CTCs), we can get useful genomic information that enable monitoring of patient’s prognosis or selecting of appropriate treatment over time by repeat biopsy. Despite their usefulness, current CTC separation techniques are limited in terms of their ability to separate high recovery and purity CTCs from peripheral blood. We aimed to separate CTCs from prostate cancer patients using microfluidic technique that based on disposable lateral magnetophoretic microseparator (named ‘assembly-disposable CTC-μChip’) and assess the gene expression that are specific for prostate cancer using mRNA from isolated CTCs measured by droplet digital PCR (ddPCR) method. Patients and Methods Total 54 samples of 45 patients who underwent treatment for prostate cancer between 06/2018 and 03/2019 were prospectively enrolled for CTC collection. Approximately 10 ml of whole blood was drawn from prostate cancer patients; Group A, 12 samples (22.2%) with localized stage II-III cancer, Group B, 5 samples (9.3%) with stage IV, Group C, 6 samples (11.1%) with metastatic hormone-sensitive prostate cancer (mHSPC) and Group D, 31 samples (57.4%) with metastatic castration-resistant prostate cancer (mCRPC). CTCs were positively isolated by using Assembly-disposable CTC-μChip. CTCs from 5 mL of blood were used for counting to identify isolated CTC numbers with immunofluorescence detection procedure. CTCs from the remaining 5 mL of blood were used for gene analysis by ddPCR. For the gene expression analysis, selected 13 patient’s samples were examined for 6 prostate cancer-related genes, such as androgen receptor (AR), androgen receptor splice variant 7 (AR-V7), prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), cytokeratin-19 (CK-19) and epithelial cell adhesion molecule (EpCAM). Results We identified the average of 3, 16, 14 and 26 CTCs per 1ml in group A, B, C and D, respectively (P&amp;lt; 0.01). Background contaminated white blood cells are indispensably isolated with CTCs that counted on average 2245.4 cells, thereby CTC purity rate was 2.56 %. The gene positive rate of AR, AR-V7, PSA, PSMA, KRT-19, and EpCAM were 2.3 % (12/13), 15.4 % (2/13), 38.5 % (5/13), 69.2 % (9/13), and 100 % (13/13), respectively. Especially, the copy number of PSA, PSAM and CK-19 increased in proportion as the clinical stage increased. All samples showed EpCAM positive, whereas AR-V7 was rare events, with only in 2 out 5 patients positive with mCRPC, but not in other stages. Conclusions This study demonstrates that assembly-disposable CTC-μChip is a suitable for CTC isolation from prostate cancer patients. Our data also showed that CTC-based multigene profiling can be analyzed using ddPCR, making it a clinically relevant biomarker. Interrogating tumor expression via CTCs isolation may allow for enhancing precision-based treatment. Citation Format: Hyungseok Cho, Ki-Ho Han, Jae-Seung Chung. Detection of circulating tumor cells (CTC) by microfluidic technology based on lateral magnetophoresis and CTC-based multigene profiling analysis in prostate cancer patient [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A089.

Flexible Three-Dimensional Net for Intravascular Fishing of Circulating Tumor Cells.

Current strategies for in vitro isolation of circulating tumor cells (CTCs) fail to detect extremely rare CTCs heterogeneously distributed in blood. It is possible to devise methods for in vivo capture of CTCs based on processing almost all of the blood in the human body to improve detection sensitivity, but the complicated manipulation, biosafety concerns, and limited capture efficiency of conventional detection strategies prohibit their implementation in the clinic. Herein, we present a flexible three-dimensional (3-D) CTC-Net probe for intravascular collection of CTCs. The CTC-Net, consisting of a 3-D elastic scaffold with an interconnected, spatially distributed network accommodates a large quantity of immobilized antibodies and provides an enhanced substrate-cell contact frequency, which results in an enhanced capture efficiency and effective detection of heterogeneous CTCs. The as-prepared CTC-Net can be readily compressed and injected into blood vessels and fully unfolded to form a 3-D "fishing-net" structure for capture of the CTCs, and then retracted for imaging and downstream gene analysis of the captured CTCs. Significant advantages for the CTC-Net over currently available in vitro and in vivo procedures are demonstrated for detection of extremely rare CTCs from wild-type rats and successful capture of CTCs and CTC clusters before metastasis in the case of tumor-bearing rats. Our research demonstrates for the first time the use of a 3-D scaffold CTC-Net probe for in vivo capture of CTCs. The method shows exceptional performance for cell capture, which is readily implemented and holds great potential in the clinic for early diagnosis of cancer.

Circulating Tumor Cell–Based Molecular Classifier for Predicting Resistance to Abiraterone and Enzalutamide in Metastatic Castration-Resistant Prostate Cancer

While circulating tumor cell (CTC)–based detection of AR-V7 has been demonstrated to predict patient response to second-generation androgen receptor therapies, the rarity of AR-V7 expression in metastatic castrate-resistant prostate cancer (mCRPC) suggests that other drivers of resistance exist. We sought to use a multiplex gene expression platform to interrogate CTCs and identify potential markers of resistance to abiraterone and enzalutamide. 37 patients with mCRPC initiating treatment with enzalutamide (n = 16) or abiraterone (n = 21) were prospectively enrolled for CTC collection and gene expression analysis using a panel of 89 prostate cancer–related genes. Gene expression from CTCs was correlated with PSA response and radioclinical progression-free survival (PFS) using Kaplan-Meier and Cox regression analyses. Twenty patients (54%) had detectable CTCs. At a median follow-up of 11.3 months, increased expression of the following genes was significantly associated with shorter PSA PFS and radioclinical PFS: AR, AR-V7, PSA, PSCA, TSPAN8, NKX3.1, and WNT5B. Additionally, high SPINK1 expression was associated with increased PFS. A predictive model including all eight genes gave an area under the curve (AUC) of 0.84 for PSA PFS and 0.86 for radioclinical PFS. In comparison, the AR-V7 only model resulted in AUC values of 0.65 and 0.64.These data demonstrate that clinically relevant information regarding gene expression can be obtained from whole blood using a CTC-based approach. Multigene classifiers in this setting may allow for the development of noninvasive predictive biomarkers to guide clinical management.

Poly(3,4-ethylenedioxythiophene)-Based Nanofiber Mats as an Organic Bioelectronic Platform for Programming Multiple Capture/Release Cycles of Circulating Tumor Cells.

In this investigation, we employed a novel one-step electrospinning process to fabricate poly(ethylene oxide) (PEO)/poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) core/shell nanofiber structures with improved water resistance and good electrochemical properties and characterized them using scanning electron microscopy, transmission electron microscopy, and time-of-flight secondary ion mass spectrometry imaging. We then integrated a biotinylated poly-(l-lysine-graft-ethylene glycol) (PLL-g-PEG-biotin) coating with three-dimensional (3D) PEDOT-based nanofiber devices for dynamic control over the capture/release performance of rare circulating tumor cells (CTCs) on-chip. The detailed capture/release behavior of the circulating tumor cells was studied using an organic bioelectronic platform comprising PEO/PEDOT:PSS nanofiber mats with 3 wt % (3-glycidyloxypropyl)trimethoxysilane as an additive. We have demonstrated that these nanofiber mats deposited on five-patterned indium tin oxide finger electrodes are excellent candidates for use as functional bioelectronic interfaces for the isolation, detection, sequential collection, and enrichment of rare CTCs through electrical activation of each single electrode. This combination behaved as an ideal model system displaying a high cell-capture yield for antibody-positive cells while resisting the adhesion of antibody-negative cells. Taking advantage of the electrochemical doping/dedoping characteristics of PEDOT:PSS materials, the captured rare cells could be electrically triggered release through the desorption phenomena of PLL-g-PEG-biotin on device surface. More than 90% of the targeted cancer cells were captured on the 3D PEDOT-based nanofiber microfluidic device; over 87% of captured cancer cells were subsequently released for collection; approximately 80% of spiked cancer cells could be collected in a 96-well plate. Therefore, this 3D PEDOT-based nanofiber approach appears to be an economical route for the large-scale preparation of systems for enhancing the downstream characterization of rare CTCs.

Abstract 3919: Isolation and characterization of viable pancreatic cancer circulating tumor cells using size-based filtration system

Abstract Introduction: Circulating tumor cells (CTCs) are important actors in metastasis, a key feature of pancreatic ductal adenocarcinoma (PDAC). The ability to isolate, characterize and culture viable CTCs holds promise for understanding biology, treatment response and resistance. Methods: Peripheral blood samples from healthy donors and patients with advanced PDAC were processed using an innovative size filtration system (Viatar LLC, Lowell MA). Normal human blood samples were spiked with varying numbers of human PDAC organoid cells. The impact of buffy coat separation was studied. Isolated CTCs were cultured in Matrigel to generate organoids as previously described. Cells were imaged with fluorescently labeled antibody to EpCAM. Data: Using spiked normal human blood samples, viable CTCs can be isolated and expanded reliably in 3D organoid culture from as few as 10 CTCs/mL. Separation and processing of the buffy coat enriched for the number of viable CTCs isolated. EpCAM positive CTCs were isolated from 6/6 blood samples from patients with advanced PDAC. Updated results will be presented. Conclusions: The current size-based filtration system allows for collection of viable CTCs from human PDAC blood samples. Current work will use this system to grow, expand and characterize human PDAC CTCs in organoid culture. Genetic, transcriptional, and epigenetic characteristics will be compared with the primary tumor. CTC characteristics will be studied as biomarker of treatment response and resistance. Citation Format: Kenneth H. Yu, Benjamin D. Krempley, Brian McCarthy. Isolation and characterization of viable pancreatic cancer circulating tumor cells using size-based filtration system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3919. doi:10.1158/1538-7445.AM2017-3919

P2.01-005 Evaluation of Circulating Tumoral Microemboli (CTM) as a Prognostic Factor in Non-Small Cell Lung Cancer (NSCLC)

Much has been studied regarding the prognostic role of circulating tumor cells (CTCs) in NSCLC. CTM (defined as clusters of 3 or more cancer cells detected in peripheral blood) relationship to prognosis was previously published for small cell lung cancer (SCLC), demonstrating worst prognosis for the presence of CTM. No relevant data, however, was published for CTM in NSCLC. The objective of this study is to define the presence of CTM as a prognostic factor for survival in NSCLC and its molecular characteristics. It was performed a retrospective evaluation of 31 metastatic NSCLC patients positive for CTC, which were previously enrolled for CTC research in a single institution. CTC and CTM were detected by ISET (Isolation by Size of Epithelial/Trophoblastic Tumor Cells, Rarecells, France®). Included patients had metastatic disease treated in multiple lines of cytotoxic treatment. Analysis included frequencies, demographic characteristics and survival variables, including Progression Free Survival (PFS) and Overall Survival (OS), with PFS as primary endpoint. The PFS and OS were calculated based on the date of first CTC collection and first progression after collection (PFS) or death (OS). Molecular characterization was performed by immunocytochemistry for Transforming Growth Factor beta receptor (TGFßR) and Matrix Metalloproteinase 2 (MMP2). The primary endpoint was not met. Presence of CTM did not have statistically significant influence on PFS or OS in our population. Eight patients were positive (CTM+) and 23 were negative (CTM-) for CTM. Median follow-up was 13.3 months (m). Median age was 65.5 years in CTM- and 69.6 years in CTM+ patients. Remaining demographic variables were balanced between groups. All patients had progressive disease and 9 were still alive at the time of analysis. Median PFS (mPFS) was 6.9m for CTM- and 4,5m for CTM+, with p=0.59. Median OS (mOS) was paradoxically greater in CTM+, without statistical significance (27.3m for CTM- and 31.6m for CTM+; p=0.83). Molecular characterization did not have any prognostic impact on both CTM- and CTM+ groups. No patient was positive for TGFßR and 2 CTM+ patients were positive for MMP2 (10/31 patients with isolated CTCs positive for MMP2). This retrospective analysis showed no impact on survival for the presence of CTM in NSCLC, which was opposite to findings of positive prognostic value of CTM for SCLC where CTM was a negative factor for survival. Molecular characterization also did not show differences between groups. The findings warrant further evaluation in dedicated research.

Microscale Laminar Vortices for High-Purity Extraction and Release of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are disseminated tumor cells that reflect the tumors of origin and can provide a liquid biopsy that would potentially enable noninvasive tumor profiling, treatment monitoring, and identification of targeted treatments. Accurate and rapid purification of CTCs holds great potential to improve cancer care but the task remains technically challenging. Microfluidic isolation of CTCs within microscale vortices enables high-throughput and size-based purification of rare CTCs from bodily fluids. Collected cells are highly pure, viable, and easily accessible, allowing seamless integration with various downstream applications. Here, we describe how to fabricate the High-Throughput Vortex Chip (Vortex-HT) and to process diluted whole blood for CTC collection. Lastly, immunostaining and imaging protocols for CTC classification and corresponding CTC image galleries are reported.

Molecular profiling of single circulating tumor cells from lung cancer patients

Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.