Changes In Circulating Tumor Cells Research Articles

Abstract PO1-20-12: CD8+ Tumor Infiltrating Lymphocytes Turn a Cold Tumor Hot in Metastatic Breast Cancer

Abstract Introduction: Turning “cold” tumors “hot” is considered essential for immune-oncology therapies to be effective. Bria-IMT is an allogenic human cancer cell line with antigen presenting cell activity (SV-BR-1-GM) designed to overcome the immune-suppressive tumor microenvironment. However, until now there has not been proof that “cold” tumors can become “hot” with this therapy. Using Zr-89 crefmirlimab berdoxam, a zirconium-89 labelled truncated antibody specific to human CD8α and developed for CD8 ImmunoPET, we image before and after Bria-IMT therapy administration to assess the baseline status as well as changes in CD8 cell presence after initiation of therapy. Here we present the first evidence that cold tumors can become hot on treatment with Bria-IMT combined with an anti-PD-1 checkpoint inhibitor (CPI), showing CD8+ cells increased over baseline using CD8 ImmunoPET. Case: After informed consent, a 61 year-old Hispanic female, enrolled in the ongoing randomized phase 2 Bria-IMT trial in advanced heavily pretreated metastatic breast cancer (MBC) (NCT03328026). Her history included MBC in May 2020, ductal type, grade 3, triple negative breast cancer (TNBC) with 6 prior lines of therapy including a Trop-2-directed antibody-drug conjugate and 6 lines of chemotherapy. Precision medicine testing identified no actionable targets. All prior therapies had progressive disease as their best response. The subject was randomized to start the CPI after 2 cycles of Bria-IMT to “train” the host immune system before proceeding with combination immune-oncology therapy. Baseline testing revealed the subject to be anergic to candida and SV-BR-1-GM inoculation. However, by cycle 3 delayed-type hypersensitivity to SV-BR-1GM developed. Interestingly the subject matched SV-BR-1-GM at 2 HLA loci: DRB3*02:02 and DRB1*11:04 which in previous publications is associated with greater clinical benefit. Tumor markers were stable or decreased slightly including CEA (25% decrease) and CA 15-3 (43% decrease). Circulating Tumor Cells (CTCs) and Cancer Associated Macrophage Like cells (CAMLs) also improved. Standard imaging at baseline showed 3 main sites of disease including a large right breast mass, multiple right axillary lymph nodes and a right lower lobe mass with CD8 ImmunoPET uptake in these areas similar to the blood pool. On the follow-up CT imaging, all of these lesions increased in size with newly appreciated pulmonary nodules. The right lower lobe mass showed increased CD8 ImmunoPET accumulation with an SUV of 5.8 (close to the liver background) as compared to an SUV of 2.2 at baseline. The lesions in the right breast and right axilla remained at blood pool levels. There was an increase in CD8 ImmunoPET uptake in bilateral inguinal lymph nodes reaching an SUV of 4.8 as compared to SUV 2.2 at baseline. In addition, there were non-enlarged lymph nodes in bilateral axillary regions that also showed increased radio-tracer accumulation reaching an SUV of 9.5 as compared to SUV 4.9 at baseline. Conclusion: This subject demonstrated a mixed response of metastatic lesions on CD8 ImmunoPET with encouraging changes in peripheral blood tumor markers and CTCs. The nonspecific nodal localization of CD8 ImmunoPET may indicate a systemic activation of CD8 positive lymphoid cells. This shows the value of CD8 ImmunoPET in identifying lesions that are progressing on treatment versus pseudo progression. It also provides support that the Bria-IMT + CPI combination immune-based therapy can result in an increase of CD8+ tumor infiltrating lymphocytes in breast cancer metastatic sites as well as an increase in lymphoid organs. This advance may aid in triaging patients, adjudicating pseudo-progression and predicting clinical benefit of immune based therapies. Citation Format: Russ Kuker, Frances Valdes-Albini, Giuseppe Del Priore, Aazim Arif, Kristin Schmiedehausen, Carmen Calfa. CD8+ Tumor Infiltrating Lymphocytes Turn a Cold Tumor Hot in Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-20-12.

Circulating tumor cells in patients with hormone receptor-positive, HER2-negative metastatic breast cancer receiving palbociclib with fulvestrant after progression on palbociclib with an aromatase inhibitor.

e13018 Background: High levels of circulating tumor cells (CTCs) at initiation of therapy in patients with metastatic breast cancer (MBC) are associated with worse prognosis. In patients with hormone receptor–positive (HR+), HER2-negative MBC, the benefit of maintaining CDK4/6 inhibitor (CDK4/6i) after progression on first-line therapy with endocrine therapy and CDK4/6i is not fully defined. To study the relationship between CTCs, tumor characteristics, and clinical outcomes, we enumerated CTC dynamics in patients with HR+ HER2- MBC enrolled on a phase II, multi-center, open-label interventional trial of palbociclib and fulvestrant after disease progression on prior palbociclib with an aromatase inhibitor (NCT02738866). Methods: Blood samples from study participants were collected at baseline (pretreatment), cycle 2 (C2, 8 weeks after initiation of fulvestrant and palbociclib), and at disease progression. Using a previously analytically validated assay (CellSearch), CTCs were enumerated. Progression-free survival (PFS) was estimated using the Kaplan Meier method. The relationship between CTC count at baseline (< 5, >5/7.5 ml Whole Blood (WB)) and PFS was analyzed using Cox proportional hazards regression. The association between PFS and CTC count among patients with baseline CTC ≥5 was estimated with a Cox proportional hazards model, adjusting for age, race, and progesterone receptor (PgR). Results: Between May 2017 and September 2022, baseline samples were collected from 52 of 60 trial participants enrolled at 5 centers in the United States, mean age 55 years (SD 14). Baseline CTC counts were <5 in 30/52 (58%) and >5 in 22/52 (42%) of participants. Among participants at baseline, only tissue PgR negativity was associated with higher likelihood of having elevated CTCs >5 (p = 0.04). Association of CTCs >5 and select patient and tumor characteristics is shown in Table 1. There was no statistically significant association between continuously measured CTC and PFS among those with elevated CTCs at baseline. Median duration of therapy among the entire 52 participant cohort was 4 months. Median PFS in participants with baseline CTC <5 vs >5/7.5 ml WB was 7.83 (95% CI [5.37, 12.60]) and 2.83 (95% CI [1.77, 4.73]) months, respectively (HR): 2.10, 95% CI [1.15, 3.82], p = 0.01). Conclusions: Baseline CTCs >5/7.5 ml WB conferred a 2-fold increased risk of disease progression. Tissue PgR negativity was associated with baseline CTCs >5. These data can be used to inform new studies in which baseline and early change in CTCs may inform second line treatment decisions in women with HR+, HER2- MBC. [Table: see text]

Perioperative change of circulating tumor cells in cytoreductive radical prostatectomy for oligometastatic hormone-sensitive prostate cancer: the preliminary safety evidence from long-term oncologic outcomes.

Surgical manipulation has a risk of triggering the shedding of circulating tumor cells (CTCs) in patients with malignancies, However, perioperative change of circulating tumor cells in cytoreductive radical prostatectomy (CRP) for patients with oligometastatic hormone-sensitive prostate cancer (omHSPC) has not yet been well documented. This study aimed to assess whether CRP is a safe procedure for patients with omHSPC by monitoring the perioperative change of CTCs and investigating its impact on long-term oncologic outcomes. We have observed a significant decrease between the median CTC counts before and after surgery (6 vs. 4, p = 0.026). Comparing preoperative and postoperative CTC levels, seven patients increased (CTC increase group), one did not change and nineteen decreased (CTC non-increase group). PSA response rates in CTC increase group were lower than those in CTC non-increase group (73.0% vs 99.8%, p = 0.162), and nadir PSA was higher in CTC increase group (0.043 vs 0.003, p = 0.072). The CTC increase was positively correlated with the nadir PSA (r = 0.386, p = 0.047). The median follow-up period was 71.6months, we found that there was no significant difference in clinical-pathological, operative variables or long-term oncologic outcomes between perioperative CTC increase and non-increase groups. In the entire cohort, the CTC level significantly decreased after surgery. There was no significant differences in long-term oncologic outcomes between the CTC increase and non-increase groups, implying that CRP potentially represents a safe procedure for the treatment of patients with omHSPC. The results need to be confirmed in a prospective large-scale clinical trial.

Abstract CT143: Whole cell antigen presenting immune stimulating cells (Bria-IMT) for the treatment of metastatic breast cancer

Abstract Background: Bria-IMT (SV-BR-1-GM) is an off-the-shelf whole tumor cell therapeutic vaccine engineered to express class I & class II HLAs, secrete GM-CSF, and function as antigen-presenting cells; with subsequent enhancements improving in-vitro characteristics. By expressing cancer antigens such as HER2/Neu and PRAME, SV-BR-1-GM also serves as the reservoir of cancer antigens to activates patient’s anti-tumor immune responses. The safety and efficacy of the ongoing clinical trial of SV-BR-1-GM in combination with a PD1 inhibitor are reported. We also report post-hoc exploratory data for patients with advanced metastatic breast cancer (aMBC) treated with SV-BR-1-GM regimens. Methods: Ongoing prospective, phase 1-2, with an expansion randomized phase 2 cohort (NCT03328026; 2018-present) using SV-BR-1-GM with a PD1 inhibitor (pembrolizumab or retifanlimab) with cycles every 3 weeks (16 patients dosed to date). SV-BR-1-GM “monotherapy” (NCT03066947; 2013-8), a completed prospective phase 1-2 results are also presented. Results: A total of 51 patients received any SV-BR-1-GM regimen; 27 in monotherapy (SV) and 24 in combination with a PD-1 inhibitor (CO) currently ongoing: 4 subjects from SV continued on with CO. Median prior regimens = 5; 61% ER or PR+; 6% ER or PR-/HER2+ and 29% triple negative. SV-BR-1-GM was well tolerated. Disease control rate was 40% with SV and 41% with CO in subjects with available assessment(s). Median PFS was 2.7 months with 6 having PFS >150 days (150-226) for SV and 2.6 months with 5 > 150 days (188-308) for CO. Per protocol, 23 patients had anergy testing prior to treatment. Among those with a normal immunologic response (n=13, 56%), disease outcomes included 4 SD and 2 PR (46% CBR) vs anergic patients (n=7, 30%) with 1 SD (14% CBR); 3 patients (13%) with unknown anergy testing had 0% CBR. All 7 anergic patients developed delayed-type hypersensitivity (DTH) injection site responses to SV-BR-1-GM. Subjects with DTH (injection site induration/erythema) had a significant reduction in the risk of progression or death (HR=0.096, 95%CI:0.02-0.50, p<0.05). Subjects with a reduction in circulating tumor cells (CTC) also had an improvement in PFS (HR 0.08, p=.0012). Median OS (uncensored) was 11.2 months in CO (n = 8). 7 out of 20 (35%) for SV and 9 out of 22 subjects (41%) for CO with available data, exceeded prior PFS. Among HLA matched subjects 4/7 (57%) and 6/9 (67%), respectively, had an increase in PFS. PFS ratio improved independent of prior number of lines, HER2 or HR status. The greatest improvement in PFS on a SV-BR-1-GM was in a HLA matched subject (>6mo). TEAEs occurred in 20/24 (83%) of subjects in CO including 9/24 (38%) with injection site AEs. There were no AEs of special interest eg cytokine storm. There were no Grade 5 and only 6 grade 4 AEs in CO. Conclusions: In an ongoing randomized phase 2, anergic patients had worse outcomes. SV-BR-1-GM was able to illicit an immune response regardless. Responses, stable disease and CBR were seen and associated with immune responses including injection site erythema/induration (DTH), CTC changes and tumor grade I/II. Patients with disease control had better quality of life. SV-BR-1-GM may be able to alter an anergic immune system for clinical benefit. Future pivotal trials will be based upon the ongoing confirmatory randomized clinical trial. Citation Format: Saranya Chumsri, Minal Barve, Chaitali Nangia, Bonnie L. Guerin, Jarrod Holmes, Karim Mohammed, John Knecht, Shaker Dakhil, William V. Williams, Charles Wiseman, Mingjin Chang, Miguel Lopez-Lago, John G. Knecht, Kendrith Rowland, Ralph V. Boccia, Giuseppe Del Priore, Carmen Calfa. Whole cell antigen presenting immune stimulating cells (Bria-IMT) for the treatment of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT143.

Abstract 2315: Temporal changes in circulating tumor cells and circulating tumor DNA in patients with resectable pancreatic ductal adenocarcinoma

Abstract Background: Circulating tumor cells (CTCs), circulating cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA) are minimally invasive biomarkers that carry vital tumor onset, progression, and metastasis information, which is crucial for cancer diagnosis, prognosis, and management. We report here our longitudinal study of these circulating biomarkers in resectable pancreatic ductal adenocarcinoma (PDAC) patients. Although the overall 5-year survival rate of PDAC is low (~6%), truly resectable patients are expected to have better outcomes than non-resectable PDAC patients, especially if liquid biopsy can help guide anticancer treatment selection. Method: We evaluated temporal changes in CTCs, cfDNA, and ctDNA in peripheral blood samples in a cohort of patients with resectable PDAC treated with pre-operative chemotherapy and surgical excision. Five PDAC survivors in complete remission for three years without evidence of disease served as negative controls. Samples of peripheral blood were collected prior to the start of treatment and every two weeks while patients underwent neoadjuvant chemotherapy, followed by surgical excision of the pancreatic tumor. CTCs were enumerated using a previously reported microfluidic device [1] that integrates immunoaffinity-based isolation with size-based filtration, while cfDNA and ctDNA were measured using quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR). Results: Among the initial seven patients who have completed the treatment sequences [2], CTCs were detectable in all patients (100%) at some points during treatment, but only 50% of patients had detectable CTCs before the start of chemotherapy. Median cfDNA concentrations were comparable to those negative controls throughout treatment. Using ddPCR to analyze peripheral blood samples, KRAS mutations were detected in in 71% of patients during anticancer therapy, but only in 29% patients prior to treatment. Overall, the majority of circulating biomarkers (81% for CTCs and 91% for ctDNA) were detected in blood samples collected after the start of neoadjuvant therapy and before surgery. Conclusion: This study suggests that a longitudinal study of circulating biomarkers provides additional data compared to single-point liquid biopsy, and that biologic “release” of biomarkers due to therapy can increase detectability. -Further study is needed to determine the clinical utility of liquid biopsy for resectable PDAC patients. [1] Chen, et al., Angew. Chem. Int. Ed., 58, 2019, 7606-7610. [2] Dopico, et al., Biosensors, 12, 2022, 206. Citation Format: Yang Zhang, Pablo J. Dopico, Minh-Chau N. Le, Zhijie Yang, Youxiang Wang, Sherise C. Rogers, Thomas J. George, Hugh Z. Fan. Temporal changes in circulating tumor cells and circulating tumor DNA in patients with resectable pancreatic ductal adenocarcinoma [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 2315.

Abstract 1040: Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry

Abstract The detection of liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have demonstrated promise as non-invasive assays for monitoring response to therapy for patients with cancer. While ctDNA cannot predict transcriptomic and proteomic changes in response to therapy, we can measure direct cell surface antigen and gene expression changes in CTCs. In pediatric settings, blood samples are often collected from multi-institutional trials. Therefore, to translate liquid biopsies into routine practice, it is important to explore variables most likely to impact the functionality of CTCs including time to process the sample, prospective cryopreservation, blood collection tube and fixative reagents. Cell stabilizer tubes such as Streck Cell-Free DNA, CellSave, CellRescue, or PAXgene ccfDNA have been designed to prevent cell lysis and release of genomic DNA into the plasma, often observed in standard EDTA tubes. In pediatrics, we are often limited by the volume of blood we can draw for research. To this end, we have established an innovative pipeline to collect ctDNA, CTCs, and matched germline DNA from a single blood sample. Blood samples were processed for plasma extraction and CTC isolation after 4h, 24h, and 48h. We isolated rare CTCs from whole blood by combining peripheral blood cell depletion with antigen-based CTC identification. We then evaluated CTC recovery rate and cell membrane integrity (cell-membrane permeabilization) across all blood collection tubes by flow or mass cytometry. Additionally, we integrated whole transcriptome analysis of RNA transcripts in individually sorted CTCs. In this study, we detected similar levels of cell-free DNA, germline DNA and ctDNA in healthy donors and patient samples collected in the DNA stabilizer tubes up to 48h after blood collection. Surprisingly, frequent clotting was observed in the cell layer of samples collected in PAXgene tubes only 24h after blood collection, preventing further analysis of CTC quality in these tubes. At 4h, EDTA tubes and stabilizer tubes seem to perform similarly in preserving CTC integrity. However, at later timepoints, CTC recovery rate, RNA quality and cell membrane integrity were significantly higher in CellRescue tubes. Finally, we showed that CTC count, and RNA quality were slightly lower in patient samples enumerated after cryopreservation compared to the paired-samples processed the same day in both EDTA and cell-stabilizing tubes. In conclusion, we validated the CellRescue tubes as the best candidate to reduce cell lysis and protect against cell membrane permeabilization and mRNA degradation for up to 48 hours, allowing sample collection from multi-institutional studies. To the best of our knowledge, this study is the first to compare the key steps in handling blood samples and how this affects transcriptomic-based assay performance. Citation Format: Anne-Florence Blandin, Sarah Finstuen-Magro, Samuel Abbou, Kelly Klega, Alejandra E. Aguilar, Mohammad Tanhaemami, Maria Ana Isabel C. De Los Santos, Joadly Duplan, Brian Crompton. Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry [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 1040.

Abstract P1-05-09: Targeting treatment to tumor response: demonstration of response monitoring with subsequent impact on treatment choices in real time utilizing a novel technology

Abstract Breast cancers, both early and advanced, are heterogeneous causing differential responses to targeted therapy. Explosion in targeted treatment choices requires real time assessment of tumor characteristics in patients initially and over time. A dynamic detection system, not a static pathology specimen, is required for continuous profiling while treatment changes; hence, the interest in circulating tumor cells (CTCs). Our novel QCDx br™ system analyzes all nucleated cells from a blood sample aliquot, morphologically intact and immobilized in hydrogel, after multiplex, immunofluorescent (IF) staining. We characterize increased numbers of CTCs (nucleated, CD45-negative cells) as single cells, in clusters and in connection with circulating inflammatory cells, stained with two, separate IF marker cocktails, denoting (1) epithelial (EpCAM, Cytokeratin CK), mesenchymal (Vimentin VIM) phenotypes and (2) therapeutic HER2, ER and TROP2 targets which are the basis for targeted therapy in breast cancer. In the ongoing, prospective CLINBREAC trial, we enrolled to date 9 neoadjuvant (early stage, ES) and 21 metastatic (late stage, LS) breast cancer patients, collecting 7.5 ml of blood at 3-month intervals or at change of therapy. We are now out over 2 years with the earliest ES and LS patients showing changes in CTCs with disease progression and in response to treatment, often pre-dating changes seen in follow up biopsies. Detection of HER2+ CTCs was of particular interest in patients with HER2 low cancers¬ (1+ or 2+). Results presented here, contain the more complete datasets from 8 ES and 11 LS patients. QCDx br™ detected CTCs in all 19 Stage I-IV patients that could exceed 100 CTCs/2500 nucleated cells in LS and 50 CTCs/2500 nucleated cells in ES patients. The table shows count averages of CTCs/2500 nucleated cells detected by different IF markers. Of note, CTCs showed combinations of IF markers (hybrid cells). For example, CTCs expressed both VIM and CK indicating epithelial to mesenchymal transition (EMT) phenotype, which may signify higher metastatic potential. ER+ CTC were seen in LS, not ES patients. All ES patients including those with HER2 low tumors, showed HER2+ CTCs. Of note, not included in the table are two, triple-positive patients with oligo-metastatic disease now 4 and 10 years out from diagnosis without evidence of disease and on maintenance HER2 and ER-directed therapy. No CTCs are seen at their first data point. We were unable to detect CTCs in 13 healthy volunteers. In ES patients, CTC numbers did not associate with tumor size, nodal involvement, ER or HER2 status. For example, one TNBC patient with a T3 tumor and positive nodes had some of the lowest numbers of CTCs. The more complete quantification of the LS patients is in process. As development of QCDx br™ continues, multiplex IF staining of 12+ markers per CTC and identification of single CTC mutational changes is expected. This powerful technology enables targeted treatment decisions with specific drugs resulting in maximal responses. Average CTC counts/2500 nucleated cells detected by phenotypic and therapeutic IF cocktails Citation Format: Susan Tannenbaum, Emily Hsu, jasmin Hundal, Amber Wilkes, Austin Fergusson, Fahmy Mamuya, Triantafyllos Tafas. Targeting treatment to tumor response: demonstration of response monitoring with subsequent impact on treatment choices in real time utilizing a novel technology [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-09.

Intelligent recognition of CTCs from gallbladder cancer by ultrasensitive electrochemical cytosensor and diagnosis of chemotherapeutic resistance

Gallbladder carcinoma (GBC) is one of most aggressive and lethal malignancies. Early diagnosis of GBC is crucial for determining appropriate treatment and improving chances of cure. Chemotherapy represents the main therapeutic regimen for unresectable GBC patients to inhibit tumor growth & metastasis. But, chemoresistance is the major cause of GBC recurrence. Thus, there is an urgent need to explore potentially non-invasive and point-of-care approaches to screen GBC and monitor their chemoresistance. Herein, we established an electrochemical cytosensor to specifically detect circulating tumor cells (CTCs) and their chemoresistance. Trilayer of CdSe/ZnS quantum dots (QDs) were cladded upon SiO2 nanoparticles (NPs), forming Tri-QDs/PEI@SiO2 electrochemical probes. Upon conjugation of anti-ENPP1, the electrochemical probes were able to specifically label captured CTCs from GBC. The detection of CTCs and chemoresistance were realized by square wave anodic stripping voltammetric (SWASV) responses to anodic stripping current of Cd 2+ ion when cadmium in electrochemical probes was dissolved and eventually electrodeposited on bismuth film-modified glassy carbon electrode (BFE). Taking use of this cytosensor, one ensured the screening of GBC and limit of detection for CTCs approaches to ～10 cells/mL. Furthermore, by monitoring phenotypic changes of CTCs after drug treatment, the diagnosis of chemoresistance was achieved by our cytosensor.

Evaluating the clinical utility of circulating tumor cells (CTC) profiling to predict selection of preferred therapeutic regimens in newly diagnosed or pretreated refractory renal cell carcinomas (RCC).

717 Background: There is an unmet need for biomarkers enabling therapeutic selection and prediction of clinical outcomes in kidney cancer . We evaluated the feasibility and utility of profiling of circulating tumor cells (CTCs) via multiplexed fluorescence immunocytochemistry (ICC) to identify liquid biopsy biomarkers linked to treatment response (or resistance) in advanced RCC patients. In addition, transcriptome analysis for 20802 genes from exosomal RNA is planned to evaluate novel prognostic and predictive signatures. Methods: Patients with either untreated or pretreated advanced RCC were eligible prior to starting a new systemic therapy regimen. IRB approved written informed consent was obtained. Serial blood samples were collected at baseline, and at 3, 6, 12 and 24 months. Primary endpoint of the study was to detect the proportion of patients with RCC in whom CTCs can be detected and profiled. Secondary endpoints are to correlate the profiling and changes in CTC with response and clinical outcomes. The study will meet its primary endpoint if the assay provides adequate detection and profiling for at least 12 patients. With an overall sample size of 50 the width of a 95% confidence interval for the rate of providing a therapeutic intervention is guaranteed to be less than 26%. Results: 44 evaluable patients have been enrolled; 11 females, 33 males. Median age was 64 years (range 40-85 years). 38 white patients, 2 black patients and 4 of other ethnicity have been enrolled.16 patients were untreated, 22 were pretreated and 6 were undergoing adjuvant therapy post nephrectomy. 37 patients had clear cell histology, 4 non clear cell and 3 unclassified histology. 42 of the 44 (95.5%) patients had detectable CTCs in the baseline sample. 21 on treatment samples have been collected to date, with detectable CTCs in all except one sample. 53 of 65 samples (81.5%) demonstrated detection of at least one biomarker by ICC. VEGFA expression was the most commonly detected biomarker on ICC (detected in 28 of 63 samples). Conclusions: The primary endpoint was met, and feasibility of the test was demonstrated with 95% of the baseline samples showing CTC detection and 81.5% showing biomarker expression. This blood-based, non-invasive liquid biopsy demonstrated high sensitivity for detection of cancer cells, and presents a potential opportunity for biomarker profiling to predict therapeutic efficacy of conventional RCC therapeutic agents. Correlation of longitudinal CTC with clinical outcomes will be reported. Transcriptome analysis is under evaluation for novel prognostic and predictive signatures and will be reported.

A phase 2 study of immunogenic cell death inducer PT-112 in patients with metastatic castration-resistant prostate cancer.

TPS292 Background: PT-112 is a novel small I-O molecule currently in development for several cancers. Preclinical studies have shown the robust induction of immunogenic cell death by PT-112, leading to an anti-cancer adaptive immune response. Biodistribution experiments in mice showed PT-112’s partial bone affinity. In two prior phase I studies, late-line metastatic castration-resistant prostate cancer patients (mCRPC pts) were treated with evidence of anti-cancer activity, including tumor volume reductions, improvements in PET and bone scans, PSA and alkaline phosphatase (ALP) reductions, and anecdotal cases of improvement in disease-related pain. This, taken together with the immunogenic and osteotropic properties observed in preclinical work, provided a strong rationale to further explore PT-112 in mCRPC, an immunologically “cold” disease with a high prevalence of bone metastases. Moreover, in an analysis of >6,000 mCRPC pts, circulating tumor cell (CTC) declines (CTC0 and CTC conversions) were correlated with improvements in survival to a greater extent than PSA declines (PSA50) (Heller et al., JCO, 36:6 2017), prompting incorporation of CTC changes as a secondary endpoint. Methods: The primary objective of the study is to define the dose regimen of PT-112 for pivotal study. Pts are randomized to one of three arms, receiving IV PT-112 on 28-day cycles via one hour infusions on (1) Days 1 and 15 at 250 mg/m2, (2) Days 1 and 15 at 360 mg/m2, or (3) Days 1 and 15 of cycle 1 at 360 mg/m2 followed by 250 mg/m2 on Day 15 of subsequent cycles. Key eligibility criteria include radiographically progressive disease at study entry; ≥3 life-prolonging therapies for metastatic disease including 1-2 taxanes, ≥1 new generation anti-androgen therapies, and other drugs FDA approved on the basis of survival; and allowing bone-only metastatic disease. Efficacy assessments comprise disease control rate at 4 months (DCR4) using RECIST and PCWG3 criteria, objective response rate, CTC0 and CTC conversion, PSA50 and ALP reductions. Additionally, T cell receptor sequencing is conducted to characterize treatment-induced changes in T cell fraction and clonal expansions, as a means of generating meaningful supportive data on the immune mechanism of PT-112 monotherapy. A Fleming two-stage design will be used to assess each arm, with a 20% DCR4 as the null hypotheses, requiring ≥6 of 25 pts responding in stage one and ≥14 of 45 in total to reject the null. In addition, other efficacy measures, such as CTC responses, as well as exposure/response and exposure/safety analyses will be applied to characterize the risk/benefit ratio and select the optimal dose regimen for PT-112. As of October 11th, 2022, 38 pts have been enrolled out of a planned maximum of 135. Clinical trial information: NCT02266745 .

Circulating tumor cell quantification during abiraterone plus prednisone therapy may estimate survival in metastatic castration-resistant prostate cancer patients.

Circulating tumor cells (CTCs) predict survival in response to different interventions in metastatic castration-resistant prostate cancer (mCRPC) patients. This study aimed to explore the dynamic change in CTCs during abiraterone plus prednisone therapy and its optimal threshold for prognostication in mCRPC patients. CTCs in blood samples from mCRPC patients (N = 98) at baseline and in the 2nd month after abiraterone plus prednisone treatment initiation (M2) were enumerated by using the CellSearch System. CTCs were detected in 64.8% of mCRPC patients at baseline with a median value (interquartile range) of 2.0 (0.0-4.0). Elevated CTC count was related to visceral metastasis (P = 0.003), high alkaline phosphatase (P = 0.043), and high lactate dehydrogenase (P = 0.007). Baseline CTC ≥ 1 (vs. < 1) was only associated with shortened radiographic progression-free survival (rPFS) (P = 0.043); additionally, baseline CTC ≥ 5 (vs. < 5) was linked with unfavorable rPFS (P = 0.037) and overall survival (OS) (P = 0.021). Following the therapy, CTCs were reduced at M2 (P < 0.001). Notably, CTC ≥ 1 (vs. < 1) (P = 0.002) and CTC ≥ 5 (vs. < 5) (P < 0.001) at M2 were related to shortened rPFS according to the Kaplan‒Meier curves, and they could independently estimate deteriorative rPFS in the multivariate Cox regression (P = 0.043 and P = 0.027, respectively). Similarly, CTC ≥ 1 (vs. < 1) (P = 0.022) and CTC ≥ 5 (vs. < 5) (P = 0.002) at M2 were related to shortened OS, whereas only CTC ≥ 5 (vs. < 5) could independently predict unfavorable OS (P = 0.017). CTC count ≥ 5 at M2 exhibits excellent prognostic value for abiraterone plus prednisone therapy in mCRPC patients.

Circulating tumor cells dynamics during chemotherapy predict survival and response in advanced non-small-cell lung cancer patients.

Circulating tumor cells (CTCs) are prognostic biomarker in non-small-cell lung cancer (NSCLC). CTCs could also be used as predictor of efficacy of systemic treatments in advanced NSCLC. We described the dynamic changes of CTCs during first-line platinum-based chemotherapy in advanced NSCLC and clarified the correlation between CTC counts and efficacy of chemotherapy. Chemotherapy is administered and blood specimens are collected at four time points from baseline to disease progression for CTC detection. This multicenter prospective study enrolled patients with previously untreated stage III or IV NSCLC fit for standard platinum-based chemotherapy. Bloods were sampled as per standard operating procedures at baseline, cycle 1 and cycle 4 of chemotherapy, and at disease progression for CTC analysis using the CellSearch system. Among 150 patients enrolled, median overall survival (OS) was 13.8, 8.4, and 7.9 months in patients with CTC-, KIT-CTC, and KIT+CTC at baseline (p = 0.002). Patients with persistent negative CTC (46.0%) had longer progression-free survival [5.7 months, 95% confidence interval (CI): 5.0-6.5 versus 3.0 months, 0.6-5.4; hazard ratio (HR): 0.34, 95% CI: 0.18-0.67) and OS (13.1 months, 10.9-15.3 versus 5.6 months, 4.1-7.1; HR: 0.17, 0.08-0.36) compared with patients with persistent positive CTC (10.7%), which was not impacted by chemotherapy. Chemotherapy decreased CTC from 36.0% (54/150) to 13.7% (13/95). CTC persistent presence during treatment represents poor prognosis and resistance to chemotherapy in advanced NSCLC. Chemotherapy could effectively eliminate CTCs. Molecular characterization and the functionalization of CTC will be warranted for further intensive investigation. NCT01740804.

Prognostic significance of circulating tumor cell measurement in the peripheral blood of patients with nasopharyngeal carcinoma

ObjectiveNasopharyngeal Carcinoma (NPC) is lethal cancer. Typically, relapse and metastasis are the outcomes of most patients. Against this backdrop, this study aimed to investigate the correlation between Circulating Tumor Cell (CTC) profiles and clinicopathological features in patients with NPC. Patients and methodsA total of 119 blood samples from 79 patients were collected from patients with NPC during treatment. CanPatrolTM CTC enrichment and RNA In Situ Hybridization (RNA-ISH) were used to characterize CTCs, including epithelial, Mesenchymal (MCTCs), and epithelial/mesenchymal mixed types according to their surface markers. ResultsThe number of CTCs and MCTCs in the pre-treatment group was significantly higher than that in the post-treatment group (p < 0.05). The total number of CTCs and MCTCs cell numbers was significant correlation with Tumor-Node-Metastasis (TNM) staging (p < 0.05), Progression-Free Survival (PFS), and Overall Survival (OS). The PFS of patients with > 7 CTCs or > 5 MCTCs per 5 mL blood was significantly shorter PFS than those patients with ≤ 7 CTCs or ≤ 5 MCTCs (p < 0.05). Patients treated with targeted therapy combined with chemoradiotherapy had poorer PFS and OS rates than those treated with chemoradiotherapy (p < 0.05). The Kaplan-Meier survival analysis also demonstrated that patients with changes in CTC > 4 were strongly associated with PFS and OS rates (p < 0.05). ConclusionCTC and MCTC number detection in patients with NPC is a useful biomarker for predicting patient progress. Patients with more than 7 CTCs or 5 MCTCs in 5 mL of blood had shorter PFS and OS rates. CTC and MCTC count changes were also significantly associated with the patient's therapy.

Single-Cell Multi-Omics of Peripheral Blood Reveals Tiding and Evolution of Responsive and Resistant Clones upon Daratumumab-Based Treatments in Multiple Myeloma and Plasma Cell Leukemia

Background: Treatment with anti-CD38 antibodies is a standard of care in newly diagnosed and relapsed/refractory multiple myeloma (MM). Clonal tiding and evolution as well as genomic heterogeneity are major drivers towards drug-resistant disease. However, modes of resistance to anti-CD38 directed therapies are currently unknown and might include loss of antigen expression and/or exhaustion of the immune system. We performed longitudinal single-cell multi-omic analyses of circulating tumor cells (CTC) and non-malignant cells to decipher clonal heterogeneity and tiding upon response and resistance to daratumumab (Dara)-based therapies in MM and plasma cell leukemia (PCL). Methods: Peripheral blood mononuclear cells (MNCs) were isolated from 7 patients (PCL n=5, MM n=2) before and after exposure to Dara. Samples were collected at four different time points: 1. before administration of Dara (CD38-naïve). 2. Four days after first application of Dara (CD38-exposed). 3. After relapse from Dara (CD38-refractory). 4. After an additional line of non-CD38 targeted therapy following relapse from Dara. In total, we analyzed 17 PB samples. To demonstrate that results from PB are comparable to bone marrow (BM), four paired PB and BM samples were included (21 samples in total). MNCs were subjected to single cell RNA (scRNAseq), B-cell (scBCRseq), and T-cell receptor sequencing (scTCRseq). Surface protein expression was measured using a custom panel of 57 DNA-barcoded antibodies. Sequencing files were processed with CellRanger (10x Genomics) and data were analyzed with the Seurat toolkit for single cell sequencing. Cell types were annotated with SingleR based on the Human Primary Cell Atlas built-in references. ScRepertoire was used for longitudinal scTCRseq and scBCRseq data. Copy number variations (CNVs) in CTCs were detected with inferCNV, and cellular interactions were investigated with CellPhoneDB. Results: In total, we sequenced 130.038 MNCs (PB: 108.236, range 414-8700/patient; BM: 21802, range 3375-8516/patient, Figure 1A). Malignant plasma cells were identified using scBCRseq and restricted light chain expression from scRNAseq. Even in MM patients without morphological evidence for CTC, we could identify and characterize single malignant plasma cells in PB. By inferring genome-wide CNVs from scRNAseq, we were able to characterize intra-patient genomic heterogeneity. Longitudinal tracking of CTC revealed tiding of clones characterized by distinct CNV profiles and transcriptomes upon Dara-exposure and resistance. Early transcriptional changes in CTCs upon Dara-exposure included inflammatory signaling (e.g. upregulation of IFI6, IFI35, IFIT1, IFITM1). Analysis of CD38 protein surface expression using DNA-barcoded antibodies showed significantly decreased levels after Dara exposure. To rule out that this might have resulted from competitive binding of the diagnostic antibody and Dara, we repeated analyses after discontinuing Dara and after application of a further line of therapy. In contrast to CD38 RNA expression, surface protein expression decreased significantly upon exposure and relapse, but recovered after an additional line of therapy. Expression of other druggable antigens (BCMA, SLAMF7, GPRCD5, FcRL5, Figure 1B) was not affected by treatment with Dara. Next, we investigated co-evolution of non-malignant cells. Longitudinal scTCRseq showed concomitant expansion and stagnation of TCRs. Receptor-ligand interaction analyses demonstrated that exposure to Dara resulted in significant differences in CD38-PECAM1-mediated interactions between CTCs with non-malignant B-cells and CD8+ T-cells. We detected recurrently increased immunosuppressive receptor-ligand interactions between malignant plasma cells and CD4+, CD8+, and regulatory T-cells via CD86-CTLA4 and NECTIN3-TIGIT axis upon Dara exposure. Conclusion: We demonstrate that single-cell multi-omic analyses of PB provide a minimal invasive approach to study genomic heterogeneity and evolution of malignant plasma cells and non-malignant cells of the immune system. Longitudinal analyses reveal different patterns of response and resistance to targeted myeloma therapies and might affect future therapeutic decision-making. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

1475P Association of circulating tumor cells (CTCs) with immune checkpoint inhibitor (ICI) responses in metastatic renal cell carcinoma (mRCC)

mRCC treatment has expanded to include several ICIs and combinations with targeted kinase inhibitors. However, only clinical criteria has been validated to predict for ICI treatment responses. We previously described a highly sensitive CTC assay with multivalent binding, e-selectin induced cell rolling, and multiple antibody ligands. We prospectively evaluated whether CTC change would predict for ICI response in mRCC.

Abstract CT156: Changes in circulating tumor associated cells predicts progression free and overall survival in metastatic TNBC patients after induction of the anti-CCR5 drug leronlimab

Abstract Background: Metastatic triple negative breast cancer (mTNBC) is a highly invasive breast cancer subtype that has limited treatment options and poor clinical outcomes for patients. Recently, the C-C chemokine receptor 5 (CCR5) was identified in preclinical models as a potential drug target that inhibits pro-migratory tumor progression in breast cancer by blocking tumor motility and subsequent tumor cell spread. Leronlimab (PRO140), a humanized IgG4κ monoclonal antibody, is a competitive inhibitor of CCR5, with 3 active clinical trials ongoing in TNBC patients. Here we report on a preliminary pooled analysis of n=28 mTNBC patients to evaluate effects of Leronlimab on circulating tumor-associated cells (TACs) found in peripheral blood as an early predictor of drug response, with end point outcomes of progression free survival (PFS) and Overall Survival (OS). Methods: Blood samples were drawn from mTNBC patients from 3 blinded prospective clinical drug studies, Phase 1b/2 (NCT03838367), Compassionate Use (NCT04313075), and Basket Study (NCT04504942) to evaluate the predictive value of TACs, measured by the LifeTracDx assay. All subjects received ≥1 dose of Leronlimab (range 1-33 doses), ranging from 350mg - 700mg, with 4 subjects having dose escalation. Anonymized blinded peripheral blood samples were available from (n=28) patients, obtained as part of the exploratory portion of the trials, prior to drug induction (BL) and after one treatment cycle (T1), ~26 days. TACs, i.e. Circulating tumor cells (CTCs) and Cancer Associated Macrophage-like cells (CAMLS), were isolated using a low-flow CellSieve microfiltration system and changes in TACs were quantified by the LifeTracDx assay. A univariate analysis was used to analyze changes in TACs to predict for PFS and OS over 12 months. Results: A total of 28 mTNBC patients were pooled from Phase 1b/2 (n=10), Compassionate Use (n =16), and Basket Study (n=2) all of which had available BL blood samples, or T1 samples. CTCs were found in 29% (n=8/28) of patients at BL, and CAMLs were found in 96% (n=27/28). Evaluating CTC change was limited, as only 5 patients had any CTC increase, although the absence or drop of CTCs at T1 was significant for better PFS (HR=13.7 CI 95% 2.0-93.8, p=0.0296) and better OS (HR=397.7 CI 95% 19.3-100+, p=0.0019). By comparison, CAMLs or CTCs were evaluable in 100% of patients, with an increases of either population at T1 also significantly predicting for worse PFS (HR=5.8 CI 95% 1.4-23.6, p=0.0354) and worse OS (HR=36.0 CI 95% 6.2-207.6, p=0.0004). Conclusions: We observed that treatment with Leronlimab resulted in rapid decreases in the presence of multiple populations of TACs in 75% of patients, which also significantly correlated with better PFS and OS after 1 year analysis. Citation Format: Daniel L. Adams, Ashvathi Raghavakaimal, Cha-Mei Tang, Kirby P. Gardner, Nitya G. Ray, Nader Pourhassan. Changes in circulating tumor associated cells predicts progression free and overall survival in metastatic TNBC patients after induction of the anti-CCR5 drug leronlimab [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 CT156.

Abstract P2-02-05: Dynamic circulating tumor cell changes in enumeration and HER2 expression during systemic therapy for metastatic breast cancer

Abstract Introduction: CTCs are tumor cells that circulate in the blood of patient with primary and MBC and, are responsible for seeing of metastasis. The monitoring of CTCs in MBC emerged as strong prognostic and possible predictive biomarker in oncology over the past couple of decades. Meanwhile, overexpression of HER2 protein has been associated with rapid cell division and worse prognosis of MBC. Here we report a significant correlation between dynamic CTCs enumeration and CTCs-HER2 expression during the systemic therapies, with potential implication to understand treatment resistance. Methods: A total of 298 whole blood samples (7.5ml/each) were collected from 149 patients with stage IV breast cancer (2016-2020) at the Northwestern University Robert H Lurie Comprehensive Cancer Center, before (Baseline) and 3 months after (Time point 2) initiation of systemic treatment. CTC enumerations were performed using the FDA approved CellSearch™ system (Menarini) which is specific for the intracellular protein cytokeratin (CK) in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. We developed a criteria for evaluation of HER2 expression by 4 different categories (0,1+,2+,3+) based on expression intensity in our lab (present in 2021 ASCO). In this study we included CTCs with all intensities of HER2 expression (1+ to 3+) which was standardized in our lab. Mann-Whitney U test was used for statistics. Results: Of the 149 baseline samples, CTC≥1 were found in 101 patients (67.8%). A change in CTCs between baseline and time point 2 for these 101 patients: Three groups were identified: Group 1: 33patients (33%) with increase CTCs; Group 2: 64 patients (63%) with decreased CTC; Group 3: 4 patients (4%) with no change The median increase of total CTCs and HER2+ CTCs in Group 1 were 7.0 and 2.0, respectively; the median decrease of total CTCs and HER2+ CTCs in Group 2 were 9.5 and 2.0, respectively. The change of HER2+ CTCs was significantly correlated with the change of total CTCs after systemic therapy, with the correlation coefficient as rs=0.662 (p&amp;lt;0.001) for these 101 patients. A significant positive correlation between HER2+ CTCs and total CTCs were found in both Group 1 and Group 2, rs=0.717 (p&amp;lt;0.001) and rs=0.604 (p&amp;lt;0.001) respectively. Furthermore, the ratio of HER2+ CTCs (HER2+ CTCs/total CTCs) in both Group 1 and Group 2 was also significantly correlated with total CTCs after therapy with the corresponding correlation coefficient as rs=0.536 (p&amp;lt;0.001) and rs=0.388 (p&amp;lt;0.001) in Group 1 and Group 2, respectively. Conclusion: Our study demonstrated that dynamic changes of CTCs after systemic therapies, are positively correlated with the HER2 expression in CTCs in different levels of baseline CTCs amounts. Observation of HER2 expression and ratio in CTCs during the course of systemic therapy is useful in monitoring therapy efficacy and potential disease progress. Citation Format: Qiang Zhang, Weijun Qin, Paolo D'Amico, Andrew A. Davis, Jianhua Jiao, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N Shah, Amir Behdad, Lisa Flaum, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Dynamic circulating tumor cell changes in enumeration and HER2 expression during systemic therapy for metastatic breast cancer [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 P2-02-05.

Circulating tumor cells may serve as a supplement to RECIST in neoadjuvant chemotherapy of patients with locally advanced breast cancer.

Circulating tumor cells (CTCs) have been shown to be associated with the response to neoadjuvant chemotherapy (NCT) and the prognosis of locally advanced breast cancer (LABC) patients. Our study aimed to investigate whether the change of CTC status during NCT could serve as a supplement to the Response Evaluation Criteria in Solid Tumors (RECIST) in the treatment and evaluation of LABC patients. 6ml of blood samples were collected before NCT, after the first cycle of NCT and after the completion of NCT, respectively. According to the change of CTC number during NCT, the patients were divided into "CTC low-response (low-R)" group and "CTC high-response (high-R)" group. Survival data of each group of patients were obtained through long-term follow-up. A total of 35 patients diagnosed with LABC were enrolled. The median follow-up for distant metastasis was 27months (range 7-36months). There was no significant difference in distant metastasis-free survival (DMFS) between PR/CR group and PD/SD group (P = 0.0914), while CTC low-R group had a worse DMFS than CTC high-R group (P = 0.0199). In PR/CR subgroup, patients with CTC low-R showed a lower DMFS compared with those with CTC high-R (P = 0.0159). However, in PD/SD subgroup, there was no significant difference in DMFS between CTC low-R and CTC high-R group (P = 0.7521). In terms of assessing response to NCT, CTC change or RECIST classification alone had an AUC of 0.533 (95% CI 0.277-0.790) and 0.700 (95% CI 0.611-0.789), respectively. When combining the two, the AUC slightly increased to 0.713 (95% CI 0.532-0.895). The change of CTC number during NCT has a potential to serve as a supplement to RECIST in the assessment of NCT efficacy and the prognosis of LABC patients.

Association of early changes of circulating cancer stem-like cells with survival among patients with metastatic breast cancer

Background:This study aimed to investigate the role of circulating tumor cells (CTCs) and circulating cancer stem-like cells (cCSCs) before and after one cycle of chemotherapy and assessed the effects of early changes in CTCs and cCSCs on the outcomes of patients with metastatic breast cancer.Methods:Patients with stage IV invasive ductal carcinoma of the breast who received first-line chemotherapy between April 2014 and January 2016 were enrolled. CTCs and cCSCs were measured before the first cycle of chemotherapy (baseline) and on day 21, before the second cycle of chemotherapy commenced; a negative selection strategy and flow cytometry protocol were employed.Results:CTC and cCSC counts declined in 68.8 and 45.5% of patients, respectively. Declines in CTCs and cCSCs following the first chemotherapy cycle were associated with superior chemotherapy responses, longer progression-free survival (PFS), and longer overall survival (OS). An early decline in cCSCs remained an independent prognostic indicator for OS and PFS in multivariate analysis.Conclusions:A cCSC decline after one cycle of chemotherapy for metastatic breast cancer is predictive of a superior chemotherapy response and longer PFS and OS, implying that cCSC dynamic monitoring may be helpful in early prediction of treatment response and prognosis.

Clinical significance of circulating tumor cells after chemotherapy in unresectable pancreatic ductal adenocarcinoma

Circulating tumor cells (CTCs) have emerged as liquid biopsy biomarker providing non-invasive assessment of cancer progression and biology. We investigated whether longitudinal analysis of CTCs could monitor disease progression, response to chemotherapy, and survival in patients with unresectable pancreatic ductal adenocarcinoma (PDAC). A total of 52 patients with PDAC were prospectively enrolled in this study. Peripheral blood samples were serially collected at the time of diagnosis and after chemotherapy with clinical assessments. CTCs were isolated through a centrifugal microfluidic disc, enumerated with immunostaining against Epithelial cell adhesion molecule (EpCAM), Cytokeratin (CK), Plectin-1 and CD45, and identified by an automated imaging system. One or more CTCs were detected in 84.62% patients with unresectable PDAC at the time of diagnosis. CTC numbers were not statistically different across tumor sizes, location and metastatic sites. The absolute number of CTCs after chemotherapy was inversely related to overall survival (OS), and the decreased number of CTCs after chemotherapy was significantly associated with longer OS in patients with PDAC. Identifying CTCs and monitoring CTC changes after chemotherapy could be a useful prognostic marker for survival in patients with unresectable PDACs.