Circulating Tumor Cells Counts Research Articles

Predictive value of circulating tumor cell counts during the treatment of cancer: interactions with the blood microenvironment.

This study aimed to evaluate the prognostic value of circulating tumor cell (CTC) in tumor patients during treatment. This study retrospectively analyzed clinical data obtained from 174 cancer patients during treatment. The relationship between the CTC counts and clinicopathological variables was analyzed. A ROC curve was applied to determine the optimal cut-off values and assess the predictive ability of the prognostic indicators. The overall survival (OS) for different prognostic factors was calculated using the Kaplan-Meier method, and the difference between the survival curves was then compared using the log-rank test. Cox regression model was used to investigate the effect of independent factors on patients' survival. The CTC-positive rate was positively correlated with the clinicopathological variables of TNM stage, tumordifferentiation, serum CEA level, and ki-67%. In the differential analysis of hematological microenvironment parameters in CTC-positive and CTC-negative samples, the complete blood count, blood biological chemistry, tumor markers (CEA, CA19-9, CA72-4), and lymphocyte subpopulation were statistically significant. The results of the ROC curve analysis indicated that the serum CEA level was the best diagnostic indicator to discriminate the CTC count in tumor patients. Additionally, the results of the univariate and multivariate analyses of OS in relation to clinical variables revealed that the CTC counts were an independent prognostic factor for unfavorable OS. The CTC counts in patients with tumors undergoing treatment were significantly correlated with hematological microenvironment parameters. The detection of CTCs may therefore be used as an indicator of tumor prognosis.

Circulating tumor cells (CTCs) and hTERT gene expression in CTCs for radiotherapy effect with lung cancer

BackgroundCirculating tumor cells (CTCs) are important biological indicators of the lung cancer prognosis, and CTC counting and typing may provide helpful biological information for the diagnosis and treatment of lung cancer.MethodsThe CTC count in blood before and after radiotherapy was detected by the CanPatrol™ CTC analysis system, and the CTC subtypes and the expression of hTERT before and after radiotherapy were detected by multiple in situ hybridization. The CTC count was calculated as the number of cells per 5 mL of blood.ResultsThe CTC positivity rate in patients with tumors before radiotherapy was 98.44%. Epithelial–mesenchymal CTCs (EMCTCs) were more common in patients with lung adenocarcinoma and squamous carcinoma than in patients with small cell lung cancer (P = 0.027). The total CTCs (TCTCs), EMCTCs, and mesenchymal CTCs (MCTCs) counts were significantly higher in patients with TNM stage III and IV tumors (P < 0.001, P = 0.005, and P < 0.001, respectively). The TCTCs and MCTCs counts were significantly higher in patients with an ECOG score of > 1 (P = 0.022 and P = 0.024, respectively). The TCTCs and EMCTCs counts before and after radiotherapy affected the overall response rate (ORR) (P < 0.05). TCTCs and ECTCs with positive hTERT expression were associated with the ORR of radiotherapy (P = 0.002 and P = 0.038, respectively), as were TCTCs with high hTERT expression (P = 0.012). ECOG score (P = 0.006) and post-radiation TCTCs count (P = 0.011) were independent factors for progression-free survival (PFS) and TNM stage (P = 0.054) and pre-radiation EMCTCs count (P = 0.009) were independent factors of overall survival (OS).ConclusionThis study showed a high rate of positive CTC detection in patients with lung cancer, and the number, subtype, and hTERT-positive expression of CTCs were closely related to patients’ ORR, PFS, and OS with radiotherapy. EMCTCs, hTERT-positive expression of CTCs are expected to be important biological indicators for predicting radiotherapy efficacy and the prognosis in patients with lung cancer. These results may be useful in improving disease stratification for future clinical trials and may help in clinical decision-making.

Longitudinal change of circulating tumour cell count and its relation to prognosis in advanced intrahepatic cholangiocarcinoma patients

Circulating tumour cells (CTCs) are related to poor prognosis in hepatobiliary cancers, including hepatocellular carcinoma and gallbladder carcinoma, but their value in intrahepatic cholangiocarcinoma (ICC) is obscure. This study aimed to investigate the change in CTCs during chemotherapy and its correlation with clinical features, treatment response and survival profile in advanced ICC patients. Fifty-one unresectable, advanced ICC patients who underwent chemotherapy were consecutively enrolled. Peripheral blood samples were collected at diagnosis and 2 months (M2) after chemotherapy initiation for CTC detection via the ISET method. The mean and median CTC counts at diagnosis were 7.4 ± 12.2 and 4.0 (range: 0.0-68.0), respectively, with 92.2% of patients having more than one CTC. A higher CTC count at diagnosis was correlated with elevated lymph node metastasis (p = 0.005), distant metastasis (p = 0.005) and TNM stage (p = 0.001) but no other characteristics. In addition, the CTC count at diagnosis was higher in nonobjective-response patients than in objective-response patients (p = 0.002), and a CTC count at diagnosis above 3 correlated with worse progression-free survival (PFS) (p = 0.007) and overall survival (OS) (p = 0.036). At M2, the CTC count was greatly decreased (p < 0.001). CTC count at M2 also correlated with lower treatment response (p < 0.001), and CTC counts above 3 were associated with poor PFS (p = 0.003) and OS (p = 0.017). After multivariate Cox analyses, CTC counts at diagnosis above 3 and CTC count increase from diagnosis to M2 independently predicted PFS and OS (p < 0.05). Detection of CTCs before and during chemotherapy is useful for prognostication in advanced ICC patients.

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.

Prognostic value of preoperative circulating tumor cells for hepatocellular carcinoma with portal vein tumor thrombosis: A propensity score analysis

PurposeThe role of circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT) is not fully understood.MethodsIn this retrospective analysis, we included 316 HCC patients who underwent hepatectomy and preoperative CTC detection. We selected 41 pairs of matched HCC patients with and without PVTT using propensity score matching (PSM) analysis. We compared the preoperative CTC counts in patients from both the full cohort and the PSM model. We also analyzed their associations with disease-free survival (DFS) and overall survival (OS).ResultsBefore and after PSM analysis, the preoperative CTC counts in the HCC with PVTT group were substantially higher than in the HCC without PVTT group. In both the full cohort of patients and the PSM model, patients with CTC ≥ 2 had significantly shorter OS and DFS than patients with CTC < 2. The outcomes of HCC patients with PVTT could be well differentiated by preoperative CTC levels. HCC patients with CTC ≥ 2 had noticeably shorter OS (9.9 months vs. 24.6 months, P = 0.0003) and DFS (6.0 months vs. 12.3 months, P = 0.0041) than those with CTC < 2. Moreover, preoperative CTC ≥ 2 remained an independent predictor in all groups’ multivariate analysis.ConclusionWe discovered a link between preoperative CTC counts and the occurrence of PVTT and confirmed the prognostic significance of preoperative CTC in HCC patients with PVTT. These findings suggest that preoperative CTC counts have the potential to assist in identifying patients with HCC and PVTT who may benefit from surgery.

Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0-27), 101.8 (0-255), and 3.2 (0-10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0-255), and 0.2 (0-1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.

Single-cell metabolic fingerprints discover a cluster of circulating tumor cells with distinct metastatic potential

Circulating tumor cells (CTCs) are recognized as direct seeds of metastasis. However, CTC count may not be the “best” indicator of metastatic risk because their heterogeneity is generally neglected. In this study, we develop a molecular typing system to predict colorectal cancer metastasis potential based on the metabolic fingerprints of single CTCs. After identification of the metabolites potentially related to metastasis using mass spectrometry-based untargeted metabolomics, setup of a home-built single-cell quantitative mass spectrometric platform for target metabolite analysis in individual CTCs and use of a machine learning method composed of non-negative matrix factorization and logistic regression, CTCs are divided into two subgroups, C1 and C2, based on a 4-metabolite fingerprint. Both in vitro and in vivo experiments demonstrate that CTC count in C2 subgroup is closely associated with metastasis incidence. This is an interesting report on the presence of a specific population of CTCs with distinct metastatic potential at the single-cell metabolite level.

Circulating tumor cell abundance in head and neck squamous cell carcinoma decreases with successful chemoradiation and cetuximab treatment

Head and neck squamous cell carcinoma (HNSCC) is a common and deadly cancer. Circulating tumor cell (CTC) abundance may a valuable, prognostic biomarker in low- and intermediate-risk patients. However, few technologies have demonstrated success in detecting CTCs in these populations. We prospectively collected longitudinal CTC counts from two cohorts of patients receiving treatments at our institution using a highly sensitive device that purifies CTCs using biomimetic cell rolling and dendrimer-conjugated antibodies. In patients with intermediate risk human papillomavirus (HPV)-positive HNSCC, elevated CTC counts were detected in 13 of 14 subjects at screening with a median of 17 CTC/ml (range 0.2–2986.5). A second cohort of non-metastatic, HPV- HNSCC subjects received cetuximab monotherapy followed by surgical resection. In this cohort, all subjects had elevated baseline CTC counts median of 73 CTC/ml (range 5.4–332.9) with statistically significant declines during treatment. Interestingly, two patients with recurrent disease had elevated CTC counts during and following treatment, which also correlated with growth of size and ki67 expression in the primary tumor. The results suggest that our device may be a valuable tool for evaluating the success of less intensive treatment regimens.

Clinical implications and perspectives of portal venous circulating tumor cells in pancreatic cancer

Despite recent improvements in the diagnosis and treatment of pancreatic cancer (PC), clinical outcomes remain dismal. Moreover, there are no effective prognostic or predictive biomarkers or options beyond carbohydrate antigen 19-9 for personalized and precise treatment. Circulating tumor cells (CTCs), as a member of the liquid biopsy family, could be a promising biomarker; however, the rarity of CTCs in peripheral venous blood limits their clinical use. Because the first venous drainage of PC is portal circulation, the portal vein can be a more suitable location for the detection of CTCs. Endoscopic ultrasound-guided portal venous sampling of CTCs is both feasible and safe. Several studies have suggested that the detection rate and number of CTCs may be higher in the portal blood than in the peripheral blood. CTC counts in the portal blood are highly associated with hepatic metastasis, recurrence after surgery, and survival. The phenotypic and genotypic properties measured in the captured portal CTCs can help us to understand tumor heterogeneity and predict the prognosis of PC. Small sample sizes and heterogeneous CTC detection methods limit the studies to date. Therefore, a large number of prospective studies are needed to corroborate portal CTCs as a valid biomarker in PC.

Abstract 3370: Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer

Abstract Aggressive variants of prostate cancer (AVPC) emerge from prostate adenocarcinoma following enduring therapeutic pressure. They grow independent of the androgen receptor, progress rapidly and can show traits of neuroendocrine (NE) differentiation. As progression to AVPC is not well detected by prostate specific antigen monitoring, new biomarkers are urgently needed. The aim of this study is to evaluate the detection of circulating tumor cells (CTCs) in combination with gene expression analysis of NE genes to identify AVPC patients with neuroendocrine traits. Blood samples were collected from patients suffering from AVPC (n=78, including 25 patients with histological evidence of NEPC) as well as from patients with hormone-sensitive prostate cancer (HSPC) as controls (n=12) at the University Medical Center Hamburg-Eppendorf. CTC counts were determined by CellSearch analysis. In parallel, CTCs were immuno-magnetically enriched using the AdnaTest and bulk gene expression of prostate-specific and NE genes was analyzed by semi-quantitative PCR. From a subset of patients, CTCs were additionally enriched by Parsortix to compare gene expression of CTCs with EpCAM-based or size-based CTC enrichment. Using CellSearch analysis, CTCs were found in 88.9 % of AVPC patients. CTC counts ranged between 0 – 20,000 CTCs/7.5 ml of blood with a median of 32 CTCs/7.5 ml. Similarly, 81.8 % of NEPC patients were positive for CTCs with a median count of 35 CTCs/7.5 ml (range 0-13,000). In contrast, CTCs were found in only 50 % of HSPC patients at lower concentrations (median count of 0.5 CTCs/7.5 ml, range 0-34). CTC counts were significantly elevated in AVPC and NEPC patients compared to HSPC (p = 0.002, p = 0.012), but no significant differences were found between AVPC and NEPC samples.In 93.8 % of AVPC samples, CTCs expressed prostate-specific genes such as KLK3 or PSMA. At least one of the NE transcripts was detected in 37.5 % of AVPC patients. In CTCs from NEPC patients, the positivity for prostate markers was significantly reduced to 66.6 % (p = 0.01) and the positivity for NE markers was increased to 61.1 %. A random Forrest model trained on all analyzed transcripts allowed for a distinction of HSPC and NEPC with an AUC of 79.5 %. Comparison of CTC enrichment strategies in a subset of 13 patients showed similar NE marker detection by size-based enrichment (53.8 % NE-positive) compared to the AdnaTest (61.5 % NE-positive). AVPC and NEPC patients show a high CTC burden that will allow subsequent molecular analyses, and CTC counts allowed a distinction between blood samples from HSPC and NEPC patients. Gene expression analysis revealed a high degree of inter-patient heterogeneity for neuroendocrine-specific transcripts with a reduction of prostate markers in NEPC patients. Future molecular characterization and longitudinal monitoring of CTCs will shed more light on the evolution of AVPC and NEPC. Citation Format: Lina Merkens, Sarah Greimeier, Sabine Riethdorf, Klaus Pantel, Gunhild von Amsberg, Stefan Werner. Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate 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 3370.

Abstract 5454: Peripheral blood biomarkers in Phase II study of pembrolizumab in combination with oral binimetinib in patients with unresectable locally advanced or metastatic triple-negative breast cancer

Abstract Background: Activation of the RAS/MAPK pathway is associated with reduced tumor-infiltrating lymphocytes and poor outcomes in triple-negative breast cancer (TNBC). This trial evaluated the efficacy of pembrolizumab and MEK inhibitor, binimetinib. Here we evaluated potential biomarkers in peripheral blood to predict response. Methods: Patients with unresectable locally advanced or metastatic TNBC with ≤ 3 prior lines of therapy were enrolled. Treatment includes a 2-week run-in with binimetinib followed by pembrolizumab. There were 2 dose levels (DL) with binimetinib at 45 mg at DL 0 and 30 mg at DL -1. A standard 3+3 design was used in phase I, and Simon’s two-stage Optimal design was used in phase II. Circulating tumor cells (CTC) and circulating cancer-associated macrophage-like cells (CAML) were isolated using CellSieve microfilters and immunofluorescently labeled with PD-L1 and p-ERK. Wilcoxon rank sum test and Cox regression model were used for analysis. Results: A total of 22 patients were enrolled, with a median age of 58 years old. Dose-limiting toxicity (DLT) was observed in 2 out of 4 patients in DL 0, with grade 3 ALT abnormality, flank pain, and nausea. In the next 6 patients in DL -1, there was 1 DLT with grade 3 AST/ALT abnormality. There were 17 patients treated with DL -1 and were evaluable for response. The objective response rate (ORR) was 29.41% (95% CI: 10.31-55.9) with 1 complete response (CR) and 4 partial responses (PR). The clinical benefit rate (CBR ≥ 24 weeks) was 35.29% (95% CI: 14.21-61.67). ORR in patients without liver metastases was 55.56% (95% CI: 21.20 - 86.30), and CBR was 66.67% (95% CI: 29.93-92.51). There was no response observed in all 5 patients with liver metastases. Baseline mean CTC count was 1.3 cells, and CAML count was 8.9 cells/7.5 mL. Baseline PD-L1 in CAML (p 0.04) and decline in CAML size (p 0.02) after 1 cycle were significantly associated with CBR. However, baseline CTC count, CAML count (p 0.64), CAML size (p 0.46), p-ERK in CAML (p 0.23), and changes in CTC count, CAML count (p 0.83), p-ERK (p 0.07), and PD-L1 (p 0.08) in CAML were not significantly associated with responses. Using Cox regression analysis, a reduction in CAML count (p 0.02), CAML size (p 0.01), and PD-L1 in CAML (p 0.03) were associated with significant improvement in overall survival but not the reduction in p-ERK (p 0.6). Conclusions: Pembrolizumab and binimetinib at 30 mg are safe with manageable toxicities. Promising activity was observed in patients without liver metastases. Baseline PD-L1 expression, early reduction in CAML count, size, and PD-L1 expression were significantly associated with subsequent responses, providing potential noninvasive biomarkers to predict response to this combination. Future larger clinical trials are warranted to further evaluate the efficacy of this combination. Citation Format: Saranya Chumsri, Joseph J. Larson, Daniel L. Adams, Kathleen S. Tenner, Cha-Mei Tang, Morgan T. Weidner, Amanda N. Arnold, Dana L. Haley, Pooja Advani, Kostandinos Sideras, Alvaro Moreno-aspitia, Edith A. Perez, Keith L. Knutson. Peripheral blood biomarkers in Phase II study of pembrolizumab in combination with oral binimetinib in patients with unresectable locally advanced or metastatic triple-negative breast 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 5454.

Abstract 3364: Combined enrichment of plasma EpCAM-positive extracellular vesicles (EVs) and circulating tumor cells (CTCs) from a single tube of cancer patient blood samples, for subsequent molecular analysis

Abstract Background: EVs are secreted by cells in physiological and pathological conditions and carry selected molecules, such as proteins, DNA and RNA; specific EVs released by neoplastic cells are defined as “tumor-derived extracellular vesicles (tdEVs)”. tdEVs have been demonstrated to be involved in promoting cell growth and survival, shaping the tumor microenvironment and increasing invasiveness and metastatic activity. Thanks to their systemic availability in body fluids and their role in tumor pathogenesis, tdEVs have gained attention in the liquid biopsy area, as a possible biomarker for tumor diagnosis, prognosis and monitoring and also as possible therapeutic targets. Beyond being a target for the capture of CTCs, the Epithelial Cell Adhesion Molecule (EpCAM) was also recently employed for the detection of EVs released by tumors. Here we present a method (for research use only) based on the CellMag™ technology (Menarini Silicon Biosystems, MSB) to carry out parallel enrichment of EVs and CTCs from whole blood through an EpCAM-based magnetic enrichment. Method: Whole blood samples from stage III/IV prostate (n=5) or breast cancer patients (n=6) and healthy donors (n=4) were collected in CellRescue™ tubes (MSB). After centrifugation, EpCAM+ EVs and CTCs were enriched from the plasma and cell fraction respectively, using the CellMag™ Epithelial CTC kit (MSB); CTCs were counted at the CellTracks Analyzer II (MSB). EVs were enriched for qualitative evaluation by Transmission Electron Microscopy (TEM) and for miRNA analysis by Real-time PCR, using the TaqMan™ Advanced miRNA Human Serum/Plasma panel. Results: CTCs counts ranged from 0 to 68; TEM analysis showed a higher density of EpCAM+ extracellular microvesicles (0,1-1µm) in breast cancer patients’ samples (n=2) compared to controls (n=2). Endogenous hsa-miR-16-5p control was detected in all analyzed samples (n=9 patients’ samples, n=2 controls). One control and two patients’ samples were tested for a panel of 188 serum/plasma miRNAs. In both the breast and the prostate cancer patients a higher percentage of miRNAs was found with respect to healthy control (31%, 32% vs. 17%). Among these, several oncogenic miRNAs with diagnostic and prognostic value (such as hsa-miR-125b-5p or hsa-miR-26a-5p) were exclusively found in patients’ samples, and further confirmed to be related to ErbB2 pathway in breast cancer using the miRPathDB 2.0 database, despite the CTCs count was 0 for both these patients’ samples. Conclusions: The described multi-target liquid biopsy approach enabled the combined enrichment of EpCAM+ CTCs and plasma EpCAM+ EVs from a single blood sample, along with their molecular analysis. Citation Format: Giulia Bregola, Martina Masotti, Valentina Mattei, Martina Moras, Claudio Ratti, Francesco Picardo, Cecilia Simonelli, Nicolò Manaresi, Francesca Fontana. Combined enrichment of plasma EpCAM-positive extracellular vesicles (EVs) and circulating tumor cells (CTCs) from a single tube of cancer patient blood samples, for subsequent molecular analysis [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 3364.

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.

Metastasis risk stratification and response prediction through dynamic viable circulating tumor cell counts for rectal cancer in a neoadjuvant setting.

Distant metastasis (DM) and neoadjuvant treatment response prediction remain critical challenges in the management of locally advanced rectal cancer (LARC). The aim of this study was to investigate the clinical relevance of viable circulating tumor cells (CTCs) for DM or response in patients with LARC in a neoadjuvant setting. The detection of viable CTCs at different stages of treatment was planned for consecutive patients from a prospective trial. The Kaplan-Meier method, Cox proportional hazards model, and logistic regression model were utilized to analyze factors associated with DM or pathological complete response (pCR) and clinical complete response (cCR). Between December 2016 and July 2018, peripheral blood samples from 83 patients were collected before any treatment (median follow-up time, 49.3 months). CTCs were present in 76 of 83 patients (91.6%) at baseline, and more than three CTCs detected in the blood sample was considered high risk. Only the CTC risk group was significantly associated with 3-year metastasis-free survival (MFS) (high risk vs. low risk, 57.1% (95% CI, 41.6-72.6) vs. 78.3% (95% CI, 65.8-90.8), p = 0.018, log-rank test). When all the important variables were entered into the Cox model, the CTC risk group remained the only significant independent factor for DM (hazard ratio (HR), 2.74; 95% CI, 1.17-6.45, p = 0.021). The pCR and continuous cCR rates were higher in patients with a decreased number of CTCs of more than one after radiotherapy (HR, 4.00; 95% CI, 1.09-14.71, P = 0.037). The dynamic detection of viable CTCs may strengthen pretreatment risk assessment and postradiotherapy decision making for LARC. This observation requires further validation in a prospective study.

MP29-10 PROGNOSTIC VALUE OF NEUTROPHIL-TO-LYMPHOCYTE RATIO (NLR) IN PATIENTS WITH METASTATIC CASTRATION-RESISTANT PROSTATE CANCER (MCRPC) RECEIVING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) TARGETED RADIONUCLIDE THERAPY (TRT)

You have accessJournal of UrologyCME1 Apr 2023MP29-10 PROGNOSTIC VALUE OF NEUTROPHIL-TO-LYMPHOCYTE RATIO (NLR) IN PATIENTS WITH METASTATIC CASTRATION-RESISTANT PROSTATE CANCER (MCRPC) RECEIVING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) TARGETED RADIONUCLIDE THERAPY (TRT) Judith Stangl-Kremser, Michael Sun, Benedict Ho, Joseph Thomas, Jones Nauseef, Joseph Osborne, Ana Molina, Cora Sternberg, David Nanus, Neil Bander, and Scott Tagawa Judith Stangl-KremserJudith Stangl-Kremser More articles by this author , Michael SunMichael Sun More articles by this author , Benedict HoBenedict Ho More articles by this author , Joseph ThomasJoseph Thomas More articles by this author , Jones NauseefJones Nauseef More articles by this author , Joseph OsborneJoseph Osborne More articles by this author , Ana MolinaAna Molina More articles by this author , Cora SternbergCora Sternberg More articles by this author , David NanusDavid Nanus More articles by this author , Neil BanderNeil Bander More articles by this author , and Scott TagawaScott Tagawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003257.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PSMA TRT is a promising therapy for patients with mCRPC. Known prognostic factors for mCRPC include CALGB (Halabi) risk group and baseline circulating tumor cell (CTC) count. Here, we report associations between NLR and PSA response and overall survival (OS) in men with mCRPC treated on sequential prospective clinical trials at a single institution. METHODS: Patients treated on prospective studies from 2000 to 2021 were included in this analysis. Variables assessed: baseline neutrophil count:lymphocyte count ratio, baseline CTC count (CellSearch, favorable vs unfavorable), CALGB risk group, and administered radioactivity level. We used a Cox proportional hazards model to investigate the association between NLR and OS and logistic regression to determine the association between NLR and >50% PSA decline (PSA50). RESULTS: 180 patients were included in this analysis. Median age 71 (IQR 66-77.5), PSA 57.3 ng/mL (21.3-294.9), 80 (44.4%) prior chemotherapy, 86 (47.7%) prior abiraterone or androgen receptor signaling inhibitor, 167 (92.8%) bone metastases, 141 (78.3%) nodal metastases, 113 (62.8%) CALGB high-risk group. 94 (52.2%) received 177Lu-J591, 51 (28.3%) 177Lu-PSMA-617, 28 (15.6%) 225Ac-J591, 7 (3.9%) 90Y-J591. Median ANC 4.1 (IQR 3.1-5.1), ALC 1 (0.75-1.5), NLR 3.75 (2.63-5.92). On univariate analysis, higher NLR was associated with worse OS (HR 1.06, 95%CI 1.02-1.09, p=0.002) and trended toward lower rates of PSA50 (HR 1.08; 95% CI 0.99-1.17, p=0.067). On multivariable analysis, after controlling for CTC count and CALGB risk group, the relationship between higher NLR and worse OS persisted (HR 1.05; 95% CI 1.003-1.11, p=0.036). Stratifying patients by low vs high NLR (relative to median NLR), patients with low NLR had longer OS (23.7 vs 15 months, p=0.024). CONCLUSIONS: For patients with mCRPC receiving PSMA TRT, NLR may have prognostic implications with higher NLR associating with lower likelihood of PSA response and shorter overall survival. Source of Funding: NIH, Department of Defense, Weill Cornell Medicine, Prostate Cancer Foundation © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e384 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Judith Stangl-Kremser More articles by this author Michael Sun More articles by this author Benedict Ho More articles by this author Joseph Thomas More articles by this author Jones Nauseef More articles by this author Joseph Osborne More articles by this author Ana Molina More articles by this author Cora Sternberg More articles by this author David Nanus More articles by this author Neil Bander More articles by this author Scott Tagawa More articles by this author Expand All Advertisement PDF downloadLoading ...

Baseline CTC Count as a Predictor of Long-Term Outcomes in High-Risk Prostate Cancer.

The aim of the present study was to verify whether the baseline circulating tumor cell (CTC) count might serve as a predictor of overall survival (OS) and metastasis-free survival (MFS) in patients with high-risk prostate cancer (PCa) during a follow-up period of at least 5 years. CTCs were enumerated using three different assay formats in 104 patients: the CellSearch® system, EPISPOT assay and GILUPI CellCollector. A total of 57 (55%) patients survived until the end of the follow-up period, with a 5 year OS of 66% (95% CI: 56-74%). The analysis of univariate Cox proportional hazard models identified a baseline CTC count ≥ 1, which was determined with the CellSearch® system, a Gleason sum ≥ 8, cT ≥ 2c and metastases at initial diagnosis as significant predictors of a worse OS in the entire cohort. The CTC count ≥ 1 was also the only significant predictor of a worse OS in a subset of 85 patients who presented with localized PCa at the baseline. The baseline CTC number did not affect the MFS. In conclusion, the baseline CTC count can be considered a determinant of survival in high-risk PCa and also in patients with a localized disease. However, determining the prognostic value of the CTC count in patients with localized PCa would optimally require longitudinal monitoring of this parameter.

Nondestructive separation/enrichment and rolling circle amplification-powered sensitive SERS enumeration of circulating tumor cells via aptamer recognition

Nondestructive separation/enrichment and reliable detection of extremely rare circulating tumor cells (CTCs) in peripheral blood are of considerable importance in tumor precision diagnosis and treatment, yet this remains a big challenge. Herein, a novel strategy for nondestructive separation/enrichment and ultra-sensitive surface-enhanced Raman scattering (SERS)-based enumeration of CTCs is proposed via aptamer recognition and rolling circle amplification (RCA). In this work the magnetic beads modified with "Aptamer (Apt)-Primer" (AP) probes were utilized to specifically capture CTCs, and then after magnetic separation/enrichment, the RCA-powered SERS counting and benzonase nuclease cleavage-assisted nondestructive release of CTCs were realized, respectively. The AP was assembled by hybridizing the EpCAM-specific aptamer with a primer, and the optimal AP contains 4 mismatched bases. The RCA enhanced SERS signal nearly 4.5-fold, and the SERS strategy has good specificity, uniformity and reproducibility. The proposed SERS detection possesses a good linear relationship with the concentration of MCF-7 cells spiked in PBS with the limit of detection (LOD) of 2 cells/mL, which shows good potential practicality for detecting CTCs in blood with recoveries ranging from 100.56% to 116.78%. Besides, the released CTCs remained good cellular activity with the normal proliferation after re-culture for 48 h and normal growth for at least three generations. The proposed strategy of nondestructive separation/enrichment and SERS-based sensitive enumeration is promising for reliable analysis of EpCAM-positive CTCs in blood, which is expected to provide a powerful tool for analysis of extremely rare circulating tumor cells in complex peripheral blood for liquid biopsy.

Value of peripheral blood circulating tumor cell detection in the diagnosis of thoracic diseases and the prediction of severity.

Circulating tumor cell (CTC) detection, as a noninvasive liquid biopsy method, has been used in the diagnosis, prognostic indication, and monitoring of a variety of cancers. In this study, we aimed to investigate whether CTC detection could be used in the early diagnosis and prediction of severity of thoracic diseases. We enrolled 168 thoracic disease patients, all of whom underwent pathological biopsy. Carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) measurement was also performed in 146 patients. There were 131 cases of malignant thoracic diseases and 37 cases of benign lesions. We detected CTCs in a 5ml peripheral blood sample with the CTCBiopsy® system and analyzed the value of CTC count for predicting disease severity. Of 131 patients with a diagnosis of thoracic malignancy, CTCs were found in blood samples from 122 patients. However, only 2 out of 37 patients with benign thoracic disease had no detectable CTCs. There was no significant correlation between CTC count and benign and malignant lesions (P = 0.986). However, among 131 patients who had been diagnosed with malignant lesions, 33 had lymph node metastasis or distant metastasis. The presence of CTCs was significantly correlated with metastasis (P = 0.016 OR = 1.14). The area under the receiver operating characteristic (ROC) curve was 0.625 (95% confidence interval (CI), 0.519 to 0.730 P = 0.032). In addition, with stage IA1 as the cutoff, all patients were further divided into an early-stage group and a late-stage group. CTC count was significantly correlated with disease progression (P = 0.031 OR = 1.11), with an area under the curve (AUC) of 0.599 (95% CI, 0.506-0.692 P = 0.47). The sensitivity and specificity of CTC detection for the diagnosis of disease stage were 72.3% and 45.5%, respectively. In addition, the cutoff of 2.5 CTCs was the same when predicting disease metastasis and staging. Furthermore, the combination of CTC count, demographic characteristics and tumor markers had better predictive significance for disease staging. CTC count can effectively indicate the stages and metastasis of thoracic diseases, but it cannot differentiate benign and malignant diseases.

The Prognostic Significance of Circulating Tumor Cells in Patients with Pancreatobiliary Cancer.

Circulating tumor cells (CTCs) are cancer cells which separate from the primary tumor and enter systemic circulation. In this study, it was aimed to examine the relationship between CTCs isolated and identified from the peripheral blood of patients with pancreatobiliary cancer, with the clinicopathological characteristics of the patients and their overall survival. A total of 21 patients were included the study. Density gradient centrifugation with the OncoQuick® assay was performed for isolation of CTCs from peripheral blood. In order to identify CTCs, enriched samples underwent flow cytometric analysis. The rate of patients with positive surgical margin in the high CTC group (CTC <15) was identified to be statistically significantly high compared to the group with low CTC (CTC ≤15) (83.3% vs. 16.7%; P = .041). Median neutrophil/lymphocyte ratio (NLR) was found to be higher in the high CTC group compared to the low CTC group, which was close to statistical significance (2.37 vs. 1.41; P = .055). Circulating tumor cells were identified to have a significant relationship with surgical margin positivity in our study for the first time, suggesting that the CTCs count in peripheral blood in preoperative patients may be a biomarker predicting positive surgical margin. Due to the very low number of studies assessing the relationship between CTCs and NLR, our study which identified relationship close to statistical significance between CTCs and NLR, significantly contributes to the literature on the topic of the possible role of lymphocytes in CTC clearance.

Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n=24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.