Circulating Tumor Cells Detection Rate Research Articles

Abstract B010: Prognostic value of circulating tumor cells in stage III and IV melanoma: A systematic review and meta-analysis

Abstract Introduction: Previous studies have demonstrated a role for assessing circulating tumor cells (CTC) in melanoma, but significant heterogeneity among studies has limited application in the clinical setting. We sought to evaluate the prognostic value of CTCs in advanced stage melanoma using a standardized CTC enumeration platform. Methods: A PubMed literature search was conducted up to March 2024. All studies evaluating CTC enumeration using CellSearchTM with outcomes data were included. Student's t-test was used to compare differences between groups. Hazard ratios (HR) were pooled using random effects model to assess the association of CTCs with overall survival (OS). I 2 statistic and Cochran’s Q test, sensitivity analyses, and Egger’s test of asymmetry were used to assess study heterogeneity, differential impact of individual studies, and publication bias, respectively. Results: Of the 12 studies identified, nine met inclusion criteria. A total of 678 patients were included, of which 337 (49.6%) were stage III and 342 (50.4%) were stage IV. The majority of studies reported CTC rates using a cut-off of ≥ 1 (6 studies, 66.7%), while three studies used ≥ 2 and five studies used both. Median follow-up time was available for six studies and ranged from 5.9-48 months (median 13.4). Average CTC detection rate (≥ 1 or 2) was 41% (SD 14.0) and was significantly higher using a cut-off of ≥ 1 (45.4%, SD 12.6) compared to ≥ 2 (26.1%, SD 2.7, p = 0.02). Pooled analysis of two studies demonstrated significantly worse OS with CTC ≥ 1 (HR 1.6, 95% CI: 1.0-2.6, p = 0.04). Similarly, pooled analysis of five studies with available HRs demonstrated significantly worse OS with CTC ≥ 2 (HR 2.1, 95% CI: 1.4-3.1, p = 0.0003). Among these five studies, moderate heterogeneity was observed (I2 = 43.4%), although this was not statistically significant (Q statistic p = 0.13). Sensitivity analyses confirmed the prognostic value of CTC ≥ 2, with pooled HRs ranging from 1.9-2.4 after consecutive omission of each study from the overall analysis. Conclusion: Regardless of the CTC cutoff used (≥ 1 or 2), presence of any CTCs was a strong prognostic marker in patients with advanced stage melanoma. Incorporation of CTC assessments in clinical practice may help risk stratify patients and guide treatment decisions. Further studies are needed to determine the optimal cut-off point for CTC status in assessing prognosis. Citation Format: Jennifer H. Chen, Hyunsoo Hwang, Bamboo Dong, Vanessa N. Sarli, Joshua R. Upshaw, Salyna Meas, Anthony Lucci. Prognostic value of circulating tumor cells in stage III and IV melanoma: A systematic review and meta-analysis [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B010.

Abstract A004: Full spectrum isolation technique-based dual circulating rare cells analysis to maximize early cancer diagnosis

Abstract Liquid biopsy (LB) is essential in precision medicine, enabling real-time tumor monitoring through repeated sampling. However, its diagnostic sensitivity and accuracy are limited by the low detection rate of circulating tumor cells (CTCs). For example, in breast cancer, the CTC detection rate exhibited only 20%, significantly limiting its clinical utility. This limitation arises since existing technologies selectively isolate specific phenotypes of CTCs, failing to capture others, such as mesenchymal CTCs, which are associated with poor prognosis. These mesenchymal CTCs often evade detection due to their lack of EpCAM expression and high deformability, making difficult to isolate them with traditional filter or EpCAM based methods. Additionally, conventional LB methods fail to provide critical insights into the tumor microenvironment (TME). To overcome these limitations, we developed a novel approach that improves LB diagnostic sensitivity and accuracy by (1) creating a technology to isolate all types of CTCs, regardless of marker expression or size, and (2) simultaneously analyzing CTCs and circulating cancer-associated fibroblasts (cCAFs). This dual approach addresses the challenges of low sensitivity and accuracy by enabling the isolation of a broader range of CTCs, including mesenchymal types, and by incorporating cCAF analysis. cCAFs, derived from the TME and found in the bloodstream, provide valuable insights into the TME that are hard to obtain through traditional methods. Analyzing cCAFs alongside CTCs offers a more comprehensive understanding of tumor biology and its microenvironment, leading to improved diagnostic accuracy, especially in early-stage cancer. In our study involving 55 breast cancer patients, we demonstrated that while CTCs were undetectable in 42.9% of early-stage patients, cCAFs were detected in all cases and were found to be 10 times more abundant than CTCs. Importantly, for the first time, this study uncovers that the diverse CAF subtypes previously identified in tissue samples also exist in the bloodstream, highlighting their critical role in providing TME-related insights. In addition, cCAF subtypes vary with hormonal receptor expression, and their marker analysis allows for 80.9% accurate classification of breast cancer subtypes. We applied this approach to early-stage breast cancer patients, for whom diagnosis through liquid biopsy is particularly difficult, and found that the combined analysis of CTCs and cCAFs achieved a 18% improvement in accuracy, with the AUC increasing from 0.6817 to 0.8009 compared to CTC analysis alone. This innovative approach significantly enhances LB by simultaneously isolating all CTC subtypes and incorporating cCAF analysis, thereby offering a more sensitive and accurate diagnostic tool. Beyond improving breast cancer diagnostics, this method has the potential to be applied to other tumor types and could also prove valuable for prognosis prediction and therapeutic response monitoring, ultimately leading to better patient outcomes and more effective cancer treatment strategies. Citation Format: Hyeongjung Woo, Simon A Joosse, Frederike Harten, Felix Hilpert, Klaus Pantel, Minseok S. Kim. Full spectrum isolation technique-based dual circulating rare cells analysis to maximize early cancer diagnosis [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A004.

Detection of circulating tumor cells in women with early breast cancer

There are multiple methods and biomarkers with different detection rates to circulating tumor cells (CTCs) in women with breast cancer (BC). However, to the best our knowledge, no study has been carried out to detect CTCs in women with early BC in Mexico. Calculate the detection rate of CTCs in peripheral blood of Mexican women with early BC by quantification of the relative expression of KRT19, MUC1 and SCGB2A2. Observational, longitudinal, analytical study, which included 62 Mexican women, over 18 years of age, with BC clinical stage 0, I and II. Cells with a density < 1.077 g/ml were isolated from 4 mL of peripheral blood obtained 24 h before surgery, RNAS was extracted and RT-qPCR was performed to quantify the relative expression of KRT19, MUC1 and SCGB2A2. The diagnosis of BC was confirmed in the definitive pathological study in 59 women. 1 case (1.7%), expressed KRT19, 18 cases (30.5%) MUC1, 2 cases (3.4%) SCGB2A2, and 19 cases (32.2%) expressed at least one of the three biomarkers. CTCs detection rates were like those reported in other populations when MUC1 was used, but lower when KRT19 and SCGB2A2 were used. Standardization of blood sample collection and processing, as well as biomarker expression analysis, is important to reduce variability in the detection of CTCs in specific populations.

Label-free detection and simultaneous viability determination of CTCs by lens-free imaging cytometry.

The detection of extremely rare circulating tumor cells (CTCs) in peripheral blood and simultaneously identifying their viabilities are significant for cancer diagnosis and prognosis as well as monitoring the efficacy of personalized treatment. A lens-free imaging system features high-resolution images taken over a large field of view (FOV), which has great potential for CTC detection and viability determination. But current still lens-free systems restrict the application for CTC detection in real samples due to the inherent limitations of lens-free technology: (1) the location of cells in the FOV will affect the imaging; (2) the extremely rare CTCs probably did not exist in one observation. In this paper, we realized the detection of CTCs in whole blood and the simultaneous determination of their viabilities by lens-free imaging cytometry. Our in-flow system plus a large FOV range of lens-free imaging highly increased the detection rate of rare CTCs with a high throughput of 150,000 cells per minute and improved the recognition efficiency for blood cells, living/dead CTCs by using a cell tracing-assisted deep learning algorithm. With this method, the average precision of blood cells, living/dead lung cancer cells A549, and living/dead colon cancer cells SW620 reached 98.80%, 97.88%, 97.93%, 97.72%, and 98.60%, respectively. Our system got a highly consistent result with the manual counting method using fluorescent staining (Pearson's r 99.93% for SW620) and can easily detect as few as 10 dead or living CTCs from 100,000 white blood cells (WBCs). Finally, real clinical samples were detected in our system. Both dead and living CTCs were found in all six advanced-stage cancer patients, and the number of living CTCs per million WBCs ranged from 13 to 39, more than that of the dead CTCs (5 to 25), while none of the CTCs were detected in six healthy control subjects. Moreover, we also found that CTCs died very quickly after leaving the human body, indicating that CTCs should be studied as soon as possible after sampling. Although this method is implemented for CTCs, it can also be used for the detection of other rare cells.

High-throughput viable circulating tumor cell isolation using tapered-slit membrane filter-based chipsets in the differential diagnosis of ovarian tumors.

To evaluate the diagnostic performance of circulating tumor cells (CTCs) using tapered-slit membrane filter (TSF)-based chipsets for the differential diagnosis of adnexal tumors. A total of 230 women with indeterminate adnexal tumors were prospectively enrolled. The sensitivity, specificity, and accuracy of the CTC-detecting chipsets were analyzed according to postoperative pathological results and compared with those of cancer antigen (CA)-125 and imaging tests. Eighty-one (40.3%) benign tumors, 31 (15.4%) borderline tumors, and 89 (44.3%) ovarian cancers were pathologically confirmed. The sensitivity, specificity, and accuracy of CTC-detecting chipsets (75.3%, 58.0%, and 67.1%) for differentiating ovarian cancer from benign tumors were similar to CA-125 (78.7%, 53.1%, and 66.5%), but lower than CT/MRI (94.2%, 77.9%, and 86.5%). "CTC or CA125" showed increased sensitivity (91.0%) and "CTC and CA-125" revealed increased specificity (77.8%), comparable to CT/MRI. CTC detection rates in stage I/II and stage III/IV ovarian cancers were 69.6% and 81.4%, respectively. The sensitivity to detect high-grade serous (HGS) cancer from benign tumors (84.6%) was higher than that to detect non-HGS cancers (68.0%). Although the diagnostic performance of the TSF platform to differentiate between ovarian cancer and benign tumors did not yield significant results, the combination of CTC and CA-125 showed promising potential in the diagnostic accuracy of ovarian cancer.

The clinical application of circulating tumor cells (CTCs) for immunotherapy in advanced pancreatic cancer.

e14541 Background: Circulating tumor cell (CTCs) has the potential utility for screening, prognostic and predictive assessment, and offers the potential for personalized management. This trial aimed to evaluate the prognostic and predictive impact of programmed cell death ligand 1 (PD-L1) positive CTCs in patients with pancreatic ductal adenocarcinoma (PDAC). Methods: Twenty-three heavily treated advanced PDAC patients were randomized to receive camrelizumab combined with apatinib or capecitabine, or placebo plus capecitabine until disease progression or unacceptable toxicity, and peripheral blood CTCs samples were collected at baseline and after treatment. Forty-nine CTCs samples from 23 recruited advanced PDAC patients were detected by subtraction enrichment and immunostaining-FISH (SE-iFISH). PD-L1 expression level also was evaluated in tumor tissue samples by Immunohistochemistry (IHC). Investigator-assessed 6-month overall survival (OS) rate and 3-month progression-free survival (PFS) rate was evaluated. Results: The positive detection rate of CTCs in 23 PDAC patients was 95.7%. The cell sub-types of sizes of CTC analysis showed that 58.6% CTCs was the small CTCs (Diameter &lt; 5μm). CTCs karyotypes analysis exhibited that triploid small CTCs were the main karyotype (41.2%) in small CTC, and pentaploid large CTCs was the major karyotype (74.7%) in large CTCs. PD-L1+ CTCs were detected in 43.5% of PDAC patients. Tissue PD-L1 positive detection rate was 50% (CPS &gt; 1%) in available tissue samples(n=10). The presence of PD-L1+ CTCs cohort exhibited a significant increase in 6-month OS rate (80% versus 38.5%, P &lt; 0.05) and 3-month PFS rate (70% versus 23.1%, P &lt; 0.05) compared to PD-L1- CTCs cohort. Compared with the PD-L1- CTCs patients, the combination therapy camrelizumab markedly improved the 6-month OS rates (75% versus 50%) and 3-month PFS rate (62.5% versus 25%) in PD-L1+ CTC patients. Conclusions: PD-L1+ CTCs could be considered as a favorable prognostic marker in advanced PDAC. These results suggest that PD-L1+ CTCs might be used as a predictor of the efficacy of cancer immunotherapy in advanced PDAC, and provide a new monitoring method for the precise treatment of advanced PDAC. The expanding sample size test is now ongoing. Clinical trial information: ChiCTR1900024095 .

Circulating tumor cells: a valuable indicator for locally advanced nasopharyngeal carcinoma.

Advancements in nasopharyngeal carcinoma (NPC) treatment have led to a focus on personalized treatment. Circulating tumor cells (CTCs) are important for liquid biopsies and personalized treatment but are not being fully utilized. This study examined how pre- and post-treatment CTC counts, EMT subtypes, clinical characteristics, and patient prognosis are related in order to support the use of liquid biopsy in managing NPC. This retrospective study included 141 patients with locally advanced NPC. All patients underwent CanPatrol™ CTC detection pre- and post-treatment and were categorized into EMT subtypes: epithelial type, mixed type, and mesenchymal type. This study analyzed CTC enumeration, EMT subtypes, and their associations with clinical characteristics and survival outcomes. The results indicated a positive correlation between the pre-treatment detection rate of CTCs and N stage (P < 0.01), alongside a positive correlation with the TNM clinical stage (P = 0.02). Additionally, the detection rate of mesenchymal CTCs post-treatment is positively associated with the N stage (P = 0.02). The enumeration of CTCs pre- and post-treatment is negatively correlated with prognosis and has statistical significance. Additionally, an investigation into the EMT subtypes of CTCs revealed a significant association between the presence of mesenchymal CTCs pre- and post-treatment and decreased overall survival (OS) (P < 0.05). Furthermore, T stage, N stage, TNM clinical stage, and Epstein-Barr virus (EBV) DNA were also significantly correlated with OS. The study found that mesenchymal CTCs pre- and post-treatment, as well as the number of CTCs, were linked to a poor prognosis.

Abstract PO5-14-02: Diagnostic leukapheresis to increase CTC detection rate in breast cancer patients after 5 years of adjuvant endocrine treatment

Abstract Purpose: Patients with early hormone receptor positive, HER2 negative breast cancer have a risk of recurrence for up to 20 years after the initial diagnosis and is therefore treated with adjuvant endocrine treatment (ET) for 5 to 10 years. As a result there is a large proportion of patients that overtreated, hence, an urgent need for better biomarkers to guide treatment decisions. In previous research, it was shown that circulating tumor cells (CTCs) are detected in 5% of patient after 5 years of adjuvant treatment and that this indicated a 13-fold higher risk of recurrence, with a median lead time of 2.8 years. However, the sensitivity and specificity of the test were moderate. This could be caused by stochastic variation, given the low numbers of CTCs detected in 7.5 ml peripheral blood. We hypothesized that increasing the screening volume, increases the test statistics. Methods: In this study, we investigated whether increasing the screening volume by diagnostic leukapheresis (DLA) can increase the detection rate of CTCs (CellSearch platform). With DLA, 5 liters of peripheral blood was screened, which in previous studies had shown to increase the CTC-yield per ml by 25-fold. To this end, we included early hormone receptor positive, HER2 negative breast cancer patients that were treated with ET for 4.5-5.5 years with no signs of disease recurrence. We compared the number of detected CTCs in the standard fraction of 7,5 mL peripheral blood (PB), to buffy coat (30 ml), to DLA sample (200 × 109 white blood cells (WBC), equal to 25-50 mL PB) and to DLA after WBC depletion. Results: Thirty two hormone receptor positive/HER2 negative breast cancer patients were included in this study after a median duration of ET of 60 months, of which 3 (9%) patients completed ET according to clinical guidelines and the remaining patients were still on treatment. Twenty eight patients (88%) had 1-3 positive lymph nodes at diagnosis, and 3 patients had 4-9 positive lymph nodes. The Clinical Treatment Score at 5 years (CTS5 score) was intermediate or high in 75%, and unknown in 19%, indicating a recurrence risk of &amp;gt;5% at years 5-10 after diagnosis. In none of the included patients, CTCs were detected in 7,5 ml PB, nor in any of the other tested blood volumes. To date, 2 patients have been diagnosed with overt metastatic disease during follow-up. DLA was well tolerated and did not cause adverse events. Conclusions: We conclude that DLA was unable to increase the detection rate of CTCs in hormone receptor positive, HER2 negative patients with early breast cancer being on treatment for a median duration of 5 years. Additional circulating tumor DNA analyses are planned. Citation Format: Noortje Verschoor, Jaco Kraan, Johanna Zuetenhorst, Mijntje Vastbinder, Jan Drooger, Stefan Sleijfer, Saskia M Wilting, John WM Martens, Agnes Jager. Diagnostic leukapheresis to increase CTC detection rate in breast cancer patients after 5 years of adjuvant endocrine treatment [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 PO5-14-02.

Abstract PO1-16-02: Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC)

Abstract Background: Circulating Tumor Cells (CTC) can be isolated in 40-80% of patients with metastatic breast cancer (MBC). High levels of CTC, defined as ≥5/7.5 ml of blood are associated with shorter progression-free survival and overall survival. CTC can be tested for clinically relevant biomarkers, such as ER, and HER2 status, and have the potential to guide therapy as well as help with disease monitoring. We investigated CTC detection rate, its correlation with clinical characteristics, pathological characteristics, and response to treatment in patients with any type of metastatic breast cancer who progressed on at least one line of therapy. Methods: Eligible patients with MBC were enrolled in the IMAGE-II study (Individualized Molecular Analyses Guide Efforts in Breast Cancer) NCT02965755, in which we obtained archival tissue as part of the standard of care, and prospectively collected serial blood samples for CTC analysis. We compared CTC levels and biomarker status with clinical factors, tissue-based pathology, and molecular biomarkers. We assessed whether changes in CTC count correlated with treatment response. Blood samples were collected at baseline (Day 1), after 1-2 weeks of therapy, after 8-12 weeks, and at subsequent restaging (every 8-12 weeks). Medical records were reviewed every 3 months for ongoing treatment response, and death. Samples were processed at the Biocept CLIA-certified Laboratory. CTC were enumerated by the presence of CD45- and DAPI+ cells. Further CTC characterization was performed by antibody staining of specific protein biomarkers (ER, HER2). Fisher’s exact test was used to evaluate the association between baseline CTC &amp;lt; 5 and ≥5/7.5ml with patient characteristics. McNemar’s test was used to assess discordance between tissue-based and CTC-defined markers. Results: Between 1/26/2018 and 12/31/22 baseline samples were collected from 70 women with a median age of 56 (36-82) who were enrolled at four study sites. CTC was detected in 59 (84%) of participants. Baseline CTC counts were &amp;lt; 5/7.5 ml in 37/59 (63%) and ≥5/7.5 ml in 22/59 (37%) of participants. Differences between breast cancer subtype and CTC-receptor status were observed (Table 1). Among the 41 patients who were ER-positive, 31 (76%) were CTC ER-negative(p&amp;lt; 0.001). Among 29 patients who were ER-negative, 4 (14%) were CTC ER-Positive (P&amp;lt; 0.001). Among the 7 patients who were HER2 positive, 2(29%) were HER2 positive on CTC. Among 63 patients who were HER2 negative, 7(11%) had HER2 expression on their CTC. The discordance in the classification of HER2 wasn't statistically significant (p=0.77). Elevated CTC at baseline was more frequently detected in younger participants (&amp;lt; 50 years old) (55% vs 27%; P=0.22), in Black women compared to White (60 % vs 29 %, P=0.27), and in participants with visceral vs non-visceral metastasis (52% vs 28 %, P=0.40). Patients with CTC ≥5 vs &amp;lt; 5/7.5mL at baseline had a shorter duration of anti-HER2-based therapy (61.5 vs 836 days, p=0.04). There were no statistically significant differences among participants who received chemotherapy agents (316.5 vs 365.5 days, P=0.88), endocrine therapy (376 vs 490 days, P=0.89), or overall therapy (272 vs 390 days, P=0.21). Conclusions: We observed significant differences in the expression of ER between tumor tissue and CTC, which can be partly due to tumor evolution over time. Additionally, participants who are young, Black, and those who have visceral metastasis may have higher CTC counts. Higher CTC counts were associated with a shorter duration of anti-HER2 therapy. Although it didn’t meet statistical significance, a similar trend was observed in patients who received chemotherapy and endocrine therapy. Table 1: MBC Tissue-Based Subtype and CTC-receptor status at baseline Citation Format: Asnakech Bayable, Tingchang Wang, Amanda Blackford, Jessica Tao, Jenna Canzoniero, Seoho Lee, Faith Too, Barbara Blouw, Mary Wilkinson, Rima Couzi, Antonio Wolff, Christie Hiton, Ben Park, Vered Stearns, Cesar Santa-Maria. Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC) [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 PO1-16-02.

Meta-Analysis of Circulating Tumor Cell PD-L1 Expression and the Association with Clinical Outcomes in Non-Small Cell Lung Cancer.

Programmed death ligand-1 (PD-L1) expression on circulating tumor cells (CTCs) has been suggested to provide prognostic information in non-small cell lung cancer (NSCLC), but consensus relative to treatment outcomes is lacking. We conducted the first comprehensive meta-analysis exploring its potential as a prognostic and predictive marker, and assessed the concordance between PD-L1 + CTCs and paired tumor tissue in NSCLC patients. A comprehensive search was applied to PubMed and EMBASE to identify 26 studies that evaluated PD-L1 + CTCs and their association with survival outcomes in 1236 NSCLC patients. The meta-analysis estimated a mean PD-L1 + CTCs detection rate of 61% (95% CI, 49-72). Subgroup analysis based on treatment showed that PD-L1 + CTCs was not significantly associated with better overall survival (OS) in NSCLC patients treated with immune checkpoint inhibitors (ICIs) (Hazard Ratio (HR) = 0.96, 95% CI, 0.35-2.65, P = 0.944), but was predictive of worse OS in those treated with other therapies (HR = 2.11, 95% CI, 1.32-3.36, P = 0.002). Similarly, PD-L1 + CTCs was not significantly associated with superior progressing free survival (PFS) in NSCLCs treated with ICIs (HR = 0.67, 95% CI, 0.41-1.09, P = 0.121), but was significantly associated with shorter PFS in patients treated with other therapies (HR = 1.91, 95% CI, 1.24-2.94, P = 0.001). The overall estimate for the concordance between PD-L1 expression on CTCs and tumor cells was 63% (95% CI, 44-80). The average detection rate of PD-L1 + CTCs was comparable to the rate of PD-L1 expression in NSCLC tumors. There was a trend towards better PFS in ICI-treated NSCLC patients with PD-L1 + CTCs. Larger longitudinal studies on the association of PD-L1 + CTCs with clinical outcomes in NSCLC patients treated with ICIs are warranted.

Evaluation of cell surface vimentin positive circulating tumor cells as a prognostic biomarker for stage III/IV colorectal cancer

Currently, little is known about the phenotypes of circulating tumor cells (CTCs), particularly epithelial and mesenchymal phenotypes, and their impact on the prognosis of colorectal cancer (CRC) patients. This study aims to investigate the CTC phenotypes and their prognostic implications in stage III/IV CRC. Patients who were diagnosed with CRC and underwent CTC detection at two hospitals were included. CTCs were detected using a mesenchymal CTC kit, and the clinical and pathological characteristics of CTCs were compared with those of cell surface vimentin-positive CTCs (CSV-CTCs). Disease-free survival (DFS) was assessed and used as an indicator of CTC phenotype-related prognosis. Univariate and multivariate Cox regression analyses were made to identify risk factors, and nomogram models were employed for prognostic prediction. A total of 82 patients were enrolled, with a CTC detection rate of 86.6%. Among the detected CTCs, 60% were CSV-CTCs. The CSV-CTC count showed a positive correlation with the T-stage, the M-stage, and the location of the primary tumor (P = 0.01, P = 0.014, and P = 0.01, respectively). Kaplan–Meier survival analysis revealed that CSV-CTCs were associated with worse DFS in patients receiving first-line oxaliplatin chemotherapy (hazard ratio (HR) = 3.78, 95% CI 1.55–9.26, p = 0.04). When the cut-off value of the CSV-CTC count was 3, the optimal prognostic prediction was achieved. Compound models considering CSV-CTCs, TNM staging, the site of the primary tumor and the Ras gene status yielded the best results in both the receiver operating characteristic (ROC) analysis and the decision curve analysis (DCA). This study indicates that CSV-CTCs predominate in CTCs of CRC patients, and a count of CSV-CTCs ≥ 3 is an independent risk factor for worse prognosis.

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.

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 1026: A novel detection system of circulating tumor cells based on size-dependent enrichment with immunofluorescent staining of CK8/18 in advanced non-small cell lung cancer

Abstract Background: Detection of circulating tumor cells (CTCs) in the blood is a potentially noninvasive tool for cancer diagnosis, monitoring and prognosis evaluation. However, the CTC detection is difficult because of the rarity of CTCs, especially in non-small cell lung cancer (NSCLC), and the detection sensitivity of CTCs was reported to be less than 50% in NSCLC patients. Herein, we developed a novel CTC detection system and evaluated its performance in advanced NSCLC patients. Methods: Whole blood samples were collected from stage IV or recurrence NSCLC patients. CTC detection was performed with a novel system (Sysmex Corporation, Hyogo, Japan) by using size-dependent enrichment for capturing CTCs and immunofluorescent staining reagents including anti-cytokeratin (CK, AE1/AE3 or CK8/18) (specific for epithelial cells), anti-CD45 (specific for leukocyte) and Hoechst (for nucleus). Fluorescence intensity and cell images of each marker were obtained by Molecular Imaging Flow Cytometry (MI-FCM). Circulating cells that were positive for Hoechst and anti-CK but negative for anti-CD45 were defined as CTCs. To confirm whether CTCs detected were derived from lung cancer origin, CTCs obtained from patients with EGFR mutations were subjected to single cell sorting, whole genome amplification and PCR analysis for the mutation detection. Results: A total of 20 NSCLC patients (10 EGFR mutant, 10 EGFR wild-type) were enrolled in this study. Of these, CTCs were detected in 16 (80%) patients (number of CTCs, 1 to 66 in 4.5ml whole blood) when CK8/18 was used as epithelial cell staining, but in only 11 (55%) patients (1 to 6 CTCs) when using AE1/AE3. The detection sensitivity of CTCs based on CK8/18 was significantly higher compared to that based on AE1/AE3 in our novel system (80% vs. 50%, p = 0.03). The number of CTCs was significantly lower in EGFR mutation-positive patients than in EGFR wild-type patients (median number of CTCs, 3 vs. 22, p = 0.01). Furthermore, among 10 EGFR mutation-positive patients, EGFR mutations were detected in CTCs derived from 6 (60%) patients. All the mutation types detected in CTCs were matched with those identified in the corresponding primary tumors. Conclusion: Our novel CTC detection system demonstrated that the detection rate of CTCs (80%) was higher than those in previous studies. Further studies are needed for the clinical application of this system. Citation Format: Tetsuya Sakai, Yoshitaka Zenke, Mami Onishi, Eri Morita, Masatoshi Yanagida, Yuji Shibata, Hiroki Izumi, Kaname Nosaki, Hibiki Udagawa, Shingo Matsumoto, Kiyotaka Yoh, Koichi Goto. A novel detection system of circulating tumor cells based on size-dependent enrichment with immunofluorescent staining of CK8/18 in advanced non-small cell lung 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 1026.

Circulating tumor cells as a prognostic biomarker in patients with hepatocellular carcinoma

Circulating tumor cells (CTCs) have been shown as a surrogate for cancer progression and prognostication. We aimed to determine an association between CTCs and survival of hepatocellular carcinoma (HCC) patients. Peripheral blood was obtained from 73 HCC patients to enumerate for epithelial CTCs/8 mL blood. CTCs were detected by immunoaffinity-based method using epithelial cell adhesion molecule (EpCAM) and mucin1 (MUC1). The CTCs detection rates of BCLC stages A, B, and C patients were 65.4% (17/26), 77.3% (17/22), and 96% (24/25), respectively, p = 0.018. Patients with CTCs < 5 cells/8 mL had significantly longer survival than those with CTCs ≥ 5 cells/8 mL (>36 vs. 4.6 months, p < 0.001). In multivariate analysis, CTP B, BCLC B, BCLC C, AFP ≥ 400 ng/mL, and CTC ≥ 5 cells/8 mL were independently associated with survival, with adjusted HRs (95%CI) of 4.1 (2.0–8.4), 3.5 (1.1–11.4), 4.7 (1.4–15.4), 2.4 (1.1–5.0), and 2.6 (1.2–8.4); p < 0.001, 0.036, 0.011, 0.025 and 0.012, respectively. The combination of CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL provided additively increased HR to 5.3 (2.5–11.1), compared to HRs of 4.0 (2.0–8.0) and 3.5 (1.8–6.7) for CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL, p < 0.001, respectively. The larger number of peripheral CTCs is correlated with higher tumor aggressive features and poorer survival of HCC patients. CTCs can potentially become novel prognostic biomarker in HCC.

Aptamer-cocktail Functionalized Nano-microfluidic Chip for Enhancing Isolation and Characterization of Circulating Cancer Cells.

Circulating tumor cells (CTCs) have been shown to have a correlation to metastasis and prognosis of patients with cancer. The enumeration and downstream gene analysis of CTCs have attracted efforts for personalized medicine. However, enumeration and phenotypic profiling in most capture devices are challenging due to the rarity and heterogeneity of CTCs. Here, we report an aptamer-cocktail strategy coupled in nano-microfluidic chip for enhancing isolation performance and characterizing the phenotypes of CTCs. Aptamer-cocktail recognizing EpCAM/Vimentin/EGFR/CD44 were bound to a nanopillar array on a nano-microfluidic chip. The recognition was validated with cancer cells by flow cytometry and the critical parameters were optimized with the nano-microfluidic chip. Finally, the system was applied to clinical samples. The proposed aptamer-cocktail showed the predominant affinity with MDA-MB-231 and SK-BR-3. When utilized to capture artificial clinical samples, it showed 71% to 83% capture efficiency. CTC detection rate was 100% in five pre-treatment and five post-treatment breast cancer patients. The enumeration data ranged from 6-33 per 2 ml. The number of CTCs in breast cancer patients before therapy was 1.27-2 times higher than that after therapy. CTCs with epithelial and mesenchymal phenotype were both detected and identified; interestingly, the mean diameter of CTCpck pos acquired in these cases was much larger than that of CTCvimentin pos Conclusion: The nano-microfluidic chip not only made it easier to phenotyping epithelial-like or mesenchymal-like CTCs, but can also be used to detect downstream genetic variation. The established platform can be applied in clinical research and facilitate auxiliary diagnosis with tumor recurrence and metastasis in advance.

Study protocol and preliminary results from a mono-centric cohort within a trial testing stereotactic body radiotherapy and abiraterone (ARTO-NCT03449719)

BackgroundARTO trial was designed to evaluate the difference in terms of outcomes between patients affected by oligo metastatic castrate resistant prostate cancer (mCRPC) treated with Abiraterone acetate and randomized to receive or not SBRT on all sites of disease. Here, we present a preliminary analysis conducted on patients enrolled at promoting institution.ObjectiveTo present a preliminary overview about population features, clinical outcomes, adverse events, quality of life and explorative translational research.Design, setting, and participantsARTO (NCT03449719) is a phase II trial including patients affected by oligo mCRPC, randomized to receive standard of care (GnRH agonist or antagonist plus abiraterone acetate 1000 mg and oral prednisone 10 mg daily) with or without SBRT on all metastatic sites of disease. All subjects have < 3 bone or nodal metastases. All patients are treated in I line mCRPC setting, no previous lines of treatment for mCRPC are allowed.Outcome measurements and statistical analysisData about a mono-centric cohort of 42 patients enrolled are presented in the current analysis, with focus on baseline population features, PSA drop at 3 months, biochemical response, and quality of life outcomes. Descriptive statistics regarding translational research are also presented.Results and limitationSignificant difference in terms of PSA drop at three months was not detected (p = 0.68). Biochemical response (PSA reduction > 50%) was reported in 73.7 versus 76.5% of patients in control vs SBRT arm, respectively (p = 0.84). All patients are alive. Progression occurred in 1 versus 0 patients in the control versus SBRT arm, respectively. After 3 months, an average decrease of 13 points in terms of Global Health Score was reported for the overall population. However, complete recovery was noticed at 6 months. Circulating tumor cells detection rate was 40%.ConclusionsSBRT + Abiraterone treatment was safe and well tolerated, non-significant trend in terms of PSA drop and biochemical response at 3 months was detected in SBRT arm. Interestingly, CTCs detection in this selected cohort of oligo-mCRPC was lower if compared to historical data of unselected mCRPC patients.

Large-scale whole-genome sequencing identifies genetic alterations in circulating hepatocellular carcinoma cells.

e15019 Background:Hepatocellular carcinoma (HCC) is one of the most lethal malignancies. Most of the patients have large tumor burden, vascular invasion, or metastasis at initial diagnosis. Liver biopsy is usually inaccessible in HCC patients due to the high risk of hemorrhage and tumor dissemination. Circulating tumor cells (CTCs) are malignant cells shed from the tumor tissue into the blood or lymphatic vessel. The fact that CTCs are live tumor cells makes them distinct from any of the existing cancer biomarkers. The biophysical property-based enrichment methods make downstream process easier as the isolated CTCs are not tagged with antibodies and the cell viability can be largely remained. Our objective is to isolate CTCs using a novel inertial focusing-base microfluidic device (CTC-100) and determine both their clinical value and the genomic features. Methods: We enriched and isolated CTCs in 155 HCC patients, 51 cirrhosis patients and 10 healthy donors. These patients were enrolled from Sep, 2020 to Feb, 2022 in the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). This method enriches CTCs according to the cell size and is label-free. The enriched CTCs are then validated via immunostaining. The CD45-EpCAM+DAPI+ cells with size larger than 15μm are considered as epithelial CTCs, and CD45-EpCAM-DAPI+ cells are considered mesenchymal. The CTCs were then micromanipulated from the slide and the pooled CTCs from each patient were then subjected to MALBAC whole genome amplification and whole genome sequencing, which has been finished in CTCs from 12 patients receiving surgery and 4 without tumor biopsy. The other samples are under processing. Results: Our data revealed that the number of total CTCs or mesenchymal CTCs were significantly higher in HCC patients than that in cirrhosis or healthy donors. The detection rate of CTC in HCC patients was 100%. The EpCAMpositive CTCs accounted only for 16.6% (298/1798) of the total CTCs. Moreover, the number of CTCs was positively associated with macrovessel invasion and tumor size (P &lt; 0.01). Most of the mutated genes in CTC samples were accordant with corresponding cancer tissues. We have detected 24 CTC-specific mutated genes, including FBXL22, USP3, MIR1827, S100A1. We then figure out the frequently mutated genes in CTC samples. Finally, we cluster the SNVs in CTCs according to clinicopathological parameters including macrovessel invasion, distant metastasis, tumor size. The results showed that part of the mutated genes might be associated with tumor invasion or metastasis. Conclusions: The novel microfluidic platform can efficiently detect CTCs in HCC patients. CTC number is associated with macrovessel invasion and tumor size. Finally, the SNV but not CNV in CTC samples is tightly consistent with that in tumor tissue. We also find out some CTC-specific mutated genes that might be involved in HCC metastasis. Clinical trial information: ChiCTR2100051584.

Prognostic Relevance of Estrogen Receptor Status in Circulating Tumor Cells in Breast Cancer Patients Treated With Endocrine Therapy.

Recently, female breast cancer (BC) has surpassed lung cancer to occupy the first place of the most commonly diagnosed cancer. The unsatisfactory prognosis of endocrine therapy for breast cancer might be attributed to the discordance in estrogen receptor (ER) status between primary tumors and corresponding metastases, as well as temporal and spatial receptor status heterogeneity at point-in-time between biopsy and treatment. The purpose of this study was to evaluate the prognostic and predictive value of ER status in circulating tumor cells (CTCs) in BC patients. We analyzed ER expression on CTCs isolated using the Pep@MNPs method in 2.0 ml of blood samples from 70 patients with BC and 67 female controls. The predictive and prognostic value of ER expression in CTCs and immunohistochemistry results of biopsies for progression-free survival (PFS) and overall survival (OS) of patients in response to therapies were assessed. The detection rate for CTCs was 95.71% (67/70 patients), with a median of 8 CTCs within 2 ml of peripheral venous blood (PVB). A concordance of 76.56% in ER status between CTCs and corresponding primary tumor and 69.23% between CTCs and corresponding metastases was observed. We also found that patients with ER-positive CTCs (CTC ER+) had longer PFS and OS than those without ER-positive CTCs (CTC ER-). Our findings suggested that ER status in CTCs of BC patients may provide valuable predictive and prognostic insights into endocrine therapies, although further evaluation in larger prospective trials is required.

A preliminary study on the association between epithelial-mesenchymal transition and circulating tumor cells in renal cell carcinoma.

BackgroundCirculating tumor cells (CTCs) are considered useful prognostic factors for various cancers, and in 2014, our research group conducted a comparative experiment of CTC detection in patients with renal cell cancer (RCC). However, the reason for the low detection rate of CTCs in cancer patients using the CellSearch® system is still unknown, although it has been hypothesized to be attributed to the likelihood that CTCs undergoing epithelial-mesenchymal transition (EMT) do not express the CTC biomarkers cytokeratin (CK)8/18/19 or epithelial cell adhesion molecule (EpCAM). The overall aim of the current study was to investigate the expression levels of CK8/18/19 and EpCAM in relation to the EMT biomarkers vimentin and E-cadherin in patients with RCC.MethodsPatients with RCC who had undergone radical nephrectomy or partial resection between May 2014 and December 2014 were initially recruited.ResultsAmong 34 RCC patients, nine co-expressed EpCAM and CK8/18/19 in primary tumor tissues. The CellSearch® results showed that CK8/18/19 was expressed in 5 of 6 patients (5/6) and EpCAM was expressed in 6 patients (6/6). However, the isolation by size of tumor cells (ISET) technique showed these were co-expressed in only four of the 10. The expression of CK8/18/19, EpCAM, vimentin, and E-cadherin was distributed unequally in different enumeration groups of CTCs (all P>0.05), and the positive expression of CK8/18/19 was correlated with neutrophil number and tumor size (P<0.05). The positive expression of vimentin was correlated with the Karnofsky Performance Status (KPS) score and clinical stage of renal cancer patients (P<0.05).ConclusionsOur results indirectly proved the occurrence of EMT in the formation of CTCs by comparing and analyzing the expression of CK8/18/19 and EpCAM in renal cancer tissues and the detection results of CTCs.