Higher Circulating Tumor Cells Counts Research Articles

Detection and surveillance of circulating tumor cells in sarcoma for predicting therapy response and prognosis.

e23573 Background: Generally sarcomas are categorized as soft tissue and bone sarcomas and represent heterogenous group of malignancies with more than 70 distinct histologic subtypes. Due to their rarity and heterogeneity, sarcomas present particular challenges for accurate diagnosis, prognosis, and treatment. The presence and persistence of circulating tumor cells (CTCs) in the peripheral blood are believed to be associated with poor prognosis and distant metastases. A blood-based CTCs test is thus greatly needed for monitoring disease progression and predicting clinical outcomes. Methods: In this study, we developed a CTCs test for the detection and surveillance of sarcoma patients’ CTCs, and subsequently explored its clinical value. 123 patients with sarcomas were enrolled as the cohort for serial CTCs tests. Dynamic CTCs counting, in combination with therapy evaluation and post-treatment follow-up, was used to explore predicting pre- and post-chemotherapy evaluation and prognosis. Results: Quantification of CTCs demonstrated a significant increase in sarcoma patients compared to healthy individuals. Furthermore, patients in stage III-IV had a higher CTC count than those in stage I-II (p<0.01), suggesting a correlation between CTCs and disease development. Remarkably, there was variation in the quantity of CTCs among different types of sarcomas. The highest number of CTCs was observed in undifferentiated small round cell sarcoma, followed by soft tissue tumor, while bone tumors had the lowest number. In addition, the number of CTCs in patients with positive PD-L1 expression was considerably greater than in those with negative expression (p<0.01). The count of CTCs shown a substantial decrease following neoadjuvant chemotherapy treatment. There was a decline in the number of CTCs both before and after postoperative adjuvant chemotherapy treatment, however, there was no significant statistical discrepancy. The number of CTCs at the baseline was shown to be considerably larger in the group of patients with progressive disease or stable disease (PD+SD) compared to the group with partial response or complete response (PR+CR) (p<0.05). This difference was particularly pronounced for interstitial-type CTCs (p<0.01). Conclusions: The rate of presence of CTCs in the peripheral blood of sarcoma patients is high, and patients with an increased percentage of CTCs after treatment have a poor treatment effect. The dynamic monitoring of changes in CTCs counts after treatment has clinical significance for the timely detection of recurrence or metastasis. CTCs also showed a clinical value in prediction of therapy efficiency or prognosis, and may be clinically valuable for the whole course management of patients.

Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.

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 < 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 < 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< 0.001). Among 29 patients who were ER-negative, 4 (14%) were CTC ER-Positive (P< 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 (< 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 < 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.

Prognostic significance of circulating tumor cells and tumor related transcripts in small cell lung cancer: A step further to clinical implementation.

Small-cell lung cancer (SCLC) is a fatal disease with limited treatment options. Circulating tumor cells (CTCs) in liquid biopsy samples may serve as predictive and prognostic biomarkers; but the analysis of CTCs is still challenging. By using microfluidic or density gradient CTC enrichment in combination with immunofluorescent (IF) staining or qPCR of CTC-related transcripts, we achieved a 60.8% to 88.0% positivity in SCLC blood samples. Epithelial and neuroendocrine transcripts including the druggable target DLL3 were associated with shorter overall survival (OS), indicating the clinical value of these markers in terms of differential diagnosis and treatment decisions. High CTC counts and the presence of CTC duplets detected by IF staining were prognostic for OS, and thus may serve as indicators of disease progression or therapy failure. In patient samples with high CTC load detected by IF staining, a concordance of the transcripts positivity in circulating free plasma RNA and CTCs was observed. Our data emphasize the role of CTCs and CTC-related transcripts and underline the clinical value of liquid biopsy analysis in SCLC.

Correlation analysis of circulating tumor cells and Claudin-4 in breast cancer.

Objective: We aimed to explore the relationship between peripheral blood circulating tumor cells (CTCs) and the expression of Claudin-4 in patients with breast cancer, and further explore the potential impact on clinical prognosis and risk assessment. Methods: We classified and enumerated circulating tumor cells in the blood of breast cancer patients by CTC-enriched in situ hybridization and the detection of Claudin-4 expression by immunohistochemistry. We carried out an analysis of the correlation between the two and the comparison of their impact on clinical parameters and prognosis. Results: There were 38 patients with a low expression of Claudin-4 and 27 patients with a high expression of Claudin-4. Compared with Claudin-4 low-expression patients, the number of CTCs was higher in patients with high Claudin-4 expression (11.7 vs. 7.4, p < 0.001). High Claudin-4 expression was associated with a lower count of epithelial CTCs (E-CTCs) (3.4 vs. 5.0, p = 0.033), higher counts of mesenchymal CTCs (M-CTC) (4.4 vs. 1.1, p < 0.001), and epithelial/mesenchymal CTCs (E/M-CTCs) (4.0 vs. 3.5, p = 0.021). The intensity of Claudin-4 was positively correlated with CTC (rs = 0.43, p = 0.001). Multivariate COX regression analysis showed that CTC counts (HR = 1.3, p < 0.001), Claudin-4 (HR = 4.6, p = 0.008), and Lymphatic metastasis (HR = 12.9, p = 0.001) were independent factors for poor prognosis. COX regression of CTC classification showed that epithelial/mesenchymal CTCs (E/M-CTC) (HR = 1.9, p = 0.001) and mesenchymal CTCs (M-CTC) (HR = 1.5, p = 0.001) were independent influencing factors of adverse reactions in breast cancer patients. Conclusion: The number of CTC in breast cancer is positively correlated with the expression of Claudin-4. High CTC counts and a high proportion of M-CTCs correlated with Claudin-4 expression. CTC counts and Claudin-4 expression were independent predictors of poor prognosis in breast cancer patients.

Phenotypic Plasticity in Circulating Tumor Cells Is Associated with Poor Response to Therapy in Metastatic Breast Cancer Patients.

Circulating tumor cells (CTCs) are indicators of metastatic spread and progression. In a longitudinal, single-center trial of patients with metastatic breast cancer starting a new line of treatment, a microcavity array was used to enrich CTCs from 184 patients at up to 9 timepoints at 3-month intervals. CTCs were analyzed in parallel samples from the same blood draw by imaging and by gene expression profiling to capture CTC phenotypic plasticity. Enumeration of CTCs by image analysis relying primarily on epithelial markers from samples obtained before therapy or at 3-month follow-up identified the patients at the highest risk of progression. CTC counts decreased with therapy, and progressors had higher CTC counts than non-progressors. CTC count was prognostic primarily at the start of therapy in univariate and multivariate analyses but had less prognostic utility at 6 months to 1 year later. In contrast, gene expression, including both epithelial and mesenchymal markers, identified high-risk patients after 6-9 months of treatment, and progressors had a shift towards mesenchymal CTC gene expression on therapy. Cross-sectional analysis showed higher CTC-related gene expression in progressors 6-15 months after baseline. Furthermore, patients with higher CTC counts and CTC gene expression experienced more progression events. Longitudinal time-dependent multivariate analysis indicated that CTC count, triple-negative status, and CTC expression of FGFR1 significantly correlated with inferior progression-free survival while CTC count and triple-negative status correlated with inferior overall survival. This highlights the utility of protein-agnostic CTC enrichment and multimodality analysis to capture the heterogeneity of CTCs.

Abstract P4-07-24: Circulating tumor cells enumeration and Health Related Quality of Life of patients treated with first-line chemotherapy for HER2 negative metastatic breast cancer

Abstract Background: In patients with metastatic breast cancer (mBC), Circulating Tumor Cells (CTC) counts have a strong prognostic impact on progression free survival (PFS) and overall survival (OS). Changes 4 weeks after the start of a new line of therapy, inform on treatment efficacy. Despite improvements in systemic treatment, metastatic BC remains mainly uncurable with alteration of health-related quality of life (HRQOL) during the course of the disease. The aim of this work was to assess impact of clinical factors and biological factors as CTC on HRQOL. Methods: The French cohort COMET is a prospective study including first line HER2 negative patients receiving weekly paclitaxel and bevacizumab according to EMA approved combination. The aim of this cohort was to evaluate clinical, biological and radiological parameters associated with patients’ outcome (CTC, CEC, serum markers, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat assessed by initial CTscan, serum estradiol level, and quality of life). HRQOL was assessed at baseline, at every cycle until progression and then every 3 months up to death using the EORTC QLQ-C30 questionnaire and its breast cancer specific module, the EORTC QLQ-BR23. Five dimensions of HRQOL were analyzed for the primary analyses: Global health status (GHS), physical functioning (PF), Emotional functioning (EF), fatigue (FA) and pain (PA). Time until definitive deterioration (TUDD) in HRQOL was defined as the interval between inclusion and the first decrease in HRQOL score ≥ 5 compared to baseline HRQOL score with no further improvement or in case of death. CTC counts were determined using the standard CellSearch system [Menarini Silicon Biosystems]. Results: Out of 510 patients included in COMET study, 432 patients with available HRQOL data were analyzed in this study. At baseline, patients reported a mean score for GHS of 57.6 (SD=22.7), for PF of 75.8 (23.2), for EF of 62.2 (25.8), for FA of 42.2 (29.60) and for PA of 38.1 (31.5). The Median TUDDs for the 5 targeted dimensions was 10.1 months [7.5-16.9] for GHS, 6.1 months [4.1-8.9] for PF, 21.6 [18.7-31.2] for EF, 10.8 [6.2-16.6] for FA and 13.6[10.1-22.5] months for PA. CTC counts were available in 261 patients at base line and in 229 patients after 4 weeks of treatment, before second cycle of chemotherapy. CTC high count was independent of main clinical and biological characteristics except lobular subtype. We confirmed the poor outcome of patients with high CTC count at base line and after one cycle of treatment with the threshold of &amp;gt; 4CTC/7.5 ml of blood. Out of the 5 dimensions of HRQOL, TUDD of EF was significantly correlated with a high CTC level at base line (p=0.0262) and even more with still an elevated count of CTC after one cycle of chemotherapy(p=0.0137). There was no association of CTC with the other dimensions of HRQOL. Conclusion: This is the first study ever reporting an analysis of QoL and CTC. We observed an association of high CTC count with one component of HRQOL scale. This suggests that CTC could be complementary to clinical factors that could influence HRQOL in HER2 negative metastatic BC treated with first line chemotherapy. Citation Format: Jean-Yves Pierga, Oumar Billa, Sandrine Dabakuyo, Jérôme Lemonnier, Frédérique Berger, Olivier Trédan, William Jacot, Anthony Gonçalves, Marc Debled, Christelle Levy, Christelle Jouannaud, Marie-Ange Mouret-Reynier, Jean-Marc Ferrero, Florence Dalenc, Fatima-Zohra Toumi, Franck Bonnetain, Francois-Clement Bidard, Shufang Renault. Circulating tumor cells enumeration and Health Related Quality of Life of patients treated with first-line chemotherapy for HER2 negative metastatic breast cancer [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 P4-07-24.

Circulating tumor cells correlating with Ki-67 predicts the prognosis of bladder cancer patients

PurposeTo investigate the value of circulating tumor cells (CTCs) and Ki-67 in predicting the prognosis of bladder cancer. Here, we tested CTC counts and Ki-67 levels to assess patient prognosis.Methods84 patients with bladder cancer who underwent surgery were included in this study. Peripheral blood CTCs were detected by SE-iFISH technology before and after surgery, and Ki-67 levels were analyzed by immunohistochemistry. The association between CTCs and Ki-67 and the combination of the two was analyzed to predict the prognosis of patients.Results76 out of 84 patients (90.5%) were positive. ROC curve analysis showed that preoperative and postoperative CTC counts = 4 and 2 were the best thresholds for predicting patient recurrence or death. In multivariate analysis, high postoperative CTC count (≥ 2) (P < 0.001) and Ki-67 high expression (≥ 15%) (P < 0.001) were independent poor prognostic factors for PFS in bladder cancer patients. In addition, the study found that Ki-67 levels were positively correlated with high postoperative CTC counts, Bladder cancer patients with Ki-67 high expression and high postoperative CTC counts were associated with extremely poor progression-free survival.ConclusionKi-67 high expression is associated with high postoperative CTC counts, both of which predict poor prognosis in bladder cancer patients.

Abstract 5124: Isolation of viable circulating tumor cells from peripheral and central venous blood using a rapid microfluidic biochip

Abstract Background: Thousands of circulating tumor cells (CTCs) may be released into the blood stream of a cancer patient each day. However, only viable CTCs can cause metastasis. These viable CTCs hold the promise of deciphering the tumor heterogeneity, uncovering the mechanism of resistances to therapies, and enabling personalized treatments. Despite the continuously mounting interest, isolation of these cells remains challenging due to the multitude of factors, including rarity of the CTCs in blood, their constant exposure to therapeutic agents, and the harsh environment in the circulation. To address this, we have developed a novel microfluidic biochip capable of CTC isolation from unprocessed whole blood. Herein, we compare isolation of CTCs from the central and peripheral blood samples of metastatic breast cancer (MBC) patients using our biochip to assess impact of blood collection location on CTC count and viability. Methods: We included 19 patients with MBC, of which 26% are white, 63% are African American, and 11% are Latino. The median age of the cohort is 58 (34-75y). All patients are undergoing therapy. A total of 36 samples (7.5 mL each) were collected from either an antecubital vein (peripheral vein) or the subcutaneous port catheter (central vein) at different time points. All samples were processed within 5 hours of blood drawn in our novel microfluidic biochip (only 40 minutes per sample, without pretreatment), followed by in situ immunostaining. CK+/EpCAM+, DAPI+ and CD45- cells were counted as CTCs. In situ live/dead assay using Calcein AM and propidium iodide was performed for some samples right after CTC isolation. Results: CTCs were detected in all the samples (36/36), with a median of 15 CTCs per sample. A &amp;gt;5 CTCs count was found in 86% of the samples (31/36). No correlation was found between CTC count and race/ethnicity. The CTC count in central venous samples was in the range of 6-105 (median 27), while the range was 1-45 (median 10) in the peripheral samples. The difference was found to be statistically significant (p&amp;lt;0.001 using ANOVA). While no correlation between CTC count and patient age was found for the overall samples, we noted a significantly higher CTC count in central venous blood of younger patients (&amp;lt;60y) than of senior patients (p&amp;lt;0.05). Live/dead assay immediately following the microfluidic isolation found live CTCs in patient samples. Conclusions: Viable CTCs were rapidly isolated and detected in MBC samples using our novel microfluidic biochip. Our results show that CTC count in blood drawn from central vein yields significantly higher numbers than from the peripheral vein, suggesting that central venous samples might be preferred for prognosis and monitoring to drug response in MBC patients. Viable CTCs may provide an opportunity to evaluate the mechanism of resistance to therapies. More studies with larger cohorts are needed to further confirm these findings. Citation Format: Jian Zhou, Qiyue Luan, Celine Macaraniag, Arielle Guzman, Maria Mantice, Oana Danciu, Kent Hoskins, Ian Papautsky. Isolation of viable circulating tumor cells from peripheral and central venous blood using a rapid microfluidic biochip [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 5124.

Abstract P2-02-02: Differences in levels of circulating tumor cells (CTC) and disseminated tumor cells (DTC) in early-stage lobular versus ductal breast cancer

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC). ILC has unique features, such as a diffuse growth pattern due to characteristic loss of E cadherin, and a different pattern of disease metastasis compared to IDC. Prior investigators have shown increased numbers of circulating tumor cells (CTCs) in patients with metastatic ILC versus IDC. We explored the distribution of CTCs and disseminated tumor cells (DTCs) in early stage ILC versus IDC. Methods: We performed a secondary data analysis of the TIPPING study, an institutional review board approved study that pre-operatively collected blood and bone marrow samples from 655 treatment-naïve early-stage breast cancer patients. We analyzed data from 284 patients who had CTCs and DTC enumerated by an EPCAM-based method involving immunomagnetic enrichment and flow cytometry (IE/FC) (61 patients with ILC and 223 patients with IDC). We compared CTC and DTC counts by histology using the Welch Two Sample t-test, linear regression models, as well as ANOVA tests. Multivariate Cox regression analyses were performed to assess association between levels of CTCs/DTCs and clinical outcomes (distant recurrence-free survival [DRFS] and breast cancer-specific survival [BCSS]). Results: ILC tumors were lower grade than IDCs and had a higher proportion of HR+HER2- subtypes (92.00% vs. 75.30%; p&amp;lt;0.001). ILC patients had significantly higher CTC counts than IDC patients (mean 2.11 vs. 0.71 CTCs/mL; p&amp;lt;0.001), a difference that retained significance after adjusting for clinical variables (p=0.003). Additionally, we identified a subset of ILC patients (n = 9; 14.75%) that have elevated CTCs, which was absent in the IDC subset. ILC patients with elevated CTC levels showed no statistically significant association between CTC as a continuous variable with nodal status and breast cancer stage (p=0.26, p=0.25, respectively). In our study, the overall median follow-up was 7.26 years for DRFS and 8.9 years for BCSS. In the subset of ILC patients with elevated CTCs, CTC level as a continuous variable did not show significant association with DRFS or BCSS in a multivariate model adjusting for clinical variables. In the IDC subset, CTC level as a continuous variable did not show significant association with DRFS or BCSS in a multivariate model adjusting for clinical variables.Furthermore, there was no difference in the number of DTCs in ILC versus IDC. DTC level as a continuous variable did not show significant association with DRFS or BCSS in a multivariate model adjusting for clinical variables in both ILC and IDC subsets. Conclusions: Early-stage ILC patients have significantly higher CTC levels than those observed in IDC patients, and we hypothesize that the reason may be due to lower cell-cell adhesion. ILC spans the spectrum of indolent (benign) to high risk (bad actor) disease. Thus, biomarkers like CTCs may allow us to identify ILC patients who are at higher risk of late recurrence and make appropriate therapeutic decisions at earlier point in time. Studies like Endocrine Optimization Pilot in I-SPY 2 are ongoing to further investigate the use of biomarkers like CTCs to inform outcomes. Due to the short follow-up period, we will conduct an additional follow-up which should give us 10 years of follow-up, which is likely needed due to the risk for late recurrence in ILC patients. Citation Format: Silver Alkhafaji, Denise Wolf, Mark Magbanua, Laura Van 't Veer, John Park, Laura J. Esserman, Rita A. Mukhtar. Differences in levels of circulating tumor cells (CTC) and disseminated tumor cells (DTC) in early-stage lobular versus ductal 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 P2-02-02.

Coagulation/fibrinolysis and circulating tumor cells in patients with advanced breast cancer

PurposeTo evaluate the relationship between circulating tumor cells (CTCs) and standard coagulation tests in both a discovery and a validation cohort of patients with advanced breast cancer.MethodsIn a retrospective (n = 77) and a prospective (n = 92) study of patients with progressive advanced breast cancer, CTC count, platelet number, fibrinogen level, D-dimers, prothrombin time, and activated partial thromboplastin time were measured. The association between these coagulation studies and CTC count was analyzed. The impact of these measurements on overall survival (OS) was assessed.ResultsIn both cohorts, results were similar; absolute CTC count was significantly associated to D-dimer level and inversely with platelet count. In the prospective cohort, quantification of tumor-derived extracellular vesicles (tdEVs) was associated with CTC count, and with coagulation abnormalities (low platelet count and increased D-dimers). tdEVs did not add to CTC count in predicting changes in platelets or D-dimers. In multivariate analysis only CTC ≥ 5 CTC/7.5 mL, ER status, HER2 status and lines of chemotherapy were associated with OS. In patients with terminally metastatic breast cancer, very high CTC counts are prevalent.ConclusionA significant association exists between increasing CTC number and increased D-dimers and decreased platelet counts, suggesting a potential role for CTCs as a direct contributor of intravascular coagulation activation. In patients with advanced and progressive breast cancer, abnormalities in routine coagulation tests is the rule. In patients with terminally advanced breast cancer a “leukemic” phase with high CTC count is prevalent.

Predictive Value of Circulating Tumor Cells in Prognosis of Stage III/IV Colorectal Cancer After Oxaliplatin-based First-line Chemotherapy.

Insufficient data exist to support the concept of the circulating tumor cell (CTC) level as a prognostic factor for platinum-based first-line chemotherapy. This study investigated the impact of CTCs on the prognosis of patients with advanced colorectal cancer (CRC) after receiving platinum-based chemotherapy. Analyses were carried out of clinicopathological features and molecular phenotypes to clarify independent risk factors for a high CTC count. Patients diagnosed with stage III/IV CRC (n=76) were included in the study. The blood samples of patients were evaluated for CTCs using the CellRich™ platform system. Immunohistochemistry (Ias used to analyze epithelial-mesenchymal transition-associated biomarkers E-cadherin and vimentin. Univariate and logistic regression analyses were then conducted to analyze the risk factors for CTC expression. Additionally, the influence of oxaliplatin on disease-free survival after first-line chemotherapy or during chemotherapy was analyzed through a 2-year follow-up. Patients in the CTC+ group experienced shorter DFS after receiving oxaliplatin first-line chemotherapy than patients in the CTC- group (p<0.01). In addition, univariate analysis revealed that the tumor M-stage, tumor location, RAS mutation, high expression of vimentin, and deletion of E-cadherin expression were correlated with a high CTC count. Multivariate analysis suggested that the presence of RAS gene mutations and high vimentin expression were independent risk factors for high CTC loads (p<0.01). CTC positivity can indicate the efficacy of first-line chemotherapy with oxaliplatin in stage III/IV colorectal cancer. This may be linked to tumor epithelial-mesenchymal transition in patients with CTCs. Moreover, RAS gene mutation and high expression of vimentin were identified as independent risk factors for a high CTC count.

Neutrophil-Lymphocyte Ratio and Circulating Tumor Cells Counts Predict Prognosis in Gastrointestinal Cancer Patients.

PurposeThe purpose of this study is to explore the prognostic value of associating pre-treatment neutrophil–lymphocyte ratio (NLR) with circulating tumor cells counts (CTCs) in patients with gastrointestinal cancer.Materials and MethodsWe collected the related data of 72 patients with gastric cancer (GC) and colorectal cancer (CRC) who received different therapies from August 2016 to October 2020, including age, gender, primary tumor location, TNM stage, tumor-differentiation, NLR, CTCs, disease-free survival (DFS) and overall survival (OS). We chose the optimal cut-off value of NLR >3.21 or NLR ≤3.21 and CTC >1 or CTC ≤1 by obtaining receiver operating characteristic (ROC) curve. The Kaplan–Meier survival analysis and Cox regression analysis were used to analyze DFS and OS. To clarify the role of the combination of NLR and CTCs counts in predicting the prognosis, we analyzed the DFS and OS when associated NLR and CTCs counts.ResultsA high NLR (>3.21) was associated with shorter DFS (P <0.0001) and OS (P <0.0001). Patients with high CTCs level (>1) had shorter DFS (P = 0.001) and OS (P = 0.0007) than patients with low CTCs level. Furthermore, patients who had both higher NLR and higher CTCs counts had obvious shorter DFS (P <0.0001) and OS (P <0.0001).ConclusionsPatients with higher NLR and more CTCs respectively tended to have poor prognosis with shorter DFS and OS, which might be regarded as predictors of gastrointestinal cancer. In particular, associating NLR and CTCs counts might be a reliable predictor in patients with gastrointestinal cancer.

Abstract 2601: Using chaperonin containing TCP1 as a marker to track clinically relevant circulating tumor cells

Abstract Current methods for assessing circulating tumor cells (CTC) in blood focus on capture and enumeration with epithelial markers that provide little information about the aggressive and invasive potential of CTC. As a result, valuable knowledge that improves monitoring of cancer recurrence and metastasis is lacking. To provide actionable information on CTC, we developed a CTC identification method based on detection of the second subunit of Chaperonin Containing TCP1 (CCT2). CCT is a macromolecular protein folding complex composed of eight subunits (CCT1-8) that drives expression of many tumorigenic factors. Bioinformatic analysis in multiple cancers, such as breast cancer, revealed increased gene expression in tumor tissue compared to normal tissue for CCT1-8 (p&amp;lt; 0.0001), with the largest differences observed in CCT2 and CCT3. Genomic alteration of CCT2 inversely correlated with cancer patient survival, and depletion of CCT2 prevented growth of murine breast tumors. In a correlative study of blood and tumor tissues from metastatic breast cancer patients, high CTC counts positively correlated with detectable CCT2 in tumor tissues. These results support CCT2 as a possible marker for CTC. To determine this, antibodies to CCT2 were validated for use in the CellSearch System (CSS), currently the only FDA approved CTC enrichment platform, and tested with breast cancer cells spiked into healthy human blood. Recovery using CCT2 for identification, led to increased collection up to 6% of spiked breast cancer cells that would have been lost by standard CTC classification using cytokeratin (CK). With prognostic thresholds around 3-5 CTC/tube of blood, increased detection of even 1-2 more CTC can be significant for assessing patients' risks. Analysis in an open software program called ACCEPT (Automated CTC Classification, Enumeration and PhenoTyping) confirmed that the majority of recovered spiked cancer cells expressed CCT2. Flow cytometry analysis of spiked cancer cells confirmed that CCT2+ enriched cells expressed cancer markers CD44 or estrogen receptor (ER). These results show that the addition of CCT2 staining to a CTC detection protocol has positive implications for enumeration of CTC from blood, such as increased detection of CTC with low expression of epithelial markers. This experiment was repeated using small cell lung cancer cell lines with similar results. The combined bioinformatics, histologic and experimental results indicate that identification of CCT2-expressing CTC can be incorporated into CTC detection platforms to monitor those patients with a heightened risk for cancer recurrence or metastasis. Citation Format: Amanda Cox, Ana Martini, Eunkyung Lee, Rebecca Moroose, Amr Khaled, Annette R. Khaled. Using chaperonin containing TCP1 as a marker to track clinically relevant circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2601.

Circulating Tumor Cell Subtypes and T-cell Populations as Prognostic Biomarkers to Combination Immunotherapy in Patients with Metastatic Genitourinary Cancer.

Circulating tumor cells (CTC) are under investigation as a minimally invasive liquid biopsy that may improve risk stratification and treatment selection. CTCs uniquely allow for digital pathology of individual malignant cell morphology and marker expression. We compared CTC features and T-cell counts with survival endpoints in a cohort of patients with metastatic genitourinary cancer treated with combination immunotherapy. Markers evaluated included pan-CK/CD45/PD-L1/DAPI for CTCs and CD4/CD8/Ki-67/DAPI for T cells. ANOVA was used to compare CTC burden and T-cell populations across timepoints. Differences in survival and disease progression were evaluated using the maximum log-rank test. From December 2016 to January 2019, 183 samples from 81 patients were tested. CTCs were found in 75% of patients at baseline. CTC burden was associated with shorter overall survival (OS) at baseline (P = 0.022), but not on-therapy. Five morphologic subtypes were detected, and the presence of two specific subtypes with unique cellular features at baseline and on-therapy was associated with worse OS (0.9-2.3 vs. 28.2 months; P < 0.0001-0.013). Increasing CTC heterogeneity on-therapy had a trend toward worse OS (P = 0.045). PD-L1+ CTCs on-therapy were associated with worse OS (P < 0.01, cycle 2). Low baseline and on-therapy CD4/CD8 counts were also associated with poor OS and response category. Shorter survival may be associated with high CTC counts at baseline, presence of specific CTC morphologic subtypes, PD-L1+ CTCs, and low %CD4/8 T cells in patients with metastatic genitourinary cancer. A future study is warranted to validate the prognostic utility of CTC heterogeneity and detection of specific CTC morphologies.

Circulating Tumor Cells Undergoing the Epithelial-Mesenchymal Transition: Influence on Prognosis in Cytokeratin 19-Positive Hepatocellular Carcinoma.

PurposeThe purpose of this study was to elucidate the relationship between cytokeratin 19 (CK19) expression and levels of circulating tumor cells (CTCs) in preoperative peripheral blood of patients with hepatocellular carcinoma (HCC), and the potential influence of that relationship on prognosis.Patients and MethodsCanPatrol™ CTC-enrichment technique and in situ hybridization (ISH) were used to enrich and classify CTCs undergoing the epithelial–mesenchymal transition (EMT) from blood samples of 105 HCC patients. CK19 immunohistochemistry staining was performed on HCC tissues and compared with demographic and clinical data.ResultsIn total, 27 of 105 (25.7%) HCC patients were CK19-positive. CK19-positive patients had significantly lower median tumor-free survival (TFS) than CK19-negative patients (5 vs 10 months, P = 0.047). In total, 98 (93.3%) patients showed pre-surgery peripheral blood CTCs (range: 0–76, median: 6), and 57 of 105 (54.3%) patients displayed CTC counts ≥6. Furthermore, CK19-positive patients with CTC count ≥6 showed significantly higher percentage than CK19-negative ones (77.8% vs 46.2%, P = 0.004). CK19-positive patients showed a significantly higher proportion of mesenchymal CTCs among CTCs undergoing EMT than CK19-negative patients (mean rank: 62.28 vs 49.79, P = 0.046). We also found that CK19-positive patients with high CTC count showed significantly shorter median tumor-free survival than CK19-negative patients with low CTC count (5 vs 16 months, P = 0.039).ConclusionHigh CTC count and high percentage of mesenchymal CTCs are closely related to the expression of CK19, which is associated with poor prognosis in HCC patients.

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

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

CTC counts as a biomarker of prognosis and response in metastatic castration-resistant prostate cancer (mCRPC) from the CARD trial.

161 Background: In the prospective CARD trial (NCT02485691), cabazitaxel significantly improved radiographic progression-free survival (rPFS) and overall survival (OS) versus abiraterone or enzalutamide in patients with metastatic castration-resistant prostate cancer who had received docetaxel and progressed within 12 months with the alternative androgen-signaling-targeted inhibitor. Here we analyzed circulating tumor cell (CTC) counts as a biomarker of prognosis and response to therapy. Methods: Blood samples collected at screening (SC), Cycle 2 (C2) Day 1, and end of therapy were sent to Epic Sciences for CTC analysis. The association between CTC counts, defined as any cytokeratin-positive, CD45-negative cell, and clinical outcomes were pre-specified and analyzed using a multivariate Cox model for time-to-events (rPFS, OS) or a Χ2 test for categorical outcomes (objective tumor response). Results: Of the 255 patients randomized, 237 (92.9%) had evaluable samples at SC and 213 (83.5%) at C2. CTCs ≥ 1 were detected in 204 samples at SC (86%) and 178 samples at C2 (84%), with a median (interquartile range) count per mL of blood of 4.1 (1.3–14.2) and 5.2 (1.5–17.3), respectively. At baseline, higher CTC counts were associated with higher values of lactate dehydrogenase (P = 0.014), alkaline phosphatase (P &lt; 0.001), and prostate-specific antigen (P &lt; 0.001). High CTC counts, measured as a continuous variable at SC (all arms combined) were associated with a shorter OS (P = 0.03), but not rPFS (P = 0.7). However, favorable changes in absolute CTC count from SC to C2, all arms combined, were associated with longer rPFS (P = 0.03). Conversion from high (≥ 3) to low (&lt; 3) CTC count at C2 or maintenance of a low CTC count at C2 was associated with objective tumor response (P = 0.007). Analysis of the associations of CTC androgen receptor variant 7, chromosomal instability, heterogeneity status (Shannon Index), single-cell genotypes (e.g. retinoblastoma/phosphatase and tensin homolog copy-loss), and the presence of CTCs with small-cell/neuroendocrine morphology is ongoing. Conclusions: This preplanned analysis of the CARD trial confirms that baseline CTC counts, measured by Epic Sciences platform, are prognostic. Moreover, early favorable changes in CTC counts from baseline to C2 are associated with response to therapy. Funding: Sanofi. Clinical trial information: NCT02485691.

Circulating Tumor Cells Correlate With Prognosis in Head and Neck Squamous Cell Carcinoma.

Background:To investigate the relationship of circulating tumor cells (CTCs) and the clinical characteristic parameters and prognosis in patients with head and neck squamous cell carcinoma (HNSCC).Methods:The retrospective clinical study included 95 patients with HNSCC who after surgery in Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine between December 2015 and December 2016. All patients were followed up for survival until the end of June 2019. The CTCs detection was performed by negative enrichment (NE) immunofluorescence-in situ hybridization (im-FISH) of chromosome 8.Results:Patients with higher CTCs counts are associated with a worse prognosis with an area under the receiver operator characteristic (ROC) curve of 0.756 [95% confidence interval (CI) 0.640-0.872, p = 0.001]. The CTCs-positive rate of HNSCC patients was 58.9% (56/95) by using the cut-point of 3. Both the chi-square test and binary logistic regression analysis showed that the N stage and clinical stage were significantly associated with CTCs-positive in patients with HNSCC (p < 0.05). Further Non-parametric test analysis indicated that more CTCs counts were detected in late N and clinical stages patients (p < 0.001). The Kaplan-Meier survival analysis indicated that CTCs-positive were correlated with shorter progression-free survival (PFS) (p < 0.001) and overall survival (OS) (p = 0.001). Further, the CTCs-positive was an independent prognostic factor for PFS and OS according to the Cox multivariate regression analysis (p < 0.05).Conclusion:More CTCs were associated with N stage, clinical stage, poor prognosis in patients with HNSCC, which might be used as a prognostic biomarker.

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

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