Presence Of Circulating Tumor Cells Research Articles

Circulating tumor cells in uveal melanoma : "The needle in the haystack"

A fundamental prerequisite for the development of clinically manifested metastases in uveal melanoma (UM) is the hematogenous dissemination of melanoma cells, which circulate in the systemic circulation, deposit in organs and can subsequently colonize the organs. This article presents a review of the literature on the detection of circulating tumor cells (CTC) from UM and their relevance for the clinical and pathophysiological aspects of the malignancy. Since the first description of CTC in UM patients 20years ago, only 20articles have been published on the detection of intact CTCs in UM. Besides demonstrating the presence of CTCs in the systemic circulation, these articles give insights into the pathophysiology and chronology of CTC dissemination, the diagnostics and risk stratification of UM. The detection of CTC in UM has proved to be beneficial in many ways. The early time of cell dissemination and also the associated factors should lead to a rethinking in the clinical routine in the context of the fatal danger of metastases.

Abstract B026: Glioblastoma multiforme disseminates outside the central nervous system

Abstract Introduction: The liquid biopsy (LB) concept has gained significant attention in the last decade and has the potential to revolutionize cancer care. However, more validation in clinical trials is required on the value of LB in medical settings. Here, we focus on glioblastoma multiforme (GBM), a highly invasive and fatal subtype of the central nervous system malignant tumors. Circulating tumor cells (CTCs) are primary tumor cells released into the peripheral blood and migrating to distant organs. CTC presence is related to tumor spread and recurrence. In GBM, factors like short patient survival, the blood-brain barrier (BBB), and lack of lymphatic drainage in brain tissue make systemic dissemination rare but possible. Thus, noninvasive CTC detection can hold potential for diagnostic, predictive, and prognostic applications. Material and Methods: For cell stability preservation before sample processing, the peripheral blood samples from curatively resected GBM patients were collected preoperatively in Cell-Free DNA BCT® tubes (Streck, Inc.). CTCs were identified using CytoTrack CT11TM (2/C), a semi-automated immunofluorescence microscopy using glial fibrillary acidic protein, vimentin, and CD45 immunostaining, and DAPI counterstaining. The preliminary Kaplan-Maier survival analysis based on CTC positivity was performed in 39 patients with a follow-up longer than two years. Results: We analyzed the CTC presence in 100 GBM patient peripheral blood samples. The CTCs or cancer cell clusters were found in 35% of GBM patients. The Kaplan-Maier survival analysis did not show a correlation between CTC presence and survival. This phenomenon may be associated with the generally short survival of these patients, which is expected to increase with advances in oncology and palliative care and/or a possible suppression of tumor cell growth outside the CNS. Conclusions: We have observed the CTCs in the peripheral blood of GBM patients. The patient enrollment and clinical data recording are still ongoing. Citation Format: Pavel Stejskal, Josef Srovnal, Alona Rehulkova, Ondrej Kalita, Marek Slachta, Marian Hajduch. Glioblastoma multiforme disseminates outside the central nervous system [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 B026.

Effect of Cyclin-Dependent Kinase 4/6 Inhibitors on Circulating Cells in Patients with Metastatic Breast Cancer.

The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is the standard-of-care for estrogen receptor (ER)-positive, HER2-negative (ER+/HER2- advanced/metastatic breast cancer (mBC). However, the impact of CDK4/6i on circulating immune cells and circulating tumor cells (CTCs) in patients receiving CDK4/6i and ET (CDK4/6i+ET) remains poorly understood. This was a prospective cohort study including 44 patients with ER+/HER2- mBC treated with CDK4/6i+ET in either first or second line. Peripheral blood samples were collected before (baseline) and 3 months (t2) after therapy. Immune cell's subsets were quantified by flow cytometry, and microfluidic-captured CTCs were counted and classified according to the expression of cytokeratin and/or vimentin. Patients were categorized according to response as responders (progression-free survival [PFS] ≥ 6.0 months; 79.1%) and non-responders (PFS < 6.0 months; 20.9%). CDK4/6i+ET resulted in significant changes in the hematological parameters, including decreased hemoglobin levels and increased mean corpuscular volume, as well as reductions in neutrophil, eosinophil, and basophil counts. Specific immune cell subsets, such as early-stage myeloid-derived suppressor cells, central memory CD4+ T cells, and Vδ2+ T cells expressing NKG2D, decreased 3 months after CDK4/6i+ET. Additionally, correlations between the presence of CTCs and immune cell populations were observed, highlighting the interplay between immune dysfunction and tumor dissemination. This study provides insights into the immunomodulatory effects of CDK4/6i+ET, underscoring the importance of considering immune dynamics in the management of ER+/HER2- mBC.

Clinical significance of circulating tumor cells in colorectal cancer with peritoneal metastases: a prospective cohort study using a novel method for monitoring treatment response, and assessing minimal residual disease.

The treatment for patients with colorectal cancer with metastases to the peritoneum is complex and may involve both surgery and chemotherapy. Circulating tumor cells (CTCs) have been poorly investigated in peritoneal metastatic colorectal cancer. The aim of the study is to examine the role of circulating tumor cells (CTCs) as a biomarker for monitoring disease progression, treatment response and residual disease using CellMate® - a new promising in vitro diagnostic platform technology. We prospectively followed clinical outcomes of 46 patients treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) for colorectal cancer with peritoneal metastases and examined whether CTCs were present the week of surgery. The CTC measurements were made with the CellMate® technology, which is a platform technology to detect CTCs based on the difference in biomechanical properties compared to blood resident cells. The study was registered online (ClinicalTrials.gov). CTCs were detected in 17 (37%) patients. The presence of CTCs was associated with shorter recurrence-free survival and overall survival after CRS and HIPEC. Both recurrence free survival (HR 4.00, 95%CI 1.15-13.9; P=0.029) and overall survival (HR 5.91; 95% CI 1.18-29.7; P=0.03) were significantly worse if CTCs were detected after neoadjuvant treatment. In the subgroup of patients with CTCs detected, adjuvant therapy tended to improve the prognosis while in CTC negative patients it did not. Pending a prospective multi-center trial to validate these findings, CTCs may in the future be used as a dynamic personalized biomarker for prognostication, predicting response to therapy, and for monitoring disease progression in colorectal cancer with metastases to the peritoneum.

Circulating Tumor Cells – An Overview of the Current Progress and Clinical Perspectives

Circulating tumor cells (CTCs) are cancerous cells that can detach from the primary tumor and circulate through the blood vessels until they reach a tissue or an organ and initiate metastasis. It is worth noting that in many types of cancer, the presence of CTCs in blood samples either independently or in clusters is considered a poor prognostic marker. This is because it indicates a lower overall survival (OS), a poorer progression-free survival (PFS), and a higher potential for metastasis. Characterising circulating tumor cells (CTCs) and monitoring their numbers can provide crucial information in managing cancer progression. Therefore, CTCs can be extremely useful in therapeutic monitoring, allowing doctors to follow treatment efficacy and make certain adjustments depending on their quantification. Research on CTCs as a liquid biopsy analyte has seen significant advancements, leading to the translational use of CTCs as potential biomarkers. However, low concentration and the lack of standardised detection methods make it challenging to detect CTCs. We offer an overview of the various phenotypic changes of CTCs and the epithelialmesenchymal transition (EMT) process that promotes the spread of cancerous cells. We also explore the biomarkers that characterise CTCs, as well as the primary isolation techniques. Finally, we highlight the clinical perspectives of CTCs and their relevance in monitoring cancer progression and response to treatment. Thus, we believe that the study of CTCs can provide a deeper understanding of the metastasis process, which could ultimately lead to improved patient outcomes.

Effect of circulating tumor cells (CTC) and CTC clusters with PD-L1 dynamic biomarker on cellular burden in patients with ovarian cancer.

e17566 Background: In the precision oncology era, monitoring response to the treatments using circulating blood-based biomarkers e.g. circulating tumor DNA (ctDNA) and Circulating Tumor Cells (CTCs) are being rapidly evaluated and established. The leading cause of ovarian cancer patient mortality is owing to the delayed diagnosis, inability of patient selection for targeted therapies, including the use of immune checkpoint blockade (ICB) agents. The treatment of ovarian cancer usually involves a combination of surgery and chemotherapy. However, post operative resection and therapy with curative intent often fails in accounting the Minimal Cellular Disease (MCD). The dissemination of Circulating Tumor Cells (CTC) remains a dogma of Minimal Residual Disease (MRD) that diffuse and cause micro-metastasis through their epithelial to mesenchymal transitions (EMT) and ‘bio-mechanistic activation’ in blood circulation. Simultaneous detection of over-expression of Programmed Cell Death-Ligand 1 (PD-L1) on CTCs as a dynamic biomarker may be expedient for assessing patients for immune checkpoint inhibitor (ICT) therapy. Methods: In retrospective analysis of real-world data, 75 ovarian cancer patient’s peripheral blood was analyzed for the presence of CTCs with and without the expression of PD-L1 and CTC clusters. CTC were positively detected using OncoDiscover platform approved by CDSCO in 1.5 ml peripheral blood. The platform is multifunctional magneto nanosystem mediated by anti-epithelial cellular adhesion molecule (EpCAM) antibody. CTCs were identified as positive if they were EpCAM +ve, CK+ve, PD-L1 +ve, DAPI +ve and CD45-ve. PD-L1 expression on the CTCs was analyzed based on the linear intensity gradients of the fluorescence signals using image acquisition on an automated Zeiss Microscope. Results: At baseline sample analysis, 86 % (n=65) of the patients showed ≥ 1CTCs per 1.5 ml of blood. The CTC distribution in this study ranged from 1-9 CTCs. Whereas, 46.15 % (n=30) patients with CTCs showed the expression of PD-L1. Noteworthy, the highest number of CTCs were observed ~ 26.7 % (n=23) in age group 41-50. Interestingly, 8 % (n=6) of total patients showed the presence of CTC clusters. The presence of CTCs with expression of PD-L1 and CTC clusters did not correlate with factors like staging, follow ups, metastasis, and DFS status. Conclusions: We observed presence of MCD and MRD in CRC patients, in spite of curative intent in ovarian cancers. Detection of CTCs, CTC clusters with over-expression of PD-L1- as a real time dynamic biomarker may be useful for assessing the patients for early metastasis, regression, and selecting patient suitable for immune checkpoint inhibitor (ICT) therapy when the tissue is inadequate or unavailable for better outcome.

Measure of minimal residual burden on circulating tumor cells with over-expression of programmed death-ligand 1 as a dynamic biomarker in patients with colorectal cancer.

e14553 Background: In stage III colorectal cancer (CRC) patients, the extent of oxaliplatin-combination as an adjuvant therapy is still undetermined. Approx. 25-50% Stage II-III CRC patients are known to develop recurrence and metastasis even after comprehensive treatment, attributed to occult disease and Minimal Residual Disease (MRD). Circulating Tumor Cells (CTCs) are bio-mechanistic extravasation source for micro-metastatic disease. CRC patients, with lower adjuvant therapy from 3 to 6 months are known to exhibit increased CTCs and positivity rate due to the emergence of resistant clones. Assays to detect CTCs and expression of Programmed Cell Death-Ligand 1 (PD-L1) as a dynamic biomarker simultaneously has wide clinical implications. Especially, when the tissue biopsy is not inadequate to detect molecular targets for immune checkpoint inhibitor (ICT) therapy. Methods: Retrospectively, 182 CRC cancer patients were analyzed for the presence of CTC and distribution at baseline and follow-ups (up to 0-4 follow ups). The peripheral blood 1.5 ml was analyzed using the OncoDiscover platform approved by CDSCO, India consisting multifunctional magneto-nanosystem mediated by anti-epithelial cellular adhesion molecule (EpCAM) antibody. CTCs were analyzed in patients with early stages- pre and post treatment, progressive disease, Disease Free Status (DFS) and in metastasis. The isolated cells were immune-stained to detect CK-18 +ve, CD45 -ve, DAPI +ve and PD-L1 +ve expression. PD-L1 expression in CTCs was validated by analyzing the linear intensity gradients of the fluorescence signals. CTCs were termed as PD-L1 -ve based on a weak or no detectable fluorescence signal and termed +ve based on a high fluorescence signal using automated image acquisition Zeiss microscope. Results: In a cohort of 182 CRC patient samples, n = 128 (70.3 %) showed the presence of CTCs. The fluorescence intensity for PD-L1 expression as a robust functional assay on CTCs for molecular characterization was developed. The distribution of CTCs ranged from 1-9. The mean fluorescence intensity value and cut-off for the expression of PD-L1 in CTCs was accounted to be ~1.02. Interestingly, in n = 54 (42.2 %) patients, CTCs showed positive expression of PD-L1. CTC +ve patients with PD-L1 expression were associated at all stages and even in early stage, progressive disease, and metastasis. Patients without any CTCs n = 54 (29.7%) either had clinically stable disease or were in DFS with no radio-graphical image. Conclusions: The presence of PD-L1 over-expression on CTCs is a dynamic biomarker in blood, showing the progression of disease even in patients with DFS status. CTC enumeration and assessing the PD-L1 expression on CTCs could offer highly individualized treatment plans in CRC patients.

Impact of circulating tumor DNA genomic mutations and circulating tumor cells biomarker duo on clinical concordance in localized, progressive, and metastatic disease.

e14546 Background: Genomic mutations have been identified from circulating tumor DNA (ctDNA) that correlates positively with clinical disease status. EGFR and intracellular cell progression/proliferation pathway-BRCA1/2 and TP53 genes drive high ctDNA load in progressive cancer patients. Circulating tumor cells (CTC) indicate Cellular Residual Disease (CRD). ctDNA and CTC as dual biomarkers offer prediction of tumor progression and metastasis implicative for early detection and treatment modifications. Methods: Retrospectively in 96 cancer patients (lung, colon-rectal, breast, stomach, etc.) who recently underwent treatment were investigated for presence of CTCs and genomic mutations from ctDNA using OncoMonitor Test. Libraries were prepared using a hybridization-capture method covering 1000 targets with mean sequencing depth 5000X on Illumina Nextseq 2000. Test detected genomic alterations from single nucleotide variation (SNVs), small insertions and deletions (INDELs), copy number variations (CNVs), and translocations (fusions) using a 96 gene panel. CTCs were isolated using the OncoDiscover platform, with CK18+, PD-L1+, CD45- in1.5 ml of blood. Results: In 96 pan-cancer patients, 15.6% patients (n=15) were identified with localized progressive disease with no metastasis from radiological findings, of which 60% (n=9) patients showed at least 1 genomic finding from ctDNA.12.5% (n=12) patients were identified as metastatic from their radiological findings of which 58.3% (n=7) showed presence of at least 1 CTC. 33.3% (n=4) patients showed 2 CTCs and 5 patients had no CTCs. In addition, these metastatic patients showed the presence of ctDNA load in 66.6% (n=8) patients with at least 1 genomic finding. In metastatic disease cohort, CTC enumeration showed concordance of 58.3% (n=7) with metastatic radiological findings and genomic findings from ctDNA showed concordance in 66.6% (n=8) patients with metastatic radiological findings. 23.9% (n=23) patients from the cohort were radiological identified with stable/treatment responsive disease of which 73.9% (n=17) patients were not detected with genomic mutations from ctDNA and 26.0% (n=6) patients were CTC -ve, concordant with radiological findings. 26.0% (n=6) patients had at least 1 genomic finding contributing to discordance with the radiological findings. Genomic findings from duo offered concordance with radiological findings in 26.6% (n=4) patients with progressive disease, 41.6% (n=5) in metastatic disease, and 17.3% (n=4) in patients with stable/treatment responsive disease. Conclusions: Patients with progressive and metastatic disease from radiological findings showed concordance with the dual ctDNA and CTC biomarkers. Concordance of ctDNA in progressive disease and CTC in metastatic disease is indicative of individual significance.

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&lt;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&lt;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&lt;0.05). This difference was particularly pronounced for interstitial-type CTCs (p&lt;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.

Comprehensive characterization of circulating tumor cells and cell-free DNA in patients with metastatic melanoma.

Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head-to-head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome-wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P < 0.001), whereas the presence of CTCs in peripheral blood was associated with number of lesions on radiological imaging (P < 0.001). In conclusion, DLA tended to increase the proportion of patients with detectable CTCs but was also associated with low recovery. Both cfDNA and CTCs were correlated with clinical parameters such as LDH levels and extracranial tumor burden.

Circulating tumour cells predict recurrences and survival in head and neck squamous cell carcinoma patients

Patients with head and neck squamous cell carcinoma (HNSCC) are at a high risk of developing recurrence and secondary cancers. This study evaluates the prognostic and surveillance utilities of circulating tumour cells (CTCs) in HNSCC. A total of 154 HNSCC patients were recruited and followed up for 4.5 years. Blood samples were collected at baseline and follow-up. CTCs were isolated using a spiral microfluid device. Recurrence and death due to cancer were assessed during the follow-up period. In patients with HNSCC, the presence of CTCs at baseline was a predictor of recurrence (OR = 8.40, p < 0.0001) and death (OR= ∞, p < 0.0001). Patients with CTCs at baseline had poor survival outcomes (p < 0.0001). Additionally, our study found that patients with CTCs in a follow-up appointment were 2.5 times more likely to experience recurrence or death from HNSCC (p < 0.05) prior to their next clinical visit. Our study highlights the prognostic and monitoring utilities of CTCs’ in HNSCC patients. Early identification of CTCs facilitates precise risk assessment, guiding treatment choices and ultimately enhancing patient outcomes.

Detection of PD‑L1 expression and epithelial‑mesenchymal transition of circulating tumor cells in non‑small cell lung cancer.

The present study aimed to assess the roles of peripheral circulating tumor cell (CTC) count, CTC subtypes and programmed death ligand 1 (PD-L1) expression in the clinical staging and prognosis of patients with non-small cell lung cancer (NSCLC). A total of 100 patients with NSCLC with available tumor tissues were enrolled in the present study, and 7.5 ml peripheral blood was collected. Patients were divided into PD-L1-positive and PD-L1-negative groups according to PD-L1 immunohistochemical staining. Peripheral blood samples from both groups were analyzed to determine the CTC count, epithelial-type CTCs (E-CTCs), mesenchymal-type CTCs (M-CTCs) and PD-L1 expression. Clinical data were collected, and patients were followed up for a maximum of 36 months, with patient death as the endpoint event. Patients with PD-L1-positive tumors had a worse prognosis compared with those with PD-L1-negative tumors (P=0.045). The PD-L1-positive group exhibited significantly higher numbers of CTCs and M-CTCs compared with the PD-L1-negative group (P≤0.05). However, the number of E-CTCs did not differ significantly between the two groups (P>0.05). PD-L1-positive patients with higher CTC and M-CTC counts had relatively poorer prognoses (P≤0.05), while the number of E-CTCs had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the CTC count (P≤0.05), while E-CTC and M-CTC counts did not significantly differ between the two groups (P>0.05). The PD-L1-positive group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs compared with the PD-L1-negative group (P≤0.05), while PD-L1+ E-CTC counts did not differ significantly between the two groups (P>0.05). The PD-L1-positive patients with a higher number of PD-L1+ CTCs and PD-L1+ M-CTCs had relatively poorer prognoses (P≤0.05), while the PD-L1+ E-CTC count had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs (P≤0.05), while PD-L1+ E-CTC counts did not significantly differ between the two groups (P>0.05). Based on univariate and multivariate analyses, the number of PD-L1+ M-CTCs was identified as an independent prognostic factor for NSCLC. In conclusion, the presence of CTCs in peripheral blood, particularly PD-L1+ M-CTC subtype, indicated poorer clinical staging and prognosis in patients with NSCLC. These findings suggested that CTCs, specifically the PD-L1+ M-CTC subtype, could serve as a monitoring indicator for the clinical staging and prognosis of patients with NSCLC.

Investigating the Influence of Preoperative Trans Arterial Embolization (TAE) and Predictive Potential of Circulating Tumor Cells (CTCs) in Prognosis of Hepatocellular Carcinoma

IntroductionCirculating Tumor Cells are a promising biomarker in many malignancies. CTC dissemination during the operative procedure can lead to disease recurrence. The effect of preoperative Transarterial Embolization on the release of CTCs and miRNA panels and oncological outcomes in large hepatocellular carcinomas has been evaluated. Materials and MethodsThe study included non-metastatic HCC >5cm in size, that were completely resected after TAE (n=10). Blood was collected pre-TAE, post-TAE, postoperative (day 2,30 and 180) and analyzed for the presence of CTC and miRNA (miR885-5p, miR22-3p, miR 642b-5p). The samples were subjected to CTC enrichment, isolation and staining using the markers CD45, EpCAM and Cytokeratin (CK). The data was analyzed using Gene Expression Suite software. ResultsThe CTC enumeration resulted in three groups: Group 1- CTC present at both Pre TAE and postoperative day 30 (n=4), Group 2- CTC present Pre-TAE and clearing at postoperative day 30 (n=2), Group 3- No CTC detected at any stages (n=3). Group 2 patients had better survival as compared to the other groups. Downregulation of miRNA 22-3p also had favourable prognostic implications. ConclusionWhile preoperative TAE does not seem to impact CTC shedding, CTC clearance may prove to be a valuable biomarker in prognosticating HCC. A larger study to evaluate the significance of CTCs as a prognostic marker is warranted to further evaluate these findings.

Abstract PO5-16-05: Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, comprising only between 1-5% of all breast cancer cases in the United States. Despite its low incidence, IBC accounts for 10% of breast cancer-related deaths and is often diagnosed with metastasis, resulting in shorter survival compared to non-IBC cases. The 5-year relative survival rate for all stages of IBC is 39%, dropping to 19% for patients with distant metastasis. Liquid biopsy-based biomarkers have emerged as potential analytes for predicting prognosis and monitoring response to therapies in IBC patients. We explored the prognostic role of liquid biopsy encompassing both CTCs and cfDNA in the blood of patients with stage III/IV IBC and their clinical outcomes. Methods: CTC and cfDNA analysis was performed on 90 blood samples from 47 patients with stage III and IV IBC. Samples were collected before (pre-treatment, n=43) initiation and after (post-treatment, n=47) completion of neoadjuvant chemotherapy. CTCs were enriched and enumerated using the CellSearch® platform (Menarini-Silicon Biosystems). CfDNA was extracted from plasma using MagMax™ Cell-Free Total Nucleic Acid Isolation Kit (Applied Biosystems™) and quantified using the Qubit™ dsDNA High Sensitivity kit (Invitrogen™). Differences in cfDNA concentrations were used to plot a receiver operating characteristic (ROC) curve which was then used to determine Area Under the Curve (AUC) and a cfDNA cut-off point. ROC cut-off was calculated based on Youden Index J. Survival analysis was performed by the Kaplan-Meier estimator and Cox regression models. P-values were measured using the Student t-test. Results: Prior to neoadjuvant treatment, CTC was detected in 51% of patient samples, and the median cfDNA concentration was 5.87 ng/mL (range 0.95-24.11 ng/mL). After completion of neoadjuvant treatment, CTC was detected in 25% of patient samples, and the median cfDNA concentration was 11.19 ng/mL (range 3.41-88.13 ng/mL). The cut-off points determined for pre-treatment and post-treatment cfDNA concentration was &amp;gt;4.2 ng/mL and &amp;gt;9.5 ng/mL respectively. At pre-treatment, CTC detection and cfDNA concentration &amp;gt;4.2 ng/mL were independently associated with disease relapse (P=0.007, 0.016, respectively). The combination of CTC detection and cfDNA concentration &amp;gt;4.2 ng/mL was significantly associated with disease relapse (P=0.0004), decreased relapse free survival (RFS, P=0.005) and overall survival (OS, P=0.007). At the post-neoadjuvant treatment time point, the combination of CTC detection and cfDNA concentration &amp;gt;9.5 ng/mL was significantly associated with relapse regardless of pCR status (P=0.02). Conclusion: The combined presence of CTCs and elevated cfDNA concentrations in patients with stage III/IV IBC after completion of neoadjuvant chemotherapy was associated with disease relapse regardless of pCR status. A combination of elevated cfDNA and CTC detection signatures after completion of neoadjuvant chemotherapy may be useful for risk stratification and adjuvant systemic therapy planning. Citation Format: Sridevi Addanki, Salyna Meas, Vanessa Sarli, Abdulkader Almosa, Wendy Woodward, Dhruvajyoti Roy, Anthony Lucci. Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-05.

CTC together with Shh and Nrf2 are prospective diagnostic markers for HNSCC

BackgroundThe lack of appropriate prognostic biomarkers remains a significant obstacle in the early detection of Head and Neck Squamous Cell Carcinoma (HNSCC), a cancer type with a high mortality rate. Despite considerable advancements in treatment, the success in diagnosing HNSCC at an early stage still needs to be improved. Nuclear factor erythroid 2-related factor 2 (Nrf2) and Sonic Hedgehog (Shh) are overexpressed in various cancers, including HNSCC, and have recently been proposed as possible therapeutic targets for HNSCC. Circulating Tumor Cell (CTC) is a novel concept used for the early detection of cancers, and studies have suggested that a higher CTC count is associated with the aggressiveness of HNSCC and poor survival rates. Therefore, we aimed to establish molecular markers for the early diagnosis of HNSCC considering Shh/Nrf2 overexpression in the background. In addition, the relation between Shh/Nrf2 and CTCs is still unexplored in HNSCC patients.MethodsIn the present study, we selected a cohort of 151 HNSCC patients and categorized them as CTC positive or negative based on the presence or absence of CTCs in their peripheral blood. Data on demographic and clinicopathological features with the survival of the patients were analyzed to select the patient cohort to study Shh/Nrf2 expression. Shh and Nrf2 expression was measured by qRT-PCR.ResultsConsidering significant demographic [smoking, betel leaf (p-value < 0.0001)] and clinicopathological risk factors [RBC count (p < 0.05), Platelet count (p < 0.05), Neutrophil count (p < 0.005), MCV (p < 0.0001), NLR (p < 0.05), MLR (p < 0.05)], patients who tested positive for CTC also exhibited significant overexpression of Shh/Nrf2 in both blood and tissue compared to CTC-negative patients. A strong association exists between CTCs and tumor grade. Following chemotherapy (a combination of Cisplatin, 5FU, and Paclitaxel), the frequency of CTCs was significantly decreased in patients with HNSCC who had tested positive for CTCs. The Kaplan–Meier plot illustrated that a higher number of CTCs is associated with poorer overall survival (OS) in patients with HNSCC.ConclusionsDetecting CTCs, and higher expression of Shh and Nrf2 in HNSCC patients’ blood, can be a promising tool for diagnosing and prognosticating HNSCC.

Trastuzumab and first-line taxane chemotherapy in metastatic breast cancer patients with a HER2-negative tumor and HER2-positive circulating tumor cells: a phase II trial

PurposeHER2 overexpressing circulating tumor cells (CTCs) are observed in up to 25% of HER2-negative metastatic breast cancer patients. Since targeted anti-HER2 therapy has drastically improved clinical outcomes of patients with HER2-positive breast cancer, we hypothesized that patients with HER2 overexpressing CTCs might benefit from the addition of trastuzumab to chemotherapy.MethodsIn this single-arm, phase II trial, patients with HER2-positive CTCs received trastuzumab as addition to first-line treatment with taxane chemotherapy. Patients with detectable CTCs but without HER2 overexpression that received taxane chemotherapy only, were used as control group. The primary outcome measure was progression-free rate at 6 months (PFR6), with a target of 80%. In November 2022, the study was terminated early due to slow patient accrual.Results63 patients were screened, of which eight patients had HER2-positive CTCs and were treated with trastuzumab. The median number of CTCs was 15 per 7.5 ml of blood (range 1–131) in patients with HER2-positive CTCs, compared to median 5 (range 1–1047) in the control group. PFR6 was 50% in the trastuzumab group and 54% in the taxane monotherapy group, with no significant difference in median PFS (8 versus 9 months, p = 0.51).ConclusionNo clinical benefit of trastuzumab was observed, although this study was performed in a limited number of patients. Additionally, we observed a strong correlation between the number of evaluable CTCs and the presence of HER2-positive CTCs. We argue that randomized studies investigating agents that are proven to be solely effective in the HER2-positive patient group in patients with HER2-positive CTCs and HER2-negative tissue are currently infeasible. Several factors contribute to this impracticality, including the need for more stringent thresholds, and the rapidly evolving landscape of cancer treatments.

Detection of Circulating Tumor Cells by Cell Block Technique in Malignant Tumors.

Cancer is a leading cause of death worldwide and is a major cause of morbidity. To deal with this magnitude of cancers and their diagnostic and prognostics, a multitude of prognostic biomarkers for various cancers have been explored over the decades, with detection of circulating tumor cells (CTCs) in the peripheral blood being one of them. This study was undertaken to explore the routine procedure of cell block in the cytopathology lab to isolate and detect CTCs. In this cross-sectional study, 112 peripheral blood samples sent for routine blood investigations of various cancer patients were utilized for the preparation of cell block. The sections from the cell block were stained routinely and evaluated for the presence of CTCs. The statistical analysis was done using Mac Statplus software version 8.0. The malignancies were tabulated as per the International Classification of Diseases for Oncology, third edition (ICD-O-3). The maximum number of cases were from C 50 (breast) - 41/112 (36.6%), followed by C15-C26 (Digestive organs) - 19/112 (16.9%), and C00-C14 (lip, oral cavity, and pharynx) - 18/112 (16.07%) cases. CTC was detected in six (5.35%) out of 112 cases, out of which three were from the breast and one each from category C6.9 (mouth), C32.0 (glottis), and C53.8 (cervix uteri). Among various advanced and molecular techniques available for the detection of CTCs, the cell block technique proves to be one of the effective methods, especially in resource-limited settings as these can further be utilized for additional diagnostic techniques similar to the ones employed for routine paraffin blocks.

A deep learning model predicts the presence of diverse cancer types using circulating tumor cells

Circulating tumor cells (CTCs) are cancer cells that detach from the primary tumor and intravasate into the bloodstream. Thus, non-invasive liquid biopsies are being used to analyze CTC-expressed genes to identify potential cancer biomarkers. In this regard, several studies have used gene expression changes in blood to predict the presence of CTC and, consequently, cancer. However, the CTC mRNA data has not been used to develop a generic approach that indicates the presence of multiple cancer types. In this study, we developed such a generic approach. Briefly, we designed two computational workflows, one using the raw mRNA data and deep learning (DL) and the other exploiting five hub gene ranking algorithms (Degree, Maximum Neighborhood Component, Betweenness Centrality, Closeness Centrality, and Stress Centrality) with machine learning (ML). Both workflows aim to determine the top genes that best distinguish cancer types based on the CTC mRNA data. We demonstrate that our automated, robust DL framework (DNNraw) more accurately indicates the presence of multiple cancer types using the CTC gene expression data than multiple ML approaches. The DL approach achieved average precision of 0.9652, recall of 0.9640, f1-score of 0.9638 and overall accuracy of 0.9640. Furthermore, since we designed multiple approaches, we also provide a bioinformatics analysis of the gene commonly identified as top-ranked by the different methods. To our knowledge, this is the first study wherein a generic approach has been developed to predict the presence of multiple cancer types using raw CTC mRNA data, as opposed to other models that require a feature selection step.

Ultrasensitive detection of circulating tumor cells via DNA walker driven by a DNA circuit synergized with MOF-on-MOF nanozyme

Circulating tumor cells (CTCs) monitoring as a means of liquid biopsy can be effective in aiding the diagnosis, treatment monitoring, and prognosis of cancer. Herein, we fabricated a dual-mode cytosensor for ultrasensitive detection of CTCs via DNA walker powered by a DNA circuit and MOF-on-MOF nanozyme. The anti-EpCAM aptamer was hybridized with complementary DNA (cDNA) and modified onto the magnetic nanospheres (MNs). In the presence of CTCs, cDNA strand can be released by cell-aptamer specific recognition and was separated through a magnet to trigger the subsequent DNA walking process. In the DNA walking process, the cDNA strand could trigger cascade toehold-mediated DNA displacement reactions by fuel nanozyme-functionalized DNA strands and recycle to further accelerate the DNA circuit thus amplifying the output signal. What’s more, since the MOF-on-MOF nanozyme had good peroxidase activity, electrochemical and colorimetric dual-mode signal output can be achieved in the presence of 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for CTCs determination through an ITO chamber. As a proof of concept, by virtue of the outstanding synergistic signal amplification of DNA walker and MOF-on-MOF nanozymes, the designed dual-mode cytosensor exhibited a comprehensive linear range of 5 to 5 × 105 cells/mL and a sensitivity of 5 cells/mL and performed well in complex environment. This work provided important references for further investigations on CTC-related diagnosis and individualized treatment.

Biological and Clinical Significance of Undetectable Circulating Tumor Cells (CTCs) in Patients (Pts) with Multiple Myeloma (MM)

Background: There is evidence suggesting that CTCs are responsible for the spreading of clonal plasma cells (PC) inside and outside the bone marrow (BM). However, some pts unexpectedly have no CTCs at diagnosis. Here, we hypothesize that the absence of CTC defines a specific MM subtype with unique biological and clinical features. To our knowledge, this has never been investigated. Aim: Define the frequency and the characteristics of MM pts with undetectable CTCs. Methods: The presence of CTCs was assessed by NGF in 1,687 blood samples from pts with MGUS (n=467), smoldering (SMM, n=368), newly diagnosed (NDMM, n=652), and relapsed refractory MM (RRMM, n=200). All pts were enrolled in prospective PETHEMA/GEM multicenter studies. BM clonal PC from pts with (n=239) and without (n=17) detectable CTCs were isolated by FACS prior exome and RNA sequencing. Results: The frequency of pts with undetectable CTCs progressively decreased ( p &amp;lt;.001) from MGUS (54%) to SMM (21%) and NDMM (8%) and RRMM (11%). In those with detectable CTCs, the median percentage was 0.0003, 0.002, 0.01 and 0.008, respectively ( p &amp;lt;.001). The 1-log increment observed in the progression from MGUS to SMM and NDMM underpins the link between CTC levels and malignant transformation. The similarity between NDMM and RRMM is probably related with the fact that the latter were analyzed in between different lines of therapy. When compared to other SMM pts, those with undetectable CTCs were more frequently staged with low risk according to the 20/2/20 IMWG model (32% vs 49%; p &amp;lt;.001). Absence of CTCs (HR: 0.38; p =.042) and the 20/2/20 IMWG model (HR: 0.46; p =.011) had independent prognostic value in a multivariate analysis. SMM pts with undetectable vs detectable CTCs showed lower risk of transformation (progression rates at 2 years of 7% vs 24%, p =.004). When compared to other NDMM pts, those with undetectable CTCs had significantly less anemia and BM infiltration. There were no differences in the incidence of lytic lesions, renal insufficiency and hypercalcemia. Only one pt without CTCs was staged with R-ISS 3. These findings, together with a higher frequency of plasmacytomas, suggest that the MM subtype with undetectable CTCs may include pts with macrofocal disease. In transplant eligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 7 years of 90% and 44% (HR: 0.14; p &amp;lt;.001), and OS rates of 97% and 72% (HR: 0.10; p =.02). In transplant ineligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 2 years of 83% and 54% (HR: 0.31; p =.02), and OS rates of 94% and 76% (HR: 0.33; p =.13). In a multivariate analysis including the R-ISS and transplant eligibility, the absence of CTCs showed independent prognostic value for PFS (HR: 0.25; p &amp;lt;.001) and OS (HR: 0.24; p =.014). Among pts with undetectable CTCs, there were no differences in PFS and OS according to the presence of standard vs high risk cytogenetic abnormalities. In contrast with pts having detectable CTCs, the PFS and OS of those without CTCs was not significantly reduced if CR or MRD negativity were not achieved after treatment. The cytogenetic profile of BM clonal PC from pts with undetectable (n=112) vs detectable (n=846) CTCs was characterized by a lower frequency of +1q21 (16% vs 45%, p &amp;lt;.001) and del(1p32) (2% vs 8%, p =.02). Other alterations were not significant. There was no correlation between the number of mutations and the percentage of CTCs. There were 66 differentially expressed genes (DEG) between BM clonal PC from pts with detectable vs undetectable CTCs. Of note, 9 of the 66 DEG were in Chr1, with up- and downregulation of genes in 1p and 1q, respectively. There was a significant correlation between CTC levels and the expression of genes associated with MM dissemination ( FLNA , LGALS1and CXCR4). These findings, together with the modest correlation between the percentage of CTCs and BM clonal PC determined by morphology (r=.45, p &amp;lt;.001) and NGF (r=.49, p &amp;lt;.001), suggest that the selective egress of tumor cells from BM into the PB might depend on specific genetic alterations and transcriptional priming more than tumor burden. Conclusions:This study uncovered that ~8% of MM pts may progress due to mechanisms different from CTC dissemination. This subgroup showed unique features and achieved unprecedented outcomes, particularly if eligible to intensive therapy. Accordingly, our results endorse the recognition of pts with undetectable CTCs as a new biological and clinical subtype of MM.