Evaluation Of Circulating Tumor Cells Research Articles

Abstract PO3-14-10: A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data

Abstract Background: The presence of liquid biomarkers such as circulating tumor cells (CTCs) among women undergoing neoadjuvant chemotherapy (NAC) for breast cancer may be associated with treatment response and/or an increased risk of recurrence, but limited data is available. Objectives: To detect and enumerate CTCs in blood samples from women with a new diagnosis of non-metastatic breast cancer of any subtype both i) at baseline (prior to commencing NAC), and ii) after completion of NAC and surgery using the Epic Sciences platform. Methods: Women with non-metastatic breast cancer of any subtype who have not yet commenced NAC were included, irrespective of age. Those with a prior history of another invasive cancer (apart from non-melanoma skin cancer identified 5+ years prior to enrollment) were excluded. Blood samples were obtained to measure CTCs prior to commencing NAC, and after completion of NAC and surgery (at least 4 weeks post-operatively). As previously described, CTC identification was based on immunofluorescence analysis using the Epic Sciences platform (Ueno et al 2017). Clinical/pathological data and clinical outcomes were abstracted from patients’ medical records. Associations between CTC detection and clinical/pathologic characteristics were evaluated using Fisher’s exact test for categorical variables and t-test or Wilcoxon rank sum tests for numerical variables. All analyses were performed using the R software package. Results: 50 participants who met eligibility criteria were included; a baseline blood sample was evaluable for CTC detection and enumeration for 47 patients. The median age at breast cancer diagnosis was 52 (29-75). Among them, 18 (38.3%) had HER2+ breast cancer, 17 (36.1%) had hormone receptor (HR)+/HER2- breast cancer and 12 (25.5%) had triple negative breast cancer (TNBC). The majority of patients (91%) received anthracycline and taxane-based NAC. A total of 68 samples were tested for CTC enumeration (5 mL equivalent per sample) including 47 pre-treatment and 21 post-treatment samples. CTCs were detected in 37 (79%) of patients for whom a baseline (pre-NAC) sample was available with a mean of 4.2 (SD 17.0) CTCs/mL and a range of 0 - 115.4 CTCs/mL. Detection of CTCs at baseline was highest among patients with TNBC (n=10/12, 83%), followed by those with HER2+ (n=14/18, 78%) and HR+/HER2- (n=13/17, 76%) breast cancer. CTCs were detected in 43% (n=9/21) of post-treatment samples, with a mean of 1.2 (SD 3.6) CTCs/mL and range of 0 – 16.4 CTCs/mL. Among the 20 patients for whom matched pre- and post-treatment CTC results were available, 16 (80%) had detectable CTCs pre-treatment and 8 (40%) had detectable levels post-NAC and surgery; of the 8 patients with post-treatment CTCs, 4 (50%) had HR+/HER2- breast cancer, 2 had HER2+ (25%) and 2 (25%) had TNBC. Three patients had numerically higher CTC levels after completion of NAC and surgery compared to baseline levels, 2 of whom had HR+/HER2- breast cancer and one of whom had TNBC. To-date, only 8 patients (19% of 43 who have undergone surgery) have achieved a pathological complete response (PCR) to NAC, among whom 3 had matched pre- and post-treatment CTC results available. None of these 3 patients had detectable CTCs post treatment. Conclusions: Approximately 4 in 5 women with non-metastatic breast cancer who undergo NAC have detectable CTCs at baseline (pre-treatment) using the Epic Sciences Platform. Given that CTCs remain detectable in a high proportion (40%) of patients after NAC and surgery, evaluation of CTCs as a potential measure of minimal residual disease warrants further evaluation in this patient population. Citation Format: Rania Chehade, Arushi Jain, Veronika Moravan, Giuseppe Di Caro, Megan Slade, Nadine Hartmann, Rick Wenstrup, Ana Elisa Lohmann, William Tran, Katarzyna Jerzak. A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data [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 PO3-14-10.

Circulating Cancer-Associated Macrophage-like Cells as a Blood-Based Biomarker of Response to Immune Checkpoint Inhibitors.

Evidence has been provided that circulating cancer-associated macrophage-like cell (CAM-L) numbers increase in response to chemotherapy, with an inverse trend compared to circulating tumor cells (CTCs). In the era of evolving cancer immunotherapy, whether CAM-Ls might have a potential role as predictive biomarkers of response has been unexplored. We evaluated whether a serial blood evaluation of CTC to CAM-L ratio might predict response to immune checkpoint inhibitors in a cohort of non-small-cell lung cancer patients. At baseline, CTCs, CAM-Ls, and the CTC/CAM-L ratio significantly correlate with both progression-free survival (PFS) and overall survival (OS). The baseline CTC/CAM-L ratio was significantly different in early progressors (4.28 ± 3.21) compared to long responders (0.42 ± 0.47) (p = 0.001). In patients treated with immune checkpoint inhibitors, a CTC/CAM-L ratio ≤ 0.25 at baseline is associated with better PFS and OS. A baseline CTC/CAM-L ratio ≤ 0.25 is statistically significant to discriminate early progressions from durable response. The results of the present pilot study suggest that CAM-Ls together with CTCs could play an important role in evaluating patients treated with cancer immunotherapy.

Dynamic monitoring of PD-L1 and Ki67 in circulating tumor cells of metastatic non-small cell lung cancer patients treated with pembrolizumab.

Programmed cell death protein ligand-1 (PD-L1) expression in non-small cell lung cancer (NSCLC) tumors guides treatment selection. PD-L1 expression in circulating tumor cells (CTCs) may provide further information. We have explored PD-L1 and marker of proliferation Ki-67 (Ki67; also known as MKI67) in CTCs in longitudinal samples of 47 advanced NSCLC patients receiving pembrolizumab. A triple immunofluorescence, against cytokeratin, PD-L1, and Ki67, was performed on peripheral blood mononuclear cells, at baseline, post-first cycle, post-third, and primary resistance (PMR). Patients displaying PMR (progression at first evaluation) were classified as progressive disease (PD) and those with clinical benefit as disease control (DC). CTCs were categorized as PD-L1high/low/medium/negative and Ki67+ or Ki67- . CTC evaluation revealed a significant increase in the PD-L1low CTC rate at PMR compared to baseline (2.5% at baseline vs. 36.5% at PMR), whereas a reduction in the PD-L1high CTC rate was observed (31.5% vs. 0%, respectively). Investigation of CTC status between PD and DC patients showed that PD patients more frequently increased total and PD-L1low CTCs after first cycle compared to DC (83% of PD vs. 37% of DC and 67% of PD vs. 8% of DC, respectively). Progression-free survival (PFS) was longer in patients with decreased total and PD-L1low CTCs after first cycle compared to those with increased CTCs (median PFS: not reached vs. 2 months). PD-L1+ patients presenting a high Ki67 index (% Ki67+ CTCs > 30%) before treatment had a shorter PFS compared to those with a low Ki67 (≤ 30%), and overall survival (OS) was shorter in PD-L1+ patients harboring Ki67+ CTCs compared to those not presenting (median OS: 11.8 months vs. 33.1 months, respectively). In sequential samples of patients with a durable benefit, a low Ki67 index was observed. Our results suggest that monitoring PD-L1 and Ki67 expression in CTCs of NSCLC patients treated with pembrolizumab may be predictive for pembrolizumab efficacy.

Clinical Relevancy of Circulating Tumor Cells in Breast Cancer: Epithelial or Mesenchymal Characteristics, Single Cells or Clusters?

Metastatic breast cancer (MBC) is typically an incurable disease with high mortality rates; thus, early identification of metastatic features and disease recurrence through precise biomarkers is crucial. Circulating tumor cells (CTCs) consisting of heterogeneous subpopulations with different morphology and genetic, epigenetic, and gene expression profiles represent promising candidate biomarkers for metastatic potential. The experimentally verified role of epithelial-to-mesenchymal transition in cancer dissemination has not been clearly described in BC patients, but the stemness features of CTCs strongly contributes to metastatic potency. Single CTCs have been shown to be protected in the bloodstream against recognition by the immune system through impaired interactions with T lymphocytes and NK cells, while associations of heterotypic CTC clusters with platelets, leucocytes, neutrophils, tumor-associated macrophages, and fibroblasts improve their tumorigenic behavior. In addition to single CTC and CTC cluster characteristics, we reviewed CTC evaluation methods and clinical studies in early and metastatic BCs. The variable CTC tests were developed based on specific principles and strategies. However, CTC count and the presence of CTC clusters were shown to be most clinically relevant in existing clinical trials. Despite the known progress in CTC research and sampling of BC patients, implementation of CTCs and CTC clusters in routine diagnostic and treatment strategies still requires improvement in detection sensitivity and precise molecular characterizations, focused predominantly on the role of CTC clusters for their higher metastatic potency.

Circulating Tumor Cells for the Staging of Patients With Newly Diagnosed Transplant-Eligible Multiple Myeloma.

Patients with multiple myeloma (MM) may show patchy bone marrow (BM) infiltration and extramedullary disease. Notwithstanding, quantification of plasma cells (PCs) continues to be performed in BM since the clinical translation of circulating tumor cells (CTCs) remains undefined. CTCs were measured in peripheral blood (PB) of 374 patients with newly diagnosed MM enrolled in the GEM2012MENOS65 and GEM2014MAIN trials. Treatment included bortezomib, lenalidomide, and dexamethasone induction followed by autologous transplant, consolidation, and maintenance. Next-generation flow cytometry was used to evaluate CTCs in PB at diagnosis and measurable residual disease (MRD) in BM throughout treatment. CTCs were detected in 92% (344 of 374) of patients with newly diagnosed MM. The correlation between the percentages of CTCs and BM PCs was modest. Increasing logarithmic percentages of CTCs were associated with inferior progression-free survival (PFS). A cutoff of 0.01% CTCs showed an independent prognostic value (hazard ratio: 2.02; 95% CI, 1.3 to 3.1; P = .001) in multivariable PFS analysis including the International Staging System, lactate dehydrogenase levels, and cytogenetics. The combination of the four prognostic factors significantly improved risk stratification. Outcomes according to the percentage of CTCs and depth of response to treatment showed that patients with undetectable CTCs had exceptional PFS regardless of complete remission and MRD status. In all other cases with detectable CTCs, only achieving MRD negativity (and not complete remission) demonstrated a statistically significant increase in PFS. Evaluation of CTCs in PB outperformed quantification of BM PCs. The detection of ≥ 0.01% CTCs could be a new risk factor in novel staging systems for patients with transplant-eligible MM.

Circulating tumor cells in neuroblastoma: Current status and future perspectives.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 10% to 20% of deaths of pediatric malignancies. Due to the poor prognosis and significant biological heterogeneity of neuroblastoma, it is essential to develop personalized therapeutics and monitor treatment response. Circulating tumor cells (CTCs), as one of the important analytes for liquid biopsy, could facilitate response assessment and outcome prediction for patients in a non-invasive way. Several methods and platforms have been used for the enrichment and detection of CTCs. The enumeration of CTCs counts and evaluation of tumor-specific mRNA transcript levels could provide prognostic information at diagnosis, during or after chemotherapy, and during the process of disease progression. So far, studies into neuroblastoma CTCs are only in the preliminary stages. The quality-controlled large prospective cohort studies are needed to evaluate the clinical significance and statistical rigor of CTC detection methods. Moreover, there remains a lot to be explored and investigated in genotyping characterization of neuroblastoma (NB) CTCs and construction of in-vitro or in-vivo functional models. CTCs and circulating tumor DNA (ctDNA) analysis will be complementary in understanding tumor heterogeneity and evolution over the course of therapy for patients with NB in the future.

Liquid Biopsy Assessment of Circulating Tumor Cell PD-L1 and IRF-1 Expression in Patients with Advanced Solid Tumors Receiving Immune Checkpoint Inhibitor.

Reliable biomarkers that can be serially monitored to predict treatment response to immune checkpoint inhibitors (ICIs) are still an unmet need. Here, we present a multiplex immunofluorescence (IF) assay that simultaneously detects circulating tumor cells (CTCs) and assesses CTC expression of programmed death ligand-1 (PD-L1) and interferon regulatory factor 1 (IRF-1) as a candidate biomarker related to ICI use. To assess the potential of CTC PD-L1 and IRF-1 expression as candidate biomarkers for patients with advanced epithelial solid tumors receiving ICIs. We tested the IF CTC assay in a pilot study of 28 patients with advanced solid tumors who were starting ICI. Blood for CTC evaluation was obtained prior to starting ICI, after a single cycle of therapy, and at the time of radiographic assessment or treatment discontinuation. At baseline, patients with 0-1 CTCs had longer progression-free survival (PFS) compared to patients with ≥ 2 CTCs (4.3 vs 1.3 months, p = 0.01). The presence of any PD-L1+ CTCs after a single dose of ICI portended shorter PFS compared to patients with no CTCs or PD-L1- CTCs (1.2 vs 4.2 months, p = 0.02); the presence of any PD-L1+ or IRF-1+ CTCs at time of imaging assessment or treatment discontinuation also was associated with shorter PFS (1.9 vs 5.5 months, p < 0.01; 1.6 vs 4.7 months, p = 0.05). CTC PD-L1 and IRF-1 expression did not correlate with tumor tissue PD-L1 or IRF-1 expression. Strong IRF-1 expression in tumor tissue was associated with durable (≥1 year) radiographic response (p = 0.02). Based on these results, CTC PD-L1 and IRF-1 expression is of interest in identifying ICI resistance and warrants further study.

Design and numerical simulation analysis of three multifunctional microfluidic chips in isolation of CTCs

Circulating tumor cells (CTCs) are tumor cells that dissociate from the origin tumors and disseminate in the bloodstream and lymphatic circulation systems. CTCs carry a remarkably similar constituent to the original tumor, which makes CTCs have a significant value in the biological molecular analysis of cancer. However, CTCs are extremely rare and heterogeneous in blood. Therefore, CTC separation is highly challenging. In the present work, we designed a fan-shaped symmetric microfluidic chip, a multifunctional capturing and co-culture chip, and an immunomagnetic chip in isolation of CTCs. The first chip is composed of two-faced fans placed symmetrically. Inside, there are arc-shaped arrays composed of numerable circular microposts. CTCs could be captured by antibody-modified microposts, and blood constituents flow away. Pressure, velocity, streamline, and shear stress simulations were performed theoretically. The second chip is a multifunctional capturing and co-culture chip. Patient blood samples are filtered by a W-shaped structure, and isolated viable CTCs could co-culture with several normal cells of the same type to mimic the real microenvironment. The third microfluidic chip is a combination of both immunomagnetic and physical-based. Many electronic coils are wound around the chip to produce a strong magnetic field when electrified. CTCs are incubated with magnetic beads. Magnetized CTCs would be oriented to be enriched. Those CTCs without conjugating beads would be captured by two interlace arrays of square microposts. These three delicate designs and theoretical explanation clarify feasibility for further experiments of CTC enumeration, clinical analysis, and evaluation of cancer therapy.

Circulating Tumor Cells (CTCs) in Smoldering and Active Multiple Myeloma (MM): Mechanism of Egression, Clinical Significance and Therapeutic Endpoints

Circulating Tumor Cells (CTCs) in Smoldering and Active Multiple Myeloma (MM): Mechanism of Egression, Clinical Significance and Therapeutic Endpoints

Long-Term Outcomes of a Prospective Clinical Trial of Proton Craniospinal Irradiation for Leptomeningeal Metastasis

Leptomeningeal metastasis (LM) is associated with limited survival and treatment options for patients with solid tumor malignancies. While photon involved-field radiotherapy (IFRT) is effective for local palliation, it lacks durability. Craniospinal irradiation (CSI), in contrast, treats the entire central nervous system (CNS) compartment, thus potentially improving disease control. We previously reported the safety and tolerability of proton CSI (pCSI) in a completed phase IB study, and we are now reporting the mature survival outcomes of these patients.We enrolled 24 patients with solid tumor LM to receive hypofractionated pCSI of 30 Gy (RBE) in 10 fractions in this phase I prospective trial between June 2018 and May 2019 (NCT03520504). The primary endpoint is dose-liming toxicity (DLT) within 1 month of treatment. Secondary endpoints, including time to CNS progression (CNS TTP), CNS progression-free survival (PFS), and overall survival (OS), were assessed using Kaplan-Meier analysis with median survival estimates and 95% confidence intervals (CI) reported. Correlations between cerebrospinal fluid (CSF) circulating tumor cells (CTCs) and survival outcomes were evaluated using Kaplan-Meier analysis. An optimal cutoff for CTCs was determined through maximally selected rank statistics using CNS PFS.Of the 24 patients enrolled, 3 did not complete treatment due to systemic disease progression and were excluded from this analysis. The majority of patients had metastatic lung (52%) and breast (33%) malignancies. The median age was 52 years (range 30-67 years) and the median Karnofsky performance status was 70 (range 60-90). The majority of patients had LM that progressed on other therapies prior to enrollment (57% vs. 43% of patients with newly diagnosed LM), and all patients with molecularly driven disease had CNS progression on small-molecule tyrosine kinase inhibitors prior to enrollment. Only 1 patient was censored at 24 months of follow-up, while 20 patients were followed until death with a median OS of 9 months (95% CI 6-22 months). The median CNS TTP was 8 months (95% CI 7-18 months), with 5 (24%) patients with durable CNS control for > 12 months after pCSI. The median CNS PFS was 7 months (95% CI 5-13 months). In the 18 patients with CSF CTCs evaluation at baseline, having a pre-pCSI CSF CTCs < 41/3mL was associated with improved CNS TTP (19 vs. 8 months, P < 0.01), CNS PFS (18 vs. 6 months, P < 0.01), and a trend toward improved OS (21 vs. 8 months, P = 0.06).We reported that pCSI is a safe treatment for patients with solid tumor LM. In this analysis, we demonstrated durable disease control and prolonged survival in some patients. Lower CSF CTCs at baseline, a potential biomarker of response, was associated with improved survival outcomes. An ongoing randomized phase II trial will determine the efficacy of pCSI compared to photon IFRT in patients with solid tumor LM.

Evaluation of circulating tumor cells in colorectal cancer using flow cytometry.

ObjectiveWe aimed to evaluate the prognostic value of circulating tumor cells (CTCs) and the impact of intraoperative tumor manipulation on CTCs in colorectal cancer (CRC) patients.MethodsWe performed a prospective study on 40 patients with CRC stages I to IV who received curative surgery using the no-touch technique. Flow cytometry was used to identify CTCs in peripheral blood samples (4 mL/sample) collected at two surgical moments: skin incision (T1) and after surgical resection (T2). A threshold of ≥4 CTCs/4 mL blood was established for considering patients CTC positive.ResultsIn the univariate analysis, CTC evaluation at T2 was correlated with female sex, vascular invasion, tumor localization in the colon and metastatic lymph nodes. In the multivariate analysis, only female sex and colon cancer maintained statistical significance. At a medium follow-up of 15 months (1–25 months), the mortality rate was 10% (n = 4), with no significant differences between the overall survival of T1 or T2 CTC-positive and CTC-negative patients.ConclusionsFlow cytometry is a feasible CTC identification technique in CRC, and although surgical manipulation has no influence on CTC numbers, CTCs may serve as a prognostic and predictive factor.

Inertial-Assisted Immunomagnetic Bioplatform towards Efficient Enrichment of Circulating Tumor Cells.

Serving as an effective biomarker in liquid biopsy, circulating tumor cells (CTCs) can provide an accessible source for cancer biology study. For the in-depth evaluation of CTCs in cancer analysis, their efficient enrichment is essential, owing to their low abundance in peripheral blood. In this paper, self-assembled immunomagnetic beads were developed to isolate CTCs from the ordered bundles of cells under the assistance of the spiral inertial effect. Parametric numerical simulations were performed to explore the velocity distribution in the cross section. Based on this chip, rare CTCs could be recovered under the throughput of 500 μL/min, making this device a valuable supplement in cancer analysis, diagnostics, and therapeutics.

Abstract PS6-15: Mammaprint risk stratification associated with presence of circulating tumor cells in breast cancer

Abstract Background: We previously reported the prognostic and predictive value of circulating tumor cells (CTCs) in early and late-stage breast cancer (BC). While the evaluation of CTCs via liquid biopsy in oncology practice guidelines is still evolving, other biomarkers are more commonly used, including multigene sequencing. The 21-gene Oncotype DX (ODX) assay predicts a risk of disease recurrence in patients with estrogen receptor (ER)-positive disease. The 70-gene MammaPrint (MP) panel, on the other hand, predicts a risk of early metastasis, and was developed using ER- and ER+ samples. We sought to understand whether these genomic assays predicted the presence of CTCs. Methods: A cohort of patients with BC and CTC data (i.e. positive or negative) was identified in this retrospective study using the National Cancer Database (NCDB) 2004-2016 registry. Clinicopathologic characteristics, including ODX and MP data, were described using frequencies/proportions. At the univariate level, chi-squared tests were used to evaluate for an association between the results of ODX (low-risk as recurrence score of &amp;lt;11, intermediate-risk as 11-25, and high-risk as &amp;gt;25), or MP (low or high risk), and the presence of CTCs. These variables were then assessed at the multivariate level using multiple logistic regression, controlling for other variables we previously showed predicted CTCs, including: race, receptor status, histology and AJCC clinical disease stage. Analyses were performed using SPSS version 26.0. Results: A total of n=4577 patients were evaluated for CTCs: n=940 (20.5%) of which underwent ODX testing, while n=282 (6.2%) underwent MP. Chi-squared analyses showed no association between ODX risk categories and the presence of CTCs (p=0.204), but a significant association between MP and CTCs (p=0.005). Through multivariate analysis, we found the association between a high-risk designation as per MP and the presence of CTCs remained significant (OR 2.65, 95% CI 1.23-5.70, p=0.013), even after controlling for race, receptor status, histology, and AJCC clinical disease stage. A similar multivariate model including ODX as a predictor of CTCs confirmed non-significance (p=0.664). Of patients with MP and CTC data, the majority were 50-70 years of age (n=164, 58.2%), White (n=244, 88.1%), with ductal carcinoma (n=211, 74.8%) on histological evaluation (Table 1). N=12 (4.6%) were diagnosed at stage 0, n=173 (66.0%) at stage I, n=63 (24.0%) at stage II, n=7 (2.7%) at stage III, and n=7 (2.7%) at stage IV. Most were hormone receptor-positive (HR+) and HER2-negative (HER2-) (n=234, 86.3%); n=11 (4.1%) were HR+/HER2+, n=5 (1.8%) were HR-/HER2, while n=21 (7.7%) were triple-negative breast cancers (TNBC). N=62 (22.0%) were assigned high-risk as per MP, while n=220 (78.0%) were assigned low-risk. Conclusion: High-risk of early metastasis, per MammaPrint, was significantly associated with the presence of CTCs. However, a significant association was not observed between Oncotype DX risk stratification and CTCs. This may suggest clinical utility in combining ODX metrics with liquid biopsy in order to maximize survival prognostication, when ODX is utilized as the biomarker. Further evaluation will be required to understand if the MP assay’s clinical value contribution is independent of CTC status. Table 1: Multiple logistic regression model predicting presence of CTCs in patients with breast cancer.VariableCirculating tumor cellsNegative (n=211)Positive (n=71)OR*95% CIp-valueMammaPrintLow-risk (ref)173 (61.3%)47 (16.7%)1--High-risk38 (13.5%)24 (8.5%)2.651.23-5.700.013**Age&amp;lt;5045 (16.0%)23 (8.2%)---50-70126 (44.7%)38 (13.5%)&amp;gt;7040 (14.2%)10 (3.5%)Race0.993White (ref)182 (65.7%)62 (22.4%)1--Black18 (6.5%)7 (2.5%)0.930.29-2.990.903Asian7 (2.5%)1 (0.4%)0.000.00-0.000.999AJCC clinical staging0.0910 (ref)10 (3.8%)2 (0.8%)1--I136 (51.9%)37 (14.1%)0.600.10-3.560.572II49 (18.7%)14 (5.3%)0.550.09-3.470.526III1 (0.4%)6 (2.3%)10.910.61-194.790.104IV3 (1.1%)4 (1.5%)3.670.273-49.520.327Estrogen receptor statusNegative (ref)21 (7.5%)6 (2.1%)1--Positive190 (67.6%)64 (22.8%)2.190.42-11.270.350Progesterone receptor statusNegative (ref)39 (13.9%)11 (3.9%)1--Positive172 (61.2%)59 (21.0%)1.550.49-4.890.457HER2 receptor statusNegative (ref)192 (69.6%)61 (22.1%)1--Positive16 (5.8%)7 (2.5%)1.580.53-4.700.412Histology0.503Ductal (ref)162 (57.4%)49 (17.4%)1--Lobular32 (11.3%)21 (7.4%)1.600.73-3.530.241Other17 (6.0%)1 (0.4%)0.00NE-NE0.998*OR predicting presence of CTCs. **Age not included in the multivariate model. Citation Format: Nadeem Bilani, Leah Elson, Hong Liang, Elizabeth Elimimian, Zeina Nahleh. Mammaprint risk stratification associated with presence of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-15.

Detection of EGFR Mutations in cfDNA and CTCs, and Comparison to Tumor Tissue in Non-Small-Cell-Lung-Cancer (NSCLC) Patients.

Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutations, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks, are not always successful and give results relevant to the timepoint of the assay. To detect secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKIs and lead to the administration of a 3rd generation drug, effective and non-invasive monitoring of EGFR mutation status is needed. Liquid biopsy analytes, such as circulating tumor cells (CTCs) and circulating tumor DNA (cfDNA), allow such monitoring over the course of the therapy. The aim of this study was to develop and optimize a workflow for the evaluation of cfDNA and CTCs in NSCLC patients all from one blood sample. Using Vortex technology and EntroGen ctEGFR assay, EGFR mutations were identified at 0.5 ng of DNA (∼83 cells), with a sensitivity ranging from 0.1 to 2.0% for a total DNA varying from 25 ng (∼4 CTCs among 4000 white blood cells, WBCs) to 1 ng (∼4 CTCs among 200 WBCs). The processing of plasma-depleted-blood provided comparable capture recovery as whole blood, confirming the possibility of a multimodality liquid biopsy analysis (cfDNA and CTC DNA) from a single tube of blood. Different anticoagulants were evaluated and compared in terms of respective performance. Blood samples from 24 NSCLC patients and 6 age-matched healthy donors were analyzed with this combined workflow to minimize blood volume needed and sample-to-sample bias, and the EGFR mutation profile detected from CTCs and cfDNA was compared to matched tumor tissues. Despite the limited size of the patient cohort, results from this non-invasive EGFR mutation analysis are encouraging and this combined workflow represents a valuable means for informing therapy selection and for monitoring treatment of patients with NSCLC.

Circulating tumor cells and drug history in primary breast cancer patients.

Aim: Different types of chronic medication may affect breast cancer prognosis. Circulating tumor cells (CTCs) play an important role in cancer metastasis formation. There is no evidence of how chronic medication affects CTCs and breast cancer prognosis. The aim of this study was to evaluate association between chronic medication and CTCs in patients with primary breast cancer.Methods: This study involved 414 patients with stage I-III primary breast cancer. Chronic drug history was collected from patients’ medical records and included all drugs that were prescribed for patients over at least the last 6 months prior to CTCs evaluation. CTCs were detected using a quantitative real-time polymerase chain reaction (qRT-PCR)-based method at the time of breast surgery.Results: There was no association between CTCs, including their different subpopulations and chronic medication. Chronic medication using angiotensin-converting-enzyme inhibitors (ACEi), metformin, and insulin were associated with inferior disease-free survival (HR = 0.49, 95%CI 0.26-0.94, P = 0.007 for ACEi; HR = 0.27, 95%CI 0.08-0.91, P < 0.001 for metformin; and HR = 0.12, 95%CI 0.01-2.91, P < 0.001 for insulin) and this was most pronounced in patients with epithelial to mesenchymal transition (CTC_EMT) phenotype. In multivariate analysis, chronic administration of metformin and/or insulin was an independent predictor of inferior outcome.Conclusion: Our findings show that there was no association between chronically used medication and CTCs in primary breast cancer patients. However, administration of ACEi, metformin, and/or insulin could negatively affect prognosis of patients with CTC_EMT.

Circulating Tumor Cells: Beyond Isolation and Detection

Circulating tumor cells (CTCs) are the precursors to metastases and increased numbers of CTCs in the peripheral circulation have been shown to correlate with decreased progression-free and overall survival. Although the current clinical utility has been focused on the prognostic significance, other clinical applications are being explored, such as determining if a patient is a candidate for treatment, determining the efficacy of treatment, evaluation for resistance to therapy, prediction of metastatic site, or as an early predictor of metastases. Current methodologies are based on quantifying CTCs and include technologies based on physical, immunological, and molecular techniques. However, these have limitations, of which most of them do not have the ability to perform morphological evaluation. Using morphological evaluation, CTCs in body fluids could be used for primary diagnosis in the setting of cancer of unknown primary (CUP) or in initial or early diagnostic scenarios. Additionally, cytological specimens have been shown to be useful for ancillary testing in patients when surgical resection specimens or biopsies are not available. Evaluation of CTCs should incorporate histological, immunehistochemical, and molecular characterization to enable clinicians to obtain the comprehensive diagnostic, prognostic and therapeutic information necessary to provide appropriate personalized care to cancer patients.

ES25.01 Liquid Biopsy: State of the Science

ES25.01 Liquid Biopsy: State of the Science

Circulating Tumor Cells in Right- and Left-Sided Colorectal Cancer.

Molecular alterations are not randomly distributed in colorectal cancer (CRC), but rather clustered on the basis of primary tumor location underlying the importance of colorectal cancer sidedness. We aimed to investigate whether circulating tumor cells (CTC) characterization might help clarify how different the patterns of dissemination might be relative to the behavior of left- (LCC) compared to right-sided (RCC) cancers. We retrospectively analyzed patients with metastatic CRC who had undergone standard baseline CTC evaluation before starting any first-line systemic treatment. Enumeration of CTC in left- and right-sided tumors were compared. The highest prognostic impact was exerted by CTC in left-sided primary cancer patients, even though the lowest median number of cells was detected in this subgroup of patients. CTC exhibit phenotypic heterogeneity, with a predominant mesenchymal phenotype found in CTC from distal compared to proximal primary tumors. Most CTC in RCC patients exhibited an apoptotic pattern. CTC in left-sided colon cancer patients exhibit a predominant mesenchymal phenotype. This might imply a substantial difference in the biology of proximal and distal cancers, associated with different patterns of tumor cells dissemination. The poor prognosis of right-sided CRC is not determined by the hematogenous dissemination of tumor cells, which appears to be predominantly a passive shedding of non-viable cells. Conversely, the subgroup of poor-prognosis left-sided CRC is reliably identified by the presence of mesenchymal CTC.

Morphologic and genomic characterization of circulating tumor cells in patients with metastatic colorectal cancer treated with combinational treatment of andecaliximab, bevacizumab and chemotherapy.

e14753 Background: The liquid biopsy is a noninvasive route to evaluate circulating tumor cells (CTCs) during the course of treatment to gain understanding of tumor biology, with potential prognostic utility. CTCs could serve as a predictive biomarker while aiding in the identification of resistance mechanisms to treatment through single cell genomic and proteomic analysis providing a longitudinal snapshots of tumor heterogeneity. Methods: Through the use of the high definition single cell assay (HD-SCA) workflow, we characterized the rare circulating cells to determine prognostic value of the liquid biopsy in monitoring the efficacy of andecaliximab in a combinational treatment as 1st or 2nd line therapy in patients with metastatic colorectal cancer (mCRC). 174 samples from 95 patients were analyzed to determine the significance of CTCs during treatment. Results: HD-CTCs were detected in 31% of samples, with 41 (43%) patients being CTC positive in at least 1 timepoint during the study. Patients receiving 1st line therapy presented with a median of 0 (range 0-346.04) and a mean of 9.49 (±14.06) HD-CTC/mL at baseline (BL). At initiation of 2nd line therapy, patients presented with a median of 0 HD-CTC/mL (range 0-277.37) and a mean of 10.94 (±15.32). There was no association between BL HD-CTC/mL and response, but for the 20 patients with &gt; 1 HD-CTC at BL, there was a trend toward response for higher HD-CTC/mL (non-response: mean 2.8, n = 12; response: mean 89.7, n = 8; p = .04). The 3 patients with &gt; 10 HD-CTC/mL at BL had undetectable HD-CTC on-treatment, which accompanied radiologic partial response; however, the 2 patients with complete radiological response had no HD-CTC detected at BL. In case studies, treatment pressure led to an observable change in HD-CTC morphology and genomic instability (single cell CNV analysis), suggesting these parameters may inform prognosis. Conclusions: Characterization of CTCs from patients with mCRC is feasible and may provide prognostic information to guide clinical decision making. Further evaluation of CTCs for pharmacodynamics and clinical monitoring in patients with mCRC is warranted.

Circulating tumor cell investigation in breast cancer patient-derived xenograft models by automated immunofluorescence staining, image acquisition, and single cell retrieval and analysis

BackgroundBreast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®–CyteFinder® system.MethodsCTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation.ResultsThe recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method.ConclusionsAnalysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte–CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression.