BIOM-02. LEPTOMENINGEAL DISEASE SECONDARY TO MELANOMA: UPDATES ON THE VALIDITY OF THE VERIDEX CELLSEARCH SYSTEM

BACKGROUND Leptomeningeal disease (LMD) is devastating with a median survival of 8-10 weeks without treatment. LMD affects approximately 5% to 25% of melanoma patients. Its pathophysiology remains unknown and effective treatments are virtually non-existent. The primary aim of this study was to evaluate the validity of Veridex CellSearch® System (VCS) compared to Gold Standard test (i.e., CSF cytology). MATERIALS AND METHODS A retrospective chart review was performed of subjects with suspected LMD from melanoma enrolled in the MCC 19332/19648 at Moffitt Cancer Center. Patients underwent standard of care with different treatments as deemed appropriate by treating physician. CSF samples were obtained from lumbar punctures, surgeries, and Ommaya reservoir. CSF was evaluated for quantification of CSF circulating tumor cells (CTCs) with the Veridex CellSearch® System (VCS). RESULTS Forty-eight patients were identified with melanoma as primary tumor, ages 29-80. Twenty-seven had LMD (median age 62) with median KPS 70. N=19 (70%) were diagnosed radiographically and n=5 (19%) with CSF cytology; n=14 (54%) had positive cytology on first LP. From 24 patients with LMD who underwent VCS, n=22 (92% patients had positive CSF CTCs. Number of CTCs/mL CSF was significantly higher in patients with LMD versus in patients without LMD (mean SD 227.6 vs. 0.07, p < 0.001). VCS sensitivity and specificity was analyzed. AUC was 0.515, with TPR 0.250 and FPR 0.286. CSF analysis and treatments were described. The median survival of those with LMD was 2.7 months. CONCLUSION These results indicate the potential value of the VCS as an additional tool to the gold standard in the diagnosis of LMD in patients with high suspicion of the disease. Future directions involve doing prospective studies to further validate this method, and to better understand this patient population to enhance diagnostic tools and management of LMD.

Development of Imaging and Liquid Biomarker Analysis for Breast Cancer Screening: A Review

Breast cancer screening tests could reduce the mortality rates for breast cancer patients. Screening and detection are the keystone of cancer prevention and may significantly minimize the death rates in breast cancer patients for long-term. In this review, we would like to present a comprehensive summary from recent publications of the current development for breast cancer screening, classification of breast cancer based on pathological diagnosis, as well as development of breast cancer detection. The sources of the articles were collected from research published in the PubMed, NCBI databases and manual searches without time restriction based on review of the title, abstract and full review of the articles, using the keywords "breast cancer", "diagnostic", "screening", "imaging", "biomarker" and the combination of these terms. The criteria excluded in selecting references were articles that are not written in English, newspapers, and posters. Of the 146 articles that were selected, there were 103 articles included. Breast cancer screening consists of imaging and pathological assessment such as invasive biopsies of tumor tissue and measurement of biomarkers. The recent development of breast cancer screening utilizing different models and methods like biomarkers were being reviewed. For imaging methods, there are mammography, digital breast tomosynthesis (3D mammography), magnetic resonance imaging (MRI), and ultrasonography. For pathological assessment, there are primary biomarker analysis for breast cancer (estrogen receptor, progesterone receptor, HER2, KI67 index) and liquid biomarker analysis from blood or saliva samples. Additionally, there are some diagnostic kit models for breast cancer screening that were in use such as NanoString nCounter®, MammaTyper®, CellSearch System™, and AdnaTest BreastCancer™. Each of these methods has its own limitations. Therefore, the development of breast cancer models should be more sensitive, reliable, approachable and less harmful.

Integration of circulating tumor cell and neutrophil-lymphocyte ratio to identify high-risk metastatic castration-resistant prostate cancer patients

Background The neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and circulating tumor cells (CTCs) have been associated with survival in castration-resistant prostate cancer (CRPC). However, no study has examined the prognostic value of NLR and PLR in the context of CTCs. Methods Baseline CTCs from mCRPC patients were enumerated using the CellSearch System. Baseline NLR and PLR values were calculated using the data from routine complete blood counts. The associations of CTC, NLR, and PLR values, individually and jointly, with progression-free survival (PFS) and overall survival (OS), were evaluated using Kaplan-Meier analysis, as well as univariate and multivariate Cox models. Results CTCs were detected in 37 (58.7%) of 63 mCRPC patients, and among them, 16 (25.4%) had ≥5 CTCs. The presence of CTCs was significantly associated with a 4.02-fold increased risk for progression and a 3.72-fold increased risk of death during a median follow-up of 17.6 months. OS was shorter among patients with high levels of NLR or PLR than those with low levels (log-rank P = 0.023 and 0.077). Neither NLR nor PLR was individually associated with PFS. Among the 37 patients with detectable CTCs, those with a high NLR had significantly shorter OS (log-rank P = 0.024); however, among the 26 patients without CTCs, the OS difference between high- and low-NLR groups was not statistically significant. Compared to the patients with CTCs and low NLR, those with CTCs and high levels of NLR had a 3.79-fold risk of death (P = 0.036). This association remained significant after adjusting for covariates (P = 0.031). Combination analyses of CTC and PLR did not yield significant results. Conclusion Among patients with detectable CTCs, the use of NLR could further classify patients into different risk groups, suggesting a complementary role for NLR in CTC-based prognostic stratification in mCRPC.

Relationship between circulating tumor cells and ZEB1 expression in tumor cells in colorectal cancer.

e15509 Background: Intravasation and circulation of tumor cells involves active invasion of cells with an enhanced migration potential as a result of the epithelial-mesenchymal transition (EMT). The ZEB1 protein is one of the key regulators of this process. Our purpose was to evaluate the association between the amount of circulating tumor cells (CTCs) in the peripheral blood of patients with different stages of colorectal cancer and the expression of ZEB1 by tumor cells. Methods: The study included 299 patients (aged 42-86 years, mean age 64.2±1.7) with stage II-IV CRC T1-4N0-2M0-1; histologically verified G1-G3 adenocarcinoma in all patients. The numbers of CTCs were measured in the peripheral blood before surgery using the Veridex CellSearch system (Janssen). CTCs were registered taking into account morphological characteristics and expression of epithelial cell adhesion markers EpCAM, CD45, cytokeratins 8,18,19. The blood sample was evaluated according to the following criteria: 0 CTCs, 1-3 CTCs, and more than 3 CTCs. Tissues of surgically removed tumors were studied with IHC analysis using rabbit polyclonal anti-ZEB1 antibodies (Biorbyt Ltd.) diluted 1:200 and the Reveal Polyvalent HRP-DAB Detection System. The percentage and the intensity of staining were assessed: 0, 1+ weak, 2+ moderate, 3+ strong. ZEB1 expression was considered positive when staining was detected in more than 10% (cut-off) tumor cells with intensities of 2+ and 3+. Statistical analysis of results was performed in the Statistica 13.0 program (StatSoftInc., USA). Results: From 1 to 402 CTCs were determined in 62.9% cases (in 188 of 299 patients); CTCs were not registered in 37.1% cases (111 of 299). Positive ZEB1 expression was observed in 80.6% (241 of 299 patients), while the negative one was much more rare – 19.4% (58 of 299 patients). The rates of CTC detection significantly increased with the positive ZEB1+ expression, compared to the negative expression (75.1% vs. 12.1%). CTCs >3 were detected in the blood in 39.0% in ZEB1+ tumors, but not in ZEB1- tumors. 1-3 CTCs were observed 3 times more often in ZEB1+ tumors (36.1% vs. 12.1; p≤0.05). Conclusions: Statistically significant association was revealed between the epithelial-mesenchymal transition marker ZEB1 expression by tumor cells and the amount of CTCs in the peripheral blood (p < 0.001).

Peripheral blood levels of tumor-infiltrating lymphocytes (TILs) and circulating tumor cells (CTCs) in patients with colorectal cancer.

e15510 Background: Tumor is a structure where malignant cells interact, among others, with cells of the adaptive and innate immune systems largely determining the tumor microenvironment. The role of TILs in the disease prognosis has been shown. The amount of CTCs is one of the factors that significantly affect the risk and rate of metastasis. The purpose of the study was to assess an association between TILs and CTCs in the peripheral blood of patients with various stages of colorectal cancer (CRC). Methods: The study included 299 patients (aged 42-86 years, mean age 64.2±1.7) with stage II-IV CRC T1-4N0-2M0-1. TILs were identified in tumor material after standard histological processing. Lymphocytes were counted per 100 epithelial cells in 5 fields of view at a magnification of x400; the result was expressed as a percentage ( < 5% weak, 5-30% moderate, >30% strong). The numbers of CTCs were measured in the peripheral blood using the Veridex CellSearch system (Janssen), taking into account the expression of epithelial cell adhesion markers EpCAM, cytokeratins 8,18,19 and absence of the CD45 expression. The blood sample was evaluated according to the following criteria: 0 CTCs, 1-3 CTCs, and more than 3 CTCs. Statistical analysis of results was performed in the Statistica 13.0 program (StatSoftInc., USA). Results: Weak lymphocytic infiltration was detected in 32.8%, moderate - in 48.1%, strong - in 19.1% of cases. CTCs were detected in 62.9% cases (in 188 of 299 patients). The percentages of patients with 1-3 CTCs and with >3 CTCs were equal - 50% (94 of 188). CTCs were not registered in 37.1% cases (111 of 299). The absence of CTCs was noted equally often in moderate and strong lymphocytic infiltration – 43.7% and 43.8%. The presence of tumor cells in the peripheral blood was most often detected in weak lymphocytic infiltration, being 1.4 times more frequent than in moderate and strong infiltration (76.5% vs. 56.3% and 56.2%) (p = 0.019, c2= 11.890). Conclusions: The study demonstrated a significant relationship between the level of CTCs and the intensity of lymphocytic infiltration in the tumor (p≤0.05), which can be used as a new prognostic approach.

POS0424 DETERMINING CIRCULATING ENDOTHELIAL CELLS USING CELLSEARCH SYSTEM IN SYSTEMIC SCLEROSIS PATIENTS

Background:Endothelial damage and fibroproliferative vasculopathy of small vessels are pathological hallmarks of Systemic Sclerosis (SSc). Detection and analysis of circulating endothelial cells (CECs) detached from affected blood vessels may be an informative tool to study vascular dysfunction and could be considered a novel biomarker of scleroderma vasculopathy. Our group first showed the presence of CECs in SSc by fluorescence-activated cell sorting (FACS), demonstrating that a raised counts of active CECs may represent direct evidence of active vascular disease in SSc. Despite these interesting data, issues related to difficulties in CEC counting through FACS analysis, due their very low concentration in peripheral blood, prevented further investigations in this field. Recently, a specific kit for the detection of CECs has been developed through the CellSearch System (CS), a semi-automated device for the standardized analysis of rare cells, such as CECs, in peripheral blood.Objectives:To assess the counts of CECs determined by the CS in SSc patients and to evaluate their clinical implication and potential as vascular biomarker in SSc.Methods:10mL of blood samples were collected from 29 subjects (19 SSc patients and 10 healthy donors - HDs) and stored in tubes containing a specific preservative, to allow the analysis of 4mL of blood within 72 hours, according to manufacturer instructions. Out of 19 SSc patients, 18 were female, 10 had the limited form and 9 the diffuse cutaneous variant of SSc. CS uses a proprietary kit containing a ferrofluid-based reagent, that target CD146 to magnetically capture CECs, and the immunofluorescent reagents to stain the CECs, defined as CD146+, CD105-PE+, DAPI+ and CD45-APC-. Clinical, laboratoristic and demographic data were also collected.Results:The mean number of CECs in patients with SSc was significantly higher in comparison to HDs (554/4mL vs. 53.5/4 mL, p=0.0042). When analyzed according to disease subset, both lcSSc and dcSSc showed significantly increased levels of CECs in comparison with HDs (p=0.003 and p=0.005, respectively). No statistical difference was observed in the mean number of CECs in patients with lcSSc compared to those with dcSSc. Regarding vascular involvement, the CECs counts strictly correlated with the presence of digital ulcers (DUs) (p=0.0001) showing a median of 863cells/4mL for the SSc patients with DUs versus a median of 276.2/4mL for the SSc patients without DUs. No statistical correlation was found between CECs and serological autoantibody pattern, skin parameters, or joint and muscle involvement. Patients with active disease, according to the EUSTAR Activity Index, showed a higher CECs value than those with inactive disease (p=0.0012).Conclusion:The amount of CECs detectable in peripheral blood has been recently proposed as a marker of endothelial damage in different vascular diseases, including SSc. However, currently no standardized method is available to determine CEC counts, which makes reported data on CECs reliable and suitable. The CS system is a commercially available semi-automated system that enables standardized determination of CECs. Thus, we examined clinical utility of CECs count by this system in SSc patients. Our results confirm that baseline CEC counts, evaluated by a new standardized method, may represent direct evidence of endothelial damage in SSc and could be a promising tool for monitoring active disease and evaluating therapeutic responses to vascular and immunosuppressive treatments.References:[1]Del Papa N, Pignataro F. Front Immunol. 2018 Jun 18;9:1383[2]De Simone C et al. J Eur Acad Dermatol Venereol. 2014 May;28(5):590-6[3]Del Papa N et al. Arthritis Rheum. 2004 Apr;50(4):1296-304Disclosure of Interests:Francesca Pignataro: None declared, Laura Zorzino: None declared, Wanda Maglione: None declared, Antonina Minniti: None declared, Giulia Clericuzio: None declared, Marco Picozzi: None declared, Cecilia Simonelli Employee of: Menarini Silicon Biosystems, Francesco Picardo Employee of: Menarini Silicon Biosystems, Roberto Caporali: None declared, Nicoletta Del Papa: None declared

A Review of Circulating Tumour Cell Enrichment Technologies

Circulating tumour cells (CTCs) are the precursor cells for the formation of metastatic disease. With a simple blood draw, liquid biopsies enable the non-invasive sampling of CTCs from the blood, which have the potential to provide important insights into cancer detection and monitoring. Since gaining FDA approval in 2004, the CellSearch system has been used to determine the prognosis of patients with metastatic breast, prostate and colorectal cancers. This utilises the cell surface marker Epithelial Cell Adhesion Molecule (EpCAM), to enrich CTCs, and many other technologies have adopted this approach. More recently, the role of mesenchymal-like CTCs in metastasis formation has come to light. It has been suggested that these cells are more aggressive metastatic precursors than their epithelial counterparts; however, mesenchymal CTCs remain undetected by EpCAM-based enrichment methods. This has prompted the development of a variety of ‘label free’ enrichment technologies, which exploit the unique physical properties of CTCs (such as size and deformability) compared to other blood components. Here, we review a wide range of both immunocapture and label free CTC enrichment technologies, summarising the most significant advantages and disadvantages of each. We also highlight the important characteristics that technologies should possess for routine clinical use, since future developments could have important clinical implications, with the potential to direct personalised therapies for patients with cancer.

Epithelial-to-mesenchymal transition of tumor cells: cancer progression and metastasis

Detection and characterization of circulating tumor cells (CTCs) with an epithelial-to-mesenchymal transition (EMT) phenotype is very important as it can contribute to the identification of high-risk for relapse and death patients. However, most of the methods are underestimating CTC numbers, due to their dependence on epithelial markers. In the current study, we evaluated the EMT phenotype in CTCs isolated from breast cancer (BC) patients, using the CellSearch system. Spiking experiments for the evaluation of the specificity and sensitivity of our method were performed using HeLa cells. Sixty-five breast cancer (BC) patients (47 early and 18 metastatic) were enrolled in the study. Vimentin is a mesenchymal marker which indicates tumoral cells acquiring invasive and malignant properties. We studied the vimentin (VIM) expression using the extra channel of the CellSearch system and an anti-vimentin antibody conjugated with FITC. In our present results, we reported the percentage of circulating tumor cells that expressed vimentin in early and in metastatic breast cancer patients. Interestingly, the incidence of cells with a CK-VIM+CD45- phenotype was detected in both settings. These cells were detected in 31.4% of CK-negative (11/35) and 82.3% of CK-positive (10/12) early BC patients. The corresponding numbers for metastatic disease were 15.4% (2/13) and 100% (5/5), respectively. Our results suggest that in CTC-negative patients, potentially undetectable tumor cells could be identified using the FDA-approved CellSearch system, based on the (CK-VIM+CD45-)-phenotype, offering additional information regarding the metastatic dissemination in cancer patients. Further experiments evaluating more biomarkers are necessary to elucidate the mechanisms that regulate tumorigenesis and metastasis.

Prognostic Value of Circulating Tumour Cells detected with the CellSearch System in Esophageal Cancer Patients: A Systematic Review and Meta-Analysis

Background: Esophageal carcinoma (EC) is the seventh-most prevalent tumor in the world, which is still one of the primary causes of tumor-related death. Identifying noteworthy biomarkers for EC is particularly significant in guiding effective treatment. Recently, circulating tumor cells (CTCs) in peripheral blood (PB) were intensively discussed as prognostic markers in patients with EC. However, an ongoing controversy still exists regarding the prognostic significance of CTCs determined by the CellSearch system in EC sufferers. This meta-analysis was designed to approach this topic. Methods: We systematically conducted searches using PubMed, Medline, Web of Science and the Cochrane Library for relevant studies, which were published through February 20, 2020. Using the random-effects model, our study was performed in Review Manager software, with odds ratios (ORs), risk ratios (RRs), hazard ratios (HRs) and 95% confidence intervals (CIs) as the effect values. Results: Totally 7 articles were finally included in this study. For clinicopathological characteristics, the pooled results on TNM stage indicated that the III/IV group had higher rate of CTCs compared with the I/II group (OR=1.36, 95% CI: 0.68-2.71, I2=0%). Incidence of CTCs was higher in patients with T3/T4 stage (OR=2.92, 95% CI: 1.31-6.51, I2=0%) and distant metastasis group (OR=5.18, 95% CI: 2.38-11.25, I2=0%) compared to patients with T1/T2 stage or non-metastatic group. The pooled analysis revealed that CTC positivity detected in EC patients was correlated with poor overall survival (OS) (HR=2.83, 95% CI:1.99-4.03, I2=0%) and relapse-free survival (RFS) (HR=4.71, 95% CI:2.73-8.13, I2=0%). When pooling the estimated RR, a poor therapeutic response to chemoradiotherapy was discovered in patients with CTC positivity (RR=1.99, 95% CI:1.73-2.29, I2=60%). Conclusions: In summary, our meta-analysis demonstrated that CTCs positivity determined by the CellSearch system are correlated with the prognosis of EC patients and might indicate a poor therapeutic response to chemotherapy in EC patients.