Platelet-Lymphocyte Ratio, Circulating Tumor Cells and Circulating Tumor Microemboli as Predictors of Thrombosis in Patients with Gastric Cancer

Background: Cancer-associated thrombosis (CAT) is a major cause of morbidity and mortality in oncology patients. There are no accurate risk assessment tools to predict venous thromboembolism (VTE). Circulating tumor cells (CTCs), circulating tumor microemboli (CTM), and high platelet-lymphocyte ratio (PLR) may predispose to VTE. Objective: To evaluate correlations of CTCs, CTM, and PLR with VTE and progression-free survival (PFS) in gastric cancer patients. Methods: Patients with gastric cancer were recruited (March 2016 to April 2017). CTCs were assayed by ISET at two timepoints: before neoadjuvant treatment (CTC1) and after surgery/before adjuvant therapy (CTC2) for patients with localized disease, and before first-line chemotherapy (CTC1) and after 6 months (CTC2) for patients with metastases. VTE incidence was determined retrospectively. PFS was estimated by Kaplan-Meier analysis. Results: We studied 93 patients. According to Khorana scores, 63 (67.7%) patients were at intermediate and 30 (32.3%) were at high risk for VTE. VTE incidence was 20.4% and CTM were found in 39.8%. VTE developed in 7/37 (18.9%) CTM-positive and in 11/50 (22%) CTM-negative patients (p=0.93). When PLR >288, VTE occurred in 7/14 patients (p=0.005). PLR also associated with poor PFS (p<0.0001). CTC2 was associated with poor PFS (p<0.0001). CTC2, PLR and VTE were independent prognostic factors for PFS (p=0.005, 0.043, and <0.0001 respectively). Conclusion: PLR is a prognostic indicator for PFS and for VTE in gastric cancer. Neither CTC, nor CTM improved risk stratification for VTE in our population.

Circulating tumor cells in breast cancer: clinical and molecular parallels

Background. Breast cancer (BC) is the most common type of malignant neoplasm among women, with a high rate of metastasis. Early non-invasive diagnosis is required to increase the effectiveness of anticancer therapy.Objective: to determine the content of circulating tumor cells (CTCs) and their derivatives in the peripheral blood using the MDA-231 aptamer, compare the results obtained with the clinical and molecular characteristics of BC.Materials and methods. The study included 22 patients with BC. Detection of CTCs and circulating tumor microemboli was carried out in 3.5 ml of the blood of BC patients with the help of the MDA-231 aptamer which is affine for breast cancer cells, labeled with the fluorescent Cy3 dye. The count of CTCs in the blood samples was performed using fluorescent and laser scanning microscopy.Results and conclusions. The content of CTCs and circulating tumor microemboli in the peripheral blood of patients with BC of various molecular subtypes was analyzed using the MDA-231 aptamer. The relationship between the number of CTCs and the molecular biological subtype was revealed. The obtained results show the possible prognostic value of CTCs use for monitoring effectiveness of anticancer therapy and control of recurrence of BC.

Profiling heterogenous sizes of circulating tumor microemboli to track therapeutic resistance and prognosis in advanced gastric cancer

Circulating tumor microemboli (CTM) aggregated by ≥ 2 circulating tumor cells (CTCs) are more migratory than single CTCs. Aside from the plasticity in their molecular characteristics, which have been considered tumor migration, CTM also possesses high size heterogeneity. This study, therefore, systematically investigated the heterogeneous sizes of CTM and their involvement in therapeutic resistance in 114 patients with advanced gastric cancer (GC) using a pre-established surface molecule-independent subtraction enrichment (SE)-iFISH strategy. CTM, which was pre-therapeutically detected in 33.3% of GC patients, can further form in another 34.78% of patients following chemo-/targeted therapies. The presence of CTM is relevant to liver metastasis as well as higher CTC levels (≥ 5/6 mL). Further size-based profiling of GC-CTM revealed that CTM with 2 CTCs (CTM2) was the dominant subtype, accounting for 50.0% of all detected GC-CTMs. However, CTM with 3–4 CTCs (CTM3–4) specifically associates with chemo-/targeted therapeutic resistance and inferior prognosis. Patients with ≥ 1 CTM3–4/6 mL have shorter median progression-free survival and median overall survival. Unlike CTM2 and CTM3–4, which are detectable in pre-therapy and post-therapy, larger aggregated CTM≥5 (CTM with ≥ 5 CTCs) was only intra-therapeutically detected in four HER2+ GC patients, of which three experienced liver metastases. Obtained results suggested that the cluster size of GC-CTM should be dynamically profiled beyond pre-therapeutic whole CTM enumeration in terms of chemo-/targeted resistance or metastasis monitoring. GC-CTM3–4 could be a potential indicator of therapeutic resistance, while the dynamic presence of GC-CTM≥5 implies liver metastasis in HER2+ GC patients.

Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients

Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0–100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31–71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30–63%), whereas primary tumor-private alterations were rare (4–12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies.

New Labyrinth Microfluidic Device Detects Circulating Tumor Cells Expressing Cancer Stem Cell Marker and Circulating Tumor Microemboli in Hepatocellular Carcinoma

Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.

Insights on CTC Biology and Clinical Impact Emerging from Advances in Capture Technology

Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) have been shown to correlate negatively with patient survival. Actual CTC counts before and after treatment can be used to aid in the prognosis of patient outcomes. The presence of circulating tumor materials (CTMat) can advertise the presence of metastasis before clinical presentation, enabling the early detection of relapse. Importantly, emerging evidence is indicating that cancer treatments can actually increase the incidence of CTCs and metastasis in pre-clinical models. Subsequently, the study of CTCs, their biology and function are of vital importance. Emerging technologies for the capture of CTC/CTMs and CTMat are elucidating vitally important biological and functional information that can lead to important alterations in how therapies are administered. This paves the way for the development of a “liquid biopsy” where treatment decisions can be informed by information gleaned from tumor cells and tumor cell debris in the blood.