Presence Of Circulating Tumor Microemboli Research Articles

Prognostic value of circulating tumor cells and analysis of clinicopathological factors in liver cancer.

The number of circulating tumor cells (CTCs) and the presence of circulating tumor microemboli (CTM) were determined in the peripheral blood of patients with liver cancer (LC). The relationship between CTCs, CTM, clinicopathologic features, and prognosis of LC was analyzed. The objective of this study was to determine the diagnostic and prognostic value of CTCs/CTM in LC. Patients with LC were enrolled between May 2013 and August 2017, and 67 patients were included in the study. Overall survival curves were built using the Kaplan-Meier method and the log-rank test to identify risk factors. The results were analyzed using a Cox proportional hazards model and expressed as hazard ratio and 95% confidence interval (95% CI). CTCs and either CTCs or CTM were detected in 27 patients (40.3%) and 29 patients (43.3%). CTM were found in four patients. One-year, 3-year, and 5-year survival rates were 42%, 20%, and 15%, respectively. Univariate Cox regression analysis showed that alpha-fetoprotein (AFP), number of CTCs, presence of CTM, and positive CTC/CTM were associated with survival time. Multivariate Cox regression analysis showed that alpha fetoprotein (AFP), number of CTCs, and presence of CTM were independent risk factors for survival in patients with LC. There was no significant correlation between the number of CTCs, the presence of CTM, and clinicopathologic factors. AFP, number of CTCs, and presence of CTM were independent risk factors for survival in patients with LC.

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.

Prognostic significance of circulating tumor microemboli in patients with pancreatic ductal adenocarcinoma.

The significance of circulating tumor microemboli (CTMs) in patients with pancreatic ductal adenocarcinoma (PDAC) is still unknown. Thus, the present study used a epithelial cellular adhesion molecule independent subtraction and immunostaining-fluorescence in situ hybridization (SET-iFISH) platform to enumerate circulating tumor cells (CTCs) and CTMs in a total of 86 peripheral blood samples from 19 patients with PDAC. The associations between CTCs and CTMs with clinicopathologic factors and prognoses were analyzed. Prior to treatment, CTCs were detected in all 19 patients, and CTMs were detected in 4 patients with different tumor-node-metastasis (TNM) stages. A total of 85 of the 86 peripheral blood samples had cytokeratin 18-negative CTCs. The number of CTCs and CTMs were significantly associated with tumor size and vascular invasion. Patients with CTMs had poorer overall survival and disease-free survival when compared with those without CTMs (7.3 vs. 25.40 months, P=0.001; and 1.80 vs. 18.97 months, P=0.037). The presence of CTMs in the peripheral blood prior to surgery was predictive of poor prognosis in PDAC patients. CTMs could be detected in patients of different TNM stage (II, III and IV). Surgery did not benefit patients with CTMs, thus, surgeons should take greater consideration when assessing the requirements for surgery in patients with CTMs.

Abstract B06: Circulating tumor cell analysis in locally and advanced colon cancer: A pilot study

Abstract Colorectal cancer presents high incidences (9.7%) and mortality rates (8.5%) worldwide in men and women. Although the early detection of colon cancer promotes high cure rates (30-40%), there are still patients who experience local recurrence as well as distant metastases. The main treatment is surgery-based and then, after risk evaluation, patients can undergo or not undergo adjuvant chemotherapy. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) seem to play an important role in these processes, being detached from tumor and circulating in blood vessels before surgery intervention. Our objective, therefore, was to evaluate the role of CTCs and CTM in stages I-III colon cancer patients. Here, we collected 10 mL of blood from presurgery colon cancer and filtrated the sample in ISET® (Isolation by Size of Epithelial Tumor cells) device. This study started on July 2016. CTCs counts were assessed and correlated with clinical and pathologic data. We included 15 patients. However, one patient was identified as metastatic after surgery and therefore excluded from these analyses. From 14 patients analyzed, 8 (57.1%) were male, with a median age of 65 years (53-87). The most prevalent cancer sites were right colon (n= 6;42.9%) and left colon (n=4;28.6%). CTCs detection rate was 85.7%, and the mean and median of CTCs were 5.1/mL and 2.6/mL, respectively (0-30.3/mL). We found CTM in 4/14 patients (28.5%). Histopathologic analysis showed that no patient had blood vessel invasion, 5 (35.7%) had lymphatic invasion, and 4 (28.6%) had perineural invasion. Four patients (28.4%) were classified as stage I and 10 (71.4%) were classified as stage II and III. From patients with RAS mutational analysis available (12), 9 (75%) presented tumor mutation. Although we present a limited sample, exact Fisher's test showed an association between lymphatic invasion and CTC number above the median (P= 0.03), and the presence of CTM was most frequent in this group of patients, although without statistical significance (P= 0.09). CTM absence was associated with pathologic T= 2 and 3 (P= 0.06), when compared to pathologic T= 4. Our next steps are to evaluate some proteins in CTCs that could confer an invasive phenotype, resistance to adjuvant treatment as well as verify the pattern of mutations in CTCs and CTMs. Moreover, this is the first study to show the efficacy of ISET method in detecting high rates of CTCs from stage I-III colon cancer patients. Citation Format: Emne Abdallah, Virgílio Silva, Bianca Flores, Alexcia Braun, Ana Urvanegia, Vanessa Alves, Marcello Fanelli, Samuel Aguiar, Ludmilla Chinen. Circulating tumor cell analysis in locally and advanced colon cancer: A pilot study [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B06.

Abstract B07: CTC and CTM detection in sarcoma patients

Abstract Background: Sarcomas represent 1% of all cancers in adults, 8% in adolescents and young people, and 10% in children. Even though it is a rare cancer, it contributes to a significant loss of years of life in comparison with other types of cancer (Amankwah et al., 2013). The presence of isolated circulating tumor cells (CTCs) or circulating tumor microemboli (CTM) in the blood of patients with sarcoma may be early markers of tumor invasion, because it is known that these cells circulate in the blood for months or years before the development of metastases (Paterlini-Brechot and Benali, 2007; Klain, 2009; Rhim et al., 2012). Objective: To isolate and quantify CTCs and CTM from sarcoma patients, evaluating their presence and correlation with progression-free survival (PFS). Methods: Blood (approximately 8 mL) was prospectively collected from patients with different types of high-degree sarcoma. Blood was collected before the beginning of chemotherapy. The samples were processed and filtered on ISET (Isolation by Size of Epithelial Tumor Cells, Rarecells, France) system for the isolation and quantification of CTCs and CTMs. Immunocytochemistry (ICC) was later performed with anti-CD45 antibody and counterstained with hematoxylin-eosyn for leucocytes population exclusion. The cutoff was estimated using the maximum of the standardized log-rank statistic proposed by Lausen and Schumacher (1992). Results: We analyzed 11 high-degree sarcoma patients after metastasis diagnosis, before the beginning of treatment. The median age was 53 years (18-77) and 54.5% were male. The median CTCs number was 2.0 CTCs/mL (1.0-11.0) at baseline. By Kaplan-Meier test, we observed that patients with CTC number below the established cutoff (4.5 CTCs/mL) had better PFS compared to those above the cutoff (7.4 vs. 1.0 months, respectively; P= 0.006). Moreover, patients without CTM had also better PFS compared to those patients with the presence of CTM (7.6 vs. 2.7 months, respectively; P= 0.13). Conclusion: This is the first study to demonstrate, besides the CTC and CTM presence in sarcoma patients, its real prognostic value, even in a limited sample. Our next step is to characterize these cells to better understand its capability to promote tumor progression. Citation Format: Alexcia C. Braun, Celso A L Mello, Marcelo P. Corassa, Alves S. Vanessa, Emne A. Abdallah, Nicolau R. Ulisses, Mônica T. M. Díaz, Bianca T. C. P. Flores, Marcelo F. Fanelli, Patrizia Paterlini-Brechót, Ludmilla T. D. Chinen. CTC and CTM detection in sarcoma patients [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B07.

Effect of circulating tumor cell aggregate configuration on hemodynamic transport and wall contact

Selectin-mediated adhesion of circulating tumor cells (CTCs) to the endothelium is a critical step in cancer metastasis, a major factor contributing to the mortality of cancer. The formation of tethers between tumor cells and endothelial selectins initiates cell rolling, which can lead to firm adhesion, extravasation and the formation of secondary metastases. Tumor cells travel through the bloodstream as single cells, or as aggregates known as circulating tumor microemboli (CTM). CTM have increased survivability and metastatic potential relative to CTCs, and the presence of CTM is associated with worse patient prognosis. The motion of cells and cellular aggregates in flow is a function of their size and shape, and these differences influence the frequency and strength of their contact with the endothelium. In this study, a computational model consisting of the hydrodynamic component of the Multiparticle Adhesive Dynamics simulation analyzed the effects of model aggregate conformation and orientation on adhesive binding potential. Model aggregates of the Colo205 colorectal cancer cell line were created, consisting of two, three, and four cells in simple geometrical conformations. Contact time, contact area, and time integral of contact area were measured as a function of fluid shear rate, initial centroid height, and initial orientation for model aggregates that experienced hydrodynamic collisions with the plane wall. It was found that larger CTM conformations with intermediate nonsphericities had the highest adhesion potential. The results of this study shed light on the correlation between environmental conditions and extravasation efficiency, which could inform the development of new anti-metastatic drugs.

Abstract 1728: CTCs and CTM as prognostic factors in sarcoma patients

Abstract Background: Sarcomas represent 1% of all cancers in adults, 8% in adolescents and young people, and 10% in children. Even though it is a rare cancer, it contributes to a significant loss of years of life in comparison with other types of cancer (Amankwah, et al., 2013). The presence of isolated Circulating Tumor Cells (CTCs) or Circulating Tumor Microemboli (CTM) in the blood of patients with sarcoma may be early markers of tumor invasion, because it is known that these cells circulate in the blood for months or years before the development of metastases (Paterlini-Brechot and Benali, 2007; Klain, 2009; Rhim et al., 2012). Objective: To isolate and quantify CTCs and CTM from sarcoma patients, evaluating their presence and correlation with progression free survival (PFS). Methods: The blood (approximately 8mL) was prospectively collected from patients with different types of high degree sarcoma. Blood was collected before the beginning of chemotherapy. The samples were processed and filtered on ISET (Isolation by Size of Epithelial Tumor Cells, Rarecells, France) system for the isolation and quantification of CTCs and CTMs. Later, was performed immunocytochemistry (ICC) with anti-CD45 antibody and counterstained with hematoxylin-eosyn for leucocytes population exclusion. The cutoff was estimated using the maximum of the standardized log-rank statistic proposed by Lausen & Schumacher (1992). Results: we analyzed 11 high degree sarcoma patients before the beginning of treatment (any treatment line). The median age was 53 years-old (18-77) and 54,5% were male. The median CTCs number was 2.0 CTCs/mL (1.0-11.0) at baseline. By Kaplan-Meier test, we observed that patients with CTC number below the established cutoff (4.5 CTCs/mL) had better PFS compared to those above the cutoff (7.4 vs. 1.0 months respectively; P= 0.006). Moreover, patients without CTM had also better PFS compared to those patients with the presence of CTM (7.6 vs. 2.7 months respectively; P= 0.13). Conclusion: this is the first study to demonstrate, besides the CTC and CTM presence in sarcoma patients, its real prognostic value, even in a limited sample. Our next step is to characterize these cells to better understand its capability to promote tumor progression. Citation Format: Alexcia C. Braun, Celso A. Mello, Marcelo P. Corassa, Vanessa S. Alves, Emne A. Abdallah, Ulisses R. Nicolau, Mônica Taiane de Macedo Díaz, Bianca de Cássia Troncarelli de Campos Parra Flores, Marcelo F. Fanelli, Patrizia Paternili-Brechót, Ludmilla T. Chinen. CTCs and CTM as prognostic factors in sarcoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2017-1728

Associations between the Epithelial-Mesenchymal Transition Phenotypes of Circulating Tumor Cells and the Clinicopathological Features of Patients with Colorectal Cancer.

In this study, we identified CTCs using the previously reported CanPatrol CTC enrichment technique from peripheral blood samples of 126 patients with colorectal cancer (CRC) and found that CTCs could be classified into three subpopulations based on expression of epithelial cell adhesion molecule (EpCAM) (E-CTCs), the mesenchymal cell marker vimentin (M-CTCs), or both EpCAM and vimentin (biphenotypic E/M-CTCs). Circulating tumor microemboli (CTMs) were also identified in peripheral blood samples. Meanwhile, E-CTCs, M-CTCs, E/M-CTCs, and CTMs were detected in 76.98%, 42.06%, 56.35%, and 36.51% of the 126 patients, respectively. Interestingly, the presence of CTMs and each CTC subpopulation was significantly associated with blood lymphocyte counts and tumor-node-metastasis stage (P < 0.001). Lymphocyte counts and the neutrophil-to-lymphocyte ratio (NLR) in patients lacking CTCs were significantly different from those in patients testing positive for CTMs and each CTC subpopulation (P < 0.001). Our results indicate that tumor metastasis is more significantly associated with the presence of CTMs and M-CTCs than with other CTC subpopulations and suggest that EMT may be involved in CTC evasion of lymphocyte-mediated clearance.

High expression of TRF2, SOX10, and CD10 in circulating tumor microemboli detected in metastatic melanoma patients. A potential impact for the assessment of disease aggressiveness.

Circulating tumors cells (CTCs) can be detected in the blood of metastatic melanoma patients (MMPs) both as isolated circulating tumor cells (iCTCs) and circulating tumor microemboli (CTMs), but their clinical significance remains unknown. The aim of this work was to evaluate the prognostic impact in metastatic cutaneous melanoma of CTMs and iCTCs identified by a cytomorphological approach using the isolation by size of tumor cell (ISET) method. We characterized the phenotype of CTCs using anti‐PS100, anti‐SOX10, anti‐CD10, and anti‐TRF2 antibodies. 128 MMPs and 37 control healthy individuals with benign nevi were included in this study. Results were compared to the follow‐up of patients. 109/128 (85%) MMPs showed CTCs, 44/128 (34%) with 2 to 6 CTMs and 65/128 (51%) with 4 to 9 iCTCs. PS100 expression was homogeneous in iCTCs and heterogeneous in CTMs. SOX10, CD10, and TRF2 were mainly expressed in CTMs. None of the control subjects demonstrated circulating malignant tumor cells. Overall survival was significantly decreased in patients with CTMs, independently of the therapeutic strategies. In conclusion, the presence of CTMs is an independent predictor of shorter survival from the time of diagnosis of MMPs.