Cluster circulating tumor cells in surgical cases of lung cancer
Objectives
A cancer lesion sheds tumor cells into the circulating blood as circulating tumor cells (CTCs). Since cluster CTCs have been considered as precursor lesions of metastasis, their clinical implication was investigated in this study according to the preoperative status of cluster CTC detection in surgical cases of clinically early-stage lung cancer.
Methods
Among 104 surgical patients of early-stage lung cancer, CTCs were extracted from the peripheral blood before surgery using a micro-pore size selection method (ScreenCell®) and diagnosed microscopically. Implications of detecting cluster CTC were assessed according to the prognosis and clinicopathological characteristics.
Results
The status of CTC detection was not detected in 77 cases (74.0%), single CTC only detection in 7 cases (6.7%), and cluster CTC detected in 20 cases (19.2%). Patients with cluster CTCs exhibited significantly lower recurrence-free survival and overall survival than did patients of other groups. In addition, in hazard ratio analysis, the hazard ratios were independent of other predictors of poor prognosis, and detection of cluster CTCs was associated with predictors of poor prognosis.
Conclusion
Cluster CTCs were detected in cases where the original lung cancer lesion had clinical predictors of poor prognosis and were independent negative predictors of survival.
A Novel Combined Methodology for Isolation and Detection of Circulating Tumor Cells based on Flow Cytometry and Cellular Filtration Technologies
Cancer cell presents a dynamic nature that evolves over time in its microenvironment through a complicating molecular and cellular interaction network. Despite the progress already achieved in the imaging technologies and the molecular tools used for tumor cell diagnosis and treatment approaches, metastasis still remains a major hindering factor that limits the clinical outcomes. Circulating tumor cells (CTCs) present the potential in providing critical information to understand the metastasis process, the tumor cell and genome heterogeneity, in a way to overcome cancer therapy bottlenecks and improve the efficacy and safety therapeutic profiles. The experience already gained shows that CTCs as tools possess a predictive biomarker capacity by aiming the understanding of most of the known cancer cell hallmarks at the molecular level, as well as the translation of the extracted relevant knowledge in clinical practice. The efficient isolation and characterization of CTCs might reveal the intratumoral heterogeneity present in tumors while providing critical insights into cancer metastasis. Moreover, by combining the emerging single cell omics technologies, CTCs can be valuable biological source material to unveil the existed intratumoral cellular and genomic heterogeneity, as well as to pharmacologically exploit critical knowledge for the validation of prognostic and diagnostic biomarkers within the concept of precision and personalized cancer therapy. In this article, a novel combined methodology for the isolation and detection of CTCs based on flow cytometry and cellular filtration technologies is presented.
Lack of association between Screencell-detected circulating tumour cells and long-term survival of patients undergoing surgery for non-small cell lung cancer: A pilot clinical study.
Circulating tumour cells (CTCs) are cancer cells of epithelial origin that are present in peripheral blood samples. ScreenCell detection of CTCs and the association with long term survival in non‑small cell lung cancer (NSCLC) patients was evaluated in the present study. A total of 33 patients undergoing surgical resection for NSCLC were recruited. Patients were followed up for 5‑years post‑operatively. Pre‑operative patient bloods samples were processed using ScreenCell. CTCs were detected in 26 (79%) patients. In patients who were positive for CTCs, a total of 9 (35%) patients succumbed to the disease, whereas in patients negative for CTCs, a total of 4 (57%) patients succumbed to the disease (P=0.29). No association was identified between positive CTCs and poorer survival (Chi‑squared 1.47, P=0.23; hazard ratio, 0.42; 95% confidence interval: 0.1‑1.7). The presence of CTCs detected with ScreenCell does not influence prognosis in patients with NSCLC that was operated on. The high rate of CTC detection is encouraging in supporting this technology to aid early lung cancer diagnosis.
Expression Profiling of Circulating Tumor Cells in Pancreatic Ductal Adenocarcinoma Patients: Biomarkers Predicting Overall Survival
The interest in liquid biopsy is growing because it could represent a non-invasive prognostic or predictive tool for clinical outcome in patients with pancreatic ductal adenocarcinoma (PDAC), an aggressive and lethal disease. In this pilot study, circulating tumor cells (CTCs), CD16 positive atypical CTCs, and CTC clusters were captured and characterized in the blood of patients with PDAC before and after palliative first line chemotherapy by ScreenCell device, immunohistochemistry, and confocal microscopy analysis. Gene profiles were performed by digital droplet PCR in isolated CTCs, five primary PDAC tissues, and three different batches of RNA from normal human pancreatic tissue. Welsh’s t-test, Kaplan-Meier survival, and Univariate Cox regression analyses have been performed. Statistical analysis revealed that the presence of high CTC number in blood is a prognostic factor for poor overall survival and progression free survival in advanced PDAC patients, before and after first line chemotherapy. Furthermore, untreated PDAC patients with CTCs, characterized by high ALCAM, POU5F1B, and SMO mRNAs expression, have shorter progression free survival and overall survival compared with patients expressing the same biomarkers at low levels. Finally, high SHH mRNA levels are negatively associated to progression free survival, whereas high vimentin mRNA levels are correlated with the most favorable prognosis. By hierarchical clustering and correlation index analysis, two cluster gene signatures were identified in CTCs: the first, with high expression of VEGFA, NOTCH1, EPCAM, IHH, is the signature of PDAC patients before chemotherapy, whereas the second, with an enrichment in the expression of CD44, ALCAM, and POU5F1B stemness and pluripotency genes, is reported after palliative chemotherapy. Overall our data support the clinic value of the identification of CTC’s specific biomarkers to improve the prognosis and the therapy in advanced PDAC patients.
Long-Term Dynamics of Three Dimensional Telomere Profiles in Circulating Tumor Cells in High-Risk Prostate Cancer Patients Undergoing Androgen-Deprivation and Radiation Therapy
Patient-specific assessment, disease monitoring, and the development of an accurate early surrogate of the therapeutic efficacy of locally advanced prostate cancer still remain a clinical challenge. Contrary to prostate biopsies, circulating tumor cell (CTC) collection from blood is a less-invasive method and has potential as a real-time liquid biopsy and as a surrogate marker for treatment efficacy. In this study, we used size-based filtration to isolate CTCs from the blood of 100 prostate cancer patients with high-risk localized disease. CTCs from five time points: +0, +2, +6, +12 and +24 months were analyzed. Consenting treatment-naïve patients with cT3, Gleason 8-10, or prostate-specific antigen > 20 ng/mL and non-metastatic prostate cancer were included. For all time points, we performed 3D telomere-specific quantitative fluorescence in situ hybridization on a minimum of thirty isolated CTCs. The patients were divided into five groups based on the changes of number of telomeres vs telomere lengths over time and into three clusters based on all telomere parameters found on diagnosis. Group 2 was classified as non-respondent to treatment and the Cluster 3 presented more aggressive phenotype. Additionally, we compared our telomere results with the PSA levels for each patient at 6 months of ADT, at 6 months of completed RT, and at 36 months post-initial therapy. CTCs of patients with PSA levels above or equal to 0.1 ng/mL presented significant increases of nuclear volume, number of telomeres, and telomere aggregates. The 3D telomere analysis of CTCs identified disease heterogeneity among a clinically homogeneous group of patients, which suggests differences in therapeutic responses. Our finding suggests a new opportunity for better treatment monitoring of patients with localized high-risk prostate cancer.
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.
Prognostic Relevance of Circulating Tumor Cells and Circulating Cell-Free DNA Association in Metastatic Non-Small Cell Lung Cancer Treated with Nivolumab
The treatment of advanced non-small cell lung cancer (NSCLC) has been revolutionized by immune checkpoint inhibitors (ICIs). The identification of prognostic and predictive factors in ICIs-treated patients is presently challenging. Circulating tumor cells (CTCs) and cell-free DNA (cfDNA) were evaluated in 89 previously treated NSCLC patients receiving nivolumab. Blood samples were collected before therapy and at the first and second radiological response assessments. CTCs were isolated by a filtration-based method. cfDNA was extracted from plasma and estimated by quantitative PCR. Patients with baseline CTC number and cfDNA below their median values (2 and 836.5 ng from 3 mL of blood and plasma, respectively) survived significantly longer than those with higher values (p = 0.05 and p = 0.04, respectively). The two biomarkers were then used separately and jointly as time-dependent covariates in a regression model confirming their prognostic role. Additionally, a four-fold risk of death for the subgroup presenting both circulating biomarkers above the median values was observed (p < 0.001). No significant differences were found between circulating biomarkers and best response. However, progressing patients with concomitant lower CTCs and cfDNA performed clinically well (p = 0.007), suggesting that jointed CTCs and cfDNA might help discriminate a low-risk population which might benefit from continuing ICIs beyond progression.
Advancing Risk Assessment of Intermediate Risk Prostate Cancer Patients
The individual risk to progression is unclear for intermediate risk prostate cancer patients. To assess their risk to progression, we examined the level of genomic instability in circulating tumor cells (CTCs) using quantitative three-dimensional (3D) telomere analysis. Data of CTCs from 65 treatment-naïve patients with biopsy-confirmed D’Amico-defined intermediate risk prostate cancer were compared to radical prostatectomy pathology results, which provided a clinical endpoint to the study and confirmed pre-operative pathology or demonstrated upgrading. Hierarchical centroid cluster analysis of 3D pre-operative CTC telomere profiling placed the patients into three subgroups with different potential risk of aggressive disease. Logistic regression modeling of the risk of progression estimated odds ratios with 95% confidence interval (CI) and separated patients into “stable” vs. “risk of aggressive” disease. The receiver operating characteristic (ROC) curve showed an area under the curve (AUC) of 0.77, while prostate specific antigen (PSA) (AUC of 0.59) and Gleason 3 + 4 = 7 vs. 4 + 3 = 7 (p > 0.6) were unable to predict progressive or stable disease. The data suggest that quantitative 3D telomere profiling of CTCs may be a potential tool for assessing a patient’s prostate cancer pre-treatment risk.
Non-Metastatic Esophageal Adenocarcinoma: Circulating Tumor Cells in the Course of Multimodal Tumor Treatment
Background: Isolation of circulating tumor cells (CTC) holds the promise to improve response-prediction and personalization of cancer treatment. In this study, we test a filtration device for CTC isolation in patients with non-metastatic esophageal adenocarcinoma (EAC) within recent multimodal treatment protocols. Methods: Peripheral blood specimens were drawn from EAC patients before and after neoadjuvant chemotherapy (FLOT)/chemoradiation (CROSS) as well as after surgery. Filtration using ScreenCell® devices captured CTC for cytologic analysis. Giemsa-stained specimens were evaluated by a cytopathologist; the cut-off was 1 CTC/specimen (6 mL). Immunohistochemistry with epithelial (pan-CK) and mesenchymal markers (vimentin) was performed. Results: Morphologically diverse malignant CTCs were found in 12/20 patients in at least one blood specimen. CTCs were positive for both vimentin and pan-CK. More patients were CTC positive after neoadjuvant therapy (6/20 vs. 9/15) and CTCs per/ml increased in most of the CTC-positive patients. After surgery, 8/13 patients with available blood specimens were still CTC positive. In clinical follow-up, 5/9 patients who died were CTC-positive. Conclusions: Detection of CTC by filtration within multimodal treatment protocols of non-metastatic EAC is feasible. The rate of CTC positive findings and the quantity of CTCs changes in the course of multimodal neoadjuvant chemoradiation/chemotherapy and surgery.
Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in-situ hybridization.
Isolation of circulating tumor cells (CTCs) from blood of melanoma patients has been difficult owing to inconsistent expression of surface antigens. Here we report on the isolation, detection, and characterization of CTCs from blood of melanoma patients using microfiltration and fluorescent in-situ hybridization (FISH). Two tubes of blood from 15 patients with advanced melanoma were collected. These two tubes subsequently underwent filtration through a membrane with pore sizes of 7.5 μm. Isolated cells from one tube were analyzed by FISH for RREB1 (6p24), MYB (6q32), SE6 (D6Z1), and CCND1 (11q13) and the other paired specimen was analyzed by immunofluorescence for HMB45, melanoma-associated antigen recognized by T cells-1, tyrosinase and melanogenesis associated transcription factor. We identified CTCs in 10 out of 13 melanoma samples by immunofluorescence (2.5–99 CTCs/3 ml of blood) and in 13 specimens by FISH (7.2–76 CTCs/3 ml of blood) with more CTCs identified by FISH in 10 out 13 samples. Two filters failed. Our results show that CTCs are detectable in the majority of patients with advanced melanoma. These tools will be useful in characterizing treatment related changes of melanoma in CTCs.