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.
Perioperative detection of circulating tumour cells in patients with lung cancer.
Lung cancer is a leading cause of mortality and despite surgical resection a proportion of patients may develop metastatic spread. The detection of circulating tumour cells (CTCs) may allow for improved prediction of metastatic spread and survival. The current study evaluates the efficacy of the ScreenCell® filtration device, to capture, isolate and propagate CTCs in patients with primary lung cancer. Prior to assessment of CTCs, the present study detected cancer cells in a proof‑of‑principle‑ experiment using A549 human lung carcinoma cells as a model. Ten patients (five males and five females) with pathologically diagnosed primary non‑small cell lung cancer undergoing surgical resection, had their blood tested for CTCs. Samples were taken from a peripheral vessel at the baseline, from the pulmonary vein draining the lobe containing the tumour immediately prior to division, a further central sample was taken following completion of the resection, and a final peripheral sample was taken three days post‑resection. A significant increase in CTCs was observed from baseline levels following lung manipulation. No association was able to be made between increased levels of circulating tumour cells and survival or the development of metastatic deposits. Manipulation of the lung during surgical resection for non‑small cell lung carcinoma results in a temporarily increased level of CTCs; however, no clinical impact for this increase was observed. Overall, the study suggests the ScreenCell® device has the potential to be used as a CTC isolation tool, following further work, adaptations and improvements to the technology and validation of results.
Prevalence and number of circulating tumour cells and microemboli at diagnosis of advanced NSCLC
Purpose
Timing and magnitude of blood release of circulating tumour cells (CTC) and circulating tumour microemboli (CTM) from primary solid cancers are uncertain. We investigated prevalence and number of CTC and CTM at diagnosis of advanced non-small cell lung cancer (NSCLC).
Methods
Twenty-eight consecutive patients with suspected stage III–IV lung cancer gave consent to provide 15 mL of peripheral blood soon before diagnostic CT-guided fine-needle aspiration biopsy (FNAB). CTC and CTM (clusters of ≥3 CTC) were isolated by cell size filtration (ScreenCell), identified and counted by cytopathologists using morphometric criteria and (in 6 cases) immunostained for vimentin.
Results
FNAB demonstrated NSCLC in 26 cases. At least one CTC/3 mL blood (mean 6.8 ± 3.7) was detected in 17 (65 %) and one CTM (mean 4.5 ± 3.3) in 15 (58 %) of 26 NSCLC cases. No correlation between number of CTC or CTM and tumour type or stage was observed. Neoplastic cells from both FNA and CTC/CTM were positive for vimentin but heterogeneously.
Conclusions
CTC can be detected in two-thirds and CTM in more than half of patients with advanced NSCLC at diagnosis. Reasons underlying lack of CTC and CTM in some advanced lung cancers deserve further investigations.
Circulating Tumor Cells in Diagnosing Lung Cancer: Clinical and Morphologic Analysis
Background: The purpose of this study was to evaluate the value of circulating non-hematologic cells to differentiate benign from malignant lung lesions and their comparison with clinico-histologic features of corresponding primary lesions.
Methods: Circulating cells were isolated by size method from peripheral blood of 77 patients with malignant (n = 60) and benign (n = 17) lung lesions. They were morphologically classified as cells with malignant feature; cells with uncertain malignant feature; and cells with benign feature; then statistically correlated with clinico-cytopathologic characteristics of corresponding lung lesion.
Results: Malignant circulating cells were detected in 54 of 60 (90%) malignant patients, and in 1 of 17 (5%) benign patients; benign circulating cells in 1 of 60 (1%) malignant patients and in 15 of 17 (88%) benign patients; and circulating cells with uncertain malignant aspect in 5 of 60 (8%) malignant patients and 1 of 17 (5%) benign patients. For a malignant circulating cells count greater than 25, sensitivity and specificity were 89% and 100%, respectively. The count was significantly correlated with stage, size, and standard uptake value of primary tumor. In 39 of 54 (72%) cases, the malignant circulating cells allowed a specific histologic diagnosis of the corresponding primary tumor after immunohistochemical analysis.
Conclusions: Malignant circulating cells may be a valid marker in the diagnostic workup of lung lesions. However, our resuts should be corroborated by larger future studies especially for patients having small nodules.
Rapid separation of mononuclear hodgkin from multinuclear reed-sternberg cells
We describe a method to isolate small mononucleated Hodgkin (H) cells from multinucleated Reed Sternberg (RS) cells of Hodgkin lymphoma using the ScreenCell filter device. This filtration-based approach lends itself to future clinical applications in that it enables the separation of H and RS cells from lymph node biopsies, bone marrow aspirates, pleural effusions, and blood, including the isolation of monoclonal Hodgkin precursor cells from the blood.
Detection of circulating tumor cells in patients with adrenocortical carcinoma: a monocentric preliminary study
Context: Adrenocortical carcinoma (ACC) is a rare malignancy, the prognosis of which is mainly dependent on stage at diagnosis. The identification of disease-associated markers for early diagnosis and drug monitoring is mandatory. Circulating tumor cells (CTCs) are released into the bloodstream from primary tumor/metastasis. CTC detection in blood samples may have enormous potential for assisting in the diagnosis of malignancy, estimating prognosis, and monitoring the disease.
Objective: The aim of the study was to investigate the presence of CTCs in blood samples of patients with ACC or benign adrenocortical adenoma (ACA).
Setting: We conducted the study at a university hospital.
Intervention: CTC analysis was performed in blood samples from 14 ACC patients and 10 ACA patients. CTCs were isolated on the basis of cell size by filtration through ScreenCell devices, followed by identification according to validated morphometric criteria and immunocytochemistry.
Main outcome measure: We measured the difference in CTC detection between ACC and ACA.
Results: CTCs were detected in all ACC samples, but not in ACA samples. Immunocytochemistry confirmed the adrenocortical origin. When ACC patients were stratified according to the median value of tumor diameter and metastatic condition, a statistically significant difference was found in the number of CTCs detected after surgery. A significant correlation between the number of CTCs in postsurgical samples and clinical parameters was found for tumor diameter alone.
Conclusions: Our findings provide the first evidence for adrenocortical tumors that CTCs may represent a useful marker to support differential diagnosis between ACC and ACA. The correlation with some clinical parameters suggests a possible relevance of CTC analysis for prognosis and noninvasive monitoring of disease progression and drug response.
A new device for rapid isolation by size and characterization of rare circulating tumor cells
Background: Circulating tumor cells (CTCs) likely derive from clones in the primary tumor, suggesting that they can be used for all biological tests applying to the primary cells.
Materials and methods: The ScreenCell® devices are single-use and low-cost innovative devices that use a filter to isolate and sort tumor cells by size.
Results: The ScreenCell® Cyto device is able to isolate rare, fixed, tumor cells, with a high recovery rate. Cells are well preserved morphologically. Immunocytochemistry and FISH assays can be performed directly on the filter. The ScreenCell® CC device allows isolation of live cells able to grow in culture. High quality genetic materials can be obtained directly from tumor cells isolated on the ScreenCell® MB device filter.
Conclusion: Due to their reduced size, versatility, and capacity to isolate CTCs within minutes, the ScreenCell® devices may be able to simplify and improve non-invasive access to tumor cells.