Feasibility of single-cell analysis of model cancer and foetal cells in blood after isolation by cell picking

The objective of the present feasibility study was to transfer single cell line cells to either microscopy slides for downstream immune characterization or to polymerase chain reaction tubes for downstream DNA quantitation. Tumour cell lines, SKBR3 and MCF7 and trophoblast cell line JEG-3 were spiked in healthy donor blood. The CytoTrack system was used to scan the spiked blood samples to identify target cells. Individual target cells were identified, picked by use of a CytoPicker and deposited to either a microscopic slide or a polymerase chain reaction tube (PCR). Single tumour cells on microscopic slides were further immunostained with human epidermal growth factor receptor 2 (Her2) and epithelial cell adhesion molecule (EpCAM). From the picked cells in polymerase chain reaction tubes, DNA was amplified, quantified and used for Short Tandem Repeat genotyping. Depositing rare cells to microscopy slides was laborious with only five cells per hour. In this study with a trained operator, the picked cells had an 80.5% recovery rate. Depositing single trophoblast cells in PCR tubes was a faster process with 10 cells in 5 min. Immunostaining of isolated cells by both Her2 and EpCAM was possible but showed varying staining intensity. Presence of trophoblasts and contaminating white blood cells in PCR tubes after cell picking was confirmed based on DNA yield and mixed Short Tandem Repeat profiles in five out of eight samples. Using the CytoPicker tool, single tumour and trophoblast cells were successfully isolated and moved from blood samples, allowing subsequent immunostaining or Short Tandem Repeat genotyping

Clean Colorectum at Diagnostic Colonoscopy:Subsequent Detection of Extracolonic Malignancies by Plasma Protein Biomarkers?

Introduction: Most of the subjects undergoing diagnostic colonoscopy do not have neoplastic bowel lesions. Potentially, some of the symptoms may therefore be caused by extracolonic malignancy, and subjects with persisting symptoms may need subsequent examinations. Blood-based, cancer-associated biomarkers may aid in directing the examinations for other specific malignant diseases. Methods: EDTA plasma samples available from a previous prospective study of subjects undergoing diagnostic colonoscopy were used for analysis of 18 protein biomarkers. The study population of 3732 subjects included 400 patients with colorectal cancer (CRC) and 177 patients with extracolonic malignancies. Univariable analysis of the association of specific biomarkers and extracolonic cancers included those with 10 or more cases. Subsequently, reduced models of 4 or 6 biomarkers, respectively, were established by choosing those with the highest likelihood; age and sex were included as well. Results: Univariable analyses showed that CyFra21-1 had an area under curve (AUC) of 0.87 for lung cancers (n = 33), CA19-9 had an AUC of 0.85 for pancreatic cancer (n = 22), CA125 had an AUC of 0.95 for ovary cancer (n = 16), B2M had an AUC of 0.81 for non-Hodgkin lymphoma (n = 12), and total prostate-specific antigen had an AUC of 0.99 for prostate cancer (n = 10). The multivariable analysis of 4 or 6 biomarkers plus age and sex as explanatory variables showed AUCs of 0.82 to 0.85 both for extracolonic cancers and CRC. The 4 biomarkers included in the model for detection of extracolonic cancers were CA125, hsCRP, CA19-9, and CyFra21-1; the 2 additional for the 6 biomarkers model were CEA and Galectin-3. Similarly, the 4 biomarkers included in the model for detection of CRC were CEA, CyFra21-1, Ferritin, and HE4; the two additional for the 6 biomarkers model were hsCRP and Pepsinogen 2. Conclusions: Results of this study indicate that it may be possible to detect subjects that have an increased risk of extracolonic cancer following a colonoscopy without findings of neoplastic lesions. Combinations of various protein biomarkers may direct subsequent examination after colonoscopy with clean colorectum. The results, although preliminary, may form the basis for additional research directed both for primary examinations of subjects with symptoms of malignancy and subsequent examinations after colonoscopy

In vitro validation of an ultra-sensitive scanning fluorescence microscope for analysis of Circulating Tumor Cells

Analysis of circulating tumor cells (CTC) holds promise of providing liquid biopsies from patients with cancer. However, current methods include enrichment procedures. We present a method (CytoTrack®), where CTC from 7.5 mL of blood is stained, analyzed and counted by a scanning fluorescence microscope. The method was validated by breast cancer cells (MCF-7) spiked in blood from healthy donors. The number of cells spiked in each blood sample was exactly determined by cell sorter and performed in three series of three samples spiked with 10, 33 or 100 cells in addition with three control samples for each series. The recovery rate of 10, 33 and 100 tumor cells in a blood sample was 55%, 70% and 78%, percent coefficient of variation (CV%) for samples was 59%, 32% and 18%, respectively. None of the control samples contained CTC. In conclusion, the method has been validated to highly sensitively detect breast cancer cells in spiking experiments and should be tested on blood samples from breast cancer patients. The method could benefit from automation that could reduce the CV%, and further optimization of the procedure to increase the recovery