Inertia based microfluidic capture and characterisation of circulating tumour cells for the diagnosis of lung cancer

Background: Routine clinical application of circulating tumour cells (CTCs) for blood based diagnostics is yet to be established. Despite growing evidence of their clinical utility for diagnosis, prognosis and treatment monitoring, the efficacy of a robust platform and universally accepted diagnostic criteria remain uncertain. We evaluate the diagnostic performance of a microfluidic CTC isolation platform using cytomorphologic criteria in patients undergoing lung cancer surgery. Methods: Blood was processed from 51 patients undergoing surgery for known or suspected lung cancer using the ClearBridge ClearCell FX systemTM (ClearBridge Biomedics, Singapore). Captured cells were stained on slides with haematoxylin and eosin (H&E) and independently assessed by two pathologist teams. Diagnostic performance was evaluated against the pathologists reported diagnosis of cancer from surgically obtained specimens. Results: Cancer was diagnosed in 43.1% and 54.9% of all cases. In early stage primary lung cancer, between the two reporting teams, a positive diagnosis of CTCs was made for 50% and 66.7% of patients. The agreement between the reporting teams was 80.4%, corresponding to a kappa-statistic of 0.61±0.11 (P<0.001), indicating substantial agreement. Sensitivity levels for the two teams were calculated as 59% (95% CI, 41–76%) and 41% (95% CI, 24–59%), with a specificity of 53% for both. Conclusions: The performance of the tested microfluidic antibody independent device to capture CTCs using standard cytomorphological criteria provides the potential of a diagnostic blood test for lung cancer

Circulating tumor DNA outperforms circulating tumor cells for KRAS mutation detection in thoracic malignancies

Abstract BACKGROUND Circulating biomarkers, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), are both considered for blood-based mutation detection, but limited studies have compared them in a head-to-head manner. Using KRAS (Kirsten rat sarcoma viral oncogene homolog), we performed such a comparison in patients who underwent surgery for suspected lung cancer. METHODS We recruited 93 patients, including 82 with lung cancer and 11 with benign diseases of the lung. Mutations were detected in codons 12 and 13 of KRAS in DNA extracted from CTCs, plasma, and matched tumors or lung tissues with custom-designed coamplification at lower denaturation temperature (COLD)-PCR assays, high-resolution melt analysis (HRM), and commercial assays (Roche Cobas®KRAS mutation test and Qiagen Therascreen® pyrosequencing KRAS kit). RESULTS With the Cobas mutation test, we identified KRAS mutations in 21.3% of tumors. Mutation analysis in matched CTC DNA and ctDNA samples by COLD-PCR/HRM assay revealed mutations in 30.5% (ctDNA) and 23.2% (CTC DNA) of patients with lung cancer. Combined results of different tests revealed KRAS-positive cases for 28% of tumors. The diagnostic sensitivity and specificity of KRAS mutation detection in tumors achieved with ctDNA was 0.96 (95% CI 0.81–1.00) and 0.95 (0.85–0.99), respectively. The diagnostic test performance was lower for CTC DNA, at 0.52 (0.34–0.73) and 0.88 (0.79–0.95). CONCLUSIONS Our results support ctDNA as a preferential specimen type for mutation screening in thoracic malignancies vs CTC DNA, achieving greater mutation detection than either CTCs or limited amounts of tumor tissue alone. </jats:sec

Diagnostic Utility of Unbiased Circulating Tumour Cell Capture through Negative Depletion of Peripheral Blood Cells

&lt;b&gt;&lt;i&gt;Objectives:&lt;/i&gt;&lt;/b&gt; Cytological analysis of peripheral blood circulating tumour cells (CTCs) is a potential method of confirmatory clinical diagnosis of cancer. However, cell capture methods tend to be biased and captured cells are not usually portable resulting in difficulties in pathology reporting. We evaluated unbiased cell capture through depletion of unwanted normal cells and conventional clinical analyses of captured cells. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; Blood was sampled from 29 patients who underwent surgery for suspected lung cancer. It was processed using two different depletion cocktails. After depletion of unwanted cells, the resultant cell pellet was processed onto glass slides or embedded into FFPE blocks and stained using standard haematoxylin and eosin staining followed by cytopathologic assessment. Two pathologists performed the assessment independently. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; The CTCs were identified in 38-45% of cases using CD45 depletion cocktail with the cell pellet processed on a glass slide, while other combinations of methods produced poorer results. Overall, there was a good concordance between the pathologists (up to 91.3%). The sensitivity of cancer diagnosis was 42% (95% CI 23-63%), while the specificity was 100% (95% CI 29-100%). &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; Negative depletion can be used to isolate CTCs in standard clinical settings; however, more effective ways of detection are required to increase the sensitivity of the diagnosis.</jats:p

Stenting and cell technologies in the treatment of atherosclerotic renovascular hypertension Part 2. Effectiveness and safety of postnatal mobilized peripheral blood stem cell auto-transplantation into renal and vertebral arteries in ischemic kidney disease lasting over ten years

Aim. To assess effectiveness and safety of stem cell auto-transplantation (SCT) into renal and vertebral arteries among patients with renovascular hypertension (RVH) lasting over 10 years. Material and methods. Seventy-eight patients were randomized into main (MG, n=26) and placebo groups (PG, n=52). Primary end-point was systolic blood pressure (SBP) level. Secondary end-points included: restenosis incidence; glomerular filtration rate (GFR); effective renal plasma flow (ERPF); creatinine level and microalbuminuria (MAU); quality of life (QoL); renal biopsy and immuno-hystochemical assay data; renal vessel calcification; cerebral metabolism level. Results. Restenosis at 12 months was observed in 17% of both MG and CG patients; repeated revascularization was needed in 8%. Stenting resulted in average BP decrease from 181/107 to 142/93 rum Hg; after 6 weeks, no patient achieved target BP levels. After SCT into both renal arteries, BP normalization was achieved in 61% of MG participants. Only 23% of regenerated renal tissue originated from transplanted SC (trans-differentiation), the rest originated from local tissues (de-differentiation). After 50-55 weeks, in 37-39% of the patients Stage I AH was observed. At 6-8 weeks after SCT into both vertebral arteries, cerebral metabolism increased, and BP normalized, reducing from 138/90 to 119/69 mm Hg. Conclusion. SCT plays an important role in renal artery thrombosis prevention and RVH control. Renal and vertebral artery SCT solves the problems of nephron destruction and renal tissue fibrosis, atherosclerosis treatment and harmful cerebral influences. Remaining problems include target organ damage and genetic defects