Dimple Chudasama

An assessment of diagnostic performance of a filter-based antibody-independent peripheral blood circulating tumour cell capture paired with cytomorphologic criteria for the diagnosis of cancer

OBJECTIVES Circulating tumour cells (CTCs) are reported to be predictive for prognosis and response to treatment in advanced lung cancer. However, the clinical utility of the CTCs detection remains unknown for early stage lung cancer as the number of CTCs is reported as low, providing challenges in identification. We have evaluated diagnostic performance of filtration-based technology using cytomorphologic criteria in patients undergoing surgery for lung cancer. MATERIAL AND METHODS We processed blood from 76 patients undergoing surgery for known or suspected lung cancer using ScreenCell(®) Cyto filter devices. Captured cells were stained using haematoxylin and eosin and independently assessed by two pathologists for the presence of atypical cells suspicious for cancer. Diagnostic performance was evaluated against pathologist reported diagnoses of cancer from surgically obtained specimens. RESULTS Cancer was diagnosed in 57 patients (77.0%), including 32 with primary lung cancer (56.1%). The proportion of patients with early stage primary lung cancer in which CTCs were identified was 18 and 21 (56.3% and 65.6%, respectively) as reported by two pathologists. The agreement between the pathologists was 77.0% corresponding to a kappa-statistic of 53.7% indicating moderate agreement. No significant differences were found for the percentage of CTCs for primary and metastatic cancer as well as for cancer stages. On sensitivity weighted analysis, a sensitivity and specificity were 71.9% (95% CI 60.5-83.0) and 52.9% (95% CI 31.1-77.0), respectively. On specificity weighted analysis, a sensitivity and specificity were 50.9% (95% CI 39.3-64.4) and 82.4% (60.4-96.2), respectively. CONCLUSION The performance of the tested filter-based antibody-independent technology to capture CTCs using standard cytomorphologic criteria provides the potential of a diagnostic blood test for lung cancer.

Circulating tumour cells in patients with lung cancer undergoing endobronchial cryotherapy.

Early diagnosis of lung cancer still poses a major issue, with a large proportion of patients diagnosed at late stages. Therapeutic options and treatment remain limited in these patients. In most cases only palliative therapies are available to alleviate any severe symptoms. Endobronchial cryotherapy (EC) is one form of palliative treatment offered to patients with obstructive airway tumours. Although successful, the impact on circulating tumour cell (CTCs) spread has not been investigated in detail. This study recruited 20 patients awaiting EC treatment. Baseline and post EC blood samples were analysed for presence of CTCs. Results showed an increase in CTCs following EC in 75% of patients. Significant increases were noticeable in some cases. Although EC is a well-accepted modality of treatment to alleviate symptoms, it may lead to an increase in CTCs, which in turn may have implications for tumour dissemination and metastatic spread.

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.

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.

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

Objectives: 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. Methods: 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. Results: 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%). Conclusion: 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.

Prognostic value of the DNA integrity index in patients with malignant lung tumors

Introduction Lung cancer survival remains poor in the western world due to late presentation in most cases, leading to difficulty of treatment in these advanced and metastatic patients. Therefore, the development of a robust biomarker for prognosis and to monitor treatment response and relapse would be of great benefit. The use of Alu repeats and DNA Integrity Index has been shown to hold both diagnostic and prognostic value, and as it is obtained from the plasma of patients, it can serve as a non-invasive tool for routine monitoring. This study evaluates the efficiency of this technique in malignant lung cancer patients. Methods Plasma samples were collected from 48 patients, consisting of 29 lung cancer patients and 19 non-cancer controls. Alu repeat ratio and confounders were measured. Results Observations showed a higher Alu repeat ratio amongst the cancer group compared to controls (p=0.035), mean Alu ratio 0.38 (range 0.01-0.93) and 0.22 (0.007-0.44) respectively, ROC curve analysis AUC 0.61 (p=0.22). Analysis by staging was more promising, whereby a higher DNA Integrity Index was seen in advanced cases compared to both early stage and controls, p<0.0001; AUC: 0.92 (P=0.0002) and p=0.0006, AUC – 0.88 (p=0.0007) respectively, however no significant difference was observed in the early stage compared to controls. Short term survival data also showed a DNA Integrity Index of >0.5 to be associated with poorer overall survival p=0.03. Conclusion The results of this study show a potential use of Alu repeats ratios for prognostic purposes in the advanced setting for lung cancer patients.

Identification of novel cancer biomarkers of prognostic value using specific gene regulatory networks (GRN): a novel role of RAD51AP1 for ovarian and lung cancers.

We provide evidence of how RAD51AP1 can be of importance as potential biomarker since it is overexpressed in both tissue and peripheral blood of ovarian and lung cancer patients. Silencing of the gene can also lead to decrease in cell proliferation in vitro, so a potential therapeutic target.

Differential effects of rapalogues, dual kinase inhibitors on human ovarian carcinoma cells in vitro

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH-2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer.

Contents Vol. 89, 2015

A.B. Benson, Chicago, Ill. A. Chang, Singapore A.L. Cheng, Taipei J.F. Cleary, Madison, Wis. M. Dietel, Berlin M.S. Ernstoff, Cleveland, Ohio M.G. Fakih, Duarte, Calif. J.J. Grau, Barcelona H. Gronemeyer, Illkirch D.F. Hayes, Ann Arbor, Mich. C.S. Johnson, Buffalo, N.Y. M.J. Kelley, Durham, N.C. L. Kumar, New Delhi P.J. Loehrer, Indianapolis, Ind. J.R. Marshall, Buffalo, N.Y. S. Monfardini, Milan R. Nagler, Haifa R. Ohno, Nagoya B. Pestalozzi, Zurich H.M. Pinedo, Amsterdam E.A. Repasky, Buffalo, N.Y. A. Semczuk, Lublin E.F. Smit, Amsterdam C.N. Sternberg, Rome R. Stupp, Zurich M.S. Tallman, New York, N.Y. S. Tanaka, Hiroshima M. Tian, Houston, Tex. D.L. Trump, Buffalo, N.Y. T. Wiegel, Ulm W. Yasui, Hiroshima H. Zhang, Hangzhou City Editor-in-Chief