Vladimir Anikin

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.

Clinical results of microfluidic antibody-independent peripheral blood circulating tumor cell capture for the diagnosis of lung cancer

OBJECTIVES The ability to capture and characterize peripheral blood circulating tumor cells has the potential for the development of a blood test for cancer. The aim of this study was to evaluate the diagnostic performance of microfluidic technology as a proof-of-concept study. METHODS Blood from patients undergoing surgery for known or suspected lung cancer was obtained and processed using a microfluidic biochip. Diagnostic performance was evaluated against the reference of cancer identified within surgically obtained formalin-fixed paraffin embedded specimens reported by a principal pathologist. Agreement was assessed in a sample reported by a second independent pathologist. Sensitivity- and specificity-weighted analyses were undertaken. RESULTS From March 2011 to October 2012, 46 patients at our institution donated blood for research. Cancer was the underlying diagnosis in 43 (94%); 34 (79%) of the patients had primary lung cancer. The proportion of patients with cancer in which atypical cells suspicious for cancer were identified on hematoxylin and eosin staining was 16/43 (37%) by the principal pathologist and 10/17 (59%) by the second pathologist. On sensitivity-weighted analysis, the sensitivity of the biochip was 54% (95% confidence interval [CI], 37-72) and the specificity was 33% (95% CI, 2-91). On specificity-weighted analysis, the sensitivity was 43% (95% CI, 21-71) and the specificity was 100% (95% CI, 5-100). CONCLUSIONS This work highlights the potential of microfluidic technology to develop a blood test for the diagnosis of cancer using peripheral blood; conventional clinical criteria can be used as a proof-of-concept of what may be possible with todays technology.

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.

Increasing frequency of non-smoking lung cancer: Presentation of patients with early disease to a tertiary institution in the UK

BACKGROUND Never-smokers with lung cancer often present late as there are no established aetiological risk factors. The aim of the study is to define the frequency over time and characterise clinical features of never-smokers presenting sufficiently early to determine if it is possible to identify patients at risk. METHODS We retrospectively analysed data from a prospectively collected database of patients who underwent surgery. The frequency was defined as number of never-smokers versus current and ex-smokers by year. Clinical features at presentation were collated as frequency. RESULTS A total of 2170 patients underwent resection for lung cancer from March 2008 to November 2014. The annual frequency of developing lung cancer in never-smokers increased from 13% to 28%, attributable to an absolute increase in numbers and not simply a change in the ratio of never-smokers to current and ex-smokers. A total of 436 (20%) patients were never-smokers. The mean age was 60 (16 SD) years and 67% were female. Presenting features were non-specific consisting of cough in 34%, chest infections in 18% and haemoptysis in 11%. A total of 14% were detected on incidental chest film, 30% on computed tomography, 7% on positron-emission tomography/computed tomography and 1% on MRI. CONCLUSIONS We observed more than a double of the annual frequency of never-smokers in the last 7 years. Patients present with non-specific symptoms and majority were detected on incidental imaging, a modality that is likely to play an increasingly important role for early detection in this cohort that does not have any observable clinical risk factors.

Video-assisted intercostal nerve cryoablation in managing intractable chest wall pain.

FIGURE 1. Patient is in the left lateral decubitus position. Percutaneous placement of cyroprobe lateral to the spine is performed under thorascopic guidance at the premarked 10th, 11th, and 12th intercostal spaces. An endoscopic lung retractor and thoracoscope was inserted through a single anterolateral 10-mm port placed in the seventh intercostal space (white arrow). CLINICAL SUMMARY A 52-year-old women was referred to our unit with severe pain in the right side of the chest, having been kicked in the right flank by a horse 10 years earlier. At the time she sustained fractures to the right 10th, 11th, and 12th ribs and briefly required hospitalization. The pain was sharp and stabbing in nature. Standard oral analgesia including nonsteroidal antiinflammatory drugs was ineffective. She obtained little relief from opiates and was reluctant to take them long term. Some transient analgesia was obtained through epidurals and intercostal nerve blocks. She had been managed with gabapentin 800 mg twice daily and amitriptyline 25 mg daily long term. She had no relevant medical history and clinical examination was unremarkable apart from an exquisitely hypersensitive area in the right flank. No obvious deformity was noted, but she had restricted chest wall movement owing to pain. Initial baseline investigations including chest radiograph showed no abnormalities. The rib fractures had healed without deformity. Magnetic resonance imaging revealed no soft tissue abnormality. She was admitted the day before the operation and underwent an intercostal nerve block with lidocaine by the operating surgeon (V.A.) to assess exact location of intercostal nerves affected and effectiveness of blockade. The area was marked. The next day, after single lung ventilation and appropriate isolation under a general anesthetic, a single 10-mm port was inserted anterolaterally into the 7th intercostal space and the lung was retracted medially. A straight 15-cm long insulated cryoprobe with a tip diameter of 2 mm (designed and modified by one of us [O.M.] and manufactured by Spembly, Andover, United Kingdom) was inserted percutaneously close to the paraspinal junction at the previously marked sites and positioned under thoracoscopic guidance (Figure 1). The area close to the affected intercostal nerve in the 10th intercostal space was frozen until an ice ball could clearly be seen (Figure 2) for about 2 minutes. Parietal pleura remained intact. This was repeated in the 11th and 12th intercostal spaces. No signs of bleeding were noted. A single 24F chest drain was placed and removed the following day. No postoperative complica-

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.

Selection for adjuvant chemotherapy in completely resected stage I non-small cell lung cancer: external validation of a Chinese prognostic risk model

BACKGROUND The ability to sub-stratify survival within stage I is an important consideration as it is assumed that survival is heterogeneous within this sub-group. Liang et al. recently published a nomogram to predict post-operative survival in patients undergoing lung cancer surgery. The aim of our study is external validation of their published nomogram in a British cohort focusing on stages IA and IB to determine applicability in selection of adjuvant chemotherapy within stage I. METHODS Patient variables were extracted and the score individually calculated. Receiver operative characteristics curve (ROC) was calculated and compared with the original derivation cohort and the discriminatory ability was further quantified using survival plots by splitting our (external) validation cohort into three tertiles and Kaplan Meier plots were constructed and individual curves tested using Cox regression analysis on Stata 13 and R 3.1.2 respectively. RESULTS A total of 1,238 patients were included for analysis. For all patients from stage IA to IIB the mean (SD) score was 9.95 (4.2). The ROC score comparing patients who died versus those that remained alive was 0.62 (95% CI: 0.58 to 0.67). When divided into prognostic score tertiles, survival discrimination remained evident for the entire cohort, as well as those for stage IA and IB alone. The P value comparing survival between the middle and highest score with baseline (low score) was P=0.031 and P=0.034 respectively. CONCLUSIONS Our results of external validation suggested lower survival discrimination than reported by the original group; however discrimination between survival remained evident for stage I.

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.

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.

Video-assisted thoracoscopic PlasmaJet ablation for malignant pleural mesothelioma

The role of surgery in malignant pleural mesothelioma (MPM) remains debatable; nonetheless the relative advantages of different surgical approaches are frequently reassessed and reconsidered. While extensive operations and longer recovery periods can be justified for a group of carefully selected patients, many will present at an advanced stage of their disease or with associated co-morbidities which will exclude them from selection criteria for radical treatment. For these patients, minimally invasive video-assisted procedures may be considered, for purposes of cytoreduction and/or symptomatic relief. Even though there is currently not enough clinical evidence to suggest an improvement in overall survival with limited debulking procedures, it has been suggested that they can improve quality of life over drainage and pleurodesis alone. We consider video-assisted PlasmaJet ablation to potentially have a role in mesothelioma surgery, as it may be used for effective cytoreduction while minimising the risk for complications often associated with extensive pleurectomy procedures, and we report on the use of the PlasmaJet Surgical System in our centre for surgical management of a patient with MPM. After demonstrating safety and absence of major adverse events with this approach, we feel justified in offering the procedure to more of our patients as we aim to collect additional data.