Maria Leung

Circulating Tumor DNA Outperforms Circulating Tumor Cells for KRAS Mutation Detection in Thoracic Malignancies

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.

Debridement alone without decortication can achieve lung re-expansion in patients with empyema: an observational study

Decortication is widely performed for empyema, but the effectiveness in achieving lung re-expansion has never been formally reported. The aim of this study is to quantify the degree of lung re-expansion in comparison to that achieved naturally after debridement alone. A retrospective review of patients who underwent either decortication or debridement for empyema between 2007 and 2009. The change of the cavity size with time were standardized and recorded before, immediately after surgery and on follow-up. Of 25 patients who underwent surgical management of empyema, 16 (64%) underwent debridement alone and nine (36%) underwent decortication. The mean age (standard deviation) was 58 (19) years and 15 (60%) were male. On radiological follow-up at a median [interquartile range (IQR)] of 45 (36-116) days, further reduction of 36% and 34% was achieved leaving 27% and 12% of the original cavity size in the debridement and decortication groups, respectively. Procedure (debridement or decortication) was not associated with any difference to the eventual follow-up cavity size (P = 0.937). Similar follow-up results were achieved by debridement alone without decortication in patients presenting with empyema, despite the presence of an underlying trapped lung.

Test performance of PET-CT for mediastinal lymph node staging of pulmonary carcinoid tumours

Positron emission tomography-CT (PET-CT) is one of the initial mediastinal staging modality for non-small cell lung cancer; however, the clinical utility in carcinoid tumours is uncertain. We sought to determine the test performance of PET-CT for mediastinal lymph node staging of pulmonary carcinoid tumours. We collated data from seven institutions, performing a retrospective search on pathological databases for a consecutive series of patients who underwent thoracic surgery (with lymph nodal dissection) for carcinoid tumours with preoperative PET-CT staging. PET-CT results were compared with the reference standard of pathologic results obtained from lymph node dissection and test performance reported using sensitivity and specificity. From November 1999 to January 2013, 247 patients from seven institutions underwent surgery for carcinoid tumours with a corresponding preoperative PET-CT scan. The mean age of the patients was 61 (SD 15, range 73) and 84 were male patients (34%). The pathologic subtype was typical carcinoid in 217 patients (88%) and atypical carcinoid in 30 patients (12%). Results from lymph node dissection were obtained in 207 patients. The calculated sensitivity and specificity of PET-CT to identify mediastinal lymph node disease was 33% (95% CI 4% to 78%) and 94% (95% CI 89% to 97%), respectively. Our results indicate that PET-CT has a poor sensitivity but good specificity to detect the presence of mediastinal lymph node metastases in pulmonary carcinoid tumours. Mediastinal lymph node metastases cannot be ruled out with negative PET-CT uptake, and if the absence of mediastinal lymph node disease is a prerequisite for directing management, tissue sampling should be undertaken.

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.

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.

1 A comparative analysis of cancer hotspot mutation profiles in circulating tumour cells, circulating tumour DNA and matched primary lung tumour

in the development of a number of successful targeted therapies. These targeted therapies show dramatic response in a small proportion of patients who process specific genetic aberrations. Blood based mutation profile analysis is becoming an increasingly important non-invasive form of mutation screening in cancer. Whilst many have reported on single mutation comparisons between blood based and primary tumour tissue, limited information is available on multiplex comparisons between the DNA extracted from circulating tumour cells (CTC) and circulating free tumour DNA in the plasma (ctDNA) against the current standard of FFPE analysis of primary tumour and no information exists comparing next generation sequencing (NGS) profiles between the 3 different substrates within the same patient. The aim of our work is to report the concordances between CTCs and ctDNA versus the primary FFPE tumour mutations using a NGS hotspot panel. Royal Brompton and Harefield NHS Foundation Trust A comparative analysis of cancer hotspot mutation profiles in circulating tumour cells, circulating tumour DNA and matched primary lung tumour