Scope of the study Patients of lung cancer often present with mediastinal/hilar lymph nodes. These may, at times, be inadequate or indeterminate on EBUS guided FNAC. This study proposes to estimate CTCs in this group of patients with the following aims and objectives: Aim: To detect CTCs and mutations from cell-free DNA (liquid biopsies) in peripheral blood of suspected lung cancer patients with mediastinal/hilar lymphadenopathy undergoing EBUS guided FNA. Objectives: A. Primary objective: To evaluate the sensitivity and specificity of CTCs in detecting lung cancer in patients with mediastinal/hilar lymphadenopathy. Undergoing EBUS guided FNA. The fine-needle aspiration biopsy report from the EBUS will serve as the reference standard. B. Secondary objectives: 1. To evaluate the sensitivity and specificity of CTCs in lung cancer patients with inadequate or indeterminate EBUS-TBNA cytology on the first attempt at EBUS-guided FNA. Fine needle aspiration biopsy from subsequent attempts as well as a combination of CT/MRI findings, ancillary tissue diagnosis (by FNA/biopsy from a separate secondary lymph node, tissue or pleural fluid) will serve as reference test. 2. Evaluate the correlation of CTC yield with disease stage 3. Extract the DNA for detection of molecular markers from plasma of leftover blood as well as from the tumor from adequate FNA (cell-free DNA) 4. Comparison of EGFR and common targetable mutations status from DNA extracted from cell-free DNA to that extracted from tissue FNAB.
Methods Study design: Multicentre, diagnostic accuracy study Study setting: Bronchoscopy suite of participating institutions (for EBUS-TBNA) and Department of Pathology and Laboratory Medicine’s Cytology Lab at AIIMS Rishikesh (for CTC detection). Sample size: 140 patients with suspicious mediastinal/hilar nodes. Sample size calculation: The sample size was calculated assuming a prevalence of disease of 75% in the patients with suspected lung cancer (based on clinical experience), sensitivity of at least 65% (based on experience with urinary bladder carcinoma), and specificity of at least 90%. We designed the study to ensure a confidence interval width of ±10%
Inclusion criteria: Adult (greater than 18 years) patients with clinico-radiologically suspected lung cancer with mediastinal/hilar lymph nodes will be enrolled. Exclusion criteria: Denial of consent to participate History of a prior malignancy within five years of enrollment in the present study Study procedure Enrollment: Clinico-radiologically suspected patients satisfying the inclusion/exclusion criteria and undergoing EBUS-TBNA from mediastinal/hilar lymph nodes will be enrolled. Sample collection for CTC: Enrolled patients will then be subjected to collection of peripheral blood samples for CTC analysis. The first 2 ml of blood drawn from the median cubital vein will be discarded to avoid potential epithelial cell contamination, and subsequently 3 ml of peripheral blood will be collected into a BD Vacutainer tube. All blood samples will be processed within 24 hours of collection for CTC analysis. CTC estimation: CTC estimation will be done by size-based filtration using ScreenCell(Trademarked) filtration technique or equivalent filtration based technique. The cells separated will be stained with MGG stain and visualized under a microscope. The DNA will be extracted for further future analysis of prognostic factors. Correlation of results: The CTC estimation results thus obtained will then be correlated with the final diagnosis, disease clinical stage and probable disease prognosis. Extraction of DNA from EBUS guided FNAB and cell-free DNA: By special kits for extraction of cell free DNA. Microdissection of the cells from EBUS-guided FNAB slides will be attempted prior to extraction with subsequent cell-lysis. Mutation status by NGS panel: By Next-generation sequencing after extraction. KRAS, NRAS, BRAF, EGFR & PIK3CA will be other genes wihich will be included in the panel. Sequencing will be carried out to a depth of 2000x for cell-free DNA and 300x for EBUS guided FNAB . Assessment of outcome measures: The proportion of cases in which a diagnosis of lung cancer can be rendered on circulating tumor cells will be estimated. For the secondary analysis, descriptive analysis with Kruskall-Wallis test for finding correlation of CTC cell count with Disease stage. Proportion of positive cases with mutation in EBUS-guided FNAB cases which are positive for targetable mutation in cell-free DNA, as well as percentage of cases in which cell-free-DNA gives additional information will be estimated after estimation by cross-tab analysis. Data recording: Data will be recorded on a predesigned proforma and managed on an excel spread sheet. All entries will be checked for any possible keyboard error. All the patient characteristics at the time of enrolment will be compared between the two groups. Statistical analysis: For primary outcome, the proportion of cases in which a diagnosis of lung cancer can be rendered on microscopic evaluation will be calculated with 95% confidence interval. Sensitivity, specificity and positive and negative likelihood ratios of CTCs in detecting lung cancer and also in the subset of patients having inadequate/indeterminate findings in the first pass will be estimated. For secondary analysis presence and absence of circulating tumour cells will be correlated with stage (Chi-square test and descriptives), age, size (Mann-Whitney test), sensitivity, additional pick up rate (for cell-free DNA) and proportions with 95% Confidence intervals. Expected outcome: We expect that circulating tumor cells will be detectable in at least 65% of invasive lung cancers at near 100% specificity. We also expect that similar sensitivity will be maintained for inadequate/indeterminate tumors on EBUS-guided FNAB. At such sensitivity, cost-effectiveness analysis will be subsequently performed to reveal whether circulating tumor cell detection will be more economical as a first-line/second line test than EBUS-guided FNAB done initially.
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