Detection of EGFR mutations and EML4‐ALK rearrangements in lung adenocarcinomas using archived cytological slides

Abstract

BACKGROUND:Although the molecular analysis of epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) in archived lung cancer tissues is relatively well established, the genetic testing of cytological material has not yet become a routine. METHODS: The current study used cell samples that were obtained by bronchial brushing, transthoracic needle aspiration, or biopsy imprint preparation between 1993 and 2008. Islets of malignant cells were visually located on the archived cytological slides, lysed in situ by a drop of sodium dodecyl sulfate-containing buffer, and subjected to the standard DNA and RNA extraction. Examination of paraffin-embedded tissue blocks (resection specimens or biopsy material) from the same patients was performed in parallel. RESULTS: A total of 75 cytological/histological lung adenocarcinoma sample pairs underwent polymerase chain reaction analysis for the EGFR mutation. Two cytological samples and 1 morphological sample failed to produce DNA. Concordance for the wild-type and mutation status was observed in 54 of 72 and 14 of 72 informative pairs, respectively; 3 pairs and 1 pair, respectively, had mutation only in the cytological or histological material. The discrepancies could be explained by the failure to ensure a high percentage of lung cancer cells in the analyzed samples or, alternatively, by the genuine intratumoral molecular heterogeneity of some neoplasms. RNA extraction followed by reverse transcriptase-polymerase chain reaction analysis for the EML4-ALK translocation was performed for 44 EGFR mutation-negative sample pairs; failures were observed for 2 cytological and 6 histological specimens. All informative pairs were concordant either for the norm (32 of 36 pairs) or for the presence of EML4-ALK gene fusion (4 of 36 pairs). CONCLUSIONS: Archived cytological slides appear to be well suited both for EGFR and ALK analysis.

Novel ALK fusion partners in lung cancer

“…In addition, PCR ALK-analysis utilizes the same technical platform as other kinds of molecular NSCLC diagnosis, i.e. it can be performed in parallel or after EGFR testing using the same pool of nucleic acids [2,15]. A number of commercial PCR kits for detection of ALK rearrangements have been developed recently (e.g., ALK RGQ RT-PCR Kit (Qiagen); EML4-ALK Fusion Gene Detection Kit (AmoyDx); EML4 ALK Gene Fusion, PCR (Quest Diagnostics), etc.…”

EGFR T790M Mutation in TKI-Naïve Clinical Samples: Frequency, Tissue Mosaicism, Predictive Value and Awareness on Artifacts

“…table 4, www.karger.com/?DOI=491441). Similarly, we subjected 10 pairs of FFPE samples to organic extraction [37] and isolation with a Roche cobas® DNA Sample Preparation Kit. DNA was successfully obtained in 9 pairs, and no significant difference was observed (p = 0.11, Wilcoxon signed-rank test for paired samples; suppl.…”

“…Blood DNA from NSCLC patients and tumor DNA from non-NSCLC oncological patients were collected on a random basis. DNA from microdissected archival tumor tissue material or blood lymphocytes was isolated by a standard organic extraction [37,38]. To evaluate the role of DNA isolation protocols, a GE Healthcare illustra blood genomicPrep Mini Spin Kit and a Roche cobas® DNA Sample Preparation Kit were used for control experiments.…”

Molecular Testing of Non–Small Cell Lung Carcinoma Diagnosed by Endobronchial Ultrasound–Guided Transbronchial Fine-Needle Aspiration: The Cleveland Clinic Experience

“…Cytology specimens provide highquality DNA for molecular testing 7,8 and are comparable to, if not superior to, formalin-fixed, paraffin-embedded surgical pathology specimens, for the evaluation of clinically relevant mutations. [8][9][10][11] Although cell blocks are frequently used for molecular testing of non-small cell lung carcinomas, at our institution they were suboptimal for analysis in a high proportion of cases. An alternate strategy for EGFR and ALK testing was therefore developed, using material from the liquid-based cytology sample for EGFR mutation testing by nextgeneration sequencing (NGS), and the ThinPrep slide for fluorescence in situ hybridization (FISH) testing for ALK gene rearrangements.…”