EGFR and KRAS Mutation Analysis in Cytologic Samples of Lung Adenocarcinoma Enabled by Laser Capture Microdissection

Sinchita Roy Chowdhuri; Liqiang Xi; Trinh Hoc-Tran Pham; Jeffrey Hanson; Jaime Rodriguez-Canales; Arlene Berman; Arun Rajan; Giuseppe Giaccone; Michael Emmert-Buck; Mark Raffeld; Armando C Filie


Mod Pathol. 2012;25(4):548-555. 

In This Article

Abstract and Introduction


The discovery of activating mutations in EGFR and KRAS in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Fine-needle aspirates and other cytologic procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used laser capture microdissection to isolate small numbers of tumor cells to assess for EGFR and KRAS mutations from cell block sections of 19 cytology samples from patients with known lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient's initial clinical work-up. Not only were we able to detect the identical EGFR or KRAS mutation that was present in the patient's prior molecular assay in every case, but we were also able to consistently detect the mutation from as few as 50 microdissected tumor cells. Furthermore, isolating a more pure population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from laser capture microdissection-enriched cases where the tumor load was low and traditional methods of whole slide scraping failed. Therefore, this method can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR and KRAS mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.


The emergence of targeted therapeutics in lung adenocarcinoma has revolutionized the field of personalized medicine and established a prognostic and predictive role for molecular analysis in conjunction with morphologic diagnosis in determining clinical outcomes of patients with advanced-stage disease.[1–3] The epidermal growth factor receptor (EGFR) is known to play a role in the development and progression of cancer, and somatic mutations within the tyrosine kinase domain of EGFR have been identified in a subset of lung adenocarcinomas. The most common mutations are a point mutation c.2573T>G (L858R) in exon 21 and small in-frame deletions in exon 19, which result in constitutive activation of tyrosine kinase.[4] What has made targeted therapy an exciting and developing field is that several clinical trials using tyrosine kinase inhibitors, gefitinib and erlotinib, have shown that patients with advanced lung adenocarcinomas harboring an EGFR mutation have a longer progression-free survival and response to tyrosine kinase inhibitors as a first-line therapy, whereas patients without these mutations have better outcomes with chemotherapy.[2,4–10] Approximately 15–30% of lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, most frequently found in codons 12 and 13 of exon 2. Mutations in EGFR and KRAS are mutually exclusive and recent studies indicate that patients with mutant KRAS tumors do not respond to tyrosine kinase inhibitors.[11,12]

A large fraction of lung carcinoma patients are diagnosed by cytology on fine-needle aspirates, pleural fluids, bronchial washes/brushes and bronchoalveolar lavages,[13] and often cytology samples may be the only available material for molecular analysis. The majority of these patients who present with advanced-stage disease due to unresectable tumor or metastatic disease are increasingly being managed by targeted therapy. Minimally invasive procedures like endobronchial ultrasound-guided transbronchial needle aspiration are gaining popularity in the staging of advanced lung carcinoma patients because of the multitude of information that can be obtained from the aspirated material, including pathologic diagnosis and molecular testing.[14,15] These minimally invasive techniques also allow for serial sampling of a patient's tumor to assess therapeutic response as well as identify additional molecular markers of resistance. With this recent paradigm shift in lung cancer diagnosis and management, cytology has moved away from being a screening modality or ancillary technique and has established itself as an independent diagnostic procedure that plays a predictive role in determining clinical management.[13,16]

Mutation analysis, however, has been discouraged in cytology specimens because of scant material,[17] and cytology specimens are often underutilized for decisions regarding targeted cancer therapy.[18] In a recent study from Smouse et al[19] over a period of 2 years, from a total of 239 cases tested for EGFR mutations, only 12 were cytology material. A similar review by Clark[18] reports only 13 cytology cases from a total of 59 specimens tested for EGFR mutations. Nonetheless, as shown in the retrospective study by Smouse et al,[19] mutation analysis of cytology cell block material shows similar or higher sensitivity in comparison with surgical specimens and was likely dependent on the proportion of tumor cells present in a given specimen.

Molecular analytical techniques have rapidly gained pace in recent years and high-throughput methods have changed the study of molecular events associated with pathological processes.[20–22] However, molecular diagnosis is limited by the amount of tissue available for analysis and the number of tumor cells present within the sample. Tumor cell heterogeneity presents a challenge for molecular assays, where it is often necessary to isolate subpopulations of cells within a neoplasm to obtain a pure sample of tumor cells for DNA isolation and amplification. Laser capture microdissection provides a simple technique for rapid and accurate selection of pure populations of cells under direct microscopic visualization.[23–26]

In this study, we assessed the feasibility of using laser capture microdissection to enable and facilitate EGFR and KRAS mutation detection from cytologic samples (formalin-fixed, paraffin-embedded cell blocks), and compared the performance of this technique with results from standard mutational assays that had been previously performed on surgical or cytology materials as part of the routine clinical work-up. Our data indicate that laser capture microdissection-enabled mutation detection on cytologic material provides highly accurate and reproducible data comparable or superior to standard methods, and could be invaluable particularly when the tumor sample is limited.


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