EGFR gene status in cytological samples of nonsmall cell lung carcinoma

Controversies and opportunities


  • Gilda da Cunha Santos PhD, MD, FRCPC,

    Corresponding author
    1. Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada
    2. Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada
    • Department of Laboratory Medicine and Pathobiology, University of Toronto, University Health Network, 200 Elizabeth Street, 11th Floor, Eaton Wing, Toronto, Ontario M5G 2C4
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    • Fax: (416) 340-5517

  • Mauro Ajaj Saieg MD, PhD,

    1. Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada
    2. Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada
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  • William Geddie MD, FRCPC,

    1. Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada
    2. Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada
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  • Natasha Leighl MD, FRCPC

    1. Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada
    2. Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada
    3. Department of Medical Oncology, University Health Network, Toronto, Ontario, Canada
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  • We thank the University Health Network Molecular Diagnostics Laboratory for providing the photographs from the mutation assays.



In nonsmall cell lung cancer (NSCLC), the development and clinical application of tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) has required the investigation of EGFR status by gene copy number and/or mutation analysis. This review aimed to present the current knowledge of the use of cytological specimens for EGFR testing in lung cancer.


A systematic computerized search was performed of the MEDLINE(R) and EMBASE databases to identify articles reporting the use of cytological samples for determining EGFR status in NSCLC.


Data were extracted from 30 original articles. An additional 19 reviews, consensus statements, and editorials were selected from 175 retrieved papers. Different techniques using cell blocks, scraped cells from archival slides, and fresh cells have shown promising results and include fluorescent in situ hybridization (FISH), direct sequencing, and quantitative polymerase chain reaction (PCR), with similar or higher accuracy and sensitivity than surgical specimens. Preservation and quality of the extracted DNA seem to matter more than the actual number of tumor cells present in the samples. However, major issues still reside in the amount of material, the interference from background non-neoplastic cells, and standardization of parameters for cytological samples.


This analysis provided evidence that cytological material is suitable for detecting EGFR status using several different methodologies and preparations. New prospective, clinical studies are encouraged for collection and handling of cytological samples as well as for validation of novel techniques in large cohorts. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

Lung cancer has the highest rate of cancer-related mortality worldwide, and nonsmall cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancer subtypes. Almost 70% of patients present with locally advanced or metastatic disease at the time of diagnosis, and only a small proportion of NSCLC patients are eligible for surgical resection. In addition, a substantial proportion of patients with early stage who undergo surgery develop recurrence.

Recent advances in cancer treatment have been achieved with agents that are designed to target cancer cell-specific attributes that are crucial to growth or survival (targeted therapies) and that avoid the severe side effects of conventional cytotoxic chemotherapy. The molecular characterization of tumors, including genome-wide RNA expression, DNA copy number and sequence analysis, and micro-RNA and proteomic profiling, has the potential to allow individualized selection of treatment as determined by the characteristics of the patient and the tumor.1

Basically 2 groups of agents have been developed with the ultimate objective of reversibly blocking the ATP-binding site of the EGFR kinase domain, thereby stopping the signal transduction initiated by activation of the EGFR receptor and inactivating its downstream pathways: small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Two TKIs, erlotinib (OSI-774 or Tarceva) and gefitinib (ZD1839 or Iressa) are currently in use in clinical practice around the world. Cetuximab (Erbitux) is a humanized mouse monoclonal antibody developed against the ligand-binding domain of EGFR.

Erlotinib was approved by the US Food and Drug Administration (FDA) in 2004 as the first targeted therapy for advanced NSCLC on the basis of improved survival and quality of life after chemotherapy failure in a placebo-controlled trial.2, 3 Gefitinib initially received conditional FDA approval in 2003 on the basis of dramatic responses in a subgroup of chemorefractory patients and was later withdrawn when randomized trials did not demonstrate survival benefit. However, gefitinib was approved in Asia and continues to be widely used. More recently, the European Medicines Agency (EMEA) approved gefitinib (Iressa) for use in adult patients with locally advanced or metastatic NSCLC with activating mutations of EGFR tyrosine kinase domain in all lines of therapy on the basis of results of a large trial conducted in Asia (IPASS).4

The EGFR gene, located on the short arm of chromosome 7 (7p11.2), encodes a transmembrane growth factor receptor with tyrosine kinase activity.5 EGFR is a member of the HER/erbB family of receptor tyrosine kinases (RTKs) which encompasses 4 proteins showing similar molecular structure. Intracellular downstream signaling involves 2 interrelated main pathways: the Ras-Raf-MEK-mitogen–activated protein kinases (MAPK, also known as extracytoplasmic regulated kinases, ERK1 and ERK2) and the phosphatidylinositol 3-kinase (PI3K)/PTEN/Akt and the signal transducer and activator of transcription (STAT). Activation of these pathways ultimately leads to increased proliferation, angiogenesis, metastasis, and decreased apoptosis.6

In NSCLC, the clinical application of TKIs targeting the epidermal growth factor receptor (EGFR) has required investigation of biomarkers for patient selection, to identify those most likely to respond to treatment and derive survival benefit. EGFR oncogenic mechanisms, including, among others, gene mutation, increased gene copy number, and protein overexpression, may deregulate TK activity.7 Current strategies to identify markers for monitoring EGFR-TKI therapy are based on those alterations.

Several studies have been published analyzing different candidate biomarkers as predictors for response to EGFR-TKI therapy. Because minimally invasive diagnostic procedures are most often used in a diagnostic workup of lung cancer, small tissue specimens and, more importantly, cytological samples are often the material available for analysis. This review aims to present an overview of the current knowledge of the use of cytological specimens for the evaluation of EGFR gene status in NSCLC. Although other markers have been explored, especially resistance markers for response to EGFR inhibitor therapy, this review will essentially focus on EGFR gene status.


Search Strategy/Study Eligibility

A systematic computerized search was performed of the MEDLINE(R) and EMBASE databases (last search conducted on November 08, 2010) to identify all published articles reporting the use of cytological samples for determining EGFR status. Articles were selected using the following terms and criteria: (“FNA” or “fine-needle aspiration” or “needle aspiration” or “cytology” or “cytopathology” or “cytologic material” or “cytological specimens” or “needle biopsy” or “small needle biopsy”) and (“EGFR” or “epidermal growth factor receptor”) and “lung”. The results were then filtered for English language, published from 2000-current, and duplicates were removed. The remaining 175 articles were reviewed to locate reviews or original studies that dealt primarily with use of cytological samples for determining EGFR status in lung cancer. No abstracts or conference proceedings were included. Forty-nine papers (30 original articles and 19 reviews, editorials, and consensus statements) for which we were able to obtain copies of the manuscript were selected. In addition, a manual search was conducted for selecting review articles, guidelines, and consensus papers relevant to our review.

Data Extraction

Data were obtained from 30 original articles included in the study, and results are summarized in Table 1. For this purpose, reviews, consensus statements, and editorials were excluded. The following information was obtained from each article: first author, journal and year of publication, number of patients enrolled in the study, methods used for analysis of gene copy number/polysomy and/or mutation analysis, and percentage of patients showing EGFR mutation and/or amplification/high polysomy.

Table 1. Characteristics of Findings in the Literature for the Study of EGFR Somatic Mutations and/or EGFR Amplification Status/Gene Copy Number Using Cytological Specimens
First AuthorPatientsMethodsType of Cytological SampleType of PreparationEGFR MutationaAmplification/ Polysomy
 No. of Samples   %%
  • Abbreviations: BAL, bronchoalveolar lavage; BB/W, bronchial brushing/washing; CB, cell blocks; CISH, chromogenic in-situ hybridization; FISH, fluorescence in-situ hybridization; FNA, fine-needle aspirates; HRMA, high resolution melting analysis; LCM, laser capture microdissection; LH-MSA, loop-hybrid mobility shift assay; PCE, pericardial effusion; PCR, polymerase chain reaction; PLE, pleural effusion; PNA-LNA, peptide nucleic acid, locked nucleic acid; PRE, peritoneal effusions; Scorpions ARMS, Scorpions Amplified Refractory Mutation System; TBA, transbronchial abrasion.

  • a

    Considering the number of patients using cytological specimens with available material for analysis.

  • b

    There is substantial data overlap among these studies.

  • c

    Data is shown for 25 patients.

  • d

    Study measured gene expression.

  • e

    Data shown for cytology and surgical specimens together. Study did not show separate data just for the cytological samples analyzed.

  • f

    Data shown just for CISH results.

  • g

    Does not discriminate high polysomy and amplification.

Huang, 20065729Direct sequencingPLECell pellet41
Nomoto,b 20064029HRMABB/W; TBNA; FNA; PLE; PCEArchived slides48.27
Asano, 20064620Mutant-enriched PCR; Direct sequencingPLEFresh cells35
Oshita, 20064450LH-MSATBAArchived slides44c
Shih, 20065362Direct sequencingPLE; PRE; FNACB47
Tanaka, 20074586PNA-LNA clamp/direct sequencingBB/W; PLE; Debris of bronchial biopsy; Sputum; PCEFresh cells33.72
Ceppi,d 20062970Real-time PCRFNACB
Daniele, 2007286 (42e)Direct sequencing/ FISH/CISHPLE; BB/W; FNACB16.6e35.7 (Polysomy), no gene amplificatione
Lim, 20073718Direct sequencingFNAFresh cells27.7
Horiike, 20074794Direct sequencing/ Scorpions ARMSTBNAFresh cells37.34
Takano,b 200741117 (212e)HRMATBNA; BB/W; PLE; PCE; FNA; SputumArchived slides/CB41e
Wei, 20072225FISHBB/W; FNASmears48 (Polysomy), no gene amplification
Nakajima, 20074343Direct sequencing/ LH-MSATBNACB25.58
Brachtel, 2007565Direct sequencingFNACB35.13e
Boldrini, 20075023Direct sequencingBB/W; FNA; SputumArchived slides13.04
Bozzetti, 20082331FISHFNAArchived slides61 (High polysomy), no gene amplification
Molina-Vila, 20083876 (268e)Direct sequencing/Taq man assay/Length analysisFNA; BAL; PLE; PCECB; Fresh cells/LCM17.05e
Fukui,b 20084852HRMAFNAArchived slides CB28.84
Savic, 20082684Direct sequencing/FISHTBNA; BB/W; BAL; PLELCM from archived slides5.1267.1 (52.2 High polysomy and 14.9 amplification)
Smith, 20083911Direct sequencing/HRMAFNAArchived slides27.27
Smouse, 20093618Direct sequencingPLE; FNA; BALCB63.63
Fassina, 20094277Direct sequencing/HRMATBNAFresh cells3.89
Nishimura, 2009511Direct sequencingTBNACB100
Zakowski, 2009552Direct sequencingFNA; PLECB50
Bozetti, 20102433FISHFNA; Biopsy imprintsArchived slides61 (55 High polysomy and 6 amplification)
Nicholson, 2010528EGFR29 kitTBNA; PLECB0
Simone, 20102720CISH/FISHFNACB80 (40 Polysomy, 30 trisomy and 10 amplified)f
Zlobec, 201025153FISHFNA; Exfoliative cytology; Touch prepArchived slides67.11 (FISH +)g
Schuurbiers, 20104935Direct sequencingTBNAArchived slides; CB7.4
Garcia-Olive, 20105851Real time PCRTBNA; FNA; BAL; PCELCM from CB8.57

EGFR Status and Its Clinical Relevance

The clinical relevance of EGFR status in NSCLC has been a subject of intensive study over the past decade. Clinical research efforts have focused on potential biomarkers of patient selection for EGFR-directed therapy. These include EGFR protein expression, EGFR gene copy number, and activating EGFR mutations. Most of the current understanding of the predictive value of EGFR biomarkers stems from retrospective analyses of larger clinical trials in unselected patients. These include trials of first-line platinum-based chemotherapy with or without erlotinib (TALENT, TRIBUTE), gefitinib (INTACT 1, 2), and cetuximab (FLEX, BMS-099).8-10 Placebo-controlled trials have also been conducted after chemotherapy failure with erlotinib (NCIC Clinical Trials Group BR.21) and gefitinib (ISEL), as maintenance therapy after initial platinum-based chemotherapy in those with stable or responding disease, (erlotinib in SATURN), and even in the first-line setting in poor performance status patients thought to be ineligible for chemotherapy (gefitinib vs placebo, INSTEP).3, 8, 11, 12 Further studies comparing EGFR-TKI with chemotherapy have been conducted: in the second-line setting against docetaxel (gefitinib, INTEREST), and also in the first-line setting against platinum-based chemotherapy (gefitinib vs chemotherapy, IPASS).4

EGFR Gene Copy Number (FISH or qPCR)

Studies suggest that high EGFR copy number is associated with a poor prognosis in NSCLC.13-15 However the value of high copy number as a predictive marker remains unclear. High EGFR copy number was only predictive of benefit in placebo-controlled trials of gefitinib and erlotinib after chemotherapy failure. In contrast, high copy number was not associated with better survival in the INTEREST trial, which compared second-line chemotherapy to gefitinib, nor in the first-line combination trials of platinum-based chemotherapy plus an EGFR-TKI or placebo (TALENT, TRIBUTE, INTACT 1 and 2).8 Interestingly, initial data from cetuximab-based trials in metastatic NSCLC suggested EGFR copy number is not predictive of outcome, but a phase 2 Southwestern Oncology Group (SWOG) study of chemoradiation with sequential or concurrent cetuximab suggested that those with high copy number may benefit from this agent. Currently, cetuximab is being tested in a large randomized trial of first-line platinum-based chemotherapy plus bevacizumab, plus or minus cetuximab.16, 17

EGFR Mutations

In the early clinical development of EGFR-TKIs, dramatic responses were seen in selected patients, most commonly never smokers, women, those with adenocarcinoma histology, and patients of Asian ethnicity. The presence of activating mutations appears to be both prognostic and predictive of benefit from EGFR-TKI therapy, although not EGFR monoclonal antibody therapy. Those with EGFR mutations consistently have better response rates and progression-free survival with TKI treatment than with first-line or second-line chemotherapy. In addition those with mutations treated in the maintenance setting (SATURN-erlotinib) had a large benefit in progression-free survival, although there was no difference in overall survival compared with placebo, as approximately two-thirds of patients crossed over to subsequent EGFR-TKI therapy.11 Similarly in the first-line chemotherapy versus EGFR-TKI trials, given the high rate of crossover in the chemotherapy control arms, a significant survival difference has not been seen. However, despite this, there are data that clearly support the use of EGFR-activating mutations to select patients for first-line EGFR-TKI treatment, followed by subsequent chemotherapy, with better response rates, quality of life, progression-free survival, and less toxicity.4

Over time, all patients develop resistance to EGFR-TKI. Subsequent biopsies of patients who initially responded to the therapy, but then progressed, identified secondary mutations associated with acquired resistance. The most common mutation seen in acquired resistance to EGFR-TKI therapy is the T790M.18 Studies have demonstrated that this mutation results in significantly higher affinity of the new mutant receptor for ATP, thus reducing the effectiveness of binding by the TKI. These account for approximately half of the cases that demonstrated acquired resistance. The NSCLC tumors insensitive to EGFR-TKIs include those driven by the KRAS and MET oncogenes.19 Studies on irreversible EGFR-TK inhibitors, such as afatinib, neratinib, and PF-00299804 that may target resistance mutations and overcome resistance, are showing promising clinical results.

Techniques for the Assessment of EGFR Status in Tissue Samples

Different techniques have been used for laboratory determination of EGFR status in tissue samples. EGFR protein expression can be assessed by immunohistochemistry (IHC), gene copy number can be determined by several methods including fluorescent in situ hybridization (FISH) and quantitative real time polymerase chain reaction (qRT-PCR), and gene mutations have been commonly detected by direct sequencing. Other highly sensitive techniques that do not require sequencing have been described more recently.

As reflected in the College of American Pathologists (CAP) 2010 lung cancer synoptic report, the standard way to assess EGFR status is to perform either polymerase chain reaction (PCR)-based EGFR mutational analysis or EGFR FISH assay, without specifying the sample type. The NSCLC Working Group published recommendations indicating that tissue blocks are the preferred sample type for EGFR molecular assays.20 It also stated that although cytospin preparations may be acceptable with further validation, the use of cytology smears has not been fully explored for EGFR FISH analysis. The consensus statement specified that cytology smears were often of nonuniform thickness, precluding accurate counting of FISH signals. Another argument given was that cytology specimens did not have an adequate number of tumor cells for FISH analysis.

A recent study has reported that FNA often yields insufficient material for performing 1 or multiple molecular tests.21 However, cytology may offer a suitable alternative to incisional biopsy in a variety of clinical settings in which it might otherwise be difficult to obtain material to study prognostic and predictive markers.

Results of the Search Strategy

EGFR Gene Copy Number

Cytological specimens can provide a reliable and feasible alternative to analyze EGFR FISH. However, research is needed to develop and validate criteria for the standardized interpretation of EGFR FISH results using cytological specimens.

Our review of assessment of EGFR gene copy number using cytological samples revealed 7 original articles on the subject, all of them using FISH,22-26 with 2 of these studies also using chromogenic in situ hybridization (CISH)27, 28 for gene copy number assessment, and 1 used real-time PCR to investigate expression level.29 Laser capture microdissection (LCM) was used in 1 study to enrich the population of tumor cells, and in 1 study, sections from formalin-fixed paraffin-embedded (FFPE) cell blocks were used. Types of specimens ranged from bronchial brushing/washings to pleural effusions, bronchoalveolar lavages, and fine-needle aspirations.

The majority of studies that examined the feasibility of EGFR testing using cytological specimens used the Colorado criteria for FISH scoring, which had limitations because this scoring system was developed for FFPE tissue sections. The EGFR FISH scoring system developed by the Colorado group stratifies results into 6 groups that include disomy (≤2 copies in >90% of cells), low trisomy (≤2 copies in ≥40% of cells, 3 copies in 10%-40% of the cells, ≥4 copies in <10% of cells), high trisomy (≤2 copies in ≥40% of cells, 3 copies in ≥40% of cells, ≥4 copies in <10% of cells), low polysomy (≥4 copies in 10%-40% of cells), high polysomy (≥4 copies in ≥40% of cells), and gene amplification (defined by presence of tight EGFR gene clusters and a ratio of EGFR gene to chromosome of ≥2 or ≥15 copies of EGFR per cell in ≥10% of analyzed cells). The cutoffs, based on the number of copies of the EGFR gene and frequency of tumor cells in the sample, were determined in a retrospective study of gefitinib-treated patients with advanced NSCLC. FISH-positive or high copy number cases consist of tumors showing amplification or high polysomy. FISH-negative or low copy number (disomy, low trisomy, high trisomy, and low polysomy) cases are those with no amplification or with <40% of cells showing ≥4 copies of the EGFR gene per cell.30 The association between high EGFR gene copy number by FISH (FISH-positive), as defined by the Colorado group (Colorado criteria), and clinical outcome has been validated in several studies in lung cancer and other sites.31

Different studies have attempted to redefine the Colorado criteria for use with cytological samples, with tuning of the threshold for determining samples to be FISH positive or negative. A prospective study of NSCLC samples included 84 cytological specimens (bronchial washings, pleural effusions, transbronchial fine-needle aspirates, and bronchoalveolar lavages) and 33 available corresponding biopsies. The FISH positivity rate for cytological specimens was 21 of 33 (63.6 %) versus 8 of 33 (24.2 %) for the matched histological specimens with P < .01.26 This discrepant result and significantly higher FISH positivity rate for cytological specimens could be explained by tumor heterogeneity but was attributed to the nuclear truncation artifact in histological samples. In tissue sections, the number of hybridization signals per cell was decreased. In contrast, cells in cytological specimens were not cut during slide preparation, and, therefore, the scores represented the true number of hybridization signals. By using regression analysis, the authors “tuned up” the Colorado score criteria for adequacy in cytology samples, changing the threshold for positivity in cytology samples from >4 copies of EGFR in >40% of the cells to >5 copies in >70% of the cells. More recently, a large series from the same group encompassing 153 cytology samples and 170 histological specimens with 151 matched cases recorded the FISH results using both the mean copy number (MCN) and the Colorado criteria. The mean copy number was defined as the mean number of EGFR copies per specimen. The results showed high interobserver concordance (97.4%; κ = 0.94; 95% CI, 0.88-0.99) for MCN in cytology specimens and moderate concordance between histological and cytological specimens (κ = 0.33; 95% CI, 0.15-0.51). Greatest variability occurred when cell blocks were used, suggesting they may be less suitable for EGFR FISH analysis than smears or cytospin preparations. They concluded MCN could provide a quantitative scoring system that could lead to the identification of cutoff scores.25 In another study, the authors included 33 cytological specimens with matched histological specimens and showed that 94% (31 of 33) of both specimen types could be used for EGFR FISH testing.24 This study also used the Colorado criteria and concluded that there was 87% concordance between cytological and histological specimens.

Recent findings suggest that the 6 subclasses proposed by Colorado criteria are suboptimal for the evaluation of EGFR-GCN in cytological specimens.25, 26, 32 When the Colorado criterion was applied to FISH results of cytological specimens, it did not account for tumor heterogeneity, common in NSCLC tumors. It also could not discriminate between chromosome 7 polysomy and gene amplification when the hybridization signals were complex.33

Mutation Analysis in NSCLC Cytological Samples

Assessment of EGFR mutational status has mainly focused on the detection of the most common EGFR-sensitizing mutations: the exon 19 in-frame deletions that eliminate 4 amino acids (LREA) downstream of the lysine residue at position 745 and point mutations in exon 21 (L858R and L861Q) that produce either a leucine to arginine or leucine to glutamine substitution.34 Together, these 2 types of mutations are approximately 85% to 90% of known EGFR-activating mutations.35 Screening for these mutations has most commonly been performed by using direct sequencing analysis or real-time PCR.34 Although macrodissection or microdissection may be used to enrich for tumor cells for direct sequencing, a high ratio of tumor-to-normal tissue content, and a correspondingly larger amount of FFPE sample, are required to reliably detect tumor-specific somatic mutations.20 Cytology specimens have not been widely used for sequence analysis due primarily to heterogeneity within samples, which precludes manual microdissection, and due occasionally to lack of paraffin-embedded material or sparse cellularity.36

All possible mutations in exons can be detected by direct sequencing analysis. However, this technique may lack sensitivity compared with other methods and is limited by the presence of non-neoplastic cells in tissue sample with high heterogeneity. Studies using direct sequencing in cytological samples have shown poor correlation with results from surgical specimens. One study comparing surgical-resection specimens and low-volume biopsies showed discordant results using direct sequencing. Whereas the analysis of surgical specimens showed the classical correlation of EGFR mutations to female sex and adenocarcinoma histology, the low-volume samples (FNA and core biopsies) showed EGFR mutations to be most commonly found in nonadenocarcinoma samples of male smokers.37

Real-time PCR uses oligonucleotide primers that bind specifically to flanking regions of the most common mutations, when present. One report using this method in cytological samples for the detection of in-frame deletions in exon 19, L858R point mutation in exon 21, and also the T790M mutation in exon 20 showed 100% sensitivity when compared with direct sequencing.38 Although highly sensitive as a result of preferential amplification of the mutant allele in excess of the wild-type allele, this method will identify only those mutations targeted. Rare or novel mutations will not be detected.

More recently, some simple and highly sensitive nonsequencing methods to detect EGFR mutations such as high resolution melting analysis,39-42 loop-hybrid mobility shift assay,43, 44 peptide nucleic acid-locked nucleic acid,45 mutant-enriched PCR assay,46 and Scorpions Amplified Refractory Mutation System47 have been reported. The main problem using these methods is that they are capable of detecting only known mutations. These studies testing novel methodologies for detection of EGFR mutations have used PCR followed by direct sequencing for validation and refinement of the results.38, 39, 43

In a comparison of Papanicolaou-stained slides, cytologic archived slides, and biopsy specimens, better results (percentage of samples successfully analyzed) were obtained from analyses of the first PCR products using the cytologic slides rather than the results obtained using the biopsy specimens, regardless of the amount of tumor cells examined.48 In addition, it was suggested that DNA is better preserved in the methanol-fixed samples than in the formalin-fixed specimens, with the recommendation for the use of methanol for better fixation of samples.

High-resolution melting analysis (HRMA) relies on the combination of real-time PCR and evaluation of DNA melting curves to accurately detect mutations, comparing the patterns of curves to known wild-type samples. It has the advantage of being less time and labor intensive and less expensive. The sensitivity, specificity, and accuracy of HRMA using small diagnostic specimens compared with DNA sequencing were 83.3%, 100%, and 94.2%, respectively, and all were 100% for surgical specimens.48 In a study using cytological samples from 29 patients, the sensitivity and specificity of HRMA using scraped cells from archival slides compared with DNA direct sequencing from surgical specimens were 90% and 100%, respectively.40 When manual macrodissection was used for tumor cell enrichment, sensitivity increased to 93%. Another study using Romanowsky-stained slides from 11 patients reported 100% sensitivity when compared with direct sequencing for the same cases.39 By using fresh cells obtained by transthoracic needle aspiration, 100% accuracy was reported, compared with results obtained from direct fragment analysis (Fig. 1).42

Figure 1.

Epidermal growth factor receptor mutation analysis in 2 cases of pulmonary adenocarcinoma using samples obtained by fine needle biopsy. (A1) Fine-needle aspiration (FNA) of metastatic tumour in supraclavicular lymph node (magnification, 40x). (A2) Fragment analysis showing exon 21 mutation (B1) FNA of subcutaneous metastasis (magnification, 1x). A highly cellular sample was obtained with a 27G needle. (B2) Cytomorphology showing tridimensional groups of adenocarcinoma cells (magnification, 40x). (B3) Fragment analysis showing exon 19 deletion.

Types of Cytological Preparations Used for EGFR Mutation Analysis

Our systematic review showed various types of cytological preparations used for determining EGFR mutation analysis. DNA was extracted from FFPE cell blocks in 13 studies, followed by the use of scraped cells from archival slides in 8 studies, fresh cells in 6, and cell pellet in 1 study. LCM was used as a method for tumor cell enrichment in 3 instances: 1 study using archived cytological specimens and 2 studies using sections from cell blocks (Table 1).

The use of tumor cells for determining mutation status from archived cytological slides has the advantage of retrieving information from older cases, when tissue collection for IHC or other ancillary studies was deemed not necessary, and also from cases with a small quantity of material, in which smears were the only material obtained. Furthermore, because the slides are used for diagnosis, no additional procedure is needed. In 13 studies, cell-block preparations were used, and in 8 studies, DNA was obtained from archived stained slides (Papanicolaou in 6 and Romanowsky-stained slides in 2). Although most of the studies using archived cytological smears used Papanicolaou-stained slides, Romanowsky-stained slides can also be used for DNA extraction and consequent PCR reactions, with similar results.39, 49 Our experience also shows that full destaining of the slides is not mandatory for successful DNA extraction and PCR. Liquid-based preparations also seem to yield reliable material.50 Studies describing scraping cells from archived slides show a high percentage of cases yielding sufficient DNA for mutation analysis, varying from 92.9% to 100% of cases.26, 39, 40, 50

Results using FFPE cell blocks and cell pellet are comparable to results using paraffin sections, with an overall high yield of DNA.36, 43, 51-57 When the use of cell blocks was compared with surgical specimens, it showed equivalent sensitivity. Although a lower proportion of cell blocks yielded inconclusive results, a higher percentage of cases contained relatively few tumor cells (less than 25%) and, therefore, could not be analyzed.36

Studies have also shown the feasibility of performing mutation analysis using formalin-fixed material43, 49, 58 and fresh cells47 obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), with analysis of EGFR mutation in samples from lung tumors and mediastinal lymph nodes bearing metastatic disease.

Although previous studies using different types of specimen have show an overall good recovery of the cytological material for molecular analysis, the use of fresh cells obtained at the time of the diagnosis whenever possible may be preferable, ensuring better quality material for molecular analysis with less degradation of the DNA. Studies using fresh cells rely mainly on cryopreservation for storage of the material obtained.47, 37 However, the use of cryopreservation for biobanking is expensive and may require extra storage space. Moreover, collection of these samples in distant locations may become complex, and transportation of the collected samples requires special conditions. One alternative to the use of frozen material is the FTA card. By using the card for cell collection, samples can be stored at room temperature for years, with preservation of good quality DNA. We have demonstrated the feasibility of storing, extracting, amplifying and sequencing DNA from surgically excised lung tumors aspirates stored in FTA cards (Whatman, Kent, United Kingdom). By using this approach, we could detect EGFR and KRAS mutations in 8 samples, extracting a better quality of DNA for sequencing than that obtained from cell blocks. FTA cards also have a great advantage of being inexpensive, and they are associated with simple protocols for DNA extraction, easy collection, transportation, and storage.59

Minimal Cytologic Material Necessary for EGFR Mutation Analysis

The effective number of tumor cells present in the samples may alter the recovery rate and may constitute a problem for obtaining sufficient material for molecular analysis. When using archival slides, a good quantity of DNA has been obtained even from slides with scanty cellularity (7.9 μg to 20.8 μg).39 To evaluate the minimal number of cells for successful EGFR DNA sequence analysis, the quality of DNA chromatograms from exons 18, 19, 20, and 21 of the EGFR gene obtained from 30, 50, and 100 cancer cells was compared.26 The best result was obtained when 100 cells were used, followed by 50 and 30 cells, although differences were not statistically significant. The point mutation in exon 21 (L858R), however, was identified even when only 30 cells were used. Other studies showed that mutations could be detected in samples containing as few as 1% of tumor cells, using more sensitive novel technologies such as HRMA or peptide nucleic acid-locked nucleic acid (PNA-LCA) PCR clamp.45, 41

Manual dissection can enhance the population of tumor cells, increasing the sensitivity of mutation detection.40 LCM can also be used to enrich the tumor cell population and help to overcome this problem, increasing the percentage of tumor cells present in the samples, minimizing the potential impact of contamination by the background non-neoplastic cells.26, 58 In a study using LCM to select tumor cells using fresh cells and sections from cell blocks, mutation analysis using real-time PCR was effective in 100% of samples using fresh cells, and cases with as low as 8 cells could have mutation analysis successfully performed. Mutation analysis using cell blocks after LCM was effective in 88% of the samples.38 LCM, however, may be tedious and time-consuming because there is usually a need for selection of various areas in the slide. Furthermore, the special instruments are still very expensive, and this hampers the broad implementation of the LCM technique in clinical practice.

Overall, preservation and quality of the DNA extracted seemed to matter more than the actual number of tumor cells present in the samples. In a recent consensus for EGFR mutation testing in NSCLC, there was agreement that the quality of amplifiable DNA is more important than its quantity.60 In addition, the necessity for standardization of tumor specimen handling, including fixation as a means of ensuring better preservation of the DNA structure and guaranteeing proper mutation analysis, was stressed.

Conclusions and Perspectives

As novel technologies using minimally invasive approaches are incorporated into clinical practice, extraction of information of diagnostic and prognostic relevance from small samples has become essential in routine diagnostic workups.61-65 The use of cytologic material in this new era of personalized targeted therapy in lung cancer faces some challenges.66 Cytopathologists play a critical role in assuring that the relatively small quantity of cellular material often available from cytologic samples can provide enough, and reliable, molecular data. In addition as the diagnosis of NSCLC is no longer acceptable for clinical management, the correct characterization of tumor subtype will also often require material for immunohistochemistry studies. The benefits of obtaining as much as tissue as possible during any biopsy procedure and storage of this tissue after initial pathologic diagnosis were emphasized in a recent consensus report.60 As recently discussed, the use of cytology specimens for molecular testing is both a challenge and an opportunity for pathologists to enhance patient care. The challenge lies in providing a mechanism by which adequate material can reproducibly be obtained for these important molecular studies.67 The authors commented that this could be achieved by increased use of immediate specimen evaluation for adequacy by pathologists and the use of novel specimen-preservation techniques.52, 68

Rapid advances have been seen in the development of molecularly targeted therapies for different types of cancer. Efficient clinical deployment of new therapies relies on the identification of patients who may benefit most from the drugs. However, despite efforts in early phase and more advanced trials, identifying these patient populations has been difficult. Major challenges involving trials with patients with advanced disease (including lung cancer) are related to retrospective procurement of archival samples and the low number of specimens for adequate biomarker studies, especially molecular assays. It has taken nearly a decade to establish epidermal growth factor (EGFR) activating mutations as the key biomarker in selecting patients for first-line EGFR-TKI therapy.4

Assessment of EGFR status by gene copy number and/or mutation analysis has proven to be predictive of response to anti-TKI inhibitors and, therefore, essential to the management of patients. Various techniques using different types of cytological samples and preparations have shown promising results, with similar or higher accuracy and sensitivity when compared with surgical specimens. New prospective, clinical studies are needed for 1) validation of these methods in large populations and 2) optimization of the collection and handling of cytological samples.

The incorporation of cytological sampling into clinical trials and biomarker discovery studies has the potential to dramatically increase the effective use of material from patients enrolled in clinical trials and drug development studies. Cytopathologists, either by actively participating in on-site assessment and specimen “triage” or through education of their colleagues in imaging and oncology, are in a position to facilitate and optimize tumor sampling in the era of molecular medicine and “small tissue” pathology.


No specific funding was disclosed.


Mauro A. Saieg is a research fellow supported by The Terry Fox Foundation Strategic Health Research Training Program in Cancer Research at CIHR, TGT-53,912.