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Keywords:

  • epidermal growth factor receptor;
  • cytology surgical correlation;
  • PyroMark Q24;
  • Rotor-Gene Q

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

BACKGROUND

The detection of epidermal growth factor receptor (EGFR) mutations on small biopsy or fine-needle aspiration samples is required to guide therapy in nonsmall cell lung cancer (NSCLC). In this study, the authors compared results from EGFR mutation testing on both cytologic smears and surgical specimens and also compared the performance of platforms using 2 different technologies (pyrosequencing and real-time polymerase chain reaction) for both specimen types.

METHODS

Specimens from 114 patients were divided into 2 subsets. The first subset had 60 paired cytology smears and surgical specimens, including 37 paired specimens from the same site and 23 paired specimens from different sites. The second subset consisted of nonpaired cytology smears and formalin-fixed, paraffin-embedded (FFPE) tissues (including 8 cell blocks), which were compared on the pyrosequencing and real-time polymerase chain reaction platforms. Laser-capture microscopy was used to enrich tumor in the FFPE specimens before DNA extraction.

RESULTS

All cytology smears that were used in the study were adequate for analysis on both platforms. Comparison between smears and concurrent FFPE tissues from the same anatomic site had a concordance rate of 97%. The concordance rate between the pyrosequencing platform and the real-time polymerase chain reaction platform was 84% and 85% for FFPE tissues and cytology smears, respectively.

CONCLUSIONS

The current results indicated that direct extraction and analysis of EGFR mutations from cytology smears can be performed successfully on both a pyrosequencing platform and a real-time polymerase chain reaction platform with results comparable to those achieved in matched surgical specimens. In fine-needle aspiration/endobronchial ultrasound samples with limited tissue, cytology smears can be important for molecular analysis. Cancer (Cancer Cytopathol) 2013;121:361–369. © 2012 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Lung cancer is the leading cause of death from cancer worldwide and is responsible for more than 158,000 deaths in the United States alone.[1] Patients with nonsmall cell lung cancer (NSCLC), including adenocarcinoma, routinely receive standard chemotherapy, which provides a 1-year survival rate of approximately 30%, and undergo surgery, after which, approximately 50% develop disease recurrence and die within 5 years.[2] This limited response rate has encouraged the introduction of molecular-targeted therapies to improve the survival rates of these patients.

Epidermal growth factor receptor (EGFR), which is a member of the ErbB (erythroblastic leukemia viral oncogene homolog) family of transmembrane tyrosine kinase receptor proteins, is activated by ligand binding followed by receptor dimerization and phosphorylation; and activating mutations can lead to uncontrolled cell proliferation, tumor invasion, and resistance to chemotherapy.[3] EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, occupy and prevent activation of the tyrosine kinase binding site, leading to inhibition of farther downstream effects,[8] and are particularly effective in approximately 10% to 20% of lung tumors that contain somatic mutations in the EGFR tyrosine kinase domain.[9] The 2 most common mutations, which account for 90% of all somatic EGFR mutations, consist of an in-frame deletion of exon 19 and a point mutation in exon 21 (L858R).[9, 10] Other mutations with known sensitivity to EGFR TKIs include the G719 mutations in exon 18 and the L861 mutations in exon 21.[9] Recently modified National Comprehensive Cancer Network (NCCN) guidelines recommend EGFR mutation testing for some histologic subtypes of lung cancer, including adenocarcinoma, large cell carcinoma, and (NSCLC, not otherwise specified, before instituting targeted EGFR TKI therapy.[11] A provisional clinical opinion generated by the American Society of Clinical Oncology also recommends that EGFR mutation testing take place in patients with NSCLC who mat receive a TKI as first-line therapy.[12] We explored the possibility of using stained cytology smears as a specimen of choice for detecting EGFR mutations by using paired cytology smears and surgical biopsies. We chose cytology smears, because cytopathologists quite often have very limited samples, and adequate tissue is not received to perform cell blocks. In the current study, we compared DNA yield and results using 60 cytology smears on single slides with formalin-fixed, paraffin-embedded (FFPE) surgical specimens, including biopsies and resections.

To date, EGFR mutation testing has been known primarily as a laboratory developed assay that works on platforms using different technologies, including polymerase chain reaction (PCR) amplification and sequencing, amplification-refractory mutations system (ARMS), peptide nucleic acid-locked PCR, and clamping or enriching of mutant alleles by restriction endonucleases before PCR amplification.[13] Conventional direct Sanger sequencing requires tumor enrichment at or greater than 25%,[14] whereas pyrosequencing is a real-time, quantitative sequencing technology that does not depend on electrophoresis; and studies have demonstrated that it is a sensitive method for detecting mutations, including insertions, deletions, and alterations in exons 19 and 21.[16] A recent article comparing EGFR detection by 3 methodologies has identified overall sensitivities of 67% for standard Sanger sequencing and 89% for pyrosequencing compared with next-generation sequencing. The authors also identified allele detection sensitivity of 11% for pyrosequencing compared with 21% for Sanger sequencing.[20] In addition, sequencing technologies can identify novel mutations not targeted by allele-specific PCR technologies. The recently US Food and Drug Administration (FDA)-approved Qiagen Rotor-Gene Q system (Qiagen, Valencia, Calif) uses a sensitive, real-time PCR based on scorpion primers coupled with ARMS (the Qiagen EGFR PCR Kit). It has been demonstrated that ARMS is very sensitive and can detect as few as 1% of tumor cells in lung cancer.[21] It has also been used in some of clinical trials with TKIs. In the current study, we chose to perform a comparative study using sensitive sequencing (pyrosequencing) and PCR methodologies on a second subset of available samples. Because the ARMS system has been used in trials, we chose to compare the ARMS-based Qiagen EGFR PCR Kit on the Qiagen Rotor-Gene Q platform with the Qiagen EGFR Pyro Kit on the PyroMark Q24, in both fine-needle aspiration (FNA)-derived cytology smears (including non-FFPE, direct smears) and FFPE laser-capture microdissected surgical and cell block specimens.

Unlike other targeted therapies, such as therapy with v-raf-murine sarcoma viral oncogene homolog B1 (BRAF) mutation-directed vemurafanib, EGFR mutation results were not included in the data used for therapy approval, and no FDA-approved companion diagnostic assay is currently available. Although NCCN guidelines recommend surgical biopsy specimens as the specimen of choice, cytologic samples (including FNA, pleural fluid, wash, and brush samples) also frequently permit the initial, rapid, and effective diagnosis of cancer. Several recent articles have demonstrated the adequacy of FNA cytology samples for EGFR mutation testing[1, 2, 22] but have not compared them with matched surgical specimens. We evaluated the adequacy of cytology smears with matched surgical specimens and further examined their concordance on different molecular technologies, including pyrosequencing and real-time PCR platforms.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Study Design

Demographic data

The Institutional Review Board of Scott & White Memorial Hospital approved this study. All data were obtained from 114 patients who received treatment within the Scott & White Healthcare system between February 2008 and March 2012. These included 58 men and 56 women, and they ranged in age from 44 to 89 years (mean age, 66 years). Sixty patients had matched cytology and FFPE samples. Of these, 37 pairs were true matched samples from the same site, and 23 pairs were samples from different sites in the same patients (Figure 1). Informed consent was waived with Institutional Review Board approval.

In total, 119 samples from 77 patients (54 unique patients compared with Subset 1) were used to compare the efficacy of detecting EGFR status on the PyroMark Q24 and the Rotor-Gene Q. This subset was divided into 2 groups: 1) an FFPE biopsy group, which consisted of lung resection specimens, small biopsy specimens of lung and metastatic sites (adrenal gland, lymph node, liver, kidney), and 8 cell blocks prepared from FNA material and pleural fluids; and 2) a group of cytology direct smears with FNA samples, as illustrated in Table 1.

image

Figure 1. This is a workflow chart of the current study. EGFR indicates epidermal growth factor receptor; Q24, the PyroMark Q24 platform (Qiagen, Valencia, Calif).

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Cytology smear preparation

Cytology smears consisted of both alcohol-fixed and air-dried specimens.[23] Alcohol-fixed specimens were stained with the standard Papanicolaou method, similar to that described in the literature.[22] Air-dried smears were allowed to air dry completely; then, Diff-Quik staining (Stat-Lab Medical Products, Lewisville, Tex) was performed according to the supplier's instructions. Air-dried smears were dipped 10 times in each of 3 proprietary solutions, then rinsed with 10 dips in water, and coverslipped.

Diagnoses

Of the 114 patient samples, 81 were diagnosed as adenocarcinoma, and 33 were diagnosed as NSCLC, because immunostains were either noncontributory or were not performed. Immunohistochemistry stains using a basic panel of thyroid transcription factor-1 (TTF-1), napsin A, p63, and cytokeratin 5/6 (CK5/6) were performed on the Ventana Benchmark Ultra (Ventana Medical Systems, Oro Valley, Ariz) and/or the Dako AutostainerPlusLink (Dako North America, Carpinteria, Calif) to classify 58 cases. If the quantity of tissue in the block was limited, then immunostains were not performed, and the tumor was classified as NSCLC. Cell groups from cytology smears and tumor foci on FFPE specimens were identified, circled by a team of 5 pathologists (including 3 cytopathologists), and submitted to the molecular laboratory for DNA extraction and EGFR mutation status testing. In FFPE specimens, laser-capture microscopy was performed to ensure enrichment >75%.

DNA Extraction and Molecular Testing

Pinpoint extraction

Tumor-containing slides were selected, and the tumor cells were extracted from a single slide per case using the Zymo Research Pinpoint Slide DNA Isolation System (Zymo Research Corporation, Irvine, Calif) according to manufacturer's instructions, which entailed placing the pinpoint solution over the area of interest and allowing it to completely dry at room temperature.[24] The cells were then scraped off the slide using a scalpel (Fig. 2). In most cases, only isolated tumor cells (approximately 50 per field) were isolated; and, whenever possible, at least 300 cells were used. In rare cases, intermingled lymphocytes were present, and enrichment beyond 20% of tumor cells was not possible. The tubes were centrifuged briefly; then, 50 μL extraction buffer and 5 μL proteinase K were added to the tube, and the tube was incubated at 55°C for 4 hours followed by heating at 95°C to 98°C for 10 minutes. Extraction was completed by adding 100 μL of pinpoint binding buffer and column purification.

image

Figure 2. Cytology smears are shown (Left) before and (Right) after pinpoint extraction with the Zymo Research Pinpoint Slide DNA Isolation System (Zymo Research Corporation, Irvine, Calif).

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Laser-capture microscopy

FFPE tissue sections were selected by the pathologists, and the tumor cells were isolated using the Arcturus Pixcell II laser-capture microscope (Arcturus Bioscience, Inc., Mountain View, Calif) to ensure maximum tumor enrichment. The sections were cut and air dried overnight, then the paraffin was removed from the slides using xylene. Next, the slides were stained using Mayer hematoxylin, and laser-capture microscopy was used to isolate the cells of interest from a single slide (Fig. 3). After laser capture, the captured cells were incubated at 65°C for more than 16 hours in the presence of proteinase K. When the incubation was complete, the samples were ready for PCR analysis.[23]

image

Figure 3. Formalin-fixed, paraffin-embedded (FFPE) tissue sections are shown before and after laser-capture microscopy (LCM).

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EGFR analysis by pyrosequencing: Qiagen Q24 Pyrosequencer

Samples were tested on the Q24 Pyrosequencer using the Qiagen EGFR Pyro Kit.[25] The package insert was followed for the testing using 4 master mixes to amplify the DNA targets of interest followed by 5 pyrosequencing reactions. Recommended DNA input for the pyrosequencing reactions was 50 ng of DNA per reaction. The pyrosequencing kit tests for mutations in the exon 18 codon 719 region, deletions in exon 19, mutations in the exon 20 codon 768 and codon 790 regions, and mutations in exon 21 codon regions 858 through 861. A 1.2% DNA flash gel was used to verify the presence and quality of PCR products before pyrosequencing for each of the exons. Testing on the Q24 Pyrosequencer can be completed in approximately 4 hours.

EGFR mutation analysis by real-time polymerase chain reaction: Qiagen Rotor-Gene Q

Samples were tested on the Rotor-Gene Q real-time instrument using the Qiagen EGFR PCR Kit according to instructions in the package insert.[26] Eight master mixes were prepared for each specimen and tested on the Rotor-Gene Q instrument. The real-time kit tests for 19 deletions in exon 19 (it detects the presence of any of 19 deletions but does not distinguish between them), L858R, L861Q, G719X (it detects the presence of G719S, G719A, or G719C but does not distinguish between them), S768I, and 3 insertions in exon 20 (it detects the presence of any of 3 insertions but does not distinguish between them). In addition to the mutation reactions, a control gene reaction is included in the kit to assess specimen integrity and quantity. Testing using real-time PCR can be completed in approximately 4 hours.

Both extraction and EGFR analysis methods were previously validated in the laboratory. Negative and positive tissue controls were analyzed routinely for all reactions. The mutation-positive cell lines HCC 2935, NCI-H1975, H1650, and HCC827 were used as positive controls.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Sample Adequacy

Cytology smears with 1 to 15 cell groups that had approximately 10 to 50 cells per group provided 0.11 to 244.18 ng DNA per microliter. A maximum of 5 μL was used per amplification reaction. It is noteworthy that a sample with the extremely low quantity of 0.55 ng per reaction from cytology smears amplified successfully. All cytology samples used in the current study were adequate for analysis on both the Rotor-Gene Q and PyroMark Q24 platforms. The quantity of DNA extracted from the FFPE samples was 5.22 to 127 ng, and 6 of 56 FFPE samples produced invalid results on the Rotor-Gene Q because of an insufficient quantity of DNA (less than the recommended 50 ng per reaction).

Comparison of EGFR Results on Cytology and Surgical Specimens Using Both Pyrosequencing and Real-Time Polymerase Chain Reaction Platforms

Overall, 55 of 60 pairs of specimens had identical EGFR results, for a combined concordance rate of 91% on both platforms. Only 1 of 37 pairs of specimens from the same site produced discrepant mutation results between the cytology smear and the biopsy specimen, which were obtained 1 month apart. In that patient, the cytology smear was wild type for EGFR, whereas the resection surgical specimen had an exon 18 mutation (Table 2, Patient 1).

Table 1. Specimens Used in the Current Study
FFPE SpecimensNo.Cytology SmearsNo.
  1. Abbreviations; CT, computed tomography; FFPE, formalin-fixed, paraffin-embedded; FNA, fine-needle aspirate; US, ultrasound.

Total56Total63
Surgical specimens: Lung and lymph nodes and 3 metastatic sites48FNA: Lung and lymph nodes (9 US FNA, 11 endobronchial US FNA, and 37 CT-guided FNA)57
Cell blocks: Lung, lymph node, and pleural fluid8Pleural fluids: Bronchial wash and brush material6
Table 2. Characteristics of Patients With Discordance Between Epidermal Growth Factor Receptor Results in Surgical and Cytologic Specimens
Patient No.Age, ySexSurgical SpecimenEGFR Status on Surgical Specimen (%)Cytology SpecimenEGFR Status on Cytology Specimen (%)Time Interval Between Collection of Surgical and Cytology Specimen
  1. Abbreviations, del, deletion; EGFR, epidermal growth factor receptor; FNA, fine-needle aspirate.

Same site       
175WomanLung resectionExon 18 (5.4)FNA, lungWild type1 mo
Different site       
264WomanMetastatic siteWild typeFNA of primary, lungExon 19 del (18.9)31 mo
373WomanMetastatic siteExon 20 (10.9)FNA, different metastatic site, lymph nodesWild type12 d
474ManMetastatic siteExon 21 (10.7)FNA, different metastatic site, lymph nodesWild type3 mo
568ManMetastatic siteExon 19 delFNA of primary, lungWild type39 mo

In total, 4 of 23 pairs of specimens from different sites were discrepant, for a concordance rate of 82% (Table 2, Patients 2-5). Two patients (2 and 5) had cytology smears only from a primary lung site and had a surgical biopsy specimen from a metastatic site. The other 2 patients with discrepant results had cytology smears and biopsy specimens from different metastatic sites and had no primary tumor available for analysis. In addition, the cytology smear from Patient 2 contained very few tumor cells in a predominantly lymphocytic background.

Comparison of EGFR Results on the PyroMark Q24 and Rotor-Gene Q Platforms Formalin-fixed, paraffin-embedded samples

Six cases had invalid results on the Rotor-Gene Q platform because of a paucity of DNA. No invalid results were obtained on the PyroMark Q24 platform. Sixteen of 56 cases were positive for EGFR mutations on the PyroMark Q24 platform, whereas only 9 cases were positive on the Rotor-Gene Q platform. Thus, the calculated specificity of the Rotor-Gene Q was 97% compared with the PyroMark Q24. The concordance rate noted for FFPE specimens was 84% (Table 3).

Table 3. Sensitivity and Specificity of the Rotor-Gene Q Platform for Formalin-Fixed, Paraffin-Embedded Specimens and Cytology Smears Compared With the PyroMark Q24 Platform
 PyroMark Q24
Rotor-Gene QMutation PresentWild TypeConcordance
  1. Abbreviations: FFPE, formalin-fixed, paraffin-embedded.

No. of FFPE specimens56 84%
Mutation present91 
Wild type733 
Invalid Rotor-Gene results6  
No. of cytology smears63 85%
Mutation present44 
Wild type550 
Cytology samples

The PyroMark Q24 detected 9 cases with EGFR mutations, whereas the Rotor-Gene Q detected 4 cases with EGFR mutations. The concordance rate for cytology smears was 85%.

The overall sensitivity of the EGFR PCR Kit on the Rotor-Gene Q was lower than that of the EGFR Pyro Kit on the PyroMark Q24 for both FFPE specimens and cytology smears, but the specificity was 97% and 92.5% for FFPE specimens and cytology smears, respectively. Concordance between the 2 platforms was almost identical at 84% and 85%, respectively.

In total, 26 mutations were detected in 26 patients. Exon 19 deletions were the most common (12 of 26 patients); and exon 21 mutations (9 of 26 patients), exon 20 mutations (4 of 26 patients), and an exon 18 mutation (1 of 26 patients) were the next most frequent among the remaining patients. There was 1 patient who had both an exon 19 deletion and an exon 20 mutation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

The discovery of activating EGFR mutations that can be targeted by TKIs is a major step in effective therapy for lung cancer. Cytology smears prepared from FNA are used routinely in the diagnosis of lung carcinoma. However, molecular testing for EGFR status has been recommended based on findings from biopsy or resection specimens rather than cytology smears. This has been highlighted in several studies, including Smouse et al,[8] who tested 12 of 239 samples, and Clark,[27] who tested 13 of 59 samples that represented cytologic material. A larger group of 209 cytology cases was analyzed by Billah et al,[22] but those authors did not compared their results with concurrent surgical pathology biopsies. More recently, Chowdhuri et al assessed the feasibility of using laser-capture microdissection for EGFR testing on 12 cytology samples[28]; however, laser-capture microdissection is an expensive technology that is not available to all laboratories.

In the current study, we performed a comparison between cytology smears and concurrent surgical specimens from the same anatomic site in a group of 37 patients and observed a concordance rate of 97%. The only patient in this group that had a discrepant result was Patient 1, who had both a cytology smear and a surgical resection specimen that revealed well differentiated adenocarcinoma. This discrepancy may be attributed to the sampling of different clones and innate tumor heterogeneity, as noted by Sakurada et al.[29] The second group of 23 patients had matched pairs of cytology smears and surgical specimens from different anatomic sites and produced 4 discordant results, for a concordance rate of 82%. Discrepancies in all 4 of those patients (Table 2, Patients 2-5) may be attributed to differences in metastatic tumor EGFR status. In addition, we noted that the cytology smears from Patient 2 had cell groups intimately admixed with the background lymphoid cells. The pinpoint extraction from this patient had only 1.57 ng per microliter isolated DNA, with less than 300 tumor cells. This case highlights the importance of accurate estimation of the proportion of inflammatory and stromal cells to tumor cells, as noted by Ladanyi and Pao.[9] The sensitivity of allele detection depends on the methodology used, and even sensitive pyrosequencing technologies have detection limits of 10%, which easily may be masked by a disproportionate representation of inflammatory cells. Pinpoint extraction is a relatively simple macrodissection enrichment method, but clean preparations of tumor cells can be difficult in FNA cytology smears from lymph nodes. Kalikaki et al[30] demonstrated that EGFR mutation status differed between primary tumors and corresponding metastases from 7 of 25 patients (28%). Their hypothesis for this included: 1) the inclusion of new mutations during the evolution of the metastasis, 2) the administration of TKIs, and 3) chemotherapy. In summary, the probable reasons for the discrepancy in 5 of those patients included, but were not limited to, 1) tumor heterogeneity, 2) difference in EGFR mutation status of primary and metastatic sites, 3) the effect of radiation or chemotherapy on the mutation status, and 4) an error in tumor selection with a low level of tumor content. In our study, there was a very high concordance rate (97%) for EGFR testing on specimens obtained from the same site. There was a slightly lower concordance rate of 82% for specimens obtained from different sites, similar to a previous report by Schmid et al (concordance rate, 14% using direct bidirectional sequencing),[31] who established the importance of repeat testing on new, additional metastatic sites before therapy. Exon 19 deletions (12 of 26 patients) were the most frequently noted mutations in our study, similar to previously reported mutation frequencies by Riely et al[32] and Marchetti et al.[33]

We performed EGFR testing successfully on both Papanicolaou-stained and Diff-Quik–stained smears using small groups of cells on a single slide. Because removing coverslips is time-consuming, it is more convenient for pathologists to anticipate that additional cellular material will be needed for molecular analysis and to maintain at least 1 uncoverslipped, stained slide at the time of the FNA procedure that can be immediately sent for EGFR testing. A study by Killian et al[34] suggested that a higher quality of DNA was obtained from rapid Romanowsky-stained direct smears, and the obtained DNA was stable even after 10 years of storage. Thus, the preservation of stained, uncoverslipped slides can play a key role in performing therapeutically important mutation analysis.

The clinical trials of EGFR-directed therapy published to date have not been conducted with standardized companion diagnostics, although ARMS, pyrosequencing, and Sanger sequencing all have been used. In the IPASS trial (Iressa Pan-Asia Study), testing was performed using ARMS, fluorescence in situ hybridization, and immunohistochemistry methods[35]; in the INTEREST trial (Iressa Nonsmall Cell Lung Cancer Trial Evaluating Response and Survival Against Taxotere), direct gene sequencing, fluorescence in situ hybridization, and immunohistochemistry were used.[36] It is important to note that no methodology for EGFR mutation detection currently has received FDA approval, but many molecular methods have been tested in an attempt to optimize EGFR testing. In a study performed by Horiike et al,[37] the results indicated that the EGFR Scorpions Kit (Qiagen) was superior to direct sequencing, especially for detecting the major deletion mutations in exon 19 and L858R. The Qiagen Rotor-Gene Q system uses a sensitive real-time PCR based on scorpion primers coupled with ARMS. It detects mutations within the EGFR gene but cannot quantify or distinguish which specific mutation has occurred.[38] Dufort et al compared pyrosequencing versus conventional BigDye Terminator sequencing (Invitrogen, Carlsbad, Calif) in 58 samples and noted that pyrosequencing was a sensitive method.[19] An analysis performed by Ellison et al[15] to compare ARMS and DNA sequencing for various mutation analysis (RAS [rat sarcoma], BRAF, and EGFR) concluded that ARMS was more sensitive and robust for detecting somatic mutations than standard DNA sequencing. We elected to compare direct pyrosequencing (a sensitive sequencing method) using the PyroMark Q24 platform versus the Qiagen EGFR PCR Rotor-Gene Q system (a qualitative, targeted ARMS, PCR-based test). The concordance between the 2 methods was 85% and 84% for cytology and FFPE, respectively.

It has been reported that ARMS is affected less by DNA artifacts from fragmented DNA in FFPE samples during the macrodissection steps, and ARMS has been proposed as ideal for samples in which the tumor content is very low, for example, circulating free tumor DNA in blood and in cytology samples.[15] The DNA quality and quantity is automatically estimated by the Qiagen Rotor-Gene Q system, and no failed cytology samples were identified, indicating that this was not an issue in our study with pinpoint extraction. However, in this study, the sensitivity of the Qiagen EGFR PCR Kit was lower for all samples compared with the sensitivity of pyrosequencing, with 13 of 25 positive results classified as wild type using the PCR method. A larger prospective clinical trial with documented response to therapy may be needed to fully establish the clinical validity of each platform for different specimen types in therapeutic settings.

In conclusion, direct extraction and analysis of EGFR mutations from cytology smears (which often are the source of initial diagnostic tissues) is a convenient and robust method for samples obtained from FNA and bronchial wash/brush samples. An overall 91% concordance rate was observed between EGFR mutation analysis on cytology and lung surgical specimens. The concordance rate was 97% when both samples were collected from the same anatomic site and 82% when they were collected from different anatomic sites. This high concordance rate supports the use of direct cytology samples for EGFR testing, optimizing diagnosis and rapid mutation testing. Platform selection may be vital for the accurate detection of EGFR mutation status. Based on our data, the PyroMark Q24 platform was more sensitive than the Rotor-Gene Q platform, although specificity was high (>95%), and the overall concordance between platforms was >80%.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES