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- MATERIALS AND METHODS
- FUNDING SOURCES
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. 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. 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. 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, and are particularly effective in approximately 10% to 20% of lung tumors that contain somatic mutations in the EGFR tyrosine kinase domain. 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. 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. 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. 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. Conventional direct Sanger sequencing requires tumor enrichment at or greater than 25%, 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. 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. 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. 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.
- Top of page
- MATERIALS AND METHODS
- FUNDING SOURCES
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, who tested 12 of 239 samples, and Clark, who tested 13 of 59 samples that represented cytologic material. A larger group of 209 cytology cases was analyzed by Billah et al, 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; 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. 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. 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 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), 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 and Marchetti et al.
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 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; 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. 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, 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. Dufort et al compared pyrosequencing versus conventional BigDye Terminator sequencing (Invitrogen, Carlsbad, Calif) in 58 samples and noted that pyrosequencing was a sensitive method. An analysis performed by Ellison et al 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. 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%.