GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 00, Number 00, 2015 ª Mary Ann Liebert, Inc. Pp. 1–6 DOI: 10.1089/gtmb.2015.0069
ORIGINAL ARTICLE
Nonenriched PCR Versus Mutant-Enriched PCR in Detecting Selected Epidermal Growth Factor Receptor Gene Mutations Among Nonsmall-Cell Lung Cancer Patients Moyassar Ahmad Zaki,1 Ragaa Abd El-Kader Ramadan,1 Mahmoud Ibrahim Mahmoud,2 Dalal Mohammed Nasr El-Din El-Kaffash,3 and Rami Samir Helmy Assaad1
Background: Biopsies obtained from lung cancers contain a mixture of cancerous and healthy tissues. The mutant-enriched polymerase chain reaction (ME-PCR) identifies low-level somatic DNA mutations within an excess wild-type sample. Aims: This study aimed at comparing nonenriched PCR (NE-PCR) versus ME-PCR for the detection of two epidermal growth factor receptor (EGFR) gene mutations among nonsmall cell lung cancer patients. Methods: Fifty lung tissue biopsies were screened for inframe TTAA deletions in exon-19 and the L858R point mutation in exon-21, using ME-PCR and NE-PCR, followed by capillary electrophoresis. Results: Only exon-19 deletions were detected in 22% and 18% of cases using ME-PCR and NE-PCR, respectively. Diagnostic performance of the NE-PCR versus the ME-PCR serving as a ‘‘gold standard’’ revealed a sensitivity of 82%, and a specificity of 100%, with positive and negative predictive values of 100% and 95%, respectively, and an overall accuracy of 96%. Despite a strong agreement shown between the two assays (K = 0.875), the NE-PCR showed an 18% false-negative rate in bronchoscopically obtained biopsies compared to ME-PCR. Conclusion: The false negativity encountered with NE-PCR in bronchoscopically obtained samples makes ME-PCR the technique of choice in such situations.
(TKD) responsible for mediating transphosphorylation of the carboxy-terminal tyrosine residues, which are mandatory for recruitment of adaptor/effector proteins (Bazley et al., 2005). Such a domain has been shown to be a promising target for the tyrosine kinase inhibitors (TKIs), gefitinib (Iressa) and erlotinib (Tarceva), recently introduced as a treatment modality in NSCLC cases. The fact that a relatively limited subset of NSCLC responded to TKIs (Sequist et al., 2007) became the subject for further investigations, which demonstrated the highest response rates to these TKIs in patients with somatic mutations within the EGFR gene coding for TKD, particularly exon-19 inframe deletion, exon-21 point mutation (CTG to CGG at nucleotide 2573) characterized by substitution of leucine by arginine at codon 858 (L858R), and occasionally exon-18 G719X (where X indicates A, C, or S) (Lynch et al., 2004). On the other hand, the exon-20 T790M mutation was associated with acquired resistance to TKI therapy (Pao et al., 2005). The methods used in detection of the EGFR gene mutation fall into one of the two broad categories; screening methods
Introduction
L
ung cancer remains the second most common cancer in the world, both in terms of cases and deaths, with over 1 million cases diagnosed yearly (Ferlay et al., 2010). Although lung cancer incidence rates and mortalities are still low in the Arab world compared to Europe or the United States, it is gradually increasing in the region (Salim et al., 2011). Eighty-five percent of lung cancers are nonsmall-cell lung cancers (NSCLCs), a heterogeneous group that comprised mostly squamous cell carcinomas, adenocarcinomas, and large undifferentiated carcinomas. The recent emergence of targeted lung cancer therapies directed against specific cellular alterations necessitates the most precise classification possible for NSCLCs (Brambilla et al., 2001). The epidermal growth factor receptor (EGFR) is expressed on the cell surface of a substantial percentage of NSCLCs. The gene coding for EGFR is located on the short arm (P 12) of chromosome 7 (Callaghan et al., 1993). The cytosolic portion of the EGFR contains the tyrosine kinase domain 1
Department of Chemical Pathology, Medical Research Institute, Alexandria University, Alexandria, Egypt. Departments of 2Chest Diseases and 3Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt.
1
2
ZAKI ET AL.
that detect all mutations in exons 18–21 and include sequencing, denaturing high-performance liquid chromatography, and high-resolution melting analysis (Cohen et al., 2006; Do et al., 2008; Cho et al., 2011) and targeted methods that detect specific known mutations, which are divided into polymerase chain reaction (PCR)-based and non-PCR-based ones (Yu et al., 2009; Miyamae et al., 2010; Dufort et al., 2011). The somatic nature of EGFR gene mutations dictates their presence in malignant cells only, and because clinical samples (lung tissue biopsies) contain a mixture of tumor and normal cells or genes, a highly sensitive PCR-based assay having the ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing the tumor status early in cancer patients for treatment decision. The mutantenriched PCR (ME-PCR) assay developed by Asano et al. (2006) was able to detect one mutant in the presence of 2 · 103 wild-type genes, providing a valuable method in detecting a small fraction of mutant genes in heterogeneous specimens, indicating its useful clinical application for NSCLC. We aimed to evaluate the diagnostic performance of nonenriched PCR (NE-PCR) versus ME-PCR in the detection of exon-19 inframe TTAA deletion and exon-21 L858R point mutation in EGFR gene on biopsy specimens from Egyptian NSCLC patients. Patients and Methods
Fifty consecutive cases of NSCLC were enrolled in this study from January to November 2013 from the Main University Hospital Alexandria University. All patients provided their informed consent, and institutional board permission was obtained for conducting this study. Thirty of the lung biopsies were obtained through bronchoscopic means and the rest through open thoracic surgery in the Cardiothoracic Surgery Department of the Main University Hospital. Sections from each biopsy were stained with hematoxylin and
eosin (H&E), assessed by two independent pathologists, and classified according to the TNM classification system, identifying the lung cancer type and grade. Cases were excluded if they were small-cell lung cancers or received TKIs. Demographic data were retrieved through a direct patient questionnaire. Tumor-rich areas, detected by the pathologist, were formalin fixed and paraffin embedded (FFPE). Genetic testing was done in the Chemical Pathology Department of the Medical Research Institute, Alexandria University. Freshly cut sections of FFPE tissue, each with a thickness of 5–6 mm, from biopsies were prepared. DNA was purified from the FFPE tissue using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions. The concentration and purity of the extracted DNA were measured on a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific) at 260 and 280 nm. The extracted DNA was stored at -20C till the time of PCR amplification. Some technical considerations in DNA extraction from the FFPE tissue were addressed in this study. First, discarding two to three sections exposed to air, while cutting the FFPE sample block. Second, extending incubation time of the tissue lysis buffer suspension with the proteinase-K enzyme at 56C overnight was done until the lysis was completed. Third, incubation of the lysate at 90C for 1 h in the tissue lysis buffer, to reverse formaldehyde alteration of nucleic acids and formalin crosslinking, thus resulting in more fragmented DNA. ME-PCR was carried out as described by Asano et al. (2006). The first and second rounds of PCR were carried out for both exons-19 and -21 of the EGFR gene using forward and reverse primers in concentrations of 20 pmol/mL for each primer. Details of primer sequences are supplied in Table 1. The PCRs were carried out in 0.2-mL sterile eppendorfs. Briefly, in the first round of PCR, 1 mL (10–100 ng) of extracted DNA was mixed with 4 mL of forward and reverse primers specific for each exon (Thermo-Fermentas), 12.5 mL HotStarTaq Master Mix (Qiagen, Inc.), and completed to a final reaction volume of 25 mL with nuclease-free sterile
Table 1. Primer Sequences Used for EGFR Exon-19 and Exon-21 Amplification in Both ME-PCR and NE-PCR First-round PCR Primers for exon-19 Forward primer (ex19-S1) Reverse primer (ex19-AS1) Primers for exon-21 Forward primer (ex21-S1) Reverse primer (ex21-AS1)
5¢-ATC CCA GAA GGT GAG AAA GAT AAA ATTC-3¢ 5¢-CCT GAG GTT CAG AGC CAT GGA-3¢ 5¢-CAG CCA GGA ACG TAC TGG TGA-3¢ 5¢-TCC CTG GTG TCA GGA AAA TGCT-3¢
Second-round PCR Primers for exon-19 Forward primer (ex19-S1) Reverse primer (ex19-AS1) Primers for exon-21 Forward primer (ex21-S2) Reverse primer (ex21-AS1)
5¢-ATC CCA GAA GGT GAG AAA GAT AAA ATTC-3¢ 5¢-CCT GAG GTT CAG AGC CAT GGA-3¢ 5¢-CGC AGC ATG TCA AGA TCA CAG AT-3¢ 5¢-TCC CTG GTG TCA GGA AAA TGCT-3¢
EGFR, epidermal growth factor receptor; ME-PCR, mutant-enriched PCR; NE-PCR, nonenriched PCR; PCR, polymerase chain reaction.
NE-PCR VERSUS ME-PCR IN EGFR MUTATION DETECTION
water. The cycler (Quanta Biotech 96S) conditions were as follows: an initial denaturation step of 95C for 15 min followed by 22 cycles of 20 s denaturation at 94C, 30 s annealing at 60C, and a final 20 s extension step at 72C. The integrity of the resulting PCR products was confirmed by electrophoresis on a 2% agarose gel and scored for the presence or absence of an allele-specific band. Intermittent restriction digestion of the T^TAA sequence in exon-19 and the TGG^CCA sequence in exon-21 was achieved using the fast digest enzymes MseI and MscI (Thermo-Fermentas), respectively, resulting in the enrichment of mutant-type genes. Restriction digestion was carried out separately for each exon. Briefly, in 1.5-mL sterile eppendorf tubes, 2 mL of the first-round PCR product was mixed with 2 mL of the supplied buffer (for each enzyme), 1 mL of enzyme, and completed to a final reaction volume of 20 mL with nuclease-free sterile water. Incubation was done in a thermomixer (Eppendorf, AG) at 37C for 10 min. The second round of PCR was carried out for both exons following the intermittent digestion step using primers supplied in Table 1. Briefly, 1 mL of digested DNA was mixed with 4 mL of forward and reverse primers specific for each exon (Thermo-Fermentas), 12.5 mL HotStarTaq Master Mix (Qiagen, Inc.), and completed to a final reaction volume of 25 mL with nuclease-free water. The cycler (Quanta Biotech 96S) conditions were like the first round, but with 40 cycles. Restriction digestion of second-round products for exon21 point mutation involved the use of the restriction enzyme Sau96I (Thermo-Fermentas) to digest the mutant type of codon 858 (CGG), as it digested the G^GNCC sequence, but not the wild type, and thus, even partial digestion of the PCR product indicated the presence of a mutation. Again, restriction digestion was carried out in sterile 1.5-mL eppendorf tubes utilizing 10 mL of the second-round PCR product, 2 mL of the supplied buffer, 1 mL of the enzyme, and completed to a final reaction volume of 30 mL using nuclease-free sterile water. Incubation was done at 37C for 15 min in a thermomixer (Eppendorf, AG). In the NE-PCR, all DNA yields were amplified utilizing the second-round PCR condition protocol, skipping the firstround PCR protocol and intermittent enzymatic restriction digestion of wild-type genes. Visualization of PCR products of second-round ME-PCR as well as NE-PCR products was done using capillary electrophoresis (QIAxcel Advanced System; QIAgen) according to the manufacturer’s instructions. The system enabled high resolution and sensitive separation of DNA fragments according to their molecular weight, providing electropherograms of the products by plotting the time from injection (reflecting the size) on X-axis versus the fluorescence intensity (reflecting the concentration) on Y-axis. The retention time of bands was calibrated by the manufacturer’s DNA size marker. Statistical analysis
Data were analyzed using the IBM Statistical Package for Social Sciences software (SPSS) Windows version release 20 (SPSS, Inc.). Qualitative data were described using number and percentage. Agreement of NE-PCR and ME-PCR was
3
expressed in sensitivity, specificity, positive predictive values (PPVs), negative predictive values (NPVs), and accuracy. The Kappa test for agreement was used. Significant test results were quoted as two-tailed probabilities. A p-value less than 0.05 was considered statistically significant. Results
The 50 Egyptian NSCLC patients enrolled in this study consisted of 40 males (80%) and 10 females (20%). The age in the whole group ranged from 30 to 87 years with a mean of 59.7 – 11.4 years. Histopathological examination of lung tissue biopsies revealed three groups of NSCLC; adenocarcinoma in 23 patients (46%), squamous cell carcinoma in 13 patients (26%), and undifferentiated/large-cell carcinoma in 14 patients (28%). According to tumor grading of the studied patients, 12 cases had pathological grade 1, 16 cases had grade 2, and 22 cases had grade 3. The concentrations of the extracted DNA ranged from 10 to 550 ng/mL, with a median of 76 ng/mL. Genomic DNA extracted from open (surgical) biopsies had a median of 203 ng/mL, which was significantly higher compared to the median concentration of DNA (39 ng/mL) extracted from bronchoscopic biopsies ( p < 0.001). ME-PCR was able to detect EGFR exon-19 deletions in 11 (22%) out of the 50 cases. Only 6 out of the 11 cases harboring exon-19 deletion were obtained through open thoracic surgery, while the other five cases were obtained through bronchoscopic means. When skipping the mutant enrichment step, the NE-PCR was able to detect exon-19 deletions in 9 (18%) out of 50 cases. The deleted allele was not detected in two bronchoscopically obtained biopsies and proved by MEPCR to harbor the mutation (Table 2). By capillary electrophoresis, the wild-type (full length) allele appeared in the electropherogram as a peak of 138 bp, while the mutant allele (deleted codons) appeared in the electropherogram as peaks of 129 bp (seven cases), 120 bp (two cases), 123 bp (one case), and 126 bp (one case). The diagnostic performance of NE-PCR in detection of exon-19 deletion compared to ME-PCR revealed a diagnostic sensitivity of 82%, specificity of 100%, PPV of 100%, NPV of 95%, and an accuracy of 96% (Table 3A). Test of agreement between both PCR-based assays showed a strong agreement (Kappa K = 0.875). The NE-PCR was able to detect the EGFR exon-19 deletion in 3 (10%) out of 30 bronchoscopically obtained samples, whereas the ME-PCR was able to detect, in addition to the three cases, two more cases with the exon-19 deletion mutation, thus reaching a total of 5 (17%) out of 30
Table 2. Summary of EGFR Mutations by NE-PCR and ME-PCR Assays EGFR Exon-19 deletions NSCLC cases (n = 50)
ME-PCR (%) NE-PCR (%)
Biopsy cases (n = 20) 6/20 (30%) Bronchoscopic cases (n = 30) 5/30 (17%) Total number of cases (n = 50) 11/50 (22%) NSCLC, nonsmall-cell lung cancer.
6/20 (30%) 3/30 (10%) 9/50 (18%)
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ZAKI ET AL.
Table 3. Diagnostic Performance of NE-PCR Versus ME-PCR (A) Diagnostic performance of NE-PCR versus ME-PCR in cases with EGFR mutation ME-PCR
NE-PCR
Wild Mutant
Wild
Mutant
Sensitivity
Specificity
PPV
NPV
Accuracy
39 0
2 9
82.0
100.0
100.0
95
96
(B) Diagnostic performance of NE-PCR versus ME-PCR among bronchoscopic biopsies ME-PCR
NE-PCR
Wild Mutant
Wild
Mutant
Sensitivity
Specificity
PPV
NPV
Accuracy
25 0
2 3
60.0
100.0
100.0
92.6
93.3
Kappa Interpretation: < 0, poor agreement; 0.0–0.20, slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agreement; 0.81–1.00, almost perfect agreement. NPV, negative predictive value; PPV, positive predictive value.
bronchoscopic samples. When comparing the diagnostic performance of ME-PCR versus the NE-PCR, a diagnostic sensitivity of 60%, specificity of 100%, PPV of 100%, NPV of 92.6%, and an accuracy of 93.3% were obtained by NEPCR (Table 3B). Test of agreement between both PCR-based assays among bronchoscopic biopsies showed a substantial agreement (Kappa K = 0.714). Despite the remarkable diagnostic performance of NEPCR compared to the ME-PCR, the NE-PCR failed to detect 2 (18%) out of the 11 cases known to harbor the mutation, even with repetition of the PCR. The two cases were obtained through bronchoscopic means, with genomic DNA concentrations of 148 and 93 ng/mL, respectively. As regards the L858R point mutation in exon-21 of the EGFR gene, only the intact undigested amplicon of 130 bp was detected by capillary electrophoresis in all cases using both ME-PCR and NE-PCR, indicating the absence of such a point mutation. Discussion
In our study, EGFR gene mutations in NSCLC cases, namely exon-19 deletion mutations and exon-21 L858R point mutation, were screened by ME-PCR. Exon-19 deletions were evident in 22% of the NSCLC cases, while none of the cases had the L858R point mutation in exon-21. The frequency of exon-19 deletion presented in this study was in close agreement with several studies conducted on several populations of Caucasian (Ilie et al., 2010; Dufort et al. 2011) or East Asian (Araki et al., 2011; Kozu et al., 2011; Choi et al., 2013) ethnic backgrounds using different methods demonstrating exon-19 deletions in 19–23% of NSCLC. As regards the absence of exon-21 point mutation, such a finding was in close agreement with Paez et al. (2004) and Gombos et al. (2010). Furthermore, the low frequency (4%–7%) of such a mutation was demonstrated in Mediterranean Spanish and French populations (Molina-Vila et al., 2008; Ellison et al., 2010; Borra`s et al., 2011). In contrast, several studies on East Asian populations demonstrated a high frequency of the L858R point mutation, ranging from 16% to 46% of
NSCLC cases (Yatabe et al., 2006; Ikeda et al., 2007; Kozu et al., 2011; Yang et al., 2011). This may explain the higher frequency of EGFR mutation in Asians compared to Caucasians. The highly sensitive ME-PCR assay developed by Asano et al. (2006) proved to be critical for the detection of cancerderived mutant EGFR genes among a great excess of wildtype genes. The studies performed by Asano et al. (2006) and Otani et al. (2008) correlated ME-PCR results to those obtained by direct sequencing in the detection of EGFR exon-19 deletions. Therefore, ME-PCR was considered the gold standard technique in our study, and hence, the diagnostic performance of NE-PCR was compared to it (Table 3A). Although the two techniques, ME-PCR and NE-PCR, showed statistically strong agreement (Kappa K = 0.875), the NE-PCR showed an 18% false-negative result. Such a percentage may be extremely critical in the field of personalized medicine and the decision of therapeutic plan, depriving onefifth of the patients from the appropriate drug. Ausch et al. (2009) demonstrated that in the detection of KRAS mutations in archived FFPE tissue, DNA sequencing without previous mutant enrichment failed to detect 15% of KRAS-positive samples detected by combined ME-PCR and reversehybridization of amplification products. When comparing the diagnostic performance between NEPCR and ME-PCR, among bronchoscopic biopsies only (30 samples), NE-PCR showed lower sensitivity (60%), NPV (92.6%), and accuracy (93.3%) (Table 3B). Furthermore, the two techniques showed lower statistical agreement (Kappa K = 0.714), although still a strong one. In bronchoscopic biopsies, the NE-PCR technique gave 40% (two out of five bronchoscopic samples) false-negative results in comparison to the ME-PCR technique. One possible explanation may involve the proportion of normal to malignant cells contained in the punch taken. Another intriguing explanation involves masking of the mutation in malignant cells not only by normal cells but also by other malignant cells in the same tumor specimen that do not harbor the mutation, a condition termed ‘‘mutational intra-tumor heterogeneity,’’ as demonstrated by Bai et al. (2013) on Chinese NSCLC cases. Both explanations
NE-PCR VERSUS ME-PCR IN EGFR MUTATION DETECTION
can threaten a large sector of patients, particularly those presenting with inoperable tumors that utilize bronchoscopic samples for histological tumor typing and molecular analysis, to be misdiagnosed as nonresponders to TKI. A technical issue was addressed by Asano et al. (2006) when dealing with ME-PCR; namely, the liability of destroying the restriction site of wild-type fragments by the erroneous replacement of a critical nucleotide, as a result of the high PCR cycle number, leading to a false-positive result (Dai et al., 2000). For this reason, our results were double confirmed with the ME-PCR assay, where lack of any discrepancies rendered the assay conditions appropriate. In conclusion, although NE-PCR gave a remarkable diagnostic performance when compared to the ME-PCR, the false negativity encountered with NE-PCR makes ME-PCR particularly useful in bronchoscopically obtained samples of lung cancer tissue due to the superiority in its diagnostic sensitivity over NE-PCR. Author Disclosure Statement
No competing financial interests exist. References
Araki T, Shimizu K, Nakamura T, et al. (2011) Clinical screening assay for EGFR exon 19 mutations using PNAclamp smart amplification process version 2 in lung adenocarcinoma. Oncol Rep 26:1213–1219. Asano H, Toyooka S, Tokumo M, et al. (2006) Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay. Clin Cancer Res 12:43–48. Ausch C, Buxhofer-Ausch V, Oberkanins C, et al. (2009) Sensitive detection of KRAS mutations in archived formalinfixed paraffin-embedded tissue using mutant-enriched PCR and reverse-hybridization. J Mol Diagn 11:508–513. Bai H, Wang Z, Wang Y, et al. (2013) Detection and clinical significance of intratumoral EGFR mutational heterogeneity in Chinese patients with advanced non-small cell lung cancer. PLoS One 8:e54170. Bazley LA, Gullick WJ (2005) The epidermal growth factor receptor family. Endocr Relat Cancer 12 (Suppl 1):S17–S27. Borra`s E, Jurado I, Hernan I, et al. (2011) Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing. BMC Cancer 11:406–415. Brambilla E, Travis WD, Colby TV, et al. (2001) The new World Health Organization classification of lung tumours. Eur Respir J 18:1059–1068. Callaghan T, Antczak M, Flickinger T, et al. (1993) A complete description of the EGF-receptor exon structure: implication in oncogenic activation and domain evolution. Oncogene 8:2939–2948. Cho MC, Choi CM, Kim S, et al. (2011) Direct sequencing in cytological specimens as a useful strategy for detecting EGFR mutations in non-small cell lung cancer patients. Clin Chem Lab Med 50:565–572. Choi Y, Sun J, Cho J, et al. (2013) EGFR mutation testing in patients with advanced non-small cell lung cancer: a comprehensive evaluation of real-world practice in an East Asian tertiary hospital. PLoS One 8:e56011. Cohen V, Agulnik JS, Jarry J, et al. (2006) Evaluation of denaturing high-performance liquid chromatography as a rapid detection method for identification of epidermal growth factor
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receptor mutations in non small-cell lung cancer. Cancer 107:2858–2865. Dai Y, MorishitaY, Mase K, et al. (2000) Application of the p53 and K-ras gene mutation patterns for cytologic diagnosis of recurrent lung carcinomas. Cancer 90:258–263. Do H, Krypuy M, Mitchell PL, et al. (2008) High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies. BMC Cancer 8:142–156. Dufort S, Richard MJ, Lantuejoul S, et al. (2011) Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC. J Exp Clin Cancer Res 30:57. Ellison G, Donald E, McWalter G, et al. (2010) A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples. J Exp Clin Cancer Res 29: 132–139. Ferlay J, Shin H, Bray F, et al. (2010) Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 127:2893–2917. Gombos Z, Danihel L, Puttaswamy K, et al. (2010) A comparative study of EGFR mutation screening methods in non- small cell carcinoma of lung. Bratisl Lek Listy 111: 365–368. Ikeda S, Takabe K, Inagaki M, et al. (2007) Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification. Pathol Int 57:594–599. Ilie MI, Hofman V, Bonnetaud C, et al. (2010) Usefulness of tissue microarrays for assessment of protein expression, gene copy number and mutational status of EGFR in lung adenocarcioma. Virchows Arch 457:483–495. Kozu Y, Tsuta K, Kohno T, et al. (2011) The usefulness of mutation-specific antibodies in detecting epidermal growth factor receptor mutations and in predicting response to tyrosine kinase inhibitor therapy in lung adenocarcinoma. Lung Cancer 73:45–50. Lynch TJ, Bell DW, Sordella R, et al. (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139. Miyamae Y, Shimizu K, Mitani Y, et al. (2010) Mutation detection of epidermal growth factor receptor and KRAS genes using the Smart Amplification Process version 2 from formalin-fixed, paraffin-embedded lung cancer tissue. J Mol Diagn 12:257–264. Molina-Vila MA, Bertran-Alamillo J, Reguart N, et al. (2008) A sensitive method for detecting EGFR mutations in nonsmall cell lung cancer samples with few tumor cells. J Thorac Oncol 3:1224–1235. Otani H, Toyooka S, Soh J, et al. (2008) Detection of EGFR gene mutations using the wash fluid of CT-guided biopsy needle in NSCLC patients. J Thorac Oncol 3:472–476. Paez JG, Janne PA, Lee JC, et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497–1500. Pao W, Miller VA, Politi KA, et al. (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2:e73. Salim EI, Jazieh AR, Moore MA (2011) Lung cancer incidence in the Arab league countries: risk factors and control. Asian Pac J Cancer Prev 12:17–34. Sequist LV, Bell DW, Lynch TJ, et al. (2007) Molecular predictors of response to epidermal growth factor receptor
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antagonists in non-small-cell lung cancer. J Clin Oncol 25:587–595. Yang Q, Qiu T, Wu W, et al. (2011) Simple and sensitive method for detecting point mutations of epidermal growth factor receptor using cationic conjugated polymers. ACS Appl Mater Interfaces 3:4539–4545. Yatabe Y, Hida T, Horio Y, et al. (2006) A rapid, sensitive assay to detect EGFR mutation in small biopsy specimens from lung cancer. J Mol Diagn 8:335–341. Yu J, Kane S, Wu J, et al. (2009) Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res 15:3023–3028.
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Address correspondence to: Moyassar Ahmad Zaki, MD Department of Chemical Pathology Medical Research Institute Alexandria University P.O. Box 21561 165 El-Horreya Avenue El-Hadara Alexandria 21561 Egypt E-mail: [email protected] Ragaa Abd El-Kader Ramadan, MD Department of Chemical Pathology Medical Research Institute Alexandria University P.O. Box 21561 165 El-Horreya Avenue El-Hadara Alexandria 21561 Egypt E-mail: [email protected]
ORIGINAL ARTICLE
Nonenriched PCR Versus Mutant-Enriched PCR in Detecting Selected Epidermal Growth Factor Receptor Gene Mutations Among Nonsmall-Cell Lung Cancer Patients Moyassar Ahmad Zaki,1 Ragaa Abd El-Kader Ramadan,1 Mahmoud Ibrahim Mahmoud,2 Dalal Mohammed Nasr El-Din El-Kaffash,3 and Rami Samir Helmy Assaad1
Background: Biopsies obtained from lung cancers contain a mixture of cancerous and healthy tissues. The mutant-enriched polymerase chain reaction (ME-PCR) identifies low-level somatic DNA mutations within an excess wild-type sample. Aims: This study aimed at comparing nonenriched PCR (NE-PCR) versus ME-PCR for the detection of two epidermal growth factor receptor (EGFR) gene mutations among nonsmall cell lung cancer patients. Methods: Fifty lung tissue biopsies were screened for inframe TTAA deletions in exon-19 and the L858R point mutation in exon-21, using ME-PCR and NE-PCR, followed by capillary electrophoresis. Results: Only exon-19 deletions were detected in 22% and 18% of cases using ME-PCR and NE-PCR, respectively. Diagnostic performance of the NE-PCR versus the ME-PCR serving as a ‘‘gold standard’’ revealed a sensitivity of 82%, and a specificity of 100%, with positive and negative predictive values of 100% and 95%, respectively, and an overall accuracy of 96%. Despite a strong agreement shown between the two assays (K = 0.875), the NE-PCR showed an 18% false-negative rate in bronchoscopically obtained biopsies compared to ME-PCR. Conclusion: The false negativity encountered with NE-PCR in bronchoscopically obtained samples makes ME-PCR the technique of choice in such situations.
(TKD) responsible for mediating transphosphorylation of the carboxy-terminal tyrosine residues, which are mandatory for recruitment of adaptor/effector proteins (Bazley et al., 2005). Such a domain has been shown to be a promising target for the tyrosine kinase inhibitors (TKIs), gefitinib (Iressa) and erlotinib (Tarceva), recently introduced as a treatment modality in NSCLC cases. The fact that a relatively limited subset of NSCLC responded to TKIs (Sequist et al., 2007) became the subject for further investigations, which demonstrated the highest response rates to these TKIs in patients with somatic mutations within the EGFR gene coding for TKD, particularly exon-19 inframe deletion, exon-21 point mutation (CTG to CGG at nucleotide 2573) characterized by substitution of leucine by arginine at codon 858 (L858R), and occasionally exon-18 G719X (where X indicates A, C, or S) (Lynch et al., 2004). On the other hand, the exon-20 T790M mutation was associated with acquired resistance to TKI therapy (Pao et al., 2005). The methods used in detection of the EGFR gene mutation fall into one of the two broad categories; screening methods
Introduction
L
ung cancer remains the second most common cancer in the world, both in terms of cases and deaths, with over 1 million cases diagnosed yearly (Ferlay et al., 2010). Although lung cancer incidence rates and mortalities are still low in the Arab world compared to Europe or the United States, it is gradually increasing in the region (Salim et al., 2011). Eighty-five percent of lung cancers are nonsmall-cell lung cancers (NSCLCs), a heterogeneous group that comprised mostly squamous cell carcinomas, adenocarcinomas, and large undifferentiated carcinomas. The recent emergence of targeted lung cancer therapies directed against specific cellular alterations necessitates the most precise classification possible for NSCLCs (Brambilla et al., 2001). The epidermal growth factor receptor (EGFR) is expressed on the cell surface of a substantial percentage of NSCLCs. The gene coding for EGFR is located on the short arm (P 12) of chromosome 7 (Callaghan et al., 1993). The cytosolic portion of the EGFR contains the tyrosine kinase domain 1
Department of Chemical Pathology, Medical Research Institute, Alexandria University, Alexandria, Egypt. Departments of 2Chest Diseases and 3Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt.
1
2
ZAKI ET AL.
that detect all mutations in exons 18–21 and include sequencing, denaturing high-performance liquid chromatography, and high-resolution melting analysis (Cohen et al., 2006; Do et al., 2008; Cho et al., 2011) and targeted methods that detect specific known mutations, which are divided into polymerase chain reaction (PCR)-based and non-PCR-based ones (Yu et al., 2009; Miyamae et al., 2010; Dufort et al., 2011). The somatic nature of EGFR gene mutations dictates their presence in malignant cells only, and because clinical samples (lung tissue biopsies) contain a mixture of tumor and normal cells or genes, a highly sensitive PCR-based assay having the ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing the tumor status early in cancer patients for treatment decision. The mutantenriched PCR (ME-PCR) assay developed by Asano et al. (2006) was able to detect one mutant in the presence of 2 · 103 wild-type genes, providing a valuable method in detecting a small fraction of mutant genes in heterogeneous specimens, indicating its useful clinical application for NSCLC. We aimed to evaluate the diagnostic performance of nonenriched PCR (NE-PCR) versus ME-PCR in the detection of exon-19 inframe TTAA deletion and exon-21 L858R point mutation in EGFR gene on biopsy specimens from Egyptian NSCLC patients. Patients and Methods
Fifty consecutive cases of NSCLC were enrolled in this study from January to November 2013 from the Main University Hospital Alexandria University. All patients provided their informed consent, and institutional board permission was obtained for conducting this study. Thirty of the lung biopsies were obtained through bronchoscopic means and the rest through open thoracic surgery in the Cardiothoracic Surgery Department of the Main University Hospital. Sections from each biopsy were stained with hematoxylin and
eosin (H&E), assessed by two independent pathologists, and classified according to the TNM classification system, identifying the lung cancer type and grade. Cases were excluded if they were small-cell lung cancers or received TKIs. Demographic data were retrieved through a direct patient questionnaire. Tumor-rich areas, detected by the pathologist, were formalin fixed and paraffin embedded (FFPE). Genetic testing was done in the Chemical Pathology Department of the Medical Research Institute, Alexandria University. Freshly cut sections of FFPE tissue, each with a thickness of 5–6 mm, from biopsies were prepared. DNA was purified from the FFPE tissue using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions. The concentration and purity of the extracted DNA were measured on a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific) at 260 and 280 nm. The extracted DNA was stored at -20C till the time of PCR amplification. Some technical considerations in DNA extraction from the FFPE tissue were addressed in this study. First, discarding two to three sections exposed to air, while cutting the FFPE sample block. Second, extending incubation time of the tissue lysis buffer suspension with the proteinase-K enzyme at 56C overnight was done until the lysis was completed. Third, incubation of the lysate at 90C for 1 h in the tissue lysis buffer, to reverse formaldehyde alteration of nucleic acids and formalin crosslinking, thus resulting in more fragmented DNA. ME-PCR was carried out as described by Asano et al. (2006). The first and second rounds of PCR were carried out for both exons-19 and -21 of the EGFR gene using forward and reverse primers in concentrations of 20 pmol/mL for each primer. Details of primer sequences are supplied in Table 1. The PCRs were carried out in 0.2-mL sterile eppendorfs. Briefly, in the first round of PCR, 1 mL (10–100 ng) of extracted DNA was mixed with 4 mL of forward and reverse primers specific for each exon (Thermo-Fermentas), 12.5 mL HotStarTaq Master Mix (Qiagen, Inc.), and completed to a final reaction volume of 25 mL with nuclease-free sterile
Table 1. Primer Sequences Used for EGFR Exon-19 and Exon-21 Amplification in Both ME-PCR and NE-PCR First-round PCR Primers for exon-19 Forward primer (ex19-S1) Reverse primer (ex19-AS1) Primers for exon-21 Forward primer (ex21-S1) Reverse primer (ex21-AS1)
5¢-ATC CCA GAA GGT GAG AAA GAT AAA ATTC-3¢ 5¢-CCT GAG GTT CAG AGC CAT GGA-3¢ 5¢-CAG CCA GGA ACG TAC TGG TGA-3¢ 5¢-TCC CTG GTG TCA GGA AAA TGCT-3¢
Second-round PCR Primers for exon-19 Forward primer (ex19-S1) Reverse primer (ex19-AS1) Primers for exon-21 Forward primer (ex21-S2) Reverse primer (ex21-AS1)
5¢-ATC CCA GAA GGT GAG AAA GAT AAA ATTC-3¢ 5¢-CCT GAG GTT CAG AGC CAT GGA-3¢ 5¢-CGC AGC ATG TCA AGA TCA CAG AT-3¢ 5¢-TCC CTG GTG TCA GGA AAA TGCT-3¢
EGFR, epidermal growth factor receptor; ME-PCR, mutant-enriched PCR; NE-PCR, nonenriched PCR; PCR, polymerase chain reaction.
NE-PCR VERSUS ME-PCR IN EGFR MUTATION DETECTION
water. The cycler (Quanta Biotech 96S) conditions were as follows: an initial denaturation step of 95C for 15 min followed by 22 cycles of 20 s denaturation at 94C, 30 s annealing at 60C, and a final 20 s extension step at 72C. The integrity of the resulting PCR products was confirmed by electrophoresis on a 2% agarose gel and scored for the presence or absence of an allele-specific band. Intermittent restriction digestion of the T^TAA sequence in exon-19 and the TGG^CCA sequence in exon-21 was achieved using the fast digest enzymes MseI and MscI (Thermo-Fermentas), respectively, resulting in the enrichment of mutant-type genes. Restriction digestion was carried out separately for each exon. Briefly, in 1.5-mL sterile eppendorf tubes, 2 mL of the first-round PCR product was mixed with 2 mL of the supplied buffer (for each enzyme), 1 mL of enzyme, and completed to a final reaction volume of 20 mL with nuclease-free sterile water. Incubation was done in a thermomixer (Eppendorf, AG) at 37C for 10 min. The second round of PCR was carried out for both exons following the intermittent digestion step using primers supplied in Table 1. Briefly, 1 mL of digested DNA was mixed with 4 mL of forward and reverse primers specific for each exon (Thermo-Fermentas), 12.5 mL HotStarTaq Master Mix (Qiagen, Inc.), and completed to a final reaction volume of 25 mL with nuclease-free water. The cycler (Quanta Biotech 96S) conditions were like the first round, but with 40 cycles. Restriction digestion of second-round products for exon21 point mutation involved the use of the restriction enzyme Sau96I (Thermo-Fermentas) to digest the mutant type of codon 858 (CGG), as it digested the G^GNCC sequence, but not the wild type, and thus, even partial digestion of the PCR product indicated the presence of a mutation. Again, restriction digestion was carried out in sterile 1.5-mL eppendorf tubes utilizing 10 mL of the second-round PCR product, 2 mL of the supplied buffer, 1 mL of the enzyme, and completed to a final reaction volume of 30 mL using nuclease-free sterile water. Incubation was done at 37C for 15 min in a thermomixer (Eppendorf, AG). In the NE-PCR, all DNA yields were amplified utilizing the second-round PCR condition protocol, skipping the firstround PCR protocol and intermittent enzymatic restriction digestion of wild-type genes. Visualization of PCR products of second-round ME-PCR as well as NE-PCR products was done using capillary electrophoresis (QIAxcel Advanced System; QIAgen) according to the manufacturer’s instructions. The system enabled high resolution and sensitive separation of DNA fragments according to their molecular weight, providing electropherograms of the products by plotting the time from injection (reflecting the size) on X-axis versus the fluorescence intensity (reflecting the concentration) on Y-axis. The retention time of bands was calibrated by the manufacturer’s DNA size marker. Statistical analysis
Data were analyzed using the IBM Statistical Package for Social Sciences software (SPSS) Windows version release 20 (SPSS, Inc.). Qualitative data were described using number and percentage. Agreement of NE-PCR and ME-PCR was
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expressed in sensitivity, specificity, positive predictive values (PPVs), negative predictive values (NPVs), and accuracy. The Kappa test for agreement was used. Significant test results were quoted as two-tailed probabilities. A p-value less than 0.05 was considered statistically significant. Results
The 50 Egyptian NSCLC patients enrolled in this study consisted of 40 males (80%) and 10 females (20%). The age in the whole group ranged from 30 to 87 years with a mean of 59.7 – 11.4 years. Histopathological examination of lung tissue biopsies revealed three groups of NSCLC; adenocarcinoma in 23 patients (46%), squamous cell carcinoma in 13 patients (26%), and undifferentiated/large-cell carcinoma in 14 patients (28%). According to tumor grading of the studied patients, 12 cases had pathological grade 1, 16 cases had grade 2, and 22 cases had grade 3. The concentrations of the extracted DNA ranged from 10 to 550 ng/mL, with a median of 76 ng/mL. Genomic DNA extracted from open (surgical) biopsies had a median of 203 ng/mL, which was significantly higher compared to the median concentration of DNA (39 ng/mL) extracted from bronchoscopic biopsies ( p < 0.001). ME-PCR was able to detect EGFR exon-19 deletions in 11 (22%) out of the 50 cases. Only 6 out of the 11 cases harboring exon-19 deletion were obtained through open thoracic surgery, while the other five cases were obtained through bronchoscopic means. When skipping the mutant enrichment step, the NE-PCR was able to detect exon-19 deletions in 9 (18%) out of 50 cases. The deleted allele was not detected in two bronchoscopically obtained biopsies and proved by MEPCR to harbor the mutation (Table 2). By capillary electrophoresis, the wild-type (full length) allele appeared in the electropherogram as a peak of 138 bp, while the mutant allele (deleted codons) appeared in the electropherogram as peaks of 129 bp (seven cases), 120 bp (two cases), 123 bp (one case), and 126 bp (one case). The diagnostic performance of NE-PCR in detection of exon-19 deletion compared to ME-PCR revealed a diagnostic sensitivity of 82%, specificity of 100%, PPV of 100%, NPV of 95%, and an accuracy of 96% (Table 3A). Test of agreement between both PCR-based assays showed a strong agreement (Kappa K = 0.875). The NE-PCR was able to detect the EGFR exon-19 deletion in 3 (10%) out of 30 bronchoscopically obtained samples, whereas the ME-PCR was able to detect, in addition to the three cases, two more cases with the exon-19 deletion mutation, thus reaching a total of 5 (17%) out of 30
Table 2. Summary of EGFR Mutations by NE-PCR and ME-PCR Assays EGFR Exon-19 deletions NSCLC cases (n = 50)
ME-PCR (%) NE-PCR (%)
Biopsy cases (n = 20) 6/20 (30%) Bronchoscopic cases (n = 30) 5/30 (17%) Total number of cases (n = 50) 11/50 (22%) NSCLC, nonsmall-cell lung cancer.
6/20 (30%) 3/30 (10%) 9/50 (18%)
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ZAKI ET AL.
Table 3. Diagnostic Performance of NE-PCR Versus ME-PCR (A) Diagnostic performance of NE-PCR versus ME-PCR in cases with EGFR mutation ME-PCR
NE-PCR
Wild Mutant
Wild
Mutant
Sensitivity
Specificity
PPV
NPV
Accuracy
39 0
2 9
82.0
100.0
100.0
95
96
(B) Diagnostic performance of NE-PCR versus ME-PCR among bronchoscopic biopsies ME-PCR
NE-PCR
Wild Mutant
Wild
Mutant
Sensitivity
Specificity
PPV
NPV
Accuracy
25 0
2 3
60.0
100.0
100.0
92.6
93.3
Kappa Interpretation: < 0, poor agreement; 0.0–0.20, slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agreement; 0.81–1.00, almost perfect agreement. NPV, negative predictive value; PPV, positive predictive value.
bronchoscopic samples. When comparing the diagnostic performance of ME-PCR versus the NE-PCR, a diagnostic sensitivity of 60%, specificity of 100%, PPV of 100%, NPV of 92.6%, and an accuracy of 93.3% were obtained by NEPCR (Table 3B). Test of agreement between both PCR-based assays among bronchoscopic biopsies showed a substantial agreement (Kappa K = 0.714). Despite the remarkable diagnostic performance of NEPCR compared to the ME-PCR, the NE-PCR failed to detect 2 (18%) out of the 11 cases known to harbor the mutation, even with repetition of the PCR. The two cases were obtained through bronchoscopic means, with genomic DNA concentrations of 148 and 93 ng/mL, respectively. As regards the L858R point mutation in exon-21 of the EGFR gene, only the intact undigested amplicon of 130 bp was detected by capillary electrophoresis in all cases using both ME-PCR and NE-PCR, indicating the absence of such a point mutation. Discussion
In our study, EGFR gene mutations in NSCLC cases, namely exon-19 deletion mutations and exon-21 L858R point mutation, were screened by ME-PCR. Exon-19 deletions were evident in 22% of the NSCLC cases, while none of the cases had the L858R point mutation in exon-21. The frequency of exon-19 deletion presented in this study was in close agreement with several studies conducted on several populations of Caucasian (Ilie et al., 2010; Dufort et al. 2011) or East Asian (Araki et al., 2011; Kozu et al., 2011; Choi et al., 2013) ethnic backgrounds using different methods demonstrating exon-19 deletions in 19–23% of NSCLC. As regards the absence of exon-21 point mutation, such a finding was in close agreement with Paez et al. (2004) and Gombos et al. (2010). Furthermore, the low frequency (4%–7%) of such a mutation was demonstrated in Mediterranean Spanish and French populations (Molina-Vila et al., 2008; Ellison et al., 2010; Borra`s et al., 2011). In contrast, several studies on East Asian populations demonstrated a high frequency of the L858R point mutation, ranging from 16% to 46% of
NSCLC cases (Yatabe et al., 2006; Ikeda et al., 2007; Kozu et al., 2011; Yang et al., 2011). This may explain the higher frequency of EGFR mutation in Asians compared to Caucasians. The highly sensitive ME-PCR assay developed by Asano et al. (2006) proved to be critical for the detection of cancerderived mutant EGFR genes among a great excess of wildtype genes. The studies performed by Asano et al. (2006) and Otani et al. (2008) correlated ME-PCR results to those obtained by direct sequencing in the detection of EGFR exon-19 deletions. Therefore, ME-PCR was considered the gold standard technique in our study, and hence, the diagnostic performance of NE-PCR was compared to it (Table 3A). Although the two techniques, ME-PCR and NE-PCR, showed statistically strong agreement (Kappa K = 0.875), the NE-PCR showed an 18% false-negative result. Such a percentage may be extremely critical in the field of personalized medicine and the decision of therapeutic plan, depriving onefifth of the patients from the appropriate drug. Ausch et al. (2009) demonstrated that in the detection of KRAS mutations in archived FFPE tissue, DNA sequencing without previous mutant enrichment failed to detect 15% of KRAS-positive samples detected by combined ME-PCR and reversehybridization of amplification products. When comparing the diagnostic performance between NEPCR and ME-PCR, among bronchoscopic biopsies only (30 samples), NE-PCR showed lower sensitivity (60%), NPV (92.6%), and accuracy (93.3%) (Table 3B). Furthermore, the two techniques showed lower statistical agreement (Kappa K = 0.714), although still a strong one. In bronchoscopic biopsies, the NE-PCR technique gave 40% (two out of five bronchoscopic samples) false-negative results in comparison to the ME-PCR technique. One possible explanation may involve the proportion of normal to malignant cells contained in the punch taken. Another intriguing explanation involves masking of the mutation in malignant cells not only by normal cells but also by other malignant cells in the same tumor specimen that do not harbor the mutation, a condition termed ‘‘mutational intra-tumor heterogeneity,’’ as demonstrated by Bai et al. (2013) on Chinese NSCLC cases. Both explanations
NE-PCR VERSUS ME-PCR IN EGFR MUTATION DETECTION
can threaten a large sector of patients, particularly those presenting with inoperable tumors that utilize bronchoscopic samples for histological tumor typing and molecular analysis, to be misdiagnosed as nonresponders to TKI. A technical issue was addressed by Asano et al. (2006) when dealing with ME-PCR; namely, the liability of destroying the restriction site of wild-type fragments by the erroneous replacement of a critical nucleotide, as a result of the high PCR cycle number, leading to a false-positive result (Dai et al., 2000). For this reason, our results were double confirmed with the ME-PCR assay, where lack of any discrepancies rendered the assay conditions appropriate. In conclusion, although NE-PCR gave a remarkable diagnostic performance when compared to the ME-PCR, the false negativity encountered with NE-PCR makes ME-PCR particularly useful in bronchoscopically obtained samples of lung cancer tissue due to the superiority in its diagnostic sensitivity over NE-PCR. Author Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Moyassar Ahmad Zaki, MD Department of Chemical Pathology Medical Research Institute Alexandria University P.O. Box 21561 165 El-Horreya Avenue El-Hadara Alexandria 21561 Egypt E-mail: [email protected] Ragaa Abd El-Kader Ramadan, MD Department of Chemical Pathology Medical Research Institute Alexandria University P.O. Box 21561 165 El-Horreya Avenue El-Hadara Alexandria 21561 Egypt E-mail: [email protected]
