RESEARCH ARTICLE
The Role of Immunohistochemical Analysis in the Evaluation of EML4-ALK Gene Rearrangement in Lung Cancer Harold C. Sullivan, MD, Kevin E. Fisher, MD, PhD, Anne L. Hoffa, MD, Jason Wang, MD, Debra Saxe, PhD, Momin T. Siddiqui, MD, and Cynthia Cohen, MD
Background: Among the mutations described in non–small cell lung carcinoma is a rearrangement resulting from an inversion within chromosome 2p leading to the formation of a fusion gene, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK). Fluorescence in situ hybridization (FISH) is the gold standard for the detection of ALK gene rearrangements. However, molecular methods are not readily available in all pathology laboratories. Immunohistochemistry (IHC) using an antibody directed against the EML4-ALK fusion protein provides a widely available alternative method of detection. We assessed whether IHC is a comparable and cost-effective alternative to FISH analysis for the detection of ALK gene rearrangements. Design: A total of 110 non–small cell lung carcinoma cases (63 surgical/biopsy and 47 cytology specimens), previously tested for ALK gene rearrangements by FISH [7 (6.4%) positive for the rearrangement], were probed for the EML4-ALK fusion protein using a monoclonal EML4-ALK antibody, clone 5A4. Cells were considered to stain positive for ALK if >5% of cells showed cytoplasmic staining of at least grade 1 intensity (scale: 0 to 3). A cost analysis was performed using ALK IHC as a screening test. Results: The sensitivity and specificity of the EML4-ALK IHC stain compared with ALK FISH analysis were 100% and 96%, respectively. All 7 FISH-positive cases stained positive by IHC, whereas 4 FISH-negative cases demonstrated positive staining. One of the 4 FISH-negative, IHC-positive cases harbored an EML4ALK rearrangement by RT-PCR yielding 3 false-positive results overall. The k agreement between IHC and FISH methods is 0.76 (substantial/excellent). The potential savings of implementing the ALK IHC as a screening method would be $10,418.21. Conclusions: Sensitivity of the EML4-ALK IHC stain is excellent (100%) but due to its suboptimal specificity, IHC cannot reliably supplant FISH analysis for the detection of ALK gene
Received for publication August 8, 2013; accepted March 10, 2014. From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA. The authors declare no conflict of interest. Reprints: Harold C. Sullivan, MD, Emory University Hospital, 1364 Clifton Road, NE, Room H183, Atlanta, GA 30322 (e-mail: [email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
Appl Immunohistochem Mol Morphol
rearrangements. IHC shows promise as a screening tool to prevent unnecessary costly FISH analysis. Key Words: EML4-ALK, lung, adenocarcinoma, immunohistochemistry, fluorescence in situ hybridization (Appl Immunohistochem Mol Morphol 2015;23:239–244)
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number of genetic abnormalities have been described in association with non–small cell lung carcinoma (NSCLC). One such abnormality is a rearrangement resulting from a small inversion within the short arm of chromosome 2 that leads to the formation of a fusion gene, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), which has been identified in a small percentage of patients with NSCLC.1 Studies demonstrate that lung cancers harboring EML4-ALK or kinesin family member 5B (KIF5B)ALK rearrangements are sensitive to ALK inhibitors, such as crizotinib,2 and possibly responsive to other medications such as Alimta (pemetrexed), a folate antimetabolite.3 The protocol at Emory University Hospital (EUH) at the time of the study was to reflex test all newly diagnosed epidermal growth factor receptor (EGFR) mutation-negative NSCLC for ALK rearrangements by fluorescence in situ hybridization (FISH). Presently, the FDA approved use of crizotinib for treating NSCLC requires the demonstration of an ALK gene rearrangement by FISH using molecular probes obtained from Abbott Molecular Inc (Abbott Park, IL). This method is labor intensive and expensive; for institutions that do not have the necessary expertise and facilities, the cost for referral testing can be burdensome. The recent development of antibodies directed against the fusion protein from the EML4ALK rearrangement offers an immunohistochemical (IHC) method for detection. Although IHC staining requires special equipment and technical support, most pathology laboratories perform IHC assays. Performing a single IHC stain for EML4-ALK rearrangement currently costs EUH $116.60, roughly 50% of the cost of a single FISH analysis. The purpose of this study was to evaluate the mutation-specific IHC stain as a comparable and less expensive alternative to FISH analysis in the detection of the EML4-ALK rearrangement.
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MATERIALS AND METHODS Tissue Specimens Results of 201 EML4-ALK FISH tests performed on NSCLC diagnosed at EUH were retrospectively obtained. Cases included were diagnosed from July 2001 to July 2012. Only cases that had both FISH results and corresponding paraffin-embedded tissue were included. After excluding cases in which paraffin-embedded tissue was not available, 110 specimens were included in the study. Study approval was obtained from the Emory University Investigation Review Board and all investigators were CITI certified to study material from human subjects.
FISH FISH performed at EUH was carried out using formalin-fixed, paraffin-embedded tissues. Sections (4 mm) are placed on positively charged slides. Slides are deparaffinized on a slide warmer overnight. Then slides are pretreated with protease, processed on a VP 2000. After slides are processed, 10 mL of ALK probe mixture (Abbott Molecular Inc.) is added and the slides are coverslipped and sealed with rubber cement. Slides are then placed in a hybridization chamber where the DNA of both the probe and cellular DNA are denatured at 71 degrees and then allowed to rehybridize overnight at 37 degrees. Slides are washed with 2 SSC/0.3% NP-40 and dehydrated with EtOH. Slides are counterstained with 10 mL DAPI and chilled in the freezer for at least 20 minutes. The probe is a dual color, break-apart probe in which the intact gene appears as a single fluorescent signal (yellow) and a rearranged gene appears as 2 separate fluorescent signals (green and red). A total of 200 cells are analyzed by 2 different readers each reading 100 cells. Before February 2011, EUH sent out ALK FISH to Genzyme Genetics and Clarient.
Immunohistochemistry The available 110 paraffin-embedded tissue sections (5 micron) were immunostained for ALK using monoclonal EML4-ALK, clone 5A4 (Leica Microsystems, Bannockburn, IL), at 1:20 dilution using the Dako Autostainer (Dako, Carpinteria, CA). Antigen retrieval was performed using the Trilogy antigen retrieval method with EDTA at a high pH (pH 7.8 to 9.6) using an electric pressure cooker for 3 minutes at 12 to 15 pounds per square inch (1201C), and cooled for 10 minutes before immunostaining. The EnVision+Dual Link Kit (Dako), which uses a polymer, was the detection method, used according to the manufacturer’s instructions, with diaminobenzidine as the chromogen, and hematoxylin as counterstain. A known FISH-positive lung adenocarcinoma was used as a positive control with each run. Negative controls were concurrently run, with the primary antibody replaced with buffer. ALK antigen expression was cytoplasmic. Visual quantitation was assessed. An immunostain intensity of Z1 (scale: 0 to 3) and percentage of positive-staining cells of >5% were considered positive.
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Reverse Transcriptase PCR (RT-PCR) To investigate false positives (IHC positive, FISH negative), RT-PCR was performed on IHC-positive cases to determine whether IHC detected a translocation that was missed by FISH. RNA was extracted from FFPE tissue from EML4-ALK IHC-positive patient samples using a RecoverAll RNA extraction kit (Life Biotechnologies, Carlsbad, CA) and reverse transcribed into cDNA using random hexamer primers per the manufacturer’s protocol. cDNAs were subjected to real-time PCR (qPCR) using FAST SYBR green mastermix (Life Biosciences) as the detector. RNA from normal lung was used as a negative control. The following primers were used to detect the variants: EML4-ALK variant 1-FW 50 -TAGAGCCCAC ACCTGGGAAA-30 , EML4-ALK variant 1/3-REV 50 -CG GAGCTTGCTCAGCTTGTA-30 ; EML4-ALK variant 3-FW 50 -GCATAAAGATGTCATCATCAACCAAG-30 ; KIF5B-ALK-FW 50 -TCGGCAACTTTAGCGAGTA-30 , KIF5B-ALK-REV 50 -GGACACCTGGCCTTCATAC-30 . RNA sample integrity was confirmed by GAPDH (primers: GAPDH-FW 50 -TGGGATTTCCATTGATGACAAG-30 , GAPDH-REV 50 -ATTCCACCCATGGCAAATTC-30 ). Primers were adapted from Takeuchi et al4 and Soda et al.5
Statistical Analysis Sensitivity, specificity, positive predictive value, and negative predictive value of the ALK immunostain were determined using ALK FISH as the gold standard. Percent agreement between the immunostain and FISH were calculated. In addition, k coefficient, a statistical measure of intertest agreement,6 was also determined.
Cost Analysis Cost analysis was performed to determine potential health care financial savings if the ALK immunostain was implemented as a screening test. On the basis of Medicare reimbursement prices, global cost (technical plus professional cost) to perform ALK FISH and ALK IHC at EUH is $234.79 and $116.60, respectively. The cost of the present practice of performing ALK FISH on all newly diagnosed EGFR-negative NSCLC was calculated [the product of the number of cases (110) and the cost of the test ($234.79)]. The cost of using ALK IHC as a screening test was calculated by adding the cost of performing IHC on all newly diagnosed, EGFR-negative NSCLC [product of the number of cases (110) and the cost of the test ($116.60)] to the cost of performing ALK FISH on only the cases with positive IHC [product of number of IHC-positive cases (11) and cost of the test ($234.79)]. The potential savings was then determined by subtracting the cost of the ALK IHC screening method, with FISH performed only on the IHC-positive cases, from the cost of the current method (FISH only on all cases).
RESULTS Of the 110 cases included in the study, 63 were surgical resections (n = 23) or biopsies (n = 40) and 47 were cell blocks of fine needle aspirations. Table 1 indicates specimen type and diagnosis. Eleven of these cases, all adenocarcinomas, stained positive for EML4-ALK by IHC Copyright
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TABLE 1. Specimen: Type, Location, and Diagnoses Specimen Type (n) Surgical specimens (n = 63)
Diagnosis (n)
Primary lung excisions (24) Wedge resections (12) Lobectomies (9) Pneumonectomies (3) Lung biopsies (20) Transbronchial (13) Lung mass (7) Lymph node biopsies (4) Pleural biopsies (6) Distant metastasis (9) Brain biopsies (7) Bone biopsies (2)
Adenocarcinoma (53)
Poorly differentiated carcinoma (3) Squamous cell carcinoma (2) Spindled cell carcinoma (2) Neuroendocrine carcinoma (1) Large cell carcinoma (1) Adenocarcinoma and large cell neuroendocrine (1)
Location (n) Cytology specimens (n = 47)
Lymph node (22) Lung mass (13) Pleural effusion (4) Pericardial effusion (1) Distant metastasis (7) Bone (3) Liver (1) Periesophageal mass (1) Thigh mass (1) Axillary mass (1)
(Fig. 1). Table 2 demonstrates 7 of these 11 cases were positive for EML4-ALK rearrangement by FISH, the gold standard. This represents 6.4% (7 of 110) of the total cases included in the study. Thus the IHC method detected all the true-positive cases with no false negatives but 4 false positives (IHC-positive, FISH-negative cases). Therefore, the sensitivity and specificity of the EML4-ALK IHC stain were 100% and 96%, respectively, whereas positive and negative predictive values were 64% and 100%, respectively (Table 3). Of 11 IHC-positive cases, 4 were FISH negative, representing possible false positives. RT-PCR for EML4ALK variant 1 or 3 translocations and KIF5B-ALK rearrangements was performed on 9 of the 11 IHC-positive cases (2 true positives did not have available/sufficient tissue for testing). Results of the RT-PCR revealed that one of the FISH-negative cases (a cytology specimen) harbored an EML4-ALK translocation not detected by FISH. The intensity of the staining in positive EML4-ALK FISH cases ranged from 1 to 3, with a mean of 2. Percentage of positive-staining tumor cells ranged from 60% to 100%, with a mean of 76% (Table 2). The intensity and percentage of positive-staining cells did not seem to differ between true-positive and false-positive cases as there was 1 true-positive case with low-intensity staining and 3 false-positive cases with high-intensity staining and high percent positive-staining tumor cells (90%). In terms of agreement between ALK FISH and ALK IHC, the percent agreement between positive cases was 66.7%, whereas the percent agreement between the negative cases was 100%. The kagreement was 0.76, which correlates with substantial or excellent agreement.7,8 The cost of performing the current protocol, in which all newly diagnosed, EGFR-negative NSCLC are Copyright
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Diagnosis (n) Adenocarcinoma (39) Squamous cell carcinoma (4) Poorly differentiated carcinoma (3) Adenosquamous carcinoma (1)
tested for the ALK rearrangement by FISH, for the 110 cases included is $25,826.90. The cost of performing ALK IHC on all 110 cases would be $12,826.00. The cost of performing FISH on the 11 positive IHC would be $2582.69. Thus, using the ALK IHC as a screening tool, followed by ALK FISH on the 11 ALK IHC-positive cases, would cost $15,408.69. For the 110 cases included in the study, the potential savings of implementing the ALK IHC as a screening method would be $10,418.21.
DISCUSSION We found that the EML4-ALK IHC stain used in our study had a sensitivity of 100% with a specificity of 96%. These findings are similar to other studies. Conklin and colleagues report that 2 different antibodies directed against ALK antigens, D5F3 (Cell Signaling by ADVANCE) and 5A4 (Novocastra by ADVANCE), both demonstrated a sensitivity of 100% and specificity of 75% for D5F3 and 87.5% for 5A4. As in our study, neither antibody resulted in false negatives. Thus, they concluded that IHC is a dependable screening method for detection of the EML4-ALK rearrangement in NSCLC.9 Savic et al10 also demonstrated the 5A4 clone to have high sensitivity (93.3%) and specificity (96%) in the detection of the ALK rearrangement. Most recently, Selinger et al11 compared 3 different antibodies (ALK1, 5A4, and D5F3) and showed that all 3 stains had 100% sensitivity, but slightly variable specificities given different rates of false positives. Interestingly, the 5A4 clone is designed to target an epitope that corresponds to a region, which spans the tyrosine kinase catalytic domain and part of the C-terminus of NPM-ALK transcript.12,13 However, our study and the aforementioned www.appliedimmunohist.com |
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FIGURE 1. A, Positive ALK IHC: representative image from specimen with 60% of tumor cells staining at 3+ intensity; magnification, 4. B, Positive ALK IHC: representative image from specimen with 90% of tumor cells staining at 2+ intensity; magnification, 4. C, Positive ALK IHC: representative image from specimen with 70% of tumor cells staining at 1+ intensity; magnification, 4. D, Negative ALK IHC: representative image from specimen with 0% of tumor cells staining; magnification, 4. E, ALK FISH: White arrows indicate cells with a single yellow signal, representing intact ALK genes. Yellow arrows indicate cells with a red and green signal, representing an ALK rearrangement.
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TABLE 2. ALK-positive Cases by FISH and IHC No. Cases 1 2 3 4 5 6 7 8 9 10 11
Fish Result
IHC Intensity
IHC Percentage
IHC Result
Specimen Type
Positive Positive Positive Positive Positive Positive Positive Negative Negative Negative Negative
2 3 2 2 1 2 2 3 2 2 2
80 60 95 80 70 100 50 50 90 90 10
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
Right lower lobe FNA Pleural fluid FNA Pleural biopsy Pleural biopsy Transbronchial biopsy Femur resection Cervical bone resection Scapula FNA Pneumonectomy Wedge resection Brain biopsy
ALK indicates anaplastic lymphoma kinase; FISH, fluorescence in situ hybridization; FNA, fine needle aspiration; IHC, immunohistochemistry.
studies seem to demonstrate its cross-reactivity with EML4ALK. Thus, the high sensitivity and specificity of the 5A4 antibody for ALK translocations in our cohort of NSCLC is consistent with the current literature. McLeer-Florin and colleagues showed ALK IHC to have a sensitivity of 95% and a specificity of 100%, respectively. Even without 100% sensitivity, they suggest that ALK IHC could be used as a prescreening tool before FISH analysis for the identification of the ALK rearrangement.14 A Korean study found that ALK IHC had a similar sensitivity of 100% and a specificity of 98.7%. However, unlike our study, they found a correlation between staining intensity and ALK rearrangement status with all tissues with an IHC score of 3, 84.6% of tissues with an IHC score of 2, 83.3% of tissues with an IHC score of 1, and no tissues with an IHC score of 0 being ALK FISH positive.15 Of note, RT-PCR analysis has been developed for the detection of ALK rearrangements. One of the benefits of the RT-PCR method is the detection of novel ALK rearrangement variants beyond previously identified variants 1, 2, and 3 such as KIF5B-ALK.4,16 One study did a comparison of RT-PCR, IHC, and FISH for EML4-ALK rearrangement in the identification of the ALK rearrangements. Their results showed RT-PCR to be the most sensitive and least subjective method in the evaluation of ALK rearrangement status.17 Consistent with this increase in sensitivity, RT-PCR detected 1 additional EML4-ALK rearrangement not detected by our standard FISH assay. This is likely due to the small amount of neoplastic cells present in the cytology specimen. Thus, if the IHC-positive, RT-PCR-positive, FISH-negative case is taken to represent a true positive, then IHC only detected 3 false positives. However, it is important to bear in mind that FISH and IHC allow the actual stained tissue to be visualized, which is not the case with RT-PCR. In
addition, current molecular guidelines do not recommend that RT-PCR be used in place of FISH when determining eligibility for ALK therapy.18 Although the aforementioned studies have looked into the efficacy of ALK IHC as a screening tool to ALK FISH, they have not analyzed the potential savings in terms of technical laboratory and professional costs. For the cases included in the study at EUH, we found that implementing IHC as a screening method, which would include performing ALK IHC on all newly diagnosed EGFR-negative NSCLC and ALK FISH on only the IHC-positive cases, could save $10,418.21. This total does not reflect the possible gross savings, as the 110 cases only represent 55% of the total 201 NSCLC cases that had FISH performed in the specified study period. Although the screening method would still incur cost from FISH performed on false-positive cases, the number of FISH tests performed would drastically decrease. The calculations performed only account for the financial savings, as there are potential time management benefits. If EML4-ALK IHC staining were used as the screening method, the laboratory technologist would have only had to perform 11 ALK FISH analyses. This would mean 99 less FISH analyses would have been performed. Furthermore, the process of performing IHC can be automated, and is therefore more efficient than performing FISH analyses on all specimens. Our study demonstrated that there can be substantial financial savings with our proposed methodology. We recognize that the small sample size available for this study limits the broad application of our study’s sensitivity and specificity results. Ideally, we would have liked to have had more ALK-positive cases to determine the sensitivity and specificity of ALK IHC. Nonetheless, the frequency of positive NSCLC in cases included was 6.4%
TABLE 3. Comparison of ALK IHC to ALK FISH as Gold Standard IHC positive IHC negative
FISH Positive
FISH Negative
7 0 Sensitivity 100%
4 98 Specificity 96%
Positive predictive value 64% Negative predictive value 100%
ALK indicates anaplastic lymphoma kinase; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry.
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(7/110). However, when taking into account the 201 total NSCLC with ALK FISH results, the frequency would be 3.4%. Nonetheless, both 6.4% and 3.4% fall within the incidence range of 1% to 13% reported by previous studies.4,19–28 With such a low frequency of ALK gene rearrangements, a screening test would be greatly beneficial, especially for its use in small specimens, and both surgical and cytologic specimens. Even though the specificity of the EML4-ALK IHC stain is high (96.3%), it is not sufficiently specific to supplant FISH analysis in the detection of the EML4-ALK rearrangement and would overestimate the presence of the translocation. In addition, FDA approval of Xalkori (crizotinib) for treatment of NSCLC is linked to Abbott Molecular FISH. As such, drug administration and hospital reimbursement require a positive FISH test with Abbott’s FISH probe. Presently, failure to use Abbot’s probe may hinder a patient from receiving adequate treatment and prevent proper monetary compensation by insurance companies. Furthermore, the subjectivity, interobserver variability, and visual quantitation of IHC can arguably be just as problematic when compared with FISH. However, with high sensitivity, demonstrated in the current and previous studies, ALK IHC shows promise as a screening tool to significantly decrease the number of FISH analyses needed. Indeed, some have already proposed diagnostic algorithms for using IHC to screen for ALK rearrangements.28 As mentioned previously, another advantage of using IHC is that it is particularly suitable to be used on small samples, including biopsies and cytologic specimens that may contain an abundance of benign-contaminating cells. In fact, recent guidelines from the College of American Pathologists, International Association of the Study of Lung Cancer, and Association for Molecular Pathology support the use of IHC screening as long as it has been appropriately validated.18 Consequently, screening with ALK IHC would also be financially beneficial with tens of thousands of dollars in potential health care savings. REFERENCES 1. Vasikova A. EML4-ALK fusion gene in patients with lung carcinoma: biology, diagnostics and targeted therapy. Klin Onkol. 2012;25:434–439. 2. Rodig SJ, Shapiro GI. Crizotinib, a small-molecule dual inhibitor of the c-Met and ALK receptor tyrosine kinases. Curr Opin Investig Drugs. 2010;11:1477–1490. 3. Lee HY, Ahn HK, Jeong JY, et al. Favorable clinical outcomes of pemetrexed treatment in anaplastic lymphoma kinase positive nonsmall-cell lung cancer. Lung Cancer. 2013;79:40–45. 4. Takeuchi K, Choi YL, Soda M, et al. Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res. 2008;14:6618–6624. 5. Soda M, Isobe K, Inoue A, et al. A prospective PCR-based screening for the EML4-ALK oncogene in non-small cell lung cancer. Clin Cancer Res. 2012;18:5682–5689. 6. Carletta J. Assessing agreement on classification tasks: the kappa statistic. Comput Linguist. 1996;22:249–254. 7. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159–174. 8. Fleiss JL. Statistical Methods for Rates and Proportions. 2nd ed. New York: John Wiley; 1981.
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9. Conklin CM, Craddock KJ, Have C, et al. Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent. J Thorac Oncol. 2013;8:45–51. 10. Savic S, Bode B, Diebold J, et al. Detection of ALK-positive nonsmall-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone. J Thorac Oncol. 2013;8: 1004–1011. 11. Selinger CI, Rogers TM, Russell PA, et al. Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol. 2013;26:1545–1553. 12. “Novocastra ALK.” Available at: http://www.leicabiosystems.com/ ihc-ish/novocastra-reagents/primary-antibodies/details/product/alk-1/. Accessed September 25, 2013. 13. “Anti-ALK antibody [5A4] (ab17127).” Antibodies, proteins, kits and reagents for life science. Available at: http://www.abcam.com/ alk-antibody-5a4-ab17127.html. Accessed September 25, 2013. 14. McLeer-Florin A, Moro-Sibilot D, Melis A, et al. Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study. J Thorac Oncol. 2012;7:348–354. 15. Park HS, Lee JK, Kim DW, et al. Immunohistochemical screening for anaplastic lymphoma kinase (ALK) rearrangement in advanced non-small cell lung cancer patients. Lung Cancer. 2012;77:288–292. 16. Sanders HR, Li HR, Bruey JM, et al. Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer. Cancer Genet. 2011;204:45–52. 17. Wallander ML, Geiersbach KB, Tripp SR, et al. Comparison of reverse transcription-polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization methodologies for detection of echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase fusion-positive non-small cell lung carcinoma: implications for optimal clinical testing. Arch Pathol Lab Med. 2012;136:796–803. 18. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Mol Diagn. 2013;15:415–453. 19. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448:561–566. 20. Koivunen JP, Mermel C, Zejnullahu K, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res. 2008;14:4275–4283. 21. Wong DW, Leung EL, So KK, et al. The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS. Cancer. 2009;115:1723–1733. 22. Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol. 2009;174:661–670. 23. Boland JM, Erdogan S, Vasmatzis G, et al. Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non-small cell lung carcinomas. Hum Pathol. 2009;40:1152–1158. 24. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol. 2009;27:4247–4253. 25. Perner S, Wagner PL, Demichelis F, et al. EML4-ALK fusion lung cancer: a rare acquired event. Neoplasia. 2008;10:298–302. 26. Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol. 2008;3:13–17. 27. Shinmura K, Kageyama S, Tao H, et al. EML4-ALK fusion transcripts, but no NPM-, TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas. Lung Cancer. 2008;61:163–169. 28. Takamochi K, Takeuchi K, Hayashi T, et al. A rational diagnostic algorithm for the identification of ALK rearrangement in lung cancer: a comprehensive study of surgically treated Japanese patients. PLoS One. 2013;8:e69794.
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The Role of Immunohistochemical Analysis in the Evaluation of EML4-ALK Gene Rearrangement in Lung Cancer Harold C. Sullivan, MD, Kevin E. Fisher, MD, PhD, Anne L. Hoffa, MD, Jason Wang, MD, Debra Saxe, PhD, Momin T. Siddiqui, MD, and Cynthia Cohen, MD
Background: Among the mutations described in non–small cell lung carcinoma is a rearrangement resulting from an inversion within chromosome 2p leading to the formation of a fusion gene, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK). Fluorescence in situ hybridization (FISH) is the gold standard for the detection of ALK gene rearrangements. However, molecular methods are not readily available in all pathology laboratories. Immunohistochemistry (IHC) using an antibody directed against the EML4-ALK fusion protein provides a widely available alternative method of detection. We assessed whether IHC is a comparable and cost-effective alternative to FISH analysis for the detection of ALK gene rearrangements. Design: A total of 110 non–small cell lung carcinoma cases (63 surgical/biopsy and 47 cytology specimens), previously tested for ALK gene rearrangements by FISH [7 (6.4%) positive for the rearrangement], were probed for the EML4-ALK fusion protein using a monoclonal EML4-ALK antibody, clone 5A4. Cells were considered to stain positive for ALK if >5% of cells showed cytoplasmic staining of at least grade 1 intensity (scale: 0 to 3). A cost analysis was performed using ALK IHC as a screening test. Results: The sensitivity and specificity of the EML4-ALK IHC stain compared with ALK FISH analysis were 100% and 96%, respectively. All 7 FISH-positive cases stained positive by IHC, whereas 4 FISH-negative cases demonstrated positive staining. One of the 4 FISH-negative, IHC-positive cases harbored an EML4ALK rearrangement by RT-PCR yielding 3 false-positive results overall. The k agreement between IHC and FISH methods is 0.76 (substantial/excellent). The potential savings of implementing the ALK IHC as a screening method would be $10,418.21. Conclusions: Sensitivity of the EML4-ALK IHC stain is excellent (100%) but due to its suboptimal specificity, IHC cannot reliably supplant FISH analysis for the detection of ALK gene
Received for publication August 8, 2013; accepted March 10, 2014. From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA. The authors declare no conflict of interest. Reprints: Harold C. Sullivan, MD, Emory University Hospital, 1364 Clifton Road, NE, Room H183, Atlanta, GA 30322 (e-mail: [email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
Appl Immunohistochem Mol Morphol
rearrangements. IHC shows promise as a screening tool to prevent unnecessary costly FISH analysis. Key Words: EML4-ALK, lung, adenocarcinoma, immunohistochemistry, fluorescence in situ hybridization (Appl Immunohistochem Mol Morphol 2015;23:239–244)
A
number of genetic abnormalities have been described in association with non–small cell lung carcinoma (NSCLC). One such abnormality is a rearrangement resulting from a small inversion within the short arm of chromosome 2 that leads to the formation of a fusion gene, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), which has been identified in a small percentage of patients with NSCLC.1 Studies demonstrate that lung cancers harboring EML4-ALK or kinesin family member 5B (KIF5B)ALK rearrangements are sensitive to ALK inhibitors, such as crizotinib,2 and possibly responsive to other medications such as Alimta (pemetrexed), a folate antimetabolite.3 The protocol at Emory University Hospital (EUH) at the time of the study was to reflex test all newly diagnosed epidermal growth factor receptor (EGFR) mutation-negative NSCLC for ALK rearrangements by fluorescence in situ hybridization (FISH). Presently, the FDA approved use of crizotinib for treating NSCLC requires the demonstration of an ALK gene rearrangement by FISH using molecular probes obtained from Abbott Molecular Inc (Abbott Park, IL). This method is labor intensive and expensive; for institutions that do not have the necessary expertise and facilities, the cost for referral testing can be burdensome. The recent development of antibodies directed against the fusion protein from the EML4ALK rearrangement offers an immunohistochemical (IHC) method for detection. Although IHC staining requires special equipment and technical support, most pathology laboratories perform IHC assays. Performing a single IHC stain for EML4-ALK rearrangement currently costs EUH $116.60, roughly 50% of the cost of a single FISH analysis. The purpose of this study was to evaluate the mutation-specific IHC stain as a comparable and less expensive alternative to FISH analysis in the detection of the EML4-ALK rearrangement.
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MATERIALS AND METHODS Tissue Specimens Results of 201 EML4-ALK FISH tests performed on NSCLC diagnosed at EUH were retrospectively obtained. Cases included were diagnosed from July 2001 to July 2012. Only cases that had both FISH results and corresponding paraffin-embedded tissue were included. After excluding cases in which paraffin-embedded tissue was not available, 110 specimens were included in the study. Study approval was obtained from the Emory University Investigation Review Board and all investigators were CITI certified to study material from human subjects.
FISH FISH performed at EUH was carried out using formalin-fixed, paraffin-embedded tissues. Sections (4 mm) are placed on positively charged slides. Slides are deparaffinized on a slide warmer overnight. Then slides are pretreated with protease, processed on a VP 2000. After slides are processed, 10 mL of ALK probe mixture (Abbott Molecular Inc.) is added and the slides are coverslipped and sealed with rubber cement. Slides are then placed in a hybridization chamber where the DNA of both the probe and cellular DNA are denatured at 71 degrees and then allowed to rehybridize overnight at 37 degrees. Slides are washed with 2 SSC/0.3% NP-40 and dehydrated with EtOH. Slides are counterstained with 10 mL DAPI and chilled in the freezer for at least 20 minutes. The probe is a dual color, break-apart probe in which the intact gene appears as a single fluorescent signal (yellow) and a rearranged gene appears as 2 separate fluorescent signals (green and red). A total of 200 cells are analyzed by 2 different readers each reading 100 cells. Before February 2011, EUH sent out ALK FISH to Genzyme Genetics and Clarient.
Immunohistochemistry The available 110 paraffin-embedded tissue sections (5 micron) were immunostained for ALK using monoclonal EML4-ALK, clone 5A4 (Leica Microsystems, Bannockburn, IL), at 1:20 dilution using the Dako Autostainer (Dako, Carpinteria, CA). Antigen retrieval was performed using the Trilogy antigen retrieval method with EDTA at a high pH (pH 7.8 to 9.6) using an electric pressure cooker for 3 minutes at 12 to 15 pounds per square inch (1201C), and cooled for 10 minutes before immunostaining. The EnVision+Dual Link Kit (Dako), which uses a polymer, was the detection method, used according to the manufacturer’s instructions, with diaminobenzidine as the chromogen, and hematoxylin as counterstain. A known FISH-positive lung adenocarcinoma was used as a positive control with each run. Negative controls were concurrently run, with the primary antibody replaced with buffer. ALK antigen expression was cytoplasmic. Visual quantitation was assessed. An immunostain intensity of Z1 (scale: 0 to 3) and percentage of positive-staining cells of >5% were considered positive.
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Reverse Transcriptase PCR (RT-PCR) To investigate false positives (IHC positive, FISH negative), RT-PCR was performed on IHC-positive cases to determine whether IHC detected a translocation that was missed by FISH. RNA was extracted from FFPE tissue from EML4-ALK IHC-positive patient samples using a RecoverAll RNA extraction kit (Life Biotechnologies, Carlsbad, CA) and reverse transcribed into cDNA using random hexamer primers per the manufacturer’s protocol. cDNAs were subjected to real-time PCR (qPCR) using FAST SYBR green mastermix (Life Biosciences) as the detector. RNA from normal lung was used as a negative control. The following primers were used to detect the variants: EML4-ALK variant 1-FW 50 -TAGAGCCCAC ACCTGGGAAA-30 , EML4-ALK variant 1/3-REV 50 -CG GAGCTTGCTCAGCTTGTA-30 ; EML4-ALK variant 3-FW 50 -GCATAAAGATGTCATCATCAACCAAG-30 ; KIF5B-ALK-FW 50 -TCGGCAACTTTAGCGAGTA-30 , KIF5B-ALK-REV 50 -GGACACCTGGCCTTCATAC-30 . RNA sample integrity was confirmed by GAPDH (primers: GAPDH-FW 50 -TGGGATTTCCATTGATGACAAG-30 , GAPDH-REV 50 -ATTCCACCCATGGCAAATTC-30 ). Primers were adapted from Takeuchi et al4 and Soda et al.5
Statistical Analysis Sensitivity, specificity, positive predictive value, and negative predictive value of the ALK immunostain were determined using ALK FISH as the gold standard. Percent agreement between the immunostain and FISH were calculated. In addition, k coefficient, a statistical measure of intertest agreement,6 was also determined.
Cost Analysis Cost analysis was performed to determine potential health care financial savings if the ALK immunostain was implemented as a screening test. On the basis of Medicare reimbursement prices, global cost (technical plus professional cost) to perform ALK FISH and ALK IHC at EUH is $234.79 and $116.60, respectively. The cost of the present practice of performing ALK FISH on all newly diagnosed EGFR-negative NSCLC was calculated [the product of the number of cases (110) and the cost of the test ($234.79)]. The cost of using ALK IHC as a screening test was calculated by adding the cost of performing IHC on all newly diagnosed, EGFR-negative NSCLC [product of the number of cases (110) and the cost of the test ($116.60)] to the cost of performing ALK FISH on only the cases with positive IHC [product of number of IHC-positive cases (11) and cost of the test ($234.79)]. The potential savings was then determined by subtracting the cost of the ALK IHC screening method, with FISH performed only on the IHC-positive cases, from the cost of the current method (FISH only on all cases).
RESULTS Of the 110 cases included in the study, 63 were surgical resections (n = 23) or biopsies (n = 40) and 47 were cell blocks of fine needle aspirations. Table 1 indicates specimen type and diagnosis. Eleven of these cases, all adenocarcinomas, stained positive for EML4-ALK by IHC Copyright
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TABLE 1. Specimen: Type, Location, and Diagnoses Specimen Type (n) Surgical specimens (n = 63)
Diagnosis (n)
Primary lung excisions (24) Wedge resections (12) Lobectomies (9) Pneumonectomies (3) Lung biopsies (20) Transbronchial (13) Lung mass (7) Lymph node biopsies (4) Pleural biopsies (6) Distant metastasis (9) Brain biopsies (7) Bone biopsies (2)
Adenocarcinoma (53)
Poorly differentiated carcinoma (3) Squamous cell carcinoma (2) Spindled cell carcinoma (2) Neuroendocrine carcinoma (1) Large cell carcinoma (1) Adenocarcinoma and large cell neuroendocrine (1)
Location (n) Cytology specimens (n = 47)
Lymph node (22) Lung mass (13) Pleural effusion (4) Pericardial effusion (1) Distant metastasis (7) Bone (3) Liver (1) Periesophageal mass (1) Thigh mass (1) Axillary mass (1)
(Fig. 1). Table 2 demonstrates 7 of these 11 cases were positive for EML4-ALK rearrangement by FISH, the gold standard. This represents 6.4% (7 of 110) of the total cases included in the study. Thus the IHC method detected all the true-positive cases with no false negatives but 4 false positives (IHC-positive, FISH-negative cases). Therefore, the sensitivity and specificity of the EML4-ALK IHC stain were 100% and 96%, respectively, whereas positive and negative predictive values were 64% and 100%, respectively (Table 3). Of 11 IHC-positive cases, 4 were FISH negative, representing possible false positives. RT-PCR for EML4ALK variant 1 or 3 translocations and KIF5B-ALK rearrangements was performed on 9 of the 11 IHC-positive cases (2 true positives did not have available/sufficient tissue for testing). Results of the RT-PCR revealed that one of the FISH-negative cases (a cytology specimen) harbored an EML4-ALK translocation not detected by FISH. The intensity of the staining in positive EML4-ALK FISH cases ranged from 1 to 3, with a mean of 2. Percentage of positive-staining tumor cells ranged from 60% to 100%, with a mean of 76% (Table 2). The intensity and percentage of positive-staining cells did not seem to differ between true-positive and false-positive cases as there was 1 true-positive case with low-intensity staining and 3 false-positive cases with high-intensity staining and high percent positive-staining tumor cells (90%). In terms of agreement between ALK FISH and ALK IHC, the percent agreement between positive cases was 66.7%, whereas the percent agreement between the negative cases was 100%. The kagreement was 0.76, which correlates with substantial or excellent agreement.7,8 The cost of performing the current protocol, in which all newly diagnosed, EGFR-negative NSCLC are Copyright
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Diagnosis (n) Adenocarcinoma (39) Squamous cell carcinoma (4) Poorly differentiated carcinoma (3) Adenosquamous carcinoma (1)
tested for the ALK rearrangement by FISH, for the 110 cases included is $25,826.90. The cost of performing ALK IHC on all 110 cases would be $12,826.00. The cost of performing FISH on the 11 positive IHC would be $2582.69. Thus, using the ALK IHC as a screening tool, followed by ALK FISH on the 11 ALK IHC-positive cases, would cost $15,408.69. For the 110 cases included in the study, the potential savings of implementing the ALK IHC as a screening method would be $10,418.21.
DISCUSSION We found that the EML4-ALK IHC stain used in our study had a sensitivity of 100% with a specificity of 96%. These findings are similar to other studies. Conklin and colleagues report that 2 different antibodies directed against ALK antigens, D5F3 (Cell Signaling by ADVANCE) and 5A4 (Novocastra by ADVANCE), both demonstrated a sensitivity of 100% and specificity of 75% for D5F3 and 87.5% for 5A4. As in our study, neither antibody resulted in false negatives. Thus, they concluded that IHC is a dependable screening method for detection of the EML4-ALK rearrangement in NSCLC.9 Savic et al10 also demonstrated the 5A4 clone to have high sensitivity (93.3%) and specificity (96%) in the detection of the ALK rearrangement. Most recently, Selinger et al11 compared 3 different antibodies (ALK1, 5A4, and D5F3) and showed that all 3 stains had 100% sensitivity, but slightly variable specificities given different rates of false positives. Interestingly, the 5A4 clone is designed to target an epitope that corresponds to a region, which spans the tyrosine kinase catalytic domain and part of the C-terminus of NPM-ALK transcript.12,13 However, our study and the aforementioned www.appliedimmunohist.com |
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FIGURE 1. A, Positive ALK IHC: representative image from specimen with 60% of tumor cells staining at 3+ intensity; magnification, 4. B, Positive ALK IHC: representative image from specimen with 90% of tumor cells staining at 2+ intensity; magnification, 4. C, Positive ALK IHC: representative image from specimen with 70% of tumor cells staining at 1+ intensity; magnification, 4. D, Negative ALK IHC: representative image from specimen with 0% of tumor cells staining; magnification, 4. E, ALK FISH: White arrows indicate cells with a single yellow signal, representing intact ALK genes. Yellow arrows indicate cells with a red and green signal, representing an ALK rearrangement.
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TABLE 2. ALK-positive Cases by FISH and IHC No. Cases 1 2 3 4 5 6 7 8 9 10 11
Fish Result
IHC Intensity
IHC Percentage
IHC Result
Specimen Type
Positive Positive Positive Positive Positive Positive Positive Negative Negative Negative Negative
2 3 2 2 1 2 2 3 2 2 2
80 60 95 80 70 100 50 50 90 90 10
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
Right lower lobe FNA Pleural fluid FNA Pleural biopsy Pleural biopsy Transbronchial biopsy Femur resection Cervical bone resection Scapula FNA Pneumonectomy Wedge resection Brain biopsy
ALK indicates anaplastic lymphoma kinase; FISH, fluorescence in situ hybridization; FNA, fine needle aspiration; IHC, immunohistochemistry.
studies seem to demonstrate its cross-reactivity with EML4ALK. Thus, the high sensitivity and specificity of the 5A4 antibody for ALK translocations in our cohort of NSCLC is consistent with the current literature. McLeer-Florin and colleagues showed ALK IHC to have a sensitivity of 95% and a specificity of 100%, respectively. Even without 100% sensitivity, they suggest that ALK IHC could be used as a prescreening tool before FISH analysis for the identification of the ALK rearrangement.14 A Korean study found that ALK IHC had a similar sensitivity of 100% and a specificity of 98.7%. However, unlike our study, they found a correlation between staining intensity and ALK rearrangement status with all tissues with an IHC score of 3, 84.6% of tissues with an IHC score of 2, 83.3% of tissues with an IHC score of 1, and no tissues with an IHC score of 0 being ALK FISH positive.15 Of note, RT-PCR analysis has been developed for the detection of ALK rearrangements. One of the benefits of the RT-PCR method is the detection of novel ALK rearrangement variants beyond previously identified variants 1, 2, and 3 such as KIF5B-ALK.4,16 One study did a comparison of RT-PCR, IHC, and FISH for EML4-ALK rearrangement in the identification of the ALK rearrangements. Their results showed RT-PCR to be the most sensitive and least subjective method in the evaluation of ALK rearrangement status.17 Consistent with this increase in sensitivity, RT-PCR detected 1 additional EML4-ALK rearrangement not detected by our standard FISH assay. This is likely due to the small amount of neoplastic cells present in the cytology specimen. Thus, if the IHC-positive, RT-PCR-positive, FISH-negative case is taken to represent a true positive, then IHC only detected 3 false positives. However, it is important to bear in mind that FISH and IHC allow the actual stained tissue to be visualized, which is not the case with RT-PCR. In
addition, current molecular guidelines do not recommend that RT-PCR be used in place of FISH when determining eligibility for ALK therapy.18 Although the aforementioned studies have looked into the efficacy of ALK IHC as a screening tool to ALK FISH, they have not analyzed the potential savings in terms of technical laboratory and professional costs. For the cases included in the study at EUH, we found that implementing IHC as a screening method, which would include performing ALK IHC on all newly diagnosed EGFR-negative NSCLC and ALK FISH on only the IHC-positive cases, could save $10,418.21. This total does not reflect the possible gross savings, as the 110 cases only represent 55% of the total 201 NSCLC cases that had FISH performed in the specified study period. Although the screening method would still incur cost from FISH performed on false-positive cases, the number of FISH tests performed would drastically decrease. The calculations performed only account for the financial savings, as there are potential time management benefits. If EML4-ALK IHC staining were used as the screening method, the laboratory technologist would have only had to perform 11 ALK FISH analyses. This would mean 99 less FISH analyses would have been performed. Furthermore, the process of performing IHC can be automated, and is therefore more efficient than performing FISH analyses on all specimens. Our study demonstrated that there can be substantial financial savings with our proposed methodology. We recognize that the small sample size available for this study limits the broad application of our study’s sensitivity and specificity results. Ideally, we would have liked to have had more ALK-positive cases to determine the sensitivity and specificity of ALK IHC. Nonetheless, the frequency of positive NSCLC in cases included was 6.4%
TABLE 3. Comparison of ALK IHC to ALK FISH as Gold Standard IHC positive IHC negative
FISH Positive
FISH Negative
7 0 Sensitivity 100%
4 98 Specificity 96%
Positive predictive value 64% Negative predictive value 100%
ALK indicates anaplastic lymphoma kinase; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry.
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(7/110). However, when taking into account the 201 total NSCLC with ALK FISH results, the frequency would be 3.4%. Nonetheless, both 6.4% and 3.4% fall within the incidence range of 1% to 13% reported by previous studies.4,19–28 With such a low frequency of ALK gene rearrangements, a screening test would be greatly beneficial, especially for its use in small specimens, and both surgical and cytologic specimens. Even though the specificity of the EML4-ALK IHC stain is high (96.3%), it is not sufficiently specific to supplant FISH analysis in the detection of the EML4-ALK rearrangement and would overestimate the presence of the translocation. In addition, FDA approval of Xalkori (crizotinib) for treatment of NSCLC is linked to Abbott Molecular FISH. As such, drug administration and hospital reimbursement require a positive FISH test with Abbott’s FISH probe. Presently, failure to use Abbot’s probe may hinder a patient from receiving adequate treatment and prevent proper monetary compensation by insurance companies. Furthermore, the subjectivity, interobserver variability, and visual quantitation of IHC can arguably be just as problematic when compared with FISH. However, with high sensitivity, demonstrated in the current and previous studies, ALK IHC shows promise as a screening tool to significantly decrease the number of FISH analyses needed. Indeed, some have already proposed diagnostic algorithms for using IHC to screen for ALK rearrangements.28 As mentioned previously, another advantage of using IHC is that it is particularly suitable to be used on small samples, including biopsies and cytologic specimens that may contain an abundance of benign-contaminating cells. In fact, recent guidelines from the College of American Pathologists, International Association of the Study of Lung Cancer, and Association for Molecular Pathology support the use of IHC screening as long as it has been appropriately validated.18 Consequently, screening with ALK IHC would also be financially beneficial with tens of thousands of dollars in potential health care savings. REFERENCES 1. Vasikova A. EML4-ALK fusion gene in patients with lung carcinoma: biology, diagnostics and targeted therapy. Klin Onkol. 2012;25:434–439. 2. Rodig SJ, Shapiro GI. Crizotinib, a small-molecule dual inhibitor of the c-Met and ALK receptor tyrosine kinases. Curr Opin Investig Drugs. 2010;11:1477–1490. 3. Lee HY, Ahn HK, Jeong JY, et al. Favorable clinical outcomes of pemetrexed treatment in anaplastic lymphoma kinase positive nonsmall-cell lung cancer. Lung Cancer. 2013;79:40–45. 4. Takeuchi K, Choi YL, Soda M, et al. Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res. 2008;14:6618–6624. 5. Soda M, Isobe K, Inoue A, et al. A prospective PCR-based screening for the EML4-ALK oncogene in non-small cell lung cancer. Clin Cancer Res. 2012;18:5682–5689. 6. Carletta J. Assessing agreement on classification tasks: the kappa statistic. Comput Linguist. 1996;22:249–254. 7. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159–174. 8. Fleiss JL. Statistical Methods for Rates and Proportions. 2nd ed. New York: John Wiley; 1981.
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9. Conklin CM, Craddock KJ, Have C, et al. Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent. J Thorac Oncol. 2013;8:45–51. 10. Savic S, Bode B, Diebold J, et al. Detection of ALK-positive nonsmall-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone. J Thorac Oncol. 2013;8: 1004–1011. 11. Selinger CI, Rogers TM, Russell PA, et al. Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol. 2013;26:1545–1553. 12. “Novocastra ALK.” Available at: http://www.leicabiosystems.com/ ihc-ish/novocastra-reagents/primary-antibodies/details/product/alk-1/. Accessed September 25, 2013. 13. “Anti-ALK antibody [5A4] (ab17127).” Antibodies, proteins, kits and reagents for life science. Available at: http://www.abcam.com/ alk-antibody-5a4-ab17127.html. Accessed September 25, 2013. 14. McLeer-Florin A, Moro-Sibilot D, Melis A, et al. Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study. J Thorac Oncol. 2012;7:348–354. 15. Park HS, Lee JK, Kim DW, et al. Immunohistochemical screening for anaplastic lymphoma kinase (ALK) rearrangement in advanced non-small cell lung cancer patients. Lung Cancer. 2012;77:288–292. 16. Sanders HR, Li HR, Bruey JM, et al. Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer. Cancer Genet. 2011;204:45–52. 17. Wallander ML, Geiersbach KB, Tripp SR, et al. Comparison of reverse transcription-polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization methodologies for detection of echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase fusion-positive non-small cell lung carcinoma: implications for optimal clinical testing. Arch Pathol Lab Med. 2012;136:796–803. 18. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Mol Diagn. 2013;15:415–453. 19. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448:561–566. 20. Koivunen JP, Mermel C, Zejnullahu K, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res. 2008;14:4275–4283. 21. Wong DW, Leung EL, So KK, et al. The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS. Cancer. 2009;115:1723–1733. 22. Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol. 2009;174:661–670. 23. Boland JM, Erdogan S, Vasmatzis G, et al. Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non-small cell lung carcinomas. Hum Pathol. 2009;40:1152–1158. 24. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol. 2009;27:4247–4253. 25. Perner S, Wagner PL, Demichelis F, et al. EML4-ALK fusion lung cancer: a rare acquired event. Neoplasia. 2008;10:298–302. 26. Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol. 2008;3:13–17. 27. Shinmura K, Kageyama S, Tao H, et al. EML4-ALK fusion transcripts, but no NPM-, TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas. Lung Cancer. 2008;61:163–169. 28. Takamochi K, Takeuchi K, Hayashi T, et al. A rational diagnostic algorithm for the identification of ALK rearrangement in lung cancer: a comprehensive study of surgically treated Japanese patients. PLoS One. 2013;8:e69794.
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