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ALK Status Testing in Non–Small Cell Lung Carcinoma

2013; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês

10.1016/j.jmoldx.2013.01.004

ISSN

1943-7811

Autores

Eugen C. Minca, Bryce P. Portier, Zhen Wang, Christopher Lanigan, Carol Farver, Yan Feng, C. Patrick, Valeria A. Arrossi, Nathan A. Pennell, Raymond R. Tubbs,

Tópico(s)

Lung Cancer Research Studies

Resumo

ALK gene rearrangements in advanced non–small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHC’s routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases. ALK gene rearrangements in advanced non–small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHC’s routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases. Lung cancer is the leading cause of cancer death in men and women worldwide. Traditional therapies and improved diagnosis have not significantly increased overall survival, which is estimated at 15% at 5 years.1Siegel R. Naishadham D. Jemal A. Cancer statistics, 2012.CA Cancer J Clin. 2012; 62: 10-29Crossref PubMed Scopus (10435) Google Scholar The discovery of specific molecular alterations in certain lung tumors, as in other solid cancers, provides a great opportunity for the development of new targeted therapies urgently required to improve survival for this disease. This approach proved to be successful with the finding of EGFR gene mutations in a subset of lung tumors and the subsequent improved survival after treatment with targeted kinase inhibitors in patients whose tumors harbor EGFR-activating mutations.2Paez J.G. Janne P.A. Lee J.C. Tracy S. Greulich H. Gabriel S. Herman P. Kaye F.J. Lindeman N. Boggon T.J. Naoki K. Sasaki H. Fujii Y. Eck M.J. Sellers W.R. Johnson B.E. Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8482) Google Scholar Approximately 5% of non–small cell lung carcinomas (NSCLCs) were shown to harbor rearrangements of the ALK gene.3Horn L. Pao W. EML4-ALK: honing in on a new target in non–small-cell lung cancer.J Clin Oncol. 2009; 27: 4232-4235Crossref PubMed Scopus (272) Google Scholar These most commonly consist of a small intrachromosomal inversion, inv(2)(p21;p23), with the fusion of ALK with the EML4 gene, producing the abnormal ALK/EML4, a constitutively active chimeric protein kinase with oncogenic properties.4Rikova K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li D. Zhou X. Gygi S.P. Gu T.L. Polakiewicz R.D. Rush J. Comb M.J. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.Cell. 2007; 131: 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar, 5Soda M. Choi Y.L. Enomoto M. Takada S. Yamashita Y. Ishikawa S. Fujiwara S. Watanabe H. Kurashina K. Hatanaka H. Bando M. Ohno S. Ishikawa Y. Aburatani H. Niki T. Sohara Y. Sugiyama Y. Mano H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4322) Google Scholar Novel tyrosine kinase inhibitors targeting ALK activity have proven anticancer activity, with crizotinib showing a good clinical response in advanced NSCLC patients harboring ALK rearrangements.6Kwak E.L. Bang Y.J. Camidge D.R. Shaw A.T. Solomon B. Maki R.G. Ou S.H. Dezube B.J. Janne P.A. Costa D.B. Varella-Garcia M. Kim W.H. Lynch T.J. Fidias P. Stubbs H. Engelman J.A. Sequist L.V. Tan W. Gandhi L. Mino-Kenudson M. Wei G.C. Shreeve S.M. Ratain M.J. Settleman J. Christensen J.G. Haber D.A. Wilner K. Salgia R. Shapiro G.I. Clark J.W. Iafrate A.J. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3831) Google Scholar The presence of ALK rearrangements is a prerequisite for patient eligibility to receive targeted therapy and thus needs to be accurately and reproducibly assessed in NSCLC. Certain clinical and histologic features, such as never-smoker status, mucinous cribriform, and signet-ring adenocarcinoma, have been associated with ALK rearrangements in NSCLC tumors.6Kwak E.L. Bang Y.J. Camidge D.R. Shaw A.T. Solomon B. Maki R.G. Ou S.H. Dezube B.J. Janne P.A. Costa D.B. Varella-Garcia M. Kim W.H. Lynch T.J. Fidias P. Stubbs H. Engelman J.A. Sequist L.V. Tan W. Gandhi L. Mino-Kenudson M. Wei G.C. Shreeve S.M. Ratain M.J. Settleman J. Christensen J.G. Haber D.A. Wilner K. Salgia R. Shapiro G.I. Clark J.W. Iafrate A.J. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3831) Google Scholar, 7Shaw A.T. Yeap B.Y. Mino-Kenudson M. Digumarthy S.R. Costa D.B. Heist R.S. Solomon B. Stubbs H. Admane S. McDermott U. Settleman J. Kobayashi S. Mark E.J. Rodig S.J. Chirieac L.R. Kwak E.L. Lynch T.J. Iafrate A.J. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1641) Google Scholar, 8Rodig S.J. Mino-Kenudson M. Dacic S. Yeap B.Y. Shaw A. Barletta J.A. Stubbs H. Law K. Lindeman N. Mark E. Janne P.A. Lynch T. Johnson B.E. Iafrate A.J. Chirieac L.R. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population.Clin Cancer Res. 2009; 15: 5216-5223Crossref PubMed Scopus (608) Google Scholar These characteristics are not specific, and therefore identifying the relatively small proportion of lung cancer cases with ALK rearrangements (approximately 5%)3Horn L. Pao W. EML4-ALK: honing in on a new target in non–small-cell lung cancer.J Clin Oncol. 2009; 27: 4232-4235Crossref PubMed Scopus (272) Google Scholar relies on molecular tests. In anaplastic large cell lymphoma (ALCL), the first neoplasm in which ALK rearrangements were identified,9Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Fusion of a kinase gene. ALK, to a nucleolar protein gene NPM, in non-Hodgkin’s lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1964) Google Scholar the presence of specific translocations involving ALK is commonly tested using extensively validated and highly specific methods, including karyotyping, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC).10Cataldo K.A. Jalal S.M. Law M.E. Ansell S.M. Inwards D.J. Fine M. Arber D.A. Pulford K.A. Strickler J.G. Detection of t(2;5) in anaplastic large cell lymphoma: comparison of immunohistochemical studies, FISH, and RT-PCR in paraffin-embedded tissue.Am J Surg Pathol. 1999; 23: 1386-1392Crossref PubMed Scopus (103) Google Scholar The ALK rearrangements in NSCLC are structurally different from those in ALCL. The detection of small intrachromosomal deletions or inversions involving ALK locus in NSCLC is not possible by classic karyotyping. With the approval by the US Food and Drug Administration of an in vitro diagnostic class FISH test as a companion diagnostic tool for crizotinib-based treatment eligibility (Vysis ALK Break Apart FISH Probe Kit; Abbott Molecular Inc., Des Plaines, IL), FISH is considered the current criterion standard test, and it can be performed on formalin-fixed, paraffin-embedded (FFPE) material. The commercial break-apart format contains red and green probes that flank the highly conserved translocation breakpoint within ALK, resulting in yellow fusion signals in normal cells and split red and green signals in cells harboring ALK rearrangements. Interpreting a case as positive by FISH requires that ≥15% tumor cell nuclei demonstrate isolated green and red or isolated red signals among 50 tumor nuclei scored.7Shaw A.T. Yeap B.Y. Mino-Kenudson M. Digumarthy S.R. Costa D.B. Heist R.S. Solomon B. Stubbs H. Admane S. McDermott U. Settleman J. Kobayashi S. Mark E.J. Rodig S.J. Chirieac L.R. Kwak E.L. Lynch T.J. Iafrate A.J. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1641) Google Scholar The interpretation is often subtle and challenging. Because of the probe design, distinguishing true broken-apart signal pairs from the inherently split signals can be difficult. Furthermore, the evaluation of tissue architecture and cell morphology for unambiguously distinguishing between normal and tumor cells is very limited with DAPI nuclear fluorescence. Lastly, FISH is a resource-intensive, specialized, and costly method. For these reasons, alternative, widely available, and cost-efficient screening tests for ALK status have been investigated. The utility of conventional IHC, a more affordable and accessible method, has been challenged by low expression levels of the protein encoded by ALK fusion transcripts in NSCLC. Initial studies with the ALK1 antibody clone (Dako, Carpinteria, CA) used in ALCL showed relatively modest sensitivities for IHC on NSCLC samples, which were only partially increased by secondary signal amplification protocols.8Rodig S.J. Mino-Kenudson M. Dacic S. Yeap B.Y. Shaw A. Barletta J.A. Stubbs H. Law K. Lindeman N. Mark E. Janne P.A. Lynch T. Johnson B.E. Iafrate A.J. Chirieac L.R. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population.Clin Cancer Res. 2009; 15: 5216-5223Crossref PubMed Scopus (608) Google Scholar, 11Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar More recent studies using novel engineered antibodies or signal amplification methods and simplified scoring systems have shown promising results in detecting ALK fusion product expression in NSCLC, with IHC sensitivities and specificities approaching those of FISH.11Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar, 12Takeuchi K. Choi Y.L. Togashi Y. Soda M. Hatano S. Inamura K. Takada S. Ueno T. Yamashita Y. Satoh Y. Okumura S. Nakagawa K. Ishikawa Y. Mano H. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer.Clin Cancer Res. 2009; 15: 3143-3149Crossref PubMed Scopus (630) Google Scholar We assessed a modified automated IHC method using the highly sensitive D5F3 rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA) coupled with an advanced multimer-based signal amplification and detection system (OptiView DAB IHC Detection Kit; Ventana Medical Systems) as an alternative to FISH for detecting ALK status in a NSCLC case series at our institution. We found that the modified IHC method can reliably detect ALK-encoded protein expression that results from ALK gene rearrangements in NSCLC and has a very high concordance with FISH, warranting its routine use as the initial component of an algorithmic approach to clinical ALK molecular testing in NSCLC. The study included samples from 296 patients with advanced NSCLC who were clinically referred for ALK testing at our institution (Cleveland Clinic Foundation) between July 2010 and August 2012. Specimens consisted of 318 FFPE tissue biopsy specimens, resection specimens, or cytopathology cell blocks. In 40 cases, available matched ThinPrep preparations from bronchial wash, pleural, or pericardial fluid samples were also used for FISH, which was performed according to a previously established method (Urovysion; Abbott Molecular Inc.).13Sokolova I.A. Halling K.C. Jenkins R.B. Burkhardt H.M. Meyer R.G. Seelig S.A. King W. The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine.J Mol Diagn. 2000; 2: 116-123Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 14Skacel M. Pettay J.D. Tsiftsakis E.K. Procop G.W. Biscotti C.V. Tubbs R.R. Validation of a multicolor interphase fluorescence in situ hybridization assay for detection of transitional cell carcinoma on fresh and archival thin-layer, liquid-based cytology slides.Anal Quant Cytol Histol. 2001; 23: 381-387PubMed Google Scholar Automated IHC for ALK expression was performed for all cases in a Bechmark XT staining module (Ventana Medical Systems, Tucson, AZ) on 5-μmol/L thick FFPE sections with the D5F3 rabbit anti-human CD246 monoclonal antibody (a gift from Cell Signaling Technologies). Briefly, after deparaffinization, heat-mediated antigen retrieval, and endogenous peroxidase inactivation, the primary antibody was applied at a 1:100 dilution in manufacturer antibody diluent for all samples. Detection was performed with the OptiView DAB IHC Detection Kit with signal amplification (a gift from Ventana Medical Systems), following manufacturer instructions. The ultrasensitive 2-step detection system uses a multihapten secondary antibody coupled with horseradish peroxidase multimer binding for specific signal amplification. As positive controls, we used FFPE samples from four cases of ALCL with previously documented ALK rearrangements by FISH. Negative controls consisted of the omission of the primary antibody and incubation with immunoglobulins of the same species. To increase reproducibility, IHC staining results were interpreted as either negative (no staining present) or positive (staining present) rather than scored on a semiquantitative scale.9Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Fusion of a kinase gene. ALK, to a nucleolar protein gene NPM, in non-Hodgkin’s lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1964) Google Scholar, 15Yi E.S. Boland J.M. Maleszewski J.J. Roden A.C. Oliveira A.M. Aubry M.C. Erickson-Johnson M.R. Caron B.L. Li Y. Tang H. Stoddard S. Wampfler J. Kulig K. Yang P. Correlation of IHC and FISH for ALK gene rearrangement in non-small cell lung carcinoma: iHC score algorithm for FISH.J Thorac Oncol. 2011; 6: 459-465Crossref PubMed Scopus (249) Google Scholar, 16Paik J.H. Choe G. Kim H. Choe J.Y. Lee H.J. Lee C.T. Lee J.S. Jheon S. Chung J.H. Screening of anaplastic lymphoma kinase rearrangement by immunohistochemistry in non-small cell lung cancer: correlation with fluorescence in situ hybridization.J Thorac Oncol. 2011; 6: 466-472Crossref PubMed Scopus (255) Google Scholar FISH for ALK rearrangements was performed using the Abbott Molecular Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular Inc.) following the manufacturer instructions. FISH was performed on FFPE samples in all 318 cases and on matched available ThinPrep material in 40 cases. For FFPE-FISH the previously recommended cutoff of 15% positive cells was used to interpret samples as positive or negative for ALK rearrangements,7Shaw A.T. Yeap B.Y. Mino-Kenudson M. Digumarthy S.R. Costa D.B. Heist R.S. Solomon B. Stubbs H. Admane S. McDermott U. Settleman J. Kobayashi S. Mark E.J. Rodig S.J. Chirieac L.R. Kwak E.L. Lynch T.J. Iafrate A.J. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1641) Google Scholar without prior knowledge of the IHC result. The same cutoff was also used for ThinPrep-FISH. Calculation of the 95% CIs for clinical parameters (sensitivity, specificity, positive predictive value, and negative predictive value) and χ2 analysis used to compare the proportions of uninformative samples among FFPE-FISH, ThinPrep-FISH, and IHC were performed using GraphPad Prism version 5 (GraphPad Software Inc., La Jolla, CA). Our study included 296 patients with advanced NSCLC clinically referred for ALK testing in the Cleveland Clinic Health System. A total of 318 FFPE samples were used for ALK-status testing by FISH and IHC (Table 1). FISH was informative for 235 of 318 FFPE samples (73.9%). Uninformative results on the remaining 83 of 318 samples (26.1%) were due to insufficient number of tumor cells for FISH enumeration.Table 1Breakdown of Available and Informative NSCLC Samples by Type and TestSample typeTotal no. availableNo. informative by IHCNo. informative by FISHNo. informative by FISH and IHCFFPE318304235231ThinPrep40N/A39 (21 unique, 18 complementary)34Combined318304256249 Open table in a new tab To increase the number of informative cases, we performed FISH on matched ThinPrep material, which was available for 40 of 318 FFPE samples. Of these, one sample was unsatisfactory, 18 provided ThinPrep-FISH results complementary to FFPE-FISH, and 21 were solely informative on ThinPrep-FISH, adding to a total of 256 of 318 FISH informative cases (80.5%) (Table 1). In an analysis of the 18 cases with available dual FISH data (Table 2), ThinPrep-FISH had a mean of 51% (median, 50%; range, 16% to 88%) positive cells on four FFPE-FISH–positive NSCLC cases and a mean of 4.5% (median, 4%; range, 0% to 12%; SD, 4%) on 12 FFPE-FISH–negative cases. In the remaining two cases (pleural fluid and bronchial fine-needle aspirate), the ThinPrep-FISH result was negative (2% and 10% positive cells), whereas the FFPE-FISH result was borderline positive (18% and 15%). The result discrepancy and the near-cutoff percentage of positive cells on FFPE-FISH prompted further investigation of these cases. The first patient had no clinical response to crizotinib on follow-up, whereas the lung carcinoma of the second patient harbored a confirmed EGFR mutation, which is virtually exclusive of an ALK rearrangement.17Camidge D.R. Kono S.A. Flacco A. Tan A.C. Doebele R.C. Zhou Q. Crino L. Franklin W.A. Varella-Garcia M. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment.Clin Cancer Res. 2010; 16: 5581-5590Crossref PubMed Scopus (300) Google Scholar On the basis of this information, we reclassified these cases as negative for ALK rearrangements (false-positive FFPE-FISH result).Table 2Correlation Between ThinPrep-FISH and FFPE-FISH on 18 Available Dual-Informative NSCLC SamplesThinPrep-FISHFFPE-FISHNo. positiveNo. negativeNo. positive40No. negative212Total no.18Overall agreement was 88.8%. Open table in a new tab Overall agreement was 88.8%. D5F3-IHC was informative for 304 of 318 samples (95.5%) (Table 1). All samples with positive IHC results demonstrated moderate or strong staining in at least 75% of tumor cells (Figure 1, A and B), similar to the staining obtained in ALCL-positive controls (Supplemental Figure S1). IHC staining interpretation was uninformative because of absent or insufficient tumor cells in the remaining 14 of 318 samples (4.4%), a significantly lower proportion than that of uninformative FFPE-FISH (26.1%, P < 0.001). When comparing IHC strictly with FFPE-FISH, ALK status analysis was concurrently informative on 231 samples. Of 31 samples positive by IHC, all were also positive by FFPE-FISH (Figure 1C). Of 200 samples negative by IHC, 198 were also negative by FFPE-FISH. The resulting IHC sensitivity and specificity in relation to FFPE-FISH were 94% (95% CI, 0.78 to 0.99) and 100% (95% CI, 0.97 to 1.00), respectively (Table 3). The two samples with discrepantly negative IHC and positive FFPE-FISH were those reclassified as negative on ThinPrep-FISH (false-positive FFPE-FISH result). IHC was 100% concordant with Thin-Prep FISH on 34 cases with dual-informative results: 5 positive and 29 negative (Figure 1D and Table 4). When comparing IHC with FISH overall (both FFPE and ThinPrep) and revising the two FFPE-FISH false-positive results, there was 100% concordance between the two methods on 249 samples with dual-informative analysis: 32 positive and 217 negative. Thus, IHC had 100% sensitivity (95% CI, 0.86-1.00), specificity (95% CI, 0.97-1.00), positive predictive value (95% CI, 0.86-1.00), and negative predictive value (95% CI, 0.97-1.00) in detecting ALK status in NSCLC samples (Table 5).Table 3Correlation between D5F3-IHC and FFPE-FISH on 231 Available Dual-Informative NSCLC SamplesD5F3-IHCFFPE-FISHNo. positiveNo. negativeNo. positive310No. negative2198Total no.231Sensitivity, % (95% CI)94 (78−99)Specificity, % (95% CI)100 (97–100)Positive predictive value, % (95% CI)100 (86–100)Negative predictive value, % (95% CI)99 (96−99)Overall agreement was 99%. Open table in a new tab Table 4Correlation between D5F3-IHC and ThinPrep-FISH on 34 Available Dual-Informative NSCLC SamplesD5F3-IHCThinPrep-FISHNo. positiveNo. negativeNo. positive50No. negative029Total no.34Sensitivity, % (95% CI)100 (46–100)Specificity, % (95% CI)100 (85–100)Positive predictive value, % (95% CI)100 (46–100)Negative predictive value, % (95% CI)100 (85–100)Overall agreement was 100%. Open table in a new tab Table 5Overall correlation between D5F3-IHC and FISH on 249 Available Dual-Informative NSCLC SamplesD5F3-IHCOverall FISHNo. positiveNo. negativeNo. positive320No. negative0217Total no.249Sensitivity, % (95% CI)100% (86–100)Specificity, % (95% CI)100% (97–100)Positive predictive value, % (95% CI)100% (86–100)Negative predictive value, % (95% CI)100% (97–100)Overall agreement was 100%. Open table in a new tab Overall agreement was 99%. Overall agreement was 100%. Overall agreement was 100%. Importantly, IHC was the sole informative analysis in a significant proportion of samples (55/318, 17.3%) and revealed additional ALK-positive cases (3/55) that were missed by FISH because of insufficient tumor cells for interpretation. Currently, the screening for ALK rearrangements in lung tumors is accomplished through FISH as a newly approved companion diagnostic tool for determining treatment eligibility with ALK inhibitor crizotinib. Although it is generally accepted that the FISH system detects any type of rearrangement involving ALK, this idea was challenged in a recent report, which described a crizotinib-sensitive ALK rearrangement undetected by FISH.18Peled N. Palmer G. Hirsch F.R. Wynes M.W. Ilouze M. Varella-Garcia M. Soussan-Gutman L. Otto G.A. Stephens P.J. Ross J.S. Cronin M.T. Lipson D. Miller V.A. Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small-cell lung cancer.J Thorac Oncol. 2012; 7: e14-e16Crossref PubMed Scopus (114) Google Scholar Additional shortcomings of FISH include the following: challenging interpretation due to proximity of probe binding, frequent uninformative results due to insufficient tumor cell number, limited and inaccurate evaluation of cell morphologic test results in part due to the use of Hemo-De rather than organic solvents for deparaffinization, and requirement of specialized and expensive resources. Real-time PCR, proposed as an alternative screening method for ALK status, is objective and quantitative but may not be able to detect all fusion transcript variants because several different ALK fusion products with EML4 and other partners have been described.4Rikova K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li D. Zhou X. Gygi S.P. Gu T.L. Polakiewicz R.D. Rush J. Comb M.J. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.Cell. 2007; 131: 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar, 5Soda M. Choi Y.L. Enomoto M. Takada S. Yamashita Y. Ishikawa S. Fujiwara S. Watanabe H. Kurashina K. Hatanaka H. Bando M. Ohno S. Ishikawa Y. Aburatani H. Niki T. Sohara Y. Sugiyama Y. Mano H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4322) Google Scholar, 19Wallander M.L. Geiersbach K.B. Tripp S.R. Layfield L.J. 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: 796-803Crossref PubMed Scopus (105) Google Scholar Initial IHC methods with traditional ALK antibodies had modest sensitivities8Rodig S.J. Mino-Kenudson M. Dacic S. Yeap B.Y. Shaw A. Barletta J.A. Stubbs H. Law K. Lindeman N. Mark E. Janne P.A. Lynch T. Johnson B.E. Iafrate A.J. Chirieac L.R. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population.Clin Cancer Res. 2009; 15: 5216-5223Crossref PubMed Scopus (608) Google Scholar, 11Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar due to low expression levels of ALK fusion products in NSCLC.20Martelli M.P. Sozzi G. Hernandez L. Pettirossi V. Navarro A. Conte D. Gasparini P. Perrone F. Modena P. Pastorino U. Carbone A. Fabbri A. Sidoni A. Nakamura S. Gambacorta M. Fernandez P.L. Ramirez J. Chan J.K. Grigioni W.F. Campo E. Pileri S.A. Falini B. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues.The Am J Pathol. 2009; 174: 661-670Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar With the development of more sensitive novel engineered antibodies, modified IHC versions were revisited as a potential alternative for FISH, with sensitivity and specificity results approaching those of the latter. From a practical standpoint, IHC is more accessible, significantly less expensive, and faster and can be informative on limited specimens that are not otherwise suitable for FISH. In addition, unlike FISH, IHC allows excellent correlation with the morphology. However, the limited number of studies available to compare the two methods and the lack of uniformity between protocols has precluded the adoption of IHC as a successful alternative to FISH for screening NSCLC tumors for ALK rearrangements. In this study, we performed IHC for ALK expression in NSCLC samples using a novel combination of a recently developed ALK antibody with an ultrasensitive multimer-based signal detection and amplification system. The D5F3 rabbit monoclonal antibody recognizes the C-terminus domain of ALK kinase that is preserved in all pathological ALK fusion products described to date,11Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar including those derived from complex rearrangements that are not otherwise detected by FISH.18Peled N. Palmer G. Hirsch F.R. Wynes M.W. Ilouze M. Varella-Garcia M. Soussan-Gutman L. Otto G.A. Stephens P.J. Ross J.S. Cronin M.T. Lipson D. Miller V.A. Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small-cell lung cancer.J Thorac Oncol. 2012; 7: e14-e16Crossref PubMed Scopus (114) Google Scholar The OptiView DAB detection system with signal amplification provides strong and clean signals without background staining. This allows a clear separation between negative and positive samples without the need of using a subjective IHC scoring system based on staining intensity or percentage of stained cells. Our case series included 32 NSCLC specimens confirmed positive for ALK rearrangements by FISH, representing one of the largest collections studied to date. The percentage of ALK-positive samples in our study (32/249, 12.8%) was at the upper end of the cited range (2% to 13%),3Horn L. Pao W. EML4-ALK: honing in on a new target in non–small-cell lung cancer.J Clin Oncol. 2009; 27: 4232-4235Crossref PubMed Scopus (272) Google Scholar likely due to two factors. First, the referral criteria for ALK testing may be more stringent at our tertiary care institution. Second, for several patients with ALK-positive tumors, we analyzed multiple unique specimens. The ultrasensitive D5F3-IHC method revealed a very high correlation with FISH in assessing ALK status. The 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) observed in our study surpass those reported for IHC in other studies using the same or different antibodies. The ALK1 antibody (Dako), commonly used for assessing abundant ALK expression in ALCL, showed variable 60% to 90% sensitivity in detecting low-level expression of ALK fusion transcripts in NSCLC in different series.8Rodig S.J. Mino-Kenudson M. Dacic S. Yeap B.Y. Shaw A. Barletta J.A. Stubbs H. Law K. Lindeman N. Mark E. Janne P.A. Lynch T. Johnson B.E. Iafrate A.J. Chirieac L.R. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population.Clin Cancer Res. 2009; 15: 5216-5223Crossref PubMed Scopus (608) Google Scholar, 11Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar, 15Yi E.S. Boland J.M. Maleszewski J.J. Roden A.C. Oliveira A.M. Aubry M.C. Erickson-Johnson M.R. Caron B.L. Li Y. Tang H. Stoddard S. Wampfler J. Kulig K. Yang P. Correlation of IHC and FISH for ALK gene rearrangement in non-small cell lung carcinoma: iHC score algorithm for FISH.J Thorac Oncol. 2011; 6: 459-465Crossref PubMed Scopus (249) Google Scholar The 5A4 antibody (Novocastra, Newcastle upon Tyne, UK) had 100% sensitivity and 95.8% specificity in one study16Paik J.H. Choe G. Kim H. Choe J.Y. Lee H.J. Lee C.T. Lee J.S. Jheon S. Chung J.H. Screening of anaplastic lymphoma kinase rearrangement by immunohistochemistry in non-small cell lung cancer: correlation with fluorescence in situ hybridization.J Thorac Oncol. 2011; 6: 466-472Crossref PubMed Scopus (255) Google Scholar and 95% sensitivity and 100% specificity in another study21McLeer-Florin A. Moro-Sibilot D. Melis A. Salameire D. Lefebvre C. Ceccaldi F. de Fraipont F. Brambilla E. Lantuejoul S. Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study.J Thorac Oncol. 2012; 7: 348-354Crossref PubMed Scopus (184) Google Scholar but has been used in conjunction with a four-tier scoring system because of occasional weak and focal staining. Such a system may complicate interobserver reproducibility and creates a less clearly defined equivocal category. A previous study with the D5F3 antibody on a smaller number of mostly tissue microarray samples described 100% sensitivity and 99% specificity.11Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar In that report, staining results were interpreted as either positive or negative, without the use of an IHC scoring system. Concordantly, the similar binary interpretation adopted in our study was facilitated by the moderate or strong staining of most tumor cells in all samples with positive D5F3-IHC. Although the high sensitivity of D5F3-IHC is substantiated in our work, additional studies may be of value to further validate and refine the use of this method on NSCLC samples. In our study, there were significantly fewer uninformative IHC results (4.4%) due to sample quality or quantity when compared with FFPE-FISH (4.4% versus 26.1%, P < 0.001). Furthermore, within 73 samples with uninformative FFPE-FISH and successful IHC, the latter test was positive in four cases (5.5%), of which one case was confirmed on available ThinPrep-FISH. These data indicate that IHC can be successfully used on samples that are limited or suboptimal for FFPE-FISH and can effectively detect a subset of positive cases that would be otherwise missed by FFPE-FISH analysis alone. Taken together, our data demonstrate that ultrasensitive automated IHC represents a reliable alternative to FISH for initial ALK screening in NSCLC. We propose the use of IHC as the first component within an algorithm for ALK rearrangement screening, which should include a second confirmatory step of positive cases by FISH. Such an algorithmic approach would have significant economic impact stemming from the much lower cost of screening IHC to replace FISH in the projected 90% to 95% ALK-negative cases. We used available ThinPrep material for FISH analysis in a subset of cases, including some with uninformative FFPE-FISH results. In comparison to FFPE tissue sections, cytologic preparations such as ThinPrep allow assessment of the entire tumor cell nucleus, thus avoiding signal loss by section artifacts or incomplete penetration of probes into the tissue and providing more accurate signal counts. ThinPrep-FISH was discrepant with FFPE-FISH in two cases, in which the latter returned positive results at or near the established threshold of 15% positive cells. This cutoff was chosen to be 2 SDs above the average number of split or isolated red signals that count as ALK rearrangement detected in FFPE nontumor control tissue.7Shaw A.T. Yeap B.Y. Mino-Kenudson M. Digumarthy S.R. Costa D.B. Heist R.S. Solomon B. Stubbs H. Admane S. McDermott U. Settleman J. Kobayashi S. Mark E.J. Rodig S.J. Chirieac L.R. Kwak E.L. Lynch T.J. Iafrate A.J. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1641) Google Scholar However, considering the large case series in our study, it is statistically expected that some of the true-negative samples (286/318) will display a percentage of cells with positive FISH signals exceeding 2 SDs (approximately 2.5%). Indeed, the clinical follow-up for the two patients with discrepant results was consistent with ALK-negative status (false-positive FFPE-FISH and true-negative ThinPrep-FISH and IHC results): one patient showed no clinical response to crizotinib on follow-up; the second patient harbored a confirmed EGFR mutation, an event rarely overlapping with ALK rearrangements,17Camidge D.R. Kono S.A. Flacco A. Tan A.C. Doebele R.C. Zhou Q. Crino L. Franklin W.A. Varella-Garcia M. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment.Clin Cancer Res. 2010; 16: 5581-5590Crossref PubMed Scopus (300) Google Scholar had a partial response to the EGFR inhibitor erlotinib, and was considered ALK negative clinically. The ALK status revision in these cases with borderline false-positive FFPE-FISH results allows for a more accurate representation of IHC sensitivity. ThinPrep-FISH was uninformative itself in only 1 of 40 samples, a significantly lower proportion than that of uninformative FFPE-FISH (2.5% versus 26.1%, P = 0.002). Although not currently routinely used for FISH analysis, ThinPrep material proved to be a valuable resource in our study for cases with limited FFPE samples. In conclusion, our results demonstrate that ultrasensitive IHC can reliably detect ALK-encoded protein expression resulting from ALK gene rearrangements in NSCLC. The high concordance between IHC and FISH warrants the routine use of IHC as the initial component of an algorithmic approach to clinical ALK molecular testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases. Download .pdf (.15 MB) Help with pdf files Supplemental Figure S1Comparative D5F3 IHC positive for ALK expression in ALCL (A) and NSCLC (B). Original magnification: ×100 (A and B).

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