Practice Makes Perfect Protocols: The Canadian Anaplastic Lymphoma Kinase Study
Authors: Akihiko Yoshida
Journal: Journal of Thoracic Oncology (2014)

Rearrangement of the anaplastic lymphoma kinase (ALK) gene is oncogenic in approximately 5% of lung adenocarcinomas.1Soda M Choi YL Enomoto M et al.Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4321) Google Scholar Identifying patients with this tumor subtype is important because they can significantly benefit from small-molecule ALK inhibitor therapy.2Kwak EL Bang YJ Camidge DR et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3827) Google Scholar Fluorescence in situ hybridization (FISH) is considered the standard test for this purpose and was the modality used in clinical trials to determine eligibility.2Kwak EL Bang YJ Camidge DR et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3827) Google Scholar At present, the Vysis ALK Break Apart FISH probe (Abbott Molecular, Abbott Park, IL) is the only companion diagnostic kit approved by the US Food and Drug Administration. However, other methods are also available, with ALK immunohistochemistry (IHC) considered particularly promising for clinical use, as it can detect ALK protein overexpressed as a result of gene fusion in a rapid and cost-effective manner. Nonetheless, there are challenges in using FISH and IHC for diagnosing lung cancers with ALK rearrangement. For the break-apart FISH assay, the primary difficulty is interpretational, such as distinguishing between narrow split signals in ALK-rearranged tumors arising from the proximity of ALK and its frequent partner echinoderm microtubule-associated protein like 4 (EML4), occasional split signals in ALK wild-type tumors, and admixtures of non-neoplastic elements that may dilute the signal from abnormal cells in a dark field. This can be mitigated to some extent by strict adherence to enumeration rules. In contrast, major difficulties associated with ALK IHC are technical in nature. Well-established, conventional methods using the ALK1 antibody clone have unacceptably low levels of sensitivity ranging from 9% to 67%,3Mino-Kenudson M Chirieac LR Law K et al.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,4Takeuchi K Choi YL Togashi Y et al.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 whereas excessive signal enhancement and increased antibody concentration result in reduced specificity and high background signal.3Mino-Kenudson M Chirieac LR Law K et al.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,4Takeuchi K Choi YL Togashi Y et al.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 To overcome these limitations, improved staining protocols have been developed by several groups; these involved the use of alternative primary antibodies (e.g., 5A4 and D5F3) and/or specific signal amplification steps.3Mino-Kenudson M Chirieac LR Law K et al.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, 4Takeuchi K Choi YL Togashi Y et al.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, 5Houang M Toon CW Clarkson A et al.Reflex ALK immunohistochemistry is feasible and highly specific for ALK gene rearrangements in lung cancer.Pathology. 2014; 46: 383-388Crossref PubMed Scopus (33) Google Scholar, 6To KF Tong JH Yeung KS et al.Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant.J Thorac Oncol. 2013; 8: 883-891Crossref PubMed Scopus (62) Google Scholar, 7Minca EC Portier BP Wang Z et al.ALK status testing in non-small cell lung carcinoma: correlation between ultrasensitive IHC and FISH.J Mol Diagn. 2013; 15: 341-346Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 8Yoshida A Tsuta K Nitta H et al.Bright-field dual-color chromogenic in situ hybridization for diagnosing echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase-positive lung adenocarcinomas.J Thorac Oncol. 2011; 6: 1677-1686Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar With ALK testing for lung cancers becoming the standard of care worldwide, there is growing interest in extending its benefits to different regions and communities. Test standardization and quality control issues have emerged at the center of this discussion. In this issue of Journal of Thoracic Oncology, Cutz et al.9Cutz J-C Craddock KJ Torlakovic E et al.Canadian Anaplastic Lymphoma Kinase (CALK) study: a model for multi-centre standardization and optimization of ALK testing in lung cancer.J Thorac Oncol. 2014; 9: 1255-1263Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar describe a promising model for quality assurance in ALK testing through the formation of a pathology network in the Canadian ALK (CALK) study. This structurally complex study had three phases. In phases 1 and 2, control tissue microarray slides with multiple cases of known ALK status were compiled and distributed to more than 10 institutions for FISH and locally developed IHC analyses. The results were analyzed, and feedback was provided so as to assist each center in optimizing its own IHC protocol. In phase 3, 373 cases of lung cancer were evaluated concurrently by postoptimized IHC and FISH at three centers; a perfect concordance was observed in the results obtained by the two methods if equivocal IHC staining was considered negative. This study is distinguished from other multi-institutional standardization efforts in ALK testing (summarized by Yatabe et al.10Yatabe Y Lantuéjoul S Thunnissen E et al.Tsao MS Hirsch FR Yatabe Y Guidelines and standardization studies. In: IASLC Atlas of ALK Testing in Lung Cancer. IASLC Press, Aurora CO2013: 61-66Google Scholar) by the inclusion of a protocol adjustment phase. Beyond publishing the results of initial round-robin tests, the CALK authors took active measures to improve staining methods at each institution. As a result, high background staining largely disappeared, and the interclass correlation coefficient for the 5A4 clone increased from 0.867 to 0.949. Parenthetically, such improvement alone provides evidence for the limitations of developing local methods based on publications. This type of dynamism is unprecedented; in earlier studies that compared ALK FISH and IHC, the staining protocol had already been established in-house, and it was unclear how they were previously optimized and whether they could be further improved. Another advantage of this study was that it used multiple clinical control samples rather than a single case or cell line, which allowed for fine adjustment of staining conditions. The CALK strategy is also notable for its technical flexibility in facilitating the optimization of existing approaches rather than enforcing a single, uniform, “authorized” method. Although there was a clear preference for the 5A4 clone over ALK1 or D5F3 and it was the only antibody that was prospectively validated, the CALK study demonstrated the feasibility of standardizing the effect without standardizing the means. Taken together, the CALK study provides an efficient and realistic model for establishing a diagnostic standard at the community level. Because IHC optimization thus obtained is arguably the most rigorous in the literature, the results should allow a prediction of what is expected if the process is completed. For example, the CALK study provides insight into an appropriate interpretative cutoff for IHC; staining results were binary and expressed as either unambiguously positive or negative in most instances, with a few samples (7.6%) having faint, focal staining or high background.9Cutz J-C Craddock KJ Torlakovic E et al.Canadian Anaplastic Lymphoma Kinase (CALK) study: a model for multi-centre standardization and optimization of ALK testing in lung cancer.J Thorac Oncol. 2014; 9: 1255-1263Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar In unequivocally positive cases, practically all tumor cells were stained and the staining intensity, if diffuse, did not make a significant difference to the interpretation. These results, in agreement with several previous studies,6To KF Tong JH Yeung KS et al.Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant.J Thorac Oncol. 2013; 8: 883-891Crossref PubMed Scopus (62) Google Scholar, 7Minca EC Portier BP Wang Z et al.ALK status testing in non-small cell lung carcinoma: correlation between ultrasensitive IHC and FISH.J Mol Diagn. 2013; 15: 341-346Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 8Yoshida A Tsuta K Nitta H et al.Bright-field dual-color chromogenic in situ hybridization for diagnosing echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase-positive lung adenocarcinomas.J Thorac Oncol. 2011; 6: 1677-1686Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar,11Ying J Guo L Qiu T et al.Diagnostic value of a novel fully automated immunochemistry assay for detection of ALK rearrangement in primary lung adenocarcinoma.Ann Oncol. 2013; 24: 2589-2593Crossref PubMed Scopus (118) Google Scholar are consistent with the notion that ALK fusion acts as an early driver for oncogenesis and do not support the use of a semiquantitative grading system of the kind currently applied to HER2 IHC for breast cancers. Another example is the achieved diagnostic accuracy of IHC, which was superior to that of FISH in the CALK study.9Cutz J-C Craddock KJ Torlakovic E et al.Canadian Anaplastic Lymphoma Kinase (CALK) study: a model for multi-centre standardization and optimization of ALK testing in lung cancer.J Thorac Oncol. 2014; 9: 1255-1263Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Specifically, one control case (CALK-11) in phase 2 with a discordant (IHC−/FISH+) result was reanalyzed by FISH and showed an atypical signal pattern that was uninterpretable by current criteria; reverse transcription quantitative polymerase chain reaction and RNA sequencing failed to detect any ALK fusions that confirmed the IHC result. Similarly, in all three cases that initially showed discordant results in phase 3, the original finding was contradicted after repeating the FISH testing. The initial discordance was not due to imperfect IHC performance; the study highlighted the importance of critically reanalyzing FISH data in such instances. Although FISH is a powerful diagnostic tool that allows the direct visualization of genomic changes, it is not without flaws. A small but increasing number of reports have documented falsely positive or negative errors in ALK FISH assays that were either biological or interpretative. Biologically based errors occur when FISH yields atypical signals (as in CALK-119Cutz J-C Craddock KJ Torlakovic E et al.Canadian Anaplastic Lymphoma Kinase (CALK) study: a model for multi-centre standardization and optimization of ALK testing in lung cancer.J Thorac Oncol. 2014; 9: 1255-1263Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) that defy interpretation5Houang M Toon CW Clarkson A et al.Reflex ALK immunohistochemistry is feasible and highly specific for ALK gene rearrangements in lung cancer.Pathology. 2014; 46: 383-388Crossref PubMed Scopus (33) Google Scholar,8Yoshida A Tsuta K Nitta H et al.Bright-field dual-color chromogenic in situ hybridization for diagnosing echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase-positive lung adenocarcinomas.J Thorac Oncol. 2011; 6: 1677-1686Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar,12Peled N Palmer G Hirsch FR et al.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-e16Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar,13Ren S Hirsch FR Varella-Garcia M et al.Atypical negative ALK break-apart FISH harboring a crizotinib-responsive ALK rearrangement in non-small-cell lung cancer.J Thorac Oncol. 2014; 9: e21-e23Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar or typical but incorrect patterns,5Houang M Toon CW Clarkson A et al.Reflex ALK immunohistochemistry is feasible and highly specific for ALK gene rearrangements in lung cancer.Pathology. 2014; 46: 383-388Crossref PubMed Scopus (33) Google Scholar,6To KF Tong JH Yeung KS et al.Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant.J Thorac Oncol. 2013; 8: 883-891Crossref PubMed Scopus (62) Google Scholar,11Ying J Guo L Qiu T et al.Diagnostic value of a novel fully automated immunochemistry assay for detection of ALK rearrangement in primary lung adenocarcinoma.Ann Oncol. 2013; 24: 2589-2593Crossref PubMed Scopus (118) Google Scholar,14Selinger 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: 1545-1553Crossref PubMed Scopus (131) Google Scholar,15Murakami Y Mitsudomi T Yatabe Y A Screening Method for the ALK Fusion Gene in NSCLC.Front Oncol. 2012; 2: 24Crossref PubMed Scopus (77) Google Scholar and they are likely attributable to complex and/or cryptic genomic alterations. The other, perhaps more common, cause of inaccuracy is signal misinterpretation. Although the substantial variability in FISH scoring (interclass correlation coefficient = 0.68) in the CALK study was diminished when scores were translated to the final dichotomous reading in relation to the 15% cutoff value, there were still 14 misinterpretations (11 false negatives and three false positives) of 317 readings (4.4%). Given that this assay was performed by experienced technicians who had already undergone specific hands-on training by the assay manufacturer,9Cutz J-C Craddock KJ Torlakovic E et al.Canadian Anaplastic Lymphoma Kinase (CALK) study: a model for multi-centre standardization and optimization of ALK testing in lung cancer.J Thorac Oncol. 2014; 9: 1255-1263Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar it remains to be determined whether there is room for future improvements in FISH by proficiency testing. Similar errors have also been sporadically reported by others.3Mino-Kenudson M Chirieac LR Law K et al.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,7Minca EC Portier BP Wang Z et al.ALK status testing in non-small cell lung carcinoma: correlation between ultrasensitive IHC and FISH.J Mol Diagn. 2013; 15: 341-346Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar,16Sholl LM Weremowicz S Gray SW et al.Combined use of ALK immunohistochemistry and FISH for optimal detection of ALK-rearranged lung adenocarcinomas.J Thorac Oncol. 2013; 8: 322-328Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar How will the optimized ALK IHC change the landscape of ALK testing in thoracic oncology? At a minimum, it can complement results obtained by FISH. Having reliable IHC as a backup modality is particularly useful when FISH fails (9.2% of cases in phase 3 of the CALK study9Cutz J-C Craddock KJ Torlakovic E et al.Canadian Anaplastic Lymphoma Kinase (CALK) study: a model for multi-centre standardization and optimization of ALK testing in lung cancer.J Thorac Oncol. 2014; 9: 1255-1263Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar but up to 26%7Minca EC Portier BP Wang Z et al.ALK status testing in non-small cell lung carcinoma: correlation between ultrasensitive IHC and FISH.J Mol Diagn. 2013; 15: 341-346Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar of cases in the literature), shows atypical signal patterns (the incidence of which is undetermined), or when scores fall in a borderline category (e.g., the abnormal cell range of 10–20% that was observed in 5.4% of cases in phase 3 of the CALK study). Furthermore, as the CALK study authors proposed, IHC can serve as an effective screening tool, with FISH used in cases where IHC results are unambiguously positive or equivocal; this strategy is already in place in Canada and other countries. Finally, optimized IHC may assume the diagnostic role, with FISH testing entirely omitted when there is clear staining and used only as an adjunct when IHC results are deemed inconclusive by pathologists. Although this practice may not be globally adopted until clinical trials in which cohorts are primarily defined by optimized IHC have proven to be successful, the 100% accuracy that was demonstrated for IHC by the CALK study encourages a paradigm shift in that direction. This is desirable in our opinion, in an era where a growing need for ALK testing warrants a method that is not restricted by high cost, intense labor, long turn-around time, occasional failure, and limited access. Multi-institutional initiatives for staining quality standardization, as exemplified by the CALK study, should accelerate this change. Additional requirements to that end include accreditation of laboratories that have completed staining optimization and a system that ensures periodic quality control. Wider publication is also needed to enhance awareness of the few inherent pitfalls of ALK IHC, such as the occasional detection of full-length ALK in high-grade neuroendocrine carcinomas.15Murakami Y Mitsudomi T Yatabe Y A Screening Method for the ALK Fusion Gene in NSCLC.Front Oncol. 2012; 2: 24Crossref PubMed Scopus (77) Google Scholar With all these in place, ALK IHC may finally unleash its full potential as an optimized armor in the battle against lung cancers.