Staining Performance of ALK and ROS1 Immunohistochemistry and Influence on Interpretation in Non–Small-Cell Lung Cancer

Selection of non–small-cell lung cancer patients for treatment relies on the detection of expression of anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) protein by immunohistochemistry (IHC). We evaluated staining performance for different IHC protocols and laboratory characteristics, and their influence on ALK and ROS1 interpretation during external quality assessment schemes between 2015 and 2018. Participants received five formalin-fixed, paraffin-embedded cases for staining by their routine protocol, whereafter at least two pathologists scored them simultaneously under a multihead microscope and awarded a graded expert staining score (ESS) from 1 to 5 points based on staining quality. European Conformity in Vitro Diagnostic kits (such as D5F3) revealed a better ALK ESS compared with laboratory-developed tests. ESS was indifferent to the applied antibody dilution or a recent protocol change. Lower ESSs were observed for higher antibody incubation times and temperatures. ESS for various ROS1 protocols were largely similar. Overall, for both markers, ESS improved over time and for repeated external quality assessment participation but was independent of laboratory setting or experience. Except for ROS1, ESS positively correlated with laboratory accreditation. IHC stains with lower ESS correlated with increased error rates in ALK and ROS1 interpretation and analysis failures. Laboratory characteristics differently affected staining quality and interpretation, and laboratories should assess both aspects, and less common protocols need improvement in staining performance. Selection of non–small-cell lung cancer patients for treatment relies on the detection of expression of anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) protein by immunohistochemistry (IHC). We evaluated staining performance for different IHC protocols and laboratory characteristics, and their influence on ALK and ROS1 interpretation during external quality assessment schemes between 2015 and 2018. Participants received five formalin-fixed, paraffin-embedded cases for staining by their routine protocol, whereafter at least two pathologists scored them simultaneously under a multihead microscope and awarded a graded expert staining score (ESS) from 1 to 5 points based on staining quality. European Conformity in Vitro Diagnostic kits (such as D5F3) revealed a better ALK ESS compared with laboratory-developed tests. ESS was indifferent to the applied antibody dilution or a recent protocol change. Lower ESSs were observed for higher antibody incubation times and temperatures. ESS for various ROS1 protocols were largely similar. Overall, for both markers, ESS improved over time and for repeated external quality assessment participation but was independent of laboratory setting or experience. Except for ROS1, ESS positively correlated with laboratory accreditation. IHC stains with lower ESS correlated with increased error rates in ALK and ROS1 interpretation and analysis failures. Laboratory characteristics differently affected staining quality and interpretation, and laboratories should assess both aspects, and less common protocols need improvement in staining performance. Personalized medicine by appropriate targeting of molecular targets in tumors has improved survival of patients with non–small-cell lung cancer (NSCLC). Besides testing for variants in the epidermal growth factor receptor gene, the identification of rearrangements in anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) genes is required to select patients for treatment with tyrosine kinase inhibitors.1Lindeman N.I. Cagle P.T. Aisner D.L. Arcila M.E. Beasley M.B. Bernicker E.H. Colasacco C. Dacic S. Hirsch F.R. Kerr K. Kwiatkowski D.J. Ladanyi M. Nowak J.A. Sholl L. Temple-Smolkin R. Solomon B. Souter L.H. Thunnissen E. Tsao M.S. Ventura C.B. Wynes M.W. Yatabe Y. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.Arch Pathol Lab Med. 2018; 142: 321-346Crossref PubMed Scopus (282) Google Scholar ALK and ROS1 rearrangements are mutually exclusive, and occur in approximately 3% to 7% and 1% to 2% of NSCLC cases, respectively.2Tsao M.S.H.F. Yatabe Y. IASLC Atlas of ALK and ROS1 Testing in Lung Cancer. International Association for the Study of Lung Cancer. Editorial Rx Press, North Fort Myers, FL2016Google Scholar The most common fusion partner for ALK includes the echinoderm microtubule-associated protein-like 4 gene,3Soda 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 (3914) Google Scholar whereas the CD74-ROS1 fusion occurs most frequently for ROS1. More than 20 fusion partners have been described for both ALK and ROS1, but the clinical significance of different fusion products requires further investigation. To date, five ALK tyrosine kinase inhibitors (crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib) have received by the and and the European for treatment of and and were for the treatment of Solomon in non-small-cell lung Med. PubMed Scopus Google S. R. in ROS1 non-small-cell lung analysis of PubMed Scopus Google Scholar in testing was the the detection of increased protein expression to gene by immunohistochemistry is Y. in non-small-cell lung cancer Cancer. 2018; PubMed Scopus Google M.W. Sholl M. E. Tsao M.S. Yatabe Y. Hirsch F.R. interpretation the ALK IHC antibody and a detection between ALK IHC and ALK and between PubMed Scopus Google Scholar IHC is to and in with a of and a better with clinical Y. in non-small-cell lung cancer Cancer. 2018; PubMed Scopus Google M.W. Sholl M. E. Tsao M.S. Yatabe Y. Hirsch F.R. interpretation the ALK IHC antibody and a detection between ALK IHC and ALK and between PubMed Scopus Google R. L. M. S. E. is a better for ALK than in lung PubMed Scopus Google Scholar antibody are for ALK the antibody as of the ALK IHC received for the selection of ALK IHC a compared with E. K. B. S. B. R. L. S. ALK immunohistochemistry NSCLC is but with survival treatment with Cancer. PubMed Scopus Google Scholar to a IHC of ALK staining is for score ALK or independent detection M.S.H.F. Yatabe Y. IASLC Atlas of ALK and ROS1 Testing in Lung Cancer. International Association for the Study of Lung Cancer. Editorial Rx Press, North Fort Myers, FL2016Google M. S. S. Kerr K. K. Thunnissen E. ALK immunohistochemistry in staining treatment PubMed Scopus Google E. L. M. Kerr K. H. EML4-ALK testing in of the a with PubMed Scopus Google Scholar the are as most lower of IHC compared with or L. H. Y. selection for ALK in lung a analysis on characteristics of PubMed Scopus Google Scholar are higher for compared with the ALK immunohistochemistry for ALK gene in lung a and PubMed Scopus Google Scholar ROS1, a antibody was for time the the and the ROS1 antibody were of a immunohistochemistry for ROS1 in lung Cancer. PubMed Scopus Google E. S. R. B. of a ROS1 immunohistochemistry for the identification of ROS1 rearrangements in patients with lung the PubMed Scopus Google Scholar and of the protocols and of in with have been to H. M. C. C. ROS1 immunohistochemistry for detection of lung PubMed Scopus Google Scholar and are of the ROS1 cases are by ROS1 the IHC for of but ROS1 IHC should by a molecular or N.I. Cagle P.T. Aisner D.L. Arcila M.E. Beasley M.B. Bernicker E.H. Colasacco C. Dacic S. Hirsch F.R. Kerr K. Kwiatkowski D.J. Ladanyi M. Nowak J.A. Sholl L. Temple-Smolkin R. Solomon B. Souter L.H. Thunnissen E. Tsao M.S. Ventura C.B. Wynes M.W. Yatabe Y. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.Arch Pathol Lab Med. 2018; 142: 321-346Crossref PubMed Scopus (282) Google Scholar of the of ALK and ROS1 testing are in different testing of based on the clinical or testing to detection of as as ALK or ROS1 IHC is as a N.I. Cagle P.T. Aisner D.L. Arcila M.E. Beasley M.B. Bernicker E.H. Colasacco C. Dacic S. Hirsch F.R. Kerr K. Kwiatkowski D.J. Ladanyi M. Nowak J.A. Sholl L. Temple-Smolkin R. Solomon B. Souter L.H. Thunnissen E. Tsao M.S. Ventura C.B. Wynes M.W. Yatabe Y. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.Arch Pathol Lab Med. 2018; 142: 321-346Crossref PubMed Scopus (282) Google Scholar the and of lung cancer of cancer cases and of cancer of and for in 2018; PubMed Scopus Google Scholar of is for by the identification of ALK and ROS1 rearrangements is for appropriate treatment and are are and required to laboratories to the performance of IHC for E. B. C. C. a of for of PubMed Scopus Google C. C. of immunohistochemistry for of a of and based PubMed Scopus Google S. quality assessment for from PubMed Scopus Google Scholar European external quality assessment schemes have been to the performance of ALK and ROS1 IHC and to laboratories in for improvement with to the L. K. K. L. Kerr K. E. Thunnissen E. The of external quality assessment for molecular testing for ALK lung from two for PubMed Scopus Google M. R. M. M. ALK testing in non-small-cell lung of a PubMed Scopus Google C. L. K. E. L. E. expert a in time external quality assessment improved quality of analysis in lung 2018; PubMed Scopus Google Scholar quality to with a on and protocol to identification of required for appropriate staining and their to schemes a in ALK IHC protocols and detection both M. S. S. Kerr K. K. Thunnissen E. 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Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.Arch Pathol Lab Med. 2018; 142: 321-346Crossref PubMed Scopus (282) Google Scholar with ROS1, in to ALK is in lung in targeting ROS1 in lung PubMed Scopus Google Scholar the in detection protocols applied by laboratories with different ALK in with and detection and and European Conformity in Vitro Diagnostic kits a higher ESS compared with is in with by performance for epidermal growth factor receptor IHC improved over time but compared with M. S. testing in from PubMed Scopus Google Scholar and by the laboratory to the in lower rates for ALK the of a and the laboratory to a and a protocol, and to of laboratory from the or protocol requires appropriate from the protocol have been to have on the to and the of or a PubMed Scopus Google Scholar in cases were by kits in the have to the observed in performance. for of the of on and for less common as as of by the The most and is of in antibody compared with the from and the a better performance for the antibody has been to have a lower compared with M.S.H.F. Yatabe Y. IASLC Atlas of ALK and ROS1 Testing in Lung Cancer. International Association for the Study of Lung Cancer. Editorial Rx Press, North Fort Myers, FL2016Google Scholar of by by independent has been M.S.H.F. Yatabe Y. IASLC Atlas of ALK and ROS1 Testing in Lung Cancer. International Association for the Study of Lung Cancer. Editorial Rx Press, North Fort Myers, FL2016Google Scholar compared with the of the of is and are to of less common as or in a lower is as the lower performance for and is for in N.I. Cagle P.T. Aisner D.L. Arcila M.E. Beasley M.B. Bernicker E.H. Colasacco C. Dacic S. Hirsch F.R. Kerr K. Kwiatkowski D.J. Ladanyi M. Nowak J.A. Sholl L. Temple-Smolkin R. Solomon B. Souter L.H. Thunnissen E. Tsao M.S. Ventura C.B. Wynes M.W. Yatabe Y. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.Arch Pathol Lab Med. 2018; 142: 321-346Crossref PubMed Scopus (282) Google E. Aisner D.L. Beasley M.B. Cagle P.T. S. R. M. T. K. S. Sholl Tsao M.S. M. Yatabe Y. of a from of the Pathol Lab Med. 2018; 142: PubMed Scopus Google Scholar are in with quality assessment from and lower were observed for M. S. S. Kerr K. K. Thunnissen E. ALK immunohistochemistry in staining treatment PubMed Scopus Google Scholar the ESS as on is a of both the antibody and protocol is the to the in ESS in in antibody performance to the of and detection was by the lower ESS for and the for detection of the as in with the most common detection kits IHC or detection but less common detection The with incubation time antibody a lower performance in the incubation to the between and to in as frequently in routine was in as the of laboratories was to for ALK and for their were evaluated and a of The of appropriate and by to the and of the is and has been to and M. S. S. Kerr K. K. Thunnissen E. ALK immunohistochemistry in staining treatment PubMed Scopus Google Scholar the was to the to as the schemes of the ROS1 or of a immunohistochemistry for ROS1 in lung Cancer. PubMed Scopus Google E. S. R. B. of a ROS1 immunohistochemistry for the identification of ROS1 rearrangements in patients with lung the PubMed Scopus Google Scholar were during the time of the schemes for to ALK European Conformity in Vitro Diagnostic was for ROS1 analysis at the time of the antibody of protocols were most frequently in with IHC revealed a better performance compared with compared with and is a laboratories the of the and and compared with the ROS1 testing has been than and testing were in L. R. M. Kerr K. B. Thunnissen E. Testing for ROS1 in lung a with PubMed Scopus Google Scholar less is compared with and are on the performance of detection a lower staining performance by the in increased of schemes in in further their detection protocol in improvement in their of cases in of ALK and ROS1 rearrangements in is of as a in the of to from treatment for Solomon in non-small-cell lung Med. PubMed Scopus Google Scholar on the have a to in is is required in of ALK IHC The of and observed in the schemes was with of for ALK and of for ROS1. the of ALK IHC was higher for but and was by the in the two laboratories interpretation on the of five or both and laboratories five as or a with their IHC interpretation and both laboratories the interpretation for a and or for ROS1 error laboratory was the were two laboratories with have the higher error rates in the of laboratories error of the of by on the of were for different for for ALK and for of compared with for ALK Besides or or staining to the to the staining were to the a in the protocol the analysis for the and the need for quality in of testing a in the of in cases the repeated of of from NSCLC tumors are in of the need for a in was observed for both on the of is to in the to have a score increased a higher of was for or E. C. H. setting and as to quality in testing Cancer. 2018; PubMed Scopus Google Scholar the staining quality was affected by a in the interpretation of the staining is by experience. the IHC is interpretation is to in requires with appropriate and for the various and detection kits To a ESS in the of the quality for and staining quality and in routine was to the of IHC the of protocols by kits to improved in to lower for less ALK testing to in ROS1 detection is More are to assess performance for different ROS1 IHC The between the and interpretation, and of laboratory characteristics on both aspects, the for to both the staining quality and the interpretation of the