To evaluate whether CD3 staining performed routinely on temporal artery biopsy specimens might improve the sensitivity of temporal artery biopsy in patients with biopsy-negative GCA

Cavazza, Alberto

Ciccia, Francesco

Ferrante, Angelo

Guggino, Giuliana

Rizzo, Aroldo

Salvarani, Carlo

English

Francesco Ciccia

Angelo Ferrante

Giuliana Guggino

Alberto Cavazza

Carlo Salvarani

Aroldo Rizzo

Crossref

Rheumatology

CD3 immunohistochemistry is helpful in the diagnosis of giant cell arteritis

Objective. To evaluate whether CD3 staining performed routinely on temporal artery biopsy specimens might improve the sensitivity of temporal artery biopsy in patients with biopsy-negative GCA. Methods. Two hundred and seventy biopsies were considered for this study, stained with haematoxylin and eosin and with an anti-CD3 antibody. Results. The addition of CD3 staining modified the sensibility and the specificity of the histologic examination in 89.47 and 95.00%, respectively, with a positive and negative predictive values of 97.00 and 79.78%. Conclusion. The addition of CD3 immunostaining to the classic histologic evaluation is accompanied by a significant increase in the sensibility with a comparable specificity. The Author(s) 2018

Ciccia F.

Ferrante A.

Guggino G.

Cavazza A.

Salvarani C.

Rizzo A.

Archivio istituzionale della ricerca - Università di Palermo

Archivio Istituzionale della Ricerca - Università degli Studi della Campania "Luigi Vanvitelli"

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

Concise reportCD3 immunohistochemistry is helpful in thediagnosis of giant cell arteritisFrancesco Ciccia1, Angelo Ferrante1, Giuliana Guggino1, Alberto Cavazza2,Carlo Salvarani2,* and Aroldo Rizzo3,*AbstractObjective. To evaluate whether CD3 staining performed routinely on temporal artery biopsy specimens might improvethe sensitivity of temporal artery biopsy in patients with biopsy-negative GCA.Methods. Two hundred and seventy biopsies were considered for this study, stained with haematoxylin and eosin andwith an anti-CD3 antibody.Results. The addition of CD3 staining modified the sensibility and the specificity of the histologic examination in 89.47and 95.00%, respectively, with a positive and negative predictive values of 97.00 and 79.78% .Conclusion. The addition of CD3 immunostaining to the classic histologic evaluation is accompanied by a significantincrease in the sensibility with a comparable specificity.Key words: giant cell arteritis, CD3, immunohistochemistry, diagnosisRheumatology key messages. CD3 immunohistochemistry may be useful in histological-negative arteries of patients with suspected GCA.. The addition of CD3 staining significantly increased sensibility and specificity of classic histologic examination.IntroductionGCA is a large- and medium-sized arteries systemic vas-culitis [1] resulting in a wide variety of systemic, neurologicand ophthalmologic complications. GCA is histopatho-logically characterized by transmural inflammation of theall the layers of affected arteries by monocyte-derivedmacrophages, activated T cells and multinucleated giantcells. In particular, a massive infiltration by Th1, Th9 andTh17 CD3+ effector T cells is observed in temporal arterybiopsy (TAB) specimens obtained from patients affectedby GCA with positive biopsy [2, 3]. GCA is diagnosed bycombining symptoms, clinical findings, laboratory resultsand diagnostic imaging. The gold standard for the diag-nosis of GCA is the demonstration of artery wall inflam-mation on TAB [1]. Frequently, TAB results in false-negative biopsies in a relevant number of patients, leadingto a significant delay in treatment and possible irreversiblecomplications [4]. The need to improve the diagnosis ofGCA requires the developement of new staining tech-niques of TAB specimens. Due to the important role ofCD3+ T cells in the pathogenesis of the disease weaimed to investigate whether CD3 staining performed rou-tinely on TAB specimens may improve the sensitivity ofTAB in patients with biopsy-negative GCA.MethodsPatients and immunohistochemistryFor this study, we retrospectively evaluated all the biop-sies performed in our centres from January 2003 to June2016 in patients with suspected GCA. Two hundred andseventy biopsies obtained from as many patients wereconsidered. At the time of diagnosis, 28 patients hadvision loss and PMR was associated in 45 patients. Atthe time of artery biopsy all the patients were treatment-naı̈ve or had received glucocorticoids for 41 week. Prioror current diagnosis of cancer, autoimmune disease otherthan GCA or chronic infection was carefully excluded. Allthe paraffin-embedded TAB specimens were retrieved,the tissue cut into 4-mm sections (810 per patient)and stained with haematoxylin and eosin (H&E).Immunohistochemical analysis for CD3 was also1Dipartimento Biomedico di Medicina Interna e Specialistica, Sezionedi Reumatologia, Università degli studi di Palermo, Palermo,2Arcispedale Santa Maria Nuova, Unità Operativa Complessa diReumatologia, Reggio Emilia and 3Azienda Ospedaliera OspedaliRiuniti Villa Sofia-Cervello, Unità Operativa Complessa di AnatomiaPatologica, Palermo, Italy*C. Salvarani and A. Rizzo equally contributed to this study.Correspondence to: Francesco Ciccia, Dipartimento Biomedico diMedicina Interna e Specialistica, Sezione di Reumatologia, Piazzadelle Cliniche 2, 90127 Palermo, Italy.E-mail: francescociccia@tiscali.itSubmitted 10 January 2018; revised version accepted 23 March 2018! The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.comRHEUMATOLOGYRheumatology 2018;57:13771380doi:10.1093/rheumatology/key106Advance Access publication 25 April 2018CLINICALSCIENCEDownloaded from https://academic.oup.com/rheumatology/article/57/8/1377/4985751 by guest on 26 October 2020performed on 5-mm-thick paraffin-embedded sectionsfrom artery biopsies and from tonsils (used as positivecontrols) as previously described [5]. Briefly, following re-hydration, antigen was unmasked for 45 min at 95C usingDako Target retrieval solution (pH 6; Dako, Carpinteria,CA, USA). Endogenous peroxidase was blocked for10 min with Dako peroxidase blocking reagent, and non-specific binding was blocked for 20 min with Dako proteinblock. The primary antibodies mouse monoclonal anti-human CD3 (ab5690, AbCam, Cambridge, UK) wasadded and incubated for 1 h at room temperature. Anisotype-matched irrelevant antibody was used as a nega-tive control. Following three washes with Tris-bufferedsaline, slides were incubated for 30 min with peroxidase-conjugated Dako EnVision polymer. After three furtherwashings, peroxidase activity was visualized using di-ami-nobenzidine chromogen (Dako, Carpinteria, CA, USA )and slides were lightly counterstained with haematoxylinbefore dehydration and mounting in DePex (VWRInternational, Oslo, Norway). The number of immune-reactive cells was determined by counting positivelystained cells on photomicrographs obtained from threerandom high-power microscopic fields (400 magnifica-tion) under a Leica DM2000 optical microscope, using aLeica DFC320 digital camera (Leica, Rijswijk, TheNetherlands). Two experienced pathologists (A.R. andA.C.) with an expertise in vasculitides and with noaccess to clinical data evaluated the arteries. The intra-rater agreement and the inter-rater agreement calculatedby the Cohen’s k coefficient for the two pathologists were0.81 and 0.72, respectively. In order to study the presenceof atherosclerosis, artery sections were stained withMasson’s trichrome (Sigma-Aldrich, Sigma, Saint Louis,Missouri, USA) for cellular components [smooth musclecells (SMCs): pink, and red blood cells: red], fibroustissue (blue), and fibrin and platelet-rich thrombus(purple). The study was conducted according to theDeclaration of Helsinki and Italian legislation. Consentwas obtained from all enrolled subjects after the natureof the investigation was explained and in accordance withFIG. 1 CD3 immunostaining in histology positive and negative GCA arteries(AB): representative microphotographs showing hematoxylin and eosin staining (A) and CD3 immunostaining of GCAartery showing transmural inflammation. (CD): representative microphotographs showing hematoxylin and eosinstaining (C) and CD3 immunostaining in GCA artery without apparent inflammatory infiltrates (D). (EF): representativemicrophotographs showing hematoxylin and eosin staining (E) and CD3 immunostaining in control artery (F). G: numberof CD3+ cells in GCA TAB+, GCA TAB and control arteries.1378 https://academic.oup.com/rheumatologyFrancesco Ciccia et al.Downloaded from https://academic.oup.com/rheumatology/article/57/8/1377/4985751 by guest on 26 October 2020the approved protocol from the Institutional Review Boardat the University of Palermo and the Arcispedale SantaMaria Nuova, Reggio Emilia, Italy.Statistical analysisStatistical analysis of quantitative variables was performedusing the MannWhitney rank-sum test. P < 0.05 wereconsidered significant.ResultsOne hundred and ten arteries displayed typical histolo-gical findings of GCA (90 women and 20 men, medianage 77 years, range 6280 years). Eighty patients did notdisplay any histological feature of GCA but were classifiedto be affected by GCA (65 women and 15 men, medianage 77 years, range 5783 years) according to the ACRclassification criteria [6]. Finally, 80 were histologicallynormal TAB (62 women, 18 men; median age 68 years,range 5783 years) from as many patients who did notfulfil the ACR classification criteria for GCA. In these pa-tients, the absence of GCA was confirmed by a retro-spective evaluation of all medical records fromsymptoms’ onset to the last visit. We calculated that inour cohort the sensibility and the specificity of H&E stain-ing were 57 and 100%, respectively (positive predictivevalue 100% and the negative predictive value 50%).CD3 immunohistochemistry was performed in all thearteries and the results were considered only in arterialareas not displaying evidence of atherosclerosis.According to H&E results, anti-CD3 staining was positivein all the biopsy-proven GCA patients (Fig. 1A and B;Table 1). Interestingly, CD3+ staining was also observedin 60 out of 80 patients with TAB-negative GCA (in thecontext of the adventia layer in 54 out of 60 patients andin the context of media layer in the remaining six) (Fig. 1Cand D; Table 1). Quantitative evaluation of CD3+ cellsdemonstrated a significantly higher number of CD3+cells in TAB-positive and -negative GCA arteries com-pared with controls (Fig. 1G). No significant differenceswere observed regarding ACR criteria between betweenbiopsy-negative GCA patients with or without T cells intheir biopsies. Among control arteries only 4 out of 80displayed CD3 positivity (<10 positive cells) (Fig. 1E andF; Table 1). The addition of CD3 staining to H&E evaluationmodified the sensibility and the specificity of the histologicexamination in 89.47 and 95.00%, respectively. The posi-tive and negative predictive values were 97.00 and79.78%.DiscussionThe histologic diagnosis of GCA does not require anyimmunohistochemical study of the affected arteries. Thisapproach might be considered adequate in the presenceof classic transmural inflammation. However, differentsubsets of non-classic transmural inflammation havebeen described in GCA patients and many other GCA pa-tients display no histologic inflammation [1]. GCA has longbeen considered as a T cell-mediated disease. The mas-sive infiltration of GCA-positive arteries by activated T ef-fector cells such as Th1, Th17 and Th9 highlights theimportant role of these cells in the pathogenesis of thedisease and makes T lymphocytes an interesting subjectfor diagnostic purposes [2, 3]. The presence of CD3+ infil-trating T cells has been demonstrated in TAB-negativeGCA arteries in a series of studies without, however,evaluation in a large series of GCA patients [79].In this study, we demonstrated that CD3+ staining waspresent in the majority of arteries obtained from GCA pa-tients with a negative TAB. Since arteries are immune-privileged sites, the presence of CD3+ cells in the contextof apparently histologically uninflamed non-atheroscler-otic GCA arteries may suggest the occurrence of tissueinflammation. In particular, CD3 immunostaining wasmainly observed in the context of the adventia layerwhere vasa vasorum are located. Activation of adventitialvasa vasorum has been suggested to occur in the earlystage of the disease, leading to the active recruitmentof inflammatory mononuclear cells in the inflamed arteries[1, 2].The addition of CD3 immunostaining to the classichistologic evaluation was accompanied by a significantincrease in the sensibility with a comparable specificity.Although biopsy-negative GCA characterizes a subset ofpatients who often have a different clinical spectrum of thedisease with less severe ischaemic complications thanthat of biopsy-proven GCA [10], these results seem tosuggest that CD3 immunostaining should be added tothe routine evaluation of arteries when GCA is stronglysuspected. The main limitation of the present study isthe retrospective design of the study. However, the per-fect concordance between the presence of CD3+ cellsand the classic histologic signs of GCA and the substan-tial absence of CD3+ cells in control arteries seem to con-firm that the presence of abundant CD3+ infiltrating cellsspecifically marks inflamed arteries.Funding: No specific funding was received from any fund-ing bodies in the public, commercial or not-for-profit sec-tors to carry out the work described in this manuscript.Disclosure statement: The authors have declared noconflicts of interest.References1 Salvarani C, Pipitone N, Versari A, Hunder GG. Clinicalfeatures of polymyalgia rheumatica and giant cell arteritis.Nat Rev Rheumatol 2012;8:50921.TABLE 1 Summary of the patients enrolled in the studyArterycharacterization CD3+, n (%) CD3, n (%) Total, nGCA TAB+ 110 (100) 0 110GCA TAB 60 (75) 20 (25) 80Control arteries 4 (5) 76 (95) 80https://academic.oup.com/rheumatology 1379CD3 immunohistochemistry in GCA diagnosisDownloaded from https://academic.oup.com/rheumatology/article/57/8/1377/4985751 by guest on 26 October 20202 Ciccia F, Rizzo A, Ferrante A et al. New insights into thepathogenesis of giant cell arteritis. Autoimmun Rev2017;16:67583.3 Ciccia F, Rizzo A, Guggino G et al. Difference in the ex-pression of IL-9 and IL-17 correlates with different histo-logical pattern of vascular wall injury in giant cell arteritis.Rheumatology (Oxford) 2015;54:1596604.4 Muratore F, Cavazza A, Boiardi L et al. Histopathologicfindings of patients with biopsy-negative giant cell arteritiscompared to those without arteritis: a population-basedstudy. Arthritis Care Res 2016;68:86570.5 Ciccia F, Rizzo A, Maugeri R et al. Ectopic expression ofCXCL13, BAFF, APRIL and LT-b is associated with arterytertiary lymphoid organs in giant cell arteritis. Ann RheumDis 2017;76:23543.6 Hunder GG, Bloch DA, Michel BA et al. The AmericanCollege of Rheumatology 1990 criteria for the classifica-tion of giant cell arteritis. Arthritis Rheum 1990;33:11228.7 Zhou L, Luneau K, Weyand CM et al.Clinicopathologic correlations in giant cell arteritis: aretrospective study of 107 cases. Ophthalmology2009;116:157480.8 Braun N, Fritz P, Rieth A et al. Predictors for treatmentsuccess and expression of glucocorticoid receptor in giantcell arteritis and polymyalgia rheumatica. J Rheumatol2009;36:226976.9 Stone JH, Papaliodis GN, Dunbar M, Stone JR. Case re-cords of the Massachusetts General Hospital. Case 4-2010. A 53-year-old man with arthralgias, oral ulcers,vision loss, and vocal-cord paralysis. N Engl J Med2010;362:53746.10 Gonzalez-Gay MA, Garcia-Porrua C, Llorca J, Gonzalez-Louzao C, Rodriguez-Ledo P. Biopsy-negative giant cellarteritis: clinical spectrum and predictive factors for posi-tive temporal artery biopsy. Semin Arthritis Rheum2001;30:24956.1380 https://academic.oup.com/rheumatologyFrancesco Ciccia et al.Downloaded from https://academic.oup.com/rheumatology/article/57/8/1377/4985751 by guest on 26 October 2020

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CD3 immunohistochemistry is helpful in the diagnosis of giant cell arteritis

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