Fricke, Stephan

Sack, Ulrich

English

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COMMEN T A R YCan flow cytometry outperform genetic testing in eosinophiliapatients?Ulrich Sack1 | Stephan Fricke21Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany2Department of GMP Process Development, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, GermanyCorrespondenceUlrich Sack, Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany.Email: ulrich.sack@medizin.uni-leipzig.deIn this issue (page XXX) Sedigheh Sharifzadeh and her colleagues pub-lished a paper entitled “Phosflow assessment of PDGFRA phosphory-lation state: A guide for tyrosine kinase inhibitor targeted therapy inhypereosinophilia patients.” The authors investigated to what extentflow cytometry is applicable for phosphorylated molecules in eosino-phil granulocytes and whether therapeutic consequences can bededuced for patients with hematological diseases, where clonal eosin-ophilia can be evaluated.Platelet-derived growth factor (PDGF) binds to various forms of thePDGF receptor, most commonly consisting of alpha (PDGFRA) and beta(PDGFRB) chains.1 This receptor has intracellular activating phosphoryla-tion and binding sites for signaling molecules.2 Mutations in these chainscan result in an increased level of phosphorylation due to constitutivelyactivated kinases.1 Besides other mutations, this is one of the molecularmechanisms resulting in the clinical picture of clonal hypereosinophilia.3Myeloid neoplasms with eosinophilia can be diagnosed according tostandard guidelines. The methods are complex including the determina-tion of white blood cell count, diagnostic imaging of different organs(e.g., hepato−/splenomegaly), serum markers (LDH, TnI/BNP, Tryptase,Vitamin B12, AP, IgE), bone marrow histology, detection or exclusion ofBCR1-ABL1, FIP1L1-PDGFRA by RT-PCR or FISH analysis, ETV6-ABL1by RT-PCR, performing of conventional cytogenetics from the bone mar-row (4q12, 5q31-33, 8p11, 13q12) and confirmation of a fusion gene byFISH/RT-PCR as well as determination of phenotype mutations like KITD816V, JAK2 V617F, STAT5B N642H, JAK2 ex13InDel and prognosticmutations such as ASXL1, SRSF2, RUNX1, EZH2, SETBP1, and others.4In addition, T-cell clonality by flow cytometric analysis or T-cell receptorrearrangement by PCR will be used.4,5According to the WHO classification of neoplasms with eosino-philia, we distinguish myeloid or lymphoid neoplasia with eosinophiliaand rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2(MLN-Eo), chronic eosinophil leukemia, not otherwise specified (CEL,NOS), and idiopathic hypereosinophilic syndrome (HES).5,6The measurement of phosphorylated transcription factors andreceptor molecules is today well-established in flow cytometry. Themethod was initially only a research method.7 In the meantime, appli-cations in clinical diagnostics are increasing, but to the best of ourknowledge no suitable in vitro diagnostic test kits for routinely deter-mination of the phosphorylation state of PDGFRA by flow cytometryare available.This is similar for most molecular genetic methods, and also in thiscase, the laboratories depend on their own expertise. Common applica-tions in medical diagnostics are immunodeficiencies and immunefunction.8–10 Furthermore, because STAT phosphorylation indicates acti-vation of immunological cells, phosphorylation can also be used as a sur-rogate marker for antigen-specific activation in a time-saving manner.8–10In brief, the laboratory applied a test kit for detection of intracel-lular phosphorylation and detected PDGFRA chain phosphorylationby a specific antibody. The results were compared with genetic test-ing. In addition, samples from other patients and cell lines were usedas a comparison to ensure the specificity of the method. The level ofphosphorylation was also confirmed via Western blot. Theresearchers' approach is noteworthy, given that the trend in diagnos-tics is rather more often applying genetic analyses than functionaltests. In particular in oncology, detailed molecular biological investiga-tions are now an essential basis for targeted individualized therapies.However, the results certainly show also such a functional testingis useful. Not all patients exhibit the altered function of the PDGFreceptor. Nine of 45 (20%) investigated patients with hypereosinophilia[Correction added on 17 February 2021, after first online publication: Projekt Deal fundingstatement has been added.]Received: 15 January 2021 Accepted: 19 January 2021DOI: 10.1002/cyto.a.24311This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in anymedium, provided the original work is properly cited and is not used for commercial purposes.© 2021 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.Cytometry. 2021;1–2. wileyonlinelibrary.com/journal/cytoa 1had higher levels of PDGFRA phosphorylation. In addition, in furtherinvestigations it becomes apparent that the genotype–phenotype link-age is incomplete also in this case: the authors detected PDGFRA-FIP1L1 fusion, ETV6-PDGFRA, and STRN-PDGFRA. This means thatdifferent mutations can be found with the test that cause increasedmRNA expression.This shows the possible particular relevance of this test: For ther-apy of such cases showing higher levels of PDGFRA phosphorylation,specific tyrosine kinase inhibitors can be used therapeutically andprobably can act precisely on these phosphorylation sites. Thus, a pre-diction of those pharmacological effects could be expected. However,that must be further investigated in appropriate clinical trials with dif-ferent kind of tyrosine kinase inhibitors. Since different mutations areassociated with these diseases, a faster measuring of the functionalchanges of PDGFRA is probably a superior solution.To what extent can such an examination be established in routinediagnostics? Medical laboratories must meet high quality standards(e.g., ISO 15189), and it is certainly not to be expected that an in vitrodiagnostic test kit will come onto the market for such an assay.Therefore, laboratories must be able to establish such tests them-selves (so-called laboratory-developed tests) and perform them underquality-assured conditions. For the validation of tests in routine diag-nostics, there are now updated recommendations that allow new teststo be introduced in compliance with guidelines and standards.11Here, the authors show (in this issue page XXX) a well-developedprotocol that can be established in laboratories experienced in flow cyto-metry. The volume of required blood sample is small, the processing isfast, reference intervals in a healthy reference population can be deter-mined,12 and strategies for quality assurance are available. The ROCcurve shown suggests that the procedure developed is robust enoughfor a diagnostic decision that must be figured out in clinical trials.The manuscript reports another example for methods of func-tional cytometry that no longer belong only to the research laboratorybut are indispensable in routine diagnostics. It remains a challenge forlaboratories to develop their own tests for routine diagnostics. How-ever, the diagnostic information and the therapeutic consequencesmake it quite clear that the effort is definitely worthwhile.ACKNOWLEDGMENTOpen access funding enabled and organized by Projekt DEAL.AUTHOR CONTRIBUTIONSUlrich Sack: Conceptualization; formal analysis; methodology; writing-original draft; writing-review and editing. Stephan Fricke:Conceptualization; formal analysis; methodology; writing-originaldraft; writing-review and editing.REFERENCES1. Heldin CH, Lennartsson J. Structural and functional properties ofplatelet-derived growth factor and stem cell factor receptors. ColdSpring Harb Perspect Biol. 2013;5:a009100.2. Claesson-Welsh L. Signal transduction by the PDGF receptors. ProgGrowth Factor Res. 1994;5:37–54.3. Shomali W, Gotlib J. World Health Organization-defined eosinophilicdisorders: 2019 update on diagnosis, risk stratification, and manage-ment. Am J Hematol. 2019;94:1149–67.4. Metzgeroth G, Reiter A, Goede J, Sperr WR, Valent P. MyeloischeNeoplasien mit Eosinophilie. In: Onkopedia; 2020; https://www.onkopedia.com/de/onkopedia/guidelines/myeloische-neoplasien-mit-eosinophilie-frueher-eosinophilie-assoziierte-myeloproliferative-erkrankungen-mpn-eo/@@guideline/html/index.html5. Tembhare P, Yuan CM, Xi L, Morris JC, Liewehr D, Venzon D, et al.Flow cytometric immunophenotypic assessment of T-cell clonality byVbeta repertoire analysis: detection of T-cell clonality at diagnosisand monitoring of minimal residual disease following therapy.Am J Clin Pathol. 2011;135:890–900.6. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H,Thiele J. WHO classification of tumours of haematopoietic and lym-phoid tissues. WHO, editor; 2017.7. Jacobberger JW, Sramkoski RM, Frisa PS, Ye PP, Gottlieb MA,Hedley DW, et al. Immunoreactivity of Stat5 phosphorylated on tyro-sine as a cell-based measure of Bcr/Abl kinase activity. Cytometry A.2003;54:75–88.8. Bitar M, Boettcher M, Boldt A, Hauck F, Kohl U, Liebert UG, et al.Flow cytometric measurement of STAT5 phosphorylation incytomegalovirus-stimulated T cells. Cytometry A. 2020. https://doi.org/10.1002/cyto.a.242869. Bitar M, Boldt A, Freitag MT, Gruhn B, Kohl U, Sack U. EvaluatingSTAT5 phosphorylation as a mean to assess T cell proliferation. FrontImmunol. 2019;10:722.10. Bitar M, Boldt A, Binder S, Borte M, Kentouche K, Borte S, et al.Flow cytometric measurement of STAT1 and STAT3 phosphoryla-tion in CD4(+) and CD8(+) T cells-clinical applications in primaryimmunodeficiency diagnostics. J Allergy Clin Immunol. 2017;140:1439–41.e9.11. Lambert C, Yanikkaya Demirel G, Keller T, Preijers F, Psarra K,Schiemann M, et al. Flow cytometric analyses of lymphocyte markersin immune oncology: a comprehensive guidance for validation prac-tice according to Laws and Standards. Front Immunol. 2020;11:2169.12. Özcürümez MK, Haeckel R, Gurr E, Streichert T, Sack U. Determina-tion and verification of reference interval limits in clinical chemistry.Recommendations for laboratories on behalf of the working groupguide limits of the DGKL with respect to ISO standard 15189 and theguideline of the German medical association on quality Assurance inMedical Laboratory Examinations (Rili-BAEK). J Lab Med. 2019;43:127.2 COMMENTARY

Can flow cytometry outperform genetic testing in eosinophilia patients?

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Can flow cytometry outperform genetic testing in eosinophilia patients?

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