BACKGROUND: The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS: An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS: Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS: The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes.

Centre de Recherche des Cordeliers Sorbonne Université Université Paris Cité 75006 Paris France

Department of Pathology and Molecular Genomics Providence Portland Medical Center Portland OR 97213 USA

Department of Pathology AP HP Assistance Publique Hopitaux de Paris Georges Pompidou European Hospital 75015 Paris France

Department of Pathology Cliniques Universitaires St Luc Institut de Recherche Clinique et Experimentale Université Catholique de Louvain 1348 Brussels Belgium

Department of Pathology Istituto Nazionale Tumori IRCCS Fondazione G Pascale 80131 Napoli Italy

Department of Pathology Sapporo Medical University School of Medicine Sapporo 060 8556 Japan

Department of Pathology UH Cleveland Medical Center Cleveland OH 44106 USA

Department of Pathology University of California San Francisco CA 94143 USA

Department of Pathology Weill Cornell Medicine New York NY 10021 USA

Digestive Surgery Department AP HP Assistance Publique Hopitaux de Paris Georges Pompidou European Hospital 75015 Paris France

Equipe Labellisée Ligue Contre le Cancer 75006 Paris France

Immunomonitoring Platform Laboratory of Immunology AP HP Assistance Publique Hopitaux de Paris Georges Pompidou European Hospital 75015 Paris France

Inovarion 75005 Paris France

INSERM Laboratory of Integrative Cancer Immunology 75006 Paris France

Institut Roi Albert 2 Department of Medical Oncology Cliniques Universitaires St Luc Institut de Recherche Clinique et Experimentale Université Catholique de Louvain 1030 Brussels Belgium

Institute of Pathology 1st Faculty of Medicine Charles University General University Hospital Prague 12808 Prague Czech Republic

Institute of Pathology University of Bern 3008 Bern Switzerland

Pathology Department John Hopkins Baltimore MD 21287 USA

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Galon J., Mlecnik B., Bindea G., Angell H.K., Berger A., Lagorce C., Lugli A., Zlobec I., Hartmann A., Bifulco C., et al. Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours. J. Pathol. 2014;232:199–209. doi: 10.1002/path.4287. PubMed DOI PMC

Guinney J., Dienstmann R., Wang X., de Reynies A., Schlicker A., Soneson C., Marisa L., Roepman P., Nyamundanda G., Angelino P., et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015;21:1350–1356. doi: 10.1038/nm.3967. PubMed DOI PMC

Galon J., Costes A., Sanchez-Cabo F., Kirilovsky A., Mlecnik B., Lagorce-Pages C., Tosolini M., Camus M., Berger A., Wind P., et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313:1960–1964. doi: 10.1126/science.1129139. PubMed DOI

Koelzer V.H., Dawson H., Andersson E., Karamitopoulou E., Masucci G.V., Lugli A., Zlobec I. Active immunosurveillance in the tumor microenvironment of colorectal cancer is associated with low frequency tumor budding and improved outcome. Transl. Res. 2015;166:207–217. doi: 10.1016/j.trsl.2015.02.008. PubMed DOI

Laghi L., Bianchi P., Miranda E., Balladore E., Pacetti V., Grizzi F., Allavena P., Torri V., Repici A., Santoro A., et al. CD3+ cells at the invasive margin of deeply invading (pT3-T4) colorectal cancer and risk of post-surgical metastasis: A longitudinal study. Lancet Oncol. 2009;10:877–884. doi: 10.1016/S1470-2045(09)70186-X. PubMed DOI

Lee W.S., Park S., Lee W.Y., Yun S.H., Chun H.K. Clinical impact of tumor-infiltrating lymphocytes for survival in stage II colon cancer. Cancer. 2010;116:5188–5199. doi: 10.1002/cncr.25293. PubMed DOI

Mlecnik B., Bindea G., Angell H.K., Maby P., Angelova M., Tougeron D., Church S.E., Lafontaine L., Fischer M., Fredriksen T., et al. Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival than Microsatellite Instability. Immunity. 2016;44:698–711. doi: 10.1016/j.immuni.2016.02.025. PubMed DOI

Mlecnik B., Tosolini M., Kirilovsky A., Berger A., Bindea G., Meatchi T., Bruneval P., Trajanoski Z., Fridman W.H., Pages F., et al. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J. Clin. Oncol. 2011;29:610–618. doi: 10.1200/JCO.2010.30.5425. PubMed DOI

Nosho K., Baba Y., Tanaka N., Shima K., Hayashi M., Meyerhardt J.A., Giovannucci E., Dranoff G., Fuchs C.S., Ogino S. Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer and prognosis: Cohort study and literature review. J. Pathol. 2010;222:350–366. doi: 10.1002/path.2774. PubMed DOI PMC

Ogino S., Galon J., Fuchs C.S., Dranoff G. Cancer immunology—Analysis of host and tumor factors for personalized medicine. Nat. Rev. Clin. Oncol. 2011;8:711–719. doi: 10.1038/nrclinonc.2011.122. PubMed DOI PMC

Ogino S., Nosho K., Irahara N., Meyerhardt J.A., Baba Y., Shima K., Glickman J.N., Ferrone C.R., Mino-Kenudson M., Tanaka N., et al. Lymphocytic reaction to colorectal cancer is associated with longer survival, independent of lymph node count, microsatellite instability, and CpG island methylator phenotype. Clin. Cancer Res. 2009;15:6412–6420. doi: 10.1158/1078-0432.CCR-09-1438. PubMed DOI PMC

Pages F., Berger A., Camus M., Sanchez-Cabo F., Costes A., Molidor R., Mlecnik B., Kirilovsky A., Nilsson M., Damotte D., et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N. Engl. J. Med. 2005;353:2654–2666. doi: 10.1056/NEJMoa051424. PubMed DOI

Mlecnik B., Bindea G., Angell H.K., Sasso M.S., Obenauf A.C., Fredriksen T., Lafontaine L., Bilocq A.M., Kirilovsky A., Tosolini M., et al. Functional network pipeline reveals genetic determinants associated with in situ lymphocyte proliferation and survival of cancer patients. Sci. Transl. Med. 2014;6:228ra237. doi: 10.1126/scitranslmed.3007240. PubMed DOI

Pages F., Kirilovsky A., Mlecnik B., Asslaber M., Tosolini M., Bindea G., Lagorce C., Wind P., Marliot F., Bruneval P., et al. In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. J. Clin. Oncol. 2009;27:5944–5951. doi: 10.1200/JCO.2008.19.6147. PubMed DOI

Bruni D., Angell H.K., Galon J. The immune contexture and Immunoscore in cancer prognosis and therapeutic efficacy. Nat. Rev. Cancer. 2020;20:662–680. doi: 10.1038/s41568-020-0285-7. PubMed DOI

Bindea G., Mlecnik B., Angell H.K., Galon J. The immune landscape of human tumors: Implications for cancer immunotherapy. Oncoimmunology. 2014;3:e27456. doi: 10.4161/onci.27456. PubMed DOI PMC

Bindea G., Mlecnik B., Fridman W.H., Galon J. The prognostic impact of anti-cancer immune response: A novel classification of cancer patients. Semin. Immunopathol. 2011;33:335–340. doi: 10.1007/s00281-011-0264-x. PubMed DOI PMC

Pages F., Galon J., Fridman W.H. The essential role of the in situ immune reaction in human colorectal cancer. J. Leukoc. Biol. 2008;84:981–987. doi: 10.1189/jlb.1107773. PubMed DOI

Angell H.K., Bruni D., Barrett J.C., Herbst R., Galon J. The Immunoscore: Colon Cancer and Beyond. Clin. Cancer Res. 2020;26:332–339. doi: 10.1158/1078-0432.CCR-18-1851. PubMed DOI

Galon J., Bruni D. Tumor Immunology and Tumor Evolution: Intertwined Histories. Immunity. 2020;52:55–81. doi: 10.1016/j.immuni.2019.12.018. PubMed DOI

Kirilovsky A., Marliot F., El Sissy C., Haicheur N., Galon J., Pages F. Rational bases for the use of the Immunoscore in routine clinical settings as a prognostic and predictive biomarker in cancer patients. Int. Immunol. 2016;28:373–382. doi: 10.1093/intimm/dxw021. PubMed DOI PMC

Pages F., Mlecnik B., Marliot F., Bindea G., Ou F.S., Bifulco C., Lugli A., Zlobec I., Rau T.T., Berger M.D., et al. International validation of the consensus Immunoscore for the classification of colon cancer: A prognostic and accuracy study. Lancet. 2018;391:2128–2139. doi: 10.1016/S0140-6736(18)30789-X. PubMed DOI

Zhang X., Yang J., Du L., Zhou Y., Li K. The prognostic value of Immunoscore in patients with cancer: A pooled analysis of 10,328 patients. Int. J. Biol. Markers. 2020;35:1724600820927409. doi: 10.1177/1724600820927409. PubMed DOI

Pages F., Andre T., Taieb J., Vernerey D., Henriques J., Borg C., Marliot F., Ben Jannet R., Louvet C., Mineur L., et al. Prognostic and predictive value of the Immunoscore in stage III colon cancer patients treated with oxaliplatin in the prospective IDEA France PRODIGE-GERCOR cohort study. Ann. Oncol. 2020;31:921–929. doi: 10.1016/j.annonc.2020.03.310. PubMed DOI

Sinicrope F.A., Shi Q., Hermitte F., Zemla T.J., Mlecnik B., Benson A.B., Gill S., Goldberg R.M., Kahlenberg M.S., Nair S.G., et al. Contribution of Immunoscore and Molecular Features to Survival Prediction in Stage III Colon Cancer. JNCI Cancer Spectr. 2020;4:pkaa023. doi: 10.1093/jncics/pkaa023. PubMed DOI PMC

Mlecnik B., Bifulco C., Bindea G., Marliot F., Lugli A., Lee J.J., Zlobec I., Rau T.T., Berger M.D., Nagtegaal I.D., et al. Multicenter International Society for Immunotherapy of Cancer Study of the Consensus Immunoscore for the Prediction of Survival and Response to Chemotherapy in Stage III Colon Cancer. J. Clin. Oncol. 2020;38:3638–3651. doi: 10.1200/JCO.19.03205. PubMed DOI PMC

Imen H., Amira H., Fatma K., Raja J., Mariem S., Haithem Z., Ehsene B.B., Aschraf C. Prognostic Value of Immunoscore in Colorectal Carcinomas. Int. J. Surg. Pathol. 2023;25:10668969231168357. doi: 10.1177/10668969231168357. PubMed DOI

Marliot F., Pages F., Galon J. Usefulness and robustness of Immunoscore for personalized management of cancer patients. Oncoimmunology. 2020;9:1832324. doi: 10.1080/2162402X.2020.1832324. PubMed DOI PMC

Mlecnik B., Lugli A., Bindea G., Marliot F., Bifulco C., Lee J.J., Zlobec I., Rau T.T., Berger M.D., Nagtegaal I.D., et al. Multicenter International Study of the Consensus Immunoscore for the Prediction of Relapse and Survival in Early-Stage Colon Cancer. Cancers. 2023;15:418. doi: 10.3390/cancers15020418. PubMed DOI PMC

Mlecnik B., Torigoe T., Bindea G., Popivanova B., Xu M., Fujita T., Hazama S., Suzuki N., Nagano H., Okuno K., et al. Clinical Performance of the Consensus Immunoscore in Colon Cancer in the Asian Population from the Multicenter International SITC Study. Cancers. 2022;14:4346. doi: 10.3390/cancers14184346. PubMed DOI PMC

Trabelsi M., Farah F., Zouari B., Jaafoura M.H., Kharrat M. An Immunoscore System Based on CD3+ And CD8+ Infiltrating Lymphocytes Densities to Predict the Outcome of Patients with Colorectal Adenocarcinoma. Onco Targets Ther. 2019;12:8663–8673. doi: 10.2147/OTT.S211048. PubMed DOI PMC

Wang F., Lu S., Cao D., Qian J., Li C., Zhang R., Wang F., Wu M., Liu Y., Pan Z., et al. Prognostic and predictive value of Immunoscore and its correlation with ctDNA in stage II colorectal cancer. Oncoimmunology. 2023;12:2161167. doi: 10.1080/2162402X.2022.2161167. PubMed DOI PMC

Argilés G., Tabernero J., Labianca R., Hochhauser D., Salazar R., Iveson T., Laurent-Puig P., Quirke P., Yoshino T., Taieb J., et al. Localised colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 2020;31:1291–1305. doi: 10.1016/j.annonc.2020.06.022. PubMed DOI

Yoshino T., Argilés G., Oki E., Martinelli E., Taniguchi H., Arnold D., Mishima S., Li Y., Smruti B.K., Ahn J.B., et al. Pan-Asian adapted ESMO Clinical Practice Guidelines for the diagnosis treatment and follow-up of patients with localised colon cancer. Ann. Oncol. 2021;32:1496–1510. doi: 10.1016/j.annonc.2021.08.1752. PubMed DOI

Boquet I., Kassambara A., Lui A., Tanner A., Latil M., Lovera Y., Arnoux F., Hermitte F., Galon J., Catteau A. Comparison of Immune Response Assessment in Colon Cancer by Immunoscore (Automated Digital Pathology) and Pathologist Visual Scoring. Cancers. 2022;14:1170. doi: 10.3390/cancers14051170. PubMed DOI PMC

McHugh M.L. Interrater reliability: The kappa statistic. Biochem. Med. 2012;22:276–282. doi: 10.11613/BM.2012.031. PubMed DOI PMC

Reisenbichler E.S., Han G., Bellizzi A., Bossuyt V., Brock J., Cole K., Fadare O., Hameed O., Hanley K., Harrison B.T., et al. Prospective multi-institutional evaluation of pathologist assessment of PD-L1 assays for patient selection in triple negative breast cancer. Mod. Pathol. 2020;33:1746–1752. doi: 10.1038/s41379-020-0544-x. PubMed DOI PMC

Ascierto P.A., Capone M., Urba W.J., Bifulco C.B., Botti G., Lugli A., Marincola F.M., Ciliberto G., Galon J., Fox B.A. The additional facet of immunoscore: Immunoprofiling as a possible predictive tool for cancer treatment. J. Transl. Med. 2013;11:54. doi: 10.1186/1479-5876-11-54. PubMed DOI PMC

Galon J., Bruni D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat. Rev. Drug Discov. 2019;18:197–218. doi: 10.1038/s41573-018-0007-y. PubMed DOI

Galon J., Mlecnik B., Marliot F., Ou F.S., Bifulco C.B., Lugli A., Zlobec I., Rau T., Hartmann A., Masucci G., et al. Validation of the Immunoscore (IM) as a prognostic marker in stage I/II/III colon cancer: Results of a worldwide consortium-based analysis of 1336 patients. J. Clin. Oncol. 2016;34:S3500. doi: 10.1200/JCO.2016.34.15_suppl.3500. DOI

Fridman W.H., Dieu-Nosjean M.C., Pages F., Cremer I., Damotte D., Sautes-Fridman C., Galon J. The immune microenvironment of human tumors: General significance and clinical impact. Cancer Microenviron. 2013;6:117–122. doi: 10.1007/s12307-012-0124-9. PubMed DOI PMC

Marliot F., Chen X., Kirilovsky A., Sbarrato T., El Sissy C., Batista L., Van den Eynde M., Haicheur-Adjouri N., Anitei M.G., Musina A.M., et al. Analytical validation of the Immunoscore and its associated prognostic value in patients with colon cancer. J. Immunother. Cancer. 2020;8:e000272. doi: 10.1136/jitc-2019-000272. PubMed DOI PMC

Butter R., Hondelink L.M., van Elswijk L., Blaauwgeers J.L.G., Bloemena E., Britstra R., Bulkmans N., van Gulik A.L., Monkhorst K., de Rooij M.J., et al. The impact of a pathologist’s personality on the interobserver variability and diagnostic accuracy of predictive PD-L1 immunohistochemistry in lung cancer. Lung Cancer. 2022;166:143–149. doi: 10.1016/j.lungcan.2022.03.002. PubMed DOI

Aranda F., Vacchelli E., Eggermont A., Galon J., Fridman W.H., Zitvogel L., Kroemer G., Galluzzi L. Trial Watch: Immunostimulatory monoclonal antibodies in cancer therapy. Oncoimmunology. 2014;3:e27297. doi: 10.4161/onci.27297. PubMed DOI PMC

Aranda F., Vacchelli E., Eggermont A., Galon J., Sautes-Fridman C., Tartour E., Zitvogel L., Kroemer G., Galluzzi L. Trial Watch: Peptide vaccines in cancer therapy. Oncoimmunology. 2013;2:e26621. doi: 10.4161/onci.26621. PubMed DOI PMC

Buque A., Bloy N., Aranda F., Castoldi F., Eggermont A., Cremer I., Fridman W.H., Fucikova J., Galon J., Marabelle A., et al. Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications. Oncoimmunology. 2015;4:e1008814. doi: 10.1080/2162402X.2015.1008814. PubMed DOI PMC

Galluzzi L., Vacchelli E., Fridman W.H., Galon J., Sautes-Fridman C., Tartour E., Zucman-Rossi J., Zitvogel L., Kroemer G. Trial Watch: Monoclonal antibodies in cancer therapy. Oncoimmunology. 2012;1:28–37. doi: 10.4161/onci.1.1.17938. PubMed DOI PMC

Galon J., Angell H.K., Bedognetti D., Marincola F.M. The continuum of cancer immunosurveillance: Prognostic, predictive, and mechanistic signatures. Immunity. 2013;39:11–26. doi: 10.1016/j.immuni.2013.07.008. PubMed DOI

Pol J., Bloy N., Buque A., Eggermont A., Cremer I., Sautes-Fridman C., Galon J., Tartour E., Zitvogel L., Kroemer G., et al. Trial Watch: Peptide-based anticancer vaccines. Oncoimmunology. 2015;4:e974411. doi: 10.4161/2162402X.2014.974411. PubMed DOI PMC

Pol J., Bloy N., Obrist F., Eggermont A., Galon J., Cremer I., Erbs P., Limacher J.M., Preville X., Zitvogel L., et al. Trial Watch:: Oncolytic viruses for cancer therapy. Oncoimmunology. 2014;3:e28694. doi: 10.4161/onci.28694. PubMed DOI PMC

Vacchelli E., Eggermont A., Fridman W.H., Galon J., Tartour E., Zitvogel L., Kroemer G., Galluzzi L. Trial Watch: Adoptive cell transfer for anticancer immunotherapy. Oncoimmunology. 2013;2:e24238. doi: 10.4161/onci.24238. PubMed DOI PMC

Vacchelli E., Eggermont A., Fridman W.H., Galon J., Zitvogel L., Kroemer G., Galluzzi L. Trial Watch: Immunostimulatory cytokines. Oncoimmunology. 2013;2:e24850. doi: 10.4161/onci.24850. PubMed DOI PMC

Vacchelli E., Eggermont A., Galon J., Sautes-Fridman C., Zitvogel L., Kroemer G., Galluzzi L. Trial watch: Monoclonal antibodies in cancer therapy. Oncoimmunology. 2013;2:e22789. doi: 10.4161/onci.22789. PubMed DOI PMC

Vacchelli E., Eggermont A., Sautes-Fridman C., Galon J., Zitvogel L., Kroemer G., Galluzzi L. Trial watch: Oncolytic viruses for cancer therapy. Oncoimmunology. 2013;2:e24612. doi: 10.4161/onci.24612. PubMed DOI PMC

Vacchelli E., Galluzzi L., Fridman W.H., Galon J., Sautes-Fridman C., Tartour E., Kroemer G. Trial watch: Chemotherapy with immunogenic cell death inducers. Oncoimmunology. 2012;1:179–188. doi: 10.4161/onci.1.2.19026. PubMed DOI PMC

Iribarren K., Bloy N., Buque A., Cremer I., Eggermont A., Fridman W.H., Fucikova J., Galon J., Spisek R., Zitvogel L., et al. Trial Watch: Immunostimulation with Toll-like receptor agonists in cancer therapy. Oncoimmunology. 2016;5:e1088631. doi: 10.1080/2162402X.2015.1088631. PubMed DOI PMC

Pol J., Buque A., Aranda F., Bloy N., Cremer I., Eggermont A., Erbs P., Fucikova J., Galon J., Limacher J.M., et al. Trial Watch-Oncolytic viruses and cancer therapy. Oncoimmunology. 2016;5:e1117740. doi: 10.1080/2162402X.2015.1117740. PubMed DOI PMC

Scholler N., Perbost R., Locke F.L., Jain M.D., Turcan S., Danan C., Chang E.C., Neelapu S.S., Miklos D.B., Jacobson C.A., et al. Tumor immune contexture is a determinant of anti-CD19 CAR T cell efficacy in large B cell lymphoma. Nat. Med. 2022;28:1872–1882. doi: 10.1038/s41591-022-01916-x. PubMed DOI PMC

Vacchelli E., Senovilla L., Eggermont A., Fridman W.H., Galon J., Zitvogel L., Kroemer G., Galluzzi L. Trial watch: Chemotherapy with immunogenic cell death inducers. Oncoimmunology. 2013;2:e23510. doi: 10.4161/onci.23510. PubMed DOI PMC

Marliot F., Lafontaine L., Galon J. Immunoscore assay for the immune classification of solid tumors: Technical aspects, improvements and clinical perspectives. Methods Enzymol. 2020;636:109–128. doi: 10.1016/bs.mie.2019.07.018. PubMed DOI

Antoniotti C., Rossini D., Pietrantonio F., Catteau A., Salvatore L., Lonardi S., Boquet I., Tamberi S., Marmorino F., Moretto R., et al. Upfront FOLFOXIRI plus bevacizumab with or without atezolizumab in the treatment of patients with metastatic colorectal cancer (AtezoTRIBE): A multicentre, open-label, randomised, controlled, phase 2 trial. Lancet Oncol. 2022;23:876–887. doi: 10.1016/S1470-2045(22)00274-1. PubMed DOI

Ghiringhelli F., Bibeau F., Greillier L., Fumet J.D., Ilie A., Monville F., Lauge C., Catteau A., Boquet I., Majdi A., et al. Immunoscore immune checkpoint using spatial quantitative analysis of CD8 and PD-L1 markers is predictive of the efficacy of anti-PD1/PD-L1 immunotherapy in non-small cell lung cancer. EBioMedicine. 2023;92:104633. doi: 10.1016/j.ebiom.2023.104633. PubMed DOI PMC

Pagès F., Taieb J., Laurent-Puig P., Galon J. The consensus Immunoscore in phase 3 clinical trials; potential impact on patient management decisions. Oncoimmunology. 2020;9:1812221. doi: 10.1080/2162402X.2020.1812221. PubMed DOI PMC