BACKGROUND: T cell density in colorectal cancer (CRC) has proven to be of high prognostic importance. Here, we evaluated the influence of a hyperfractionated preoperative short-term radiation protocol (25 Gy) on immune cell density in tumor samples of rectal cancer (RC) patients and on patient survival. In addition, we assessed spatial tumor heterogeneity by comparison of analogue T cell quantification on full tissue sections with digital T cell quantification on a virtually established tissue microarray (TMA). METHODS: A total of 75 RC patients (60 irradiated, 15 treatment-naïve) were defined for retrospective analysis. RC samples were processed for immunohistochemistry (CD3, CD8, PD-1, PD-L1). Analogue (score 0-3) as well as digital quantification (TMA: 2 cores vs. 6 cores, mean T cell count) of marker expression in 2 areas (central tumor, CT; invasive margin, IM) was performed. Survival was estimated on the basis of analogue as well as digital marker densities calculated from 2 cores (Immunoscore: CD3/CD8 ratio) and 6 cores per tumor area. RESULTS: Irradiated RC samples showed a significant decrease in CD3 and CD8 positive T cells, independent of quantification mode. T cell densities of 6 virtual cores approximated to T cell densities of full tissue sections, independent of individual core density or location. Survival analysis based on full tissue section quantification demonstrated that CD3 and CD8 positive T cells as well as PD-1 positive tumor infiltrating leucocytes (TILs) in the CT and the IM had a significant impact on disease-free survival (DFS) as well as overall survival (OS). In addition, CD3 and CD8 positive T cells as well as PD-1 positive TILs in the IM proved as independent prognostic factors for DFS and OS; in the CT, PD-1 positive TILs predicted DFS and CD3 and CD8 positive T cells as well as PD-1 positive TILs predicted OS. Survival analysis based on virtual TMA showed no impact on DFS or OS. CONCLUSION: Spatial tumor heterogeneity might result in inadequate quantification of immune marker expression; however, if using a TMA, 6 cores per tumor area and patient sample represent comparable amounts of T cell densities to those quantified on full tissue sections. Consistently, the tissue area used for immune marker quantification represents a crucial factor for the evaluation of prognostic and predictive biomarker potential.

CBmed Vienna Medical University of Vienna 1090 Vienna Austria

Central European Institute of Technology Masaryk University 602 00 Brno Czech Republic

Christian Doppler Laboratory for Applied Metabolomics Division of Nuclear Medicine Medical University of Vienna 1090 Vienna Austria

Comprehensive Cancer Center Medical University of Vienna 1090 Vienna Austria

Department of Experimental and Translational Pathology Clinical Institute of Pathology Medical University of Vienna 1090 Vienna Austria

Department of Pathology University of Cambridge Cambridge CB2 1QP UK

Department of Radiation Oncology Christian Doppler Laboratory for Medical Radiation Research for Radiation Oncology Comprehensive Cancer Center Medical University of Vienna 1090 Vienna Austria

Department of Surgery Medical University of Vienna 1090 Vienna Austria

Division of General Surgery Department of Surgery Medical University of Vienna 1090 Vienna Austria

INNPATH GmbH Tyrol Clinics Innsbruck 6020 Innsbruck Tyrol Austria

TissueGnostics Austria Global Headquarter TissueGnostics GmbH Vienna 1020 Vienna Austria

Unit of Laboratory Animal Pathology University of Veterinary Medicine Vienna 1210 Vienna Austria

See more in PubMed

Herter-Sprie G.S., Koyama S., Korideck H., Hai J., Deng J., Li Y.Y., Buczkowski K.A., Grant A.K., Ullas S., Rhee K., et al. Synergy of radiotherapy and PD-1 blockade in Kras-mutant lung cancer. JCI Insight. 2016:1. doi: 10.1172/jci.insight.87415. PubMed DOI PMC

Galluzzi L., Zitvogel L., Kroemer G. Immunological Mechanisms Underneath the Efficacy of Cancer Therapy. Cancer Immunol. Res. 2016;4:895–902. doi: 10.1158/2326-6066.CIR-16-0197. PubMed DOI

Golden E.B., Apetoh L. Radiotherapy and Immunogenic Cell Death. Semin. Radiat. Oncol. 2015;25:11–17. doi: 10.1016/j.semradonc.2014.07.005. PubMed DOI

Dovedi S.J., Adlard A.L., Lipowska-Bhalla G., McKenna C., Jones S., Cheadle E.J., Stratford I.J., Poon E., Morrow M., Stewart R., et al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014;74:5458–5468. doi: 10.1158/0008-5472.CAN-14-1258. PubMed DOI

Galluzzi L., Kepp O., Kroemer G. Immunogenic cell death in radiation therapy. OncoImmunology. 2013;2:e26536. doi: 10.4161/onci.26536. PubMed DOI PMC

Demaria S., Kawashima N., Yang A.M., Devitt M.L., Babb J.S., Allison J.P., Formenti S.C. Immune-Mediated Inhibition of Metastases after Treatment with Local Radiation and CTLA-4 Blockade in a Mouse Model of Breast Cancer. Clin. Cancer Res. 2005;11:728. PubMed

Stone H., Lester J., Milas L. Effect of Host Immune Capability on Radiocurability and Subsequent Transplantability of a Murine Fibrosarcoma. J. Natl. Cancer Inst. 1979;63:1229–1235. PubMed

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

Mellman I., Coukos G., Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480:480–489. doi: 10.1038/nature10673. PubMed DOI PMC

Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science. 2013;342:1432–1433. doi: 10.1126/science.342.6165.1432. PubMed DOI

Llosa N.J., Cruise M., Tam A., Wicks E.C., Hechenbleikner E.M., Taube J.M., Blosser R.L., Fan H., Wang H., Luber B.S., et al. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov. 2015;5:43–51. doi: 10.1158/2159-8290.CD-14-0863. PubMed DOI PMC

Angelova M., Charoentong P., Hackl H., Fischer M.L., Snajder R., Krogsdam A.M., Waldner M.J., Bindea G., Mlecnik B., Galon J., et al. Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy. Genome Biol. 2015;16:64. doi: 10.1186/s13059-015-0620-6. PubMed DOI PMC

Le D.T., Uram J.N., Wang H., Bartlett B.R., Kemberling H., Eyring A.D., Skora A.D., Luber B.S., Azad N.S., Laheru D., et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl. J. Med. 2015;372:2509–2520. doi: 10.1056/NEJMoa1500596. PubMed DOI PMC

Haikerwal S.J., Hagekyriakou J., MacManus M., Martin O.A., Haynes N.M. Building immunity to cancer with radiation therapy. Cancer Lett. 2015;368:198–208. doi: 10.1016/j.canlet.2015.01.009. PubMed DOI

Kang J., Demaria S., Formenti S. Current clinical trials testing the combination of immunotherapy with radiotherapy. J. Immunother Cancer. 2016;4:51. doi: 10.1186/s40425-016-0156-7. PubMed DOI PMC

Teng F., Meng X., Kong L., Mu D., Zhu H., Liu S., Zhang J., Yu J. Tumor-infiltrating lymphocytes, forkhead box P3, programmed death ligand-1, and cytotoxic T lymphocyte-associated antigen-4 expressions before and after neoadjuvant chemoradiation in rectal cancer. Transl. Res. 2015;166:721–732. doi: 10.1016/j.trsl.2015.06.019. PubMed DOI

Teng F., Mu D., Meng X., Kong L., Zhu H., Liu S., Zhang J., Yu J. Tumor infiltrating lymphocytes (TILs) before and after neoadjuvant chemoradiotherapy and its clinical utility for rectal cancer. Am. J. Cancer Res. 2015;5:2064–2074. PubMed PMC

Anitei M.G., Zeitoun G., Mlecnik B., Marliot F., Haicheur N., Todosi A.M., Kirilovsky A., Lagorce C., Bindea G., Ferariu D., et al. Prognostic and predictive values of the immunoscore in patients with rectal cancer. Clin Cancer Res. 2014;20:1891–1899. doi: 10.1158/1078-0432.CCR-13-2830. PubMed DOI

Filatenkov A., Baker J., Mueller A.M., Kenkel J., Ahn G.O., Dutt S., Zhang N., Kohrt H., Jensen K., Dejbakhsh-Jones S., et al. Ablative Tumor Radiation Can Change the Tumor Immune Cell Microenvironment to Induce Durable Complete Remissions. Clin. Cancer Res. 2015;21:3727–3739. doi: 10.1158/1078-0432.CCR-14-2824. PubMed DOI PMC

Bhargava R., Lal P., Chen B. Feasibility of Using Tissue Microarrays for the Assessment of HER-2 Gene Amplification by Fluorescence In Situ Hybridization in Breast Carcinoma. Diagn. Mol. Pathol. 2004:13. doi: 10.1097/01.pdm.0000140195.05428.1d. PubMed DOI

Derks S., Nason K.S., Liao X., Stachler M.D., Liu K.X., Liu J.B., Sicinska E., Goldberg M.S., Freeman G.J., Rodig S.J., et al. Epithelial PD-L2 Expression Marks Barrett’s Esophagus and Esophageal Adenocarcinoma. Cancer Immunol. Res. 2015;3:1123–1129. doi: 10.1158/2326-6066.CIR-15-0046. PubMed DOI PMC

Dislich B., Stein A., Seiler C.A., Kroll D., Berezowska S., Zlobec I., Galvan J., Slotta-Huspenina J., Walch A., Langer R. Expression patterns of programmed death-ligand 1 in esophageal adenocarcinomas: comparison between primary tumors and metastases. Cancer Immunol. Immunother. 2017;66:777–786. doi: 10.1007/s00262-017-1982-2. PubMed DOI PMC

Donati V., Faviana P., Dell’Omodarme M., Prati M., Camacci T., De Ieso K., Giannini R., Lucchi M., Mussi A., Pingitore R., et al. Applications of tissue microarray technology in immunohistochemistry: A study on c-kit expression in small cell lung cancer. Hum. Pathol. 2004;35:1347–1352. doi: 10.1016/j.humpath.2004.07.016. PubMed DOI

Goethals L., Perneel C., Debucquoy A., De Schutter H., Borghys D., Ectors N., Geboes K., McBride W., Haustermans K. A new approach to the validation of tissue microarrays. J. Pathol. 2006;208:607–614. doi: 10.1002/path.1934. PubMed DOI

Kononen J., Bubendorf L., Kallioniemi A., Barlund M., Schraml P., Leighton S., Torhorst J., Mihatsch M.J., Sauter G., Kallioniemi O.P. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat. Med. 1998;4:844–847. doi: 10.1038/nm0798-844. PubMed DOI

Ruiz C., Seibt S., Kuraya K.A., Siraj A.K., Mirlacher M., Schraml P., Maurer R., Spichtin H., Torhorst J., Popovska S., et al. Tissue microarrays for comparing molecular features with proliferation activity in breast cancer. Int. J. Cancer. 2006;118:2190–2194. doi: 10.1002/ijc.21581. PubMed DOI

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

Masugi Y., Nishihara R., Yang J., Mima K., da Silva A., Shi Y., Inamura K., Cao Y., Song M., Nowak J.A., et al. Tumour CD274 (PD-L1) expression and T cells in colorectal cancer. Gut. 2017;66:1463–1473. doi: 10.1136/gutjnl-2016-311421. PubMed DOI PMC

Halama N., Spille A., Lerchl T., Brand K., Herpel E., Welte S., Keim S., Lahrmann B., Klupp F., Kahlert C., et al. Hepatic metastases of colorectal cancer are rather homogeneous but differ from primary lesions in terms of immune cell infiltration. Oncoimmunology. 2013;2:e24116. doi: 10.4161/onci.24116. PubMed DOI PMC

Widder J., Herbst F., Dobrowsky W., Schmid R., Pokrajac B., Jech B., Chiari C., Stift A., Maier A., Karner-Hanusch J., et al. Preoperative short-term radiation therapy (25 Gy, 2.5 Gy twice daily) for primary resectable rectal cancer (phase II) Br. J. Cancer. 2005;92:1209–1214. doi: 10.1038/sj.bjc.6602485. PubMed DOI PMC

Pagès 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

Halama N., Zoernig I., Spille A., Michel S., Kloor M., Grauling-Halama S., Westphal K., Schirmacher P., Jäger D., Grabe N. Quantification of Prognostic Immune Cell Markers in Colorectal Cancer Using Whole Slide Imaging Tumor Maps. Anal. Quant. Cytol. Histol. 2010;32:333–340. PubMed

Hekim N., Cetin Z., Nikitaki Z., Cort A., Saygili E.I. Radiation triggering immune response and inflammation. Cancer Lett. 2015;368:156–163. doi: 10.1016/j.canlet.2015.04.016. PubMed DOI

Frey B., Rubner Y., Kulzer L., Werthmoller N., Weiss E.M., Fietkau R., Gaipl U.S. Antitumor immune responses induced by ionizing irradiation and further immune stimulation. Cancer Immunol. Immunother. 2014;63:29–36. doi: 10.1007/s00262-013-1474-y. PubMed DOI PMC

Shinto E., Hase K., Hashiguchi Y., Sekizawa A., Ueno H., Shikina A., Kajiwara Y., Kobayashi H., Ishiguro M., Yamamoto J. CD8+ and FOXP3+ tumor-infiltrating T cells before and after chemoradiotherapy for rectal cancer. Ann. Surg. Oncol. 2014;21(Suppl. 3):S414–421. doi: 10.1245/s10434-014-3584-y. PubMed DOI

Shao L., Peng Q., Du K., He J., Dong Y., Lin X., Li J., Wu J. Tumor cell PD-L1 predicts poor local control for rectal cancer patients following neoadjuvant radiotherapy. Cancer Manag. Res. 2017;9:249–258. doi: 10.2147/CMAR.S139889. PubMed DOI PMC

Saigusa S., Toiyama Y., Tanaka K., Inoue Y., Mori K., Ide S., Imaoka H., Kawamura M., Mohri Y., Kusunoki M. Implication of programmed cell death ligand 1 expression in tumor recurrence and prognosis in rectal cancer with neoadjuvant chemoradiotherapy. Int. J. Clin. Oncol. 2016;21:946–952. doi: 10.1007/s10147-016-0962-4. PubMed DOI

Hecht M., Buttner-Herold M., Erlenbach-Wunsch K., Haderlein M., Croner R., Grutzmann R., Hartmann A., Fietkau R., Distel L.V. PD-L1 is upregulated by radiochemotherapy in rectal adenocarcinoma patients and associated with a favourable prognosis. Eur. J. Cancer. 2016;65:52–60. doi: 10.1016/j.ejca.2016.06.015. PubMed DOI

Jomrich G., Silberhumer G.R., Marian B., Beer A., Mullauer L. Programmed death-ligand 1 expression in rectal cancer. Eur. Surg. 2016;48:352–356. doi: 10.1007/s10353-016-0447-8. PubMed DOI PMC

Yang L., Xue R., Pan C. Prognostic and clinicopathological value of PD-L1 in colorectal cancer: a systematic review and meta-analysis. Onco. Targets. 2019;12:3671–3682. doi: 10.2147/OTT.S190168. PubMed DOI PMC

The R Core Team . A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; Vienna, Austria: 2018.

Therneau T.M., Grambsch P.M. Modeling Survival Data: Extending the Cox Model. Springer; New York, NY, USA: 2000. pp. 39–77.

Venables W.N., Ripley B.D. Modern Applied Statistics with S-PLUS. Springer; New York, NY, USA: 2002. p. 548.

Nolte S., Zlobec I., Lugli A., Hohenberger W., Croner R., Merkel S., Hartmann A., Geppert C.I., Rau T.T. Construction and analysis of tissue microarrays in the era of digital pathology: a pilot study targeting CDX1 and CDX2 in a colon cancer cohort of 612 patients. J. Pathol. Clin. Res. 2017;3:58–70. doi: 10.1002/cjp2.62. PubMed DOI PMC

Gorris M.A.J., Halilovic A., Rabold K., van Duffelen A., Wickramasinghe I.N., Verweij D., Wortel I.M.N., Textor J.C., de Vries I.J.M., Figdor C.G. Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment. J. Immunol. 2018;200:347–354. doi: 10.4049/jimmunol.1701262. PubMed DOI

Steiner C., Ducret A., Tille J.C., Thomas M., McKee T.A., Rubbia-Brandt L., Scherl A., Lescuyer P., Cutler P. Applications of mass spectrometry for quantitative protein analysis in formalin-fixed paraffin-embedded tissues. Proteomics. 2014;14:441–451. doi: 10.1002/pmic.201300311. PubMed DOI PMC