CD8+ and Regulatory T cells Differentiate Tumor Immune Phenotypes and Predict Survival in Locally Advanced Head and Neck Cancer

Alessia Echarti, Markus Hecht, Maike Büttner-Herold, Marlen Haderlein, Arndt Hartmann, Rainer Fietkau, Luitpold Distel, Alessia Echarti, Markus Hecht, Maike Büttner-Herold, Marlen Haderlein, Arndt Hartmann, Rainer Fietkau, Luitpold Distel

Abstract

Background: The tumor immune status "inflamed", "immune excluded", and "desert" might serve as a predictive parameter. We studied these three cancer immune phenotypes while using a simple immunohistochemical algorithm.

Methods: Pre-treatment tissue samples of 280 patients with locally advanced HNSCC treated with radiochemotherapy were analyzed. A double staining of CD8+ cytotoxic T cells (CTL) and FoxP3+ (Treg) was performed and the cell density was evaluated in the intraepithelial and stromal compartment of the tumor.

Results: The classification of tumors as "immune desert" when stromal CTL were ≤ 50 cells/mm2, "inflamed" when intraepithelial CTL were > 500 cells/mm2, and as "excluded" when neither of these definitions met these cut off values allowed the best discrimination regarding overall survival. These groups had median OS periods of 37, 61, and 85 months, respectively. In "immune desert" and "immune excluded" tumors high Treg tended to worsen OS, but in "inflamed" tumors high Treg clearly improved OS.

Conclusions: We propose that, in locally advanced HNSCC, the tumor immune state "inflamed", "immune excluded", and "immune desert" can be defined by intraepithelial and stromal CTL. Tregs can further subdivide these groups. The opposing effects of Tregs in the different groups might be the reason for the inconsistency of Tregs prognostic values published earlier.

Keywords: CD8+; FoxP3+; excluded; head and neck squamous cell carcinoma; immune dessert; inflamed; regulatory T cells.

Conflict of interest statement

M.H. (Markus Hecht) participated in advisory boards for MSD, Merck Serono and BMS. M.H. (Markus Hecht) has received research support from MSD, AstraZeneca and Novartis. M.H. (Markus Hecht) received travel grants from Merck Serono, MSD and Teva. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CD8+ and FoxP3+ cell densities in head and neck cancer. Kaplan Meier plots for metastasis free rate, recurrence free rate and overall survival in the complete cohort of 280 patients (A). Tissue samples were processed into tissue microarrays using a core diameter of 2 mm. Scale bar 200 µm (B). High power views (1:400) with immunohistochemical double staining for FoxP3 (red nucleic staining) and CD8 (blue predominantly membranous staining). (Scale bars 10 µm) (C). Lymphocyte densities (cells/mm²) in the intraepithelial and stromal compartment were separately analyzed (D). Stromal/intraepithelial ratio of CD8+ and FoxP3+ cells (E). CD8+/FoxP3+ ratio in intraepithelial and stromal compartment (F). Kaplan Meier plots for densities of CD8+ lymphocytes in the stromal (G) and intraepithelial H as well as FoxP3+ cells in the stromal (J) and epithelial (K) compartment (overall survival). Cut-off values were the median.
Figure 2
Figure 2
Subgroups “immune desert”, “immune excluded” and “inflamed”. “Immune desert” was defined as having ≤ 50 CD8+ cells/mm2 in the stromal compartment, “immune excluded” ≤ 500 CD8+ cells in the intraepithelial and > 50 in the stromal compartment and “inflamed” having > 500 cells in the intraepithelial compartment. Kaplan Meier plots for overall survival in these groups (A) Tumor size (B) p16 staining (C) and tumor grade (D) were compared. Kaplan-Meier plots for the FoxP3+ cell densities in the stromal and intraepithelial compartment in the “immune desert” group (E,F), in the “immune excluded” group (G,H), and the “inflamed” group (J,K) Cut-off values were median FoxP3+ densities.
Figure 3
Figure 3
Lymphocyte densities and clinical characteristics in the “immune desert”, “immune excluded” and “inflamed” groups. Lymphocyte densities (cells mm−2) in the intraepithelial and stromal compartment of the “immune desert”. (A) the “immune excluded”. (B) and the “inflamed” group. (C) Tumor size. (D) N stage E and tumor grade. (F) in the three subgroups depending on the FoxP3+ densities are depicted. Cut-off values were the median FoxP3+ densities.

References

    1. Jenkins R.W., Barbie D.A., Flaherty K.T. Mechanisms of resistance to immune checkpoint inhibitors. Br. J. Cancer. 2018;118:9–16. doi: 10.1038/bjc.2017.434.
    1. Granier C., De Guillebon E., Blanc C., Roussel H., Badoual C., Colin E., Saldmann A., Gey A., Oudard S., Tartour E. Mechanisms of action and rationale for the use of checkpoint inhibitors in cancer. ESMO Open. 2017;2:e000213. doi: 10.1136/esmoopen-2017-000213.
    1. Ferris R.L., Blumenschein G., Jr., Fayette J., Guigay J., Colevas A.D., Licitra L., Harrington K., Kasper S., Vokes E.E., Even C., et al. Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. N. Engl. J. Med. 2016;375:1856–1867. doi: 10.1056/NEJMoa1602252.
    1. Burtness B., Harrington K.J., Greil R., Soulières D., Tahara M., Castro G.D., Psyrri A., Baste Rotllan N., Neupane P.C., Bratland A., et al. KEYNOTE-048: Phase III study of first-line pembrolizumab (P) for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) Ann. Oncol. 2018;29(Suppl. 8) doi: 10.1093/annonc/mdy424.045.
    1. Tumeh P.C., Harview C.L., Yearley J.H., Shintaku I.P., Taylor E.J., Robert L., Chmielowski B., Spasic M., Henry G., Ciobanu V., et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515:568–571. doi: 10.1038/nature13954.
    1. Chen D.S., Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature. 2017;541:321–330. doi: 10.1038/nature21349.
    1. Kansy B.A., Concha-Benavente F., Srivastava R.M., Jie H.B., Shayan G., Lei Y., Moskovitz J., Moy J., Li J., Brandau S., et al. PD-1 Status in CD8(+) T Cells Associates with Survival and Anti-PD-1 Therapeutic Outcomes in Head and Neck Cancer. Cancer Res. 2017;77:6353–6364. doi: 10.1158/0008-5472.CAN-16-3167.
    1. Hu-Lieskovan S., Lisberg A., Zaretsky J.M., Grogan T.R., Rizvi H., Wells D.K., Carroll J., Cummings A., Madrigal J., Jones B., et al. Tumor Characteristics Associated with Benefit from Pembrolizumab in Advanced Non-Small Cell Lung Cancer. Clin. Cancer Res. 2019;25:5061–5068. doi: 10.1158/1078-0432.CCR-18-4275.
    1. Badoual C., Hans S., Rodriguez J., Peyrard S., Klein C., Agueznay Nel H., Mosseri V., Laccourreye O., Bruneval P., Fridman W.H., et al. Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers. Clin. Cancer Res. 2006;12:465–472. doi: 10.1158/1078-0432.CCR-05-1886.
    1. Correale P., Rotundo M.S., Del Vecchio M.T., Remondo C., Migali C., Ginanneschi C., Tsang K.Y., Licchetta A., Mannucci S., Loiacono L., et al. Regulatory (FoxP3+) T-cell tumor infiltration is a favorable prognostic factor in advanced colon cancer patients undergoing chemo or chemoimmunotherapy. J. Immunother. 2010;33:435–441. doi: 10.1097/CJI.0b013e3181d32f01.
    1. Salama P., Phillips M., Grieu F., Morris M., Zeps N., Joseph D., Platell C., Iacopetta B. Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. J. Clin. Oncol. 2009;27:186–192. doi: 10.1200/JCO.2008.18.7229.
    1. Nagl S., Haas M., Lahmer G., Büttner-Herold M., Grabenbauer G.G., Fietkau R., Distel L. Cell-to-cell distances between tumour infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells. Oncoimmunology. 2016;5:e1127494. doi: 10.1080/2162402X.2015.1127494.
    1. Shang B., Liu Y., Jiang S.J., Liu Y. Prognostic value of tumor-infiltrating FoxP3+ regulatory T cells in cancers: A systematic review and meta-analysis. Sci. Rep. 2015;5:15179. doi: 10.1038/srep15179.
    1. Sakaguchi S., Miyara M., Costantino C.M., Hafler D.A. FOXP3+ regulatory T cells in the human immune system. Nat. Rev. Immunol. 2010;10:490–500. doi: 10.1038/nri2785.
    1. Wolf D., Sopper S., Pircher A., Gastl G., Wolf A.M. Treg(s) in Cancer: Friends or Foe? J. Cell. Physiol. 2015;230:2598–2605. doi: 10.1002/jcp.25016.
    1. Maggioni D., Pignataro L., Garavello W. T-helper and T-regulatory cells modulation in head and neck squamous cell carcinoma. Oncoimmunology. 2017;6:e1325066. doi: 10.1080/2162402X.2017.1325066.
    1. Distel L.V., Fickenscher R., Dietel K., Hung A., Iro H., Zenk J., Nkenke E., Buttner M., Niedobitek G., Grabenbauer G.G. Tumour infiltrating lymphocytes in squamous cell carcinoma of the oro- and hypopharynx: Prognostic impact may depend on type of treatment and stage of disease. Oral Oncol. 2009;45:e167–e174. doi: 10.1016/j.oraloncology.2009.05.640.
    1. Pretscher D., Distel L.V., Grabenbauer G.G., Wittlinger M., Buettner M., Niedobitek G. Distribution of immune cells in head and neck cancer: CD8+ T-cells and CD20+ B-cells in metastatic lymph nodes are associated with favourable outcome in patients with oro- and hypopharyngeal carcinoma. BMC Cancer. 2009;9:292. doi: 10.1186/1471-2407-9-292.
    1. Bron L., Jandus C., Andrejevic-Blant S., Speiser D.E., Monnier P., Romero P., Rivals J.P. Prognostic value of arginase-II expression and regulatory T-cell infiltration in head and neck squamous cell carcinoma. Int. J. Cancer. 2013;132:E85–E93. doi: 10.1002/ijc.27728.
    1. Park K., Cho K.J., Lee M., Yoon D.H., Kim S.B. Importance of FOXP3 in prognosis and its relationship with p16 in tonsillar squamous cell carcinoma. Anticancer Res. 2013;33:5667–5673.
    1. Lim K.P., Chun N.A., Ismail S.M., Abraham M.T., Yusoff M.N., Zain R.B., Ngeow W.C., Ponniah S., Cheong S.C. CD4+CD25hiCD127low regulatory T cells are increased in oral squamous cell carcinoma patients. PLoS ONE. 2014;9:e103975. doi: 10.1371/journal.pone.0103975.
    1. Strauss L., Bergmann C., Gooding W., Johnson J.T., Whiteside T.L. The frequency and suppressor function of CD4+CD25highFoxp3+ T cells in the circulation of patients with squamous cell carcinoma of the head and neck. Clin. Cancer Res. 2007;13:6301–6311. doi: 10.1158/1078-0432.CCR-07-1403.
    1. Al-Qahtani D., Anil S., Rajendran R. Tumour infiltrating CD25+ FoxP3+ regulatory T cells (Tregs) relate to tumour grade and stromal inflammation in oral squamous cell carcinoma. J. Oral Pathol. Med. 2011;40:636–642. doi: 10.1111/j.1600-0714.2011.01020.x.
    1. Liang Y.J., Liu H.C., Su Y.X., Zhang T.H., Chu M., Liang L.Z., Liao G.Q. Foxp3 expressed by tongue squamous cell carcinoma cells correlates with clinicopathologic features and overall survival in tongue squamous cell carcinoma patients. Oral Oncol. 2011;47:566–570. doi: 10.1016/j.oraloncology.2011.04.017.
    1. Balermpas P., Rodel F., Rodel C., Krause M., Linge A., Lohaus F., Baumann M., Tinhofer I., Budach V., Gkika E., et al. CD8+ tumour-infiltrating lymphocytes in relation to HPV status and clinical outcome in patients with head and neck cancer after postoperative chemoradiotherapy: A multicentre study of the German cancer consortium radiation oncology group (DKTK-ROG) Int. J. Cancer. 2016;138:171–181. doi: 10.1002/ijc.29683.
    1. Nordfors C., Grun N., Tertipis N., Ahrlund-Richter A., Haeggblom L., Sivars L., Du J., Nyberg T., Marklund L., Munck-Wikland E., et al. CD8+ and CD4+ tumour infiltrating lymphocytes in relation to human papillomavirus status and clinical outcome in tonsillar and base of tongue squamous cell carcinoma. Eur. J. cancer. 2013;49:2522–2530. doi: 10.1016/j.ejca.2013.03.019.
    1. De Ruiter E.J., Ooft M.L., Devriese L.A., Willems S.M. The prognostic role of tumor infiltrating T-lymphocytes in squamous cell carcinoma of the head and neck: A systematic review and meta-analysis. Oncoimmunology. 2017;6:e1356148. doi: 10.1080/2162402X.2017.1356148.
    1. Sun D.S., Zhao M.Q., Xia M., Li L., Jiang Y.H. The correlation between tumor-infiltrating Foxp3+ regulatory T cells and cyclooxygenase-2 expression and their association with recurrence in resected head and neck cancers. Med. Oncol. 2012;29:707–713. doi: 10.1007/s12032-011-9903-2.
    1. Posselt R., Erlenbach-Wunsch K., Haas M., Jessberger J., Buttner-Herold M., Haderlein M., Hecht M., Hartmann A., Fietkau R., Distel L.V. Spatial distribution of FoxP3+ and CD8+ tumour infiltrating T cells reflects their functional activity. Oncotarget. 2016;7:60383–60394. doi: 10.18632/oncotarget.11039.
    1. Yu N., Li X., Song W., Li D., Yu D., Zeng X., Li M., Leng X., Li X. CD4(+)CD25 (+)CD127 (low/-) T cells: A more specific Treg population in human peripheral blood. Inflammation. 2012;35:1773–1780. doi: 10.1007/s10753-012-9496-8.
    1. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 2005;6:345–352. doi: 10.1038/ni1178.
    1. Curiel T.J., Coukos G., Zou L., Alvarez X., Cheng P., Mottram P., Evdemon-Hogan M., Conejo-Garcia J.R., Zhang L., Burow M., et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 2004;10:942–949. doi: 10.1038/nm1093.
    1. Cheng H.W., Onder L., Cupovic J., Boesch M., Novkovic M., Pikor N., Tarantino I., Rodriguez R., Schneider T., Jochum W., et al. CCL19-producing fibroblastic stromal cells restrain lung carcinoma growth by promoting local antitumor T-cell responses. J. Allergy Clin. Immunol. 2018;142:1257–1271. doi: 10.1016/j.jaci.2017.12.998.
    1. Ngiow S.F., Young A., Jacquelot N., Yamazaki T., Enot D., Zitvogel L., Smyth M.J. A Threshold Level of Intratumor CD8+ T-cell PD1 Expression Dictates Therapeutic Response to Anti-PD1. Cancer Res. 2015;75:3800–3811. doi: 10.1158/0008-5472.CAN-15-1082.
    1. Oweida A., Hararah M.K., Phan A., Binder D., Bhatia S., Lennon S., Bukkapatnam S., Van Court B., Uyanga N., Darragh L., et al. Resistance to Radiotherapy and PD-L1 Blockade Is Mediated by TIM-3 Upregulation and Regulatory T-Cell Infiltration. Clin. Cancer Res. 2018;24:5368–5380. doi: 10.1158/1078-0432.CCR-18-1038.
    1. Tabachnyk M., Distel L.V., Buttner M., Grabenbauer G.G., Nkenke E., Fietkau R., Lubgan D. Radiochemotherapy induces a favourable tumour infiltrating inflammatory cell profile in head and neck cancer. Oral Oncol. 2012;48:594–601. doi: 10.1016/j.oraloncology.2012.01.024.
    1. Rave-Frank M., Tehrany N., Kitz J., Leu M., Weber H.E., Burfeind P., Schliephake H., Canis M., Beissbarth T., Reichardt H.M., et al. Prognostic value of CXCL12 and CXCR4 in inoperable head and neck squamous cell carcinoma. Strahlenther. Onkol. 2015;192:47–54. doi: 10.1007/s00066-015-0892-5.
    1. Doescher J., Jeske S., Weissinger S.E., Brunner C., Laban S., Bolke E., Hoffmann T.K., Whiteside T.L., Schuler P.J. Polyfunctionality of CD4(+) T lymphocytes is increased after chemoradiotherapy of head and neck squamous cell carcinoma. Strahlenther. Onkol. 2018;194:392–402. doi: 10.1007/s00066-018-1289-z.
    1. Preidl R.H.M., Mobius P., Weber M., Amann K., Neukam F.W., Kesting M., Geppert C.I., Wehrhan F. Long-term endothelial dysfunction in irradiated vessels: An immunohistochemical analysis. Strahlenther. Onkol. 2019;195:52–61. doi: 10.1007/s00066-018-1382-3.

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