Immune checkpoint inhibitors (ICI) are the main therapy currently used in advanced malignant melanoma (MM) and non-small cell lung cancer (NSCLC). Despite the wide variety of uses, the possibility of predicting ICI efficacy in these tumor types is scarce. The aim of our study was to find new predictive biomarkers for ICI treatment. We analyzed, by immunohistochemistry, various cell subsets, including CD3+, CD8+, CD68+, CD20+, and FoxP3+ cells, and molecules such as LAG-3, IDO1, and TGFβ. Comprehensive genomic profiles were analyzed. We evaluated 46 patients with advanced MM (31) and NSCLC (15) treated with ICI monotherapy. When analyzing the malignant melanoma group, shorter median progression-free survival (PFS) was found in tumors positive for nuclear FoxP3 in tumor-infiltrating lymphocytes (TILs) (p = 0.048, HR 3.04) and for CD68 expression (p = 0.034, HR 3.2). Longer PFS was achieved in patients with tumors with PD-L1 TPS ≥ 1 (p = 0.005, HR 0.26). In the NSCLC group, only FoxP3 positivity was associated with shorter PFS and OS. We found that FoxP3 negativity was linked with a better response to ICI in both histological groups.

Department of Comprehensive Cancer Care Faculty of Medicine Masaryk University Kamenice 753 5 625 00 Brno Czech Republic

Department of Comprehensive Cancer Care Masaryk Memorial Cancer Institute Zluty kopec 7 656 53 Brno Czech Republic

Department of Pathology Masaryk Memorial Cancer Institute Zluty kopec 7 656 53 Brno Czech Republic

Research Center for Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty kopec 7 656 53 Brno Czech Republic

Zobrazit více v PubMed

Wolchok J.D., Chiarion-Sileni V., Gonzalez R., Grob J.-J., Rutkowski P., Lao C.D., Cowey C.L., Schadendorf D., Wagstaff J., Dummer R., et al. CheckMate 067: 6.5-Year Outcomes in Patients (Pts) with Advanced Melanoma. JCO. 2021;39:9506. doi: 10.1200/JCO.2021.39.15_suppl.9506. DOI

Sezer A., Kilickap S., Gümüş M., Bondarenko I., Özgüroğlu M., Gogishvili M., Turk H.M., Cicin I., Bentsion D., Gladkov O., et al. Cemiplimab Monotherapy for First-Line Treatment of Advanced Non-Small-Cell Lung Cancer with PD-L1 of at Least 50%: A Multicentre, Open-Label, Global, Phase 3, Randomised, Controlled Trial. Lancet. 2021;397:592–604. doi: 10.1016/S0140-6736(21)00228-2. PubMed DOI

Reck M., Rodriguez-Abreu D., Robinson A., Hui R., Csoszi T., Fulop A., Gottfried M., Peled N., Tafreshi A., Cuffe S., et al. Updated Analysis of KEYNOTE-024: Pembrolizumab versus Platinum-Based Chemotherapy for Advanced Non-Small-Cell Lung Cancer with PD-L1 Tumor Proportion Score of 50% or Greater. Fac. Sci. Med. Health Pap. Part B. 2019;37:537–546. doi: 10.1200/JCO.18.00149. PubMed DOI

Herbst R.S., Giaccone G., de Marinis F., Reinmuth N., Vergnenegre A., Barrios C.H., Morise M., Felip E., Andric Z., Geater S., et al. Atezolizumab for First-Line Treatment of PD-L1–Selected Patients with NSCLC. N. Engl. J. Med. 2020;383:1328–1339. doi: 10.1056/NEJMoa1917346. PubMed DOI

Rizvi N.A., Hellmann M.D., Snyder A., Kvistborg P., Makarov V., Havel J.J., Lee W., Yuan J., Wong P., Ho T.S., et al. Mutational Landscape Determines Sensitivity to PD-1 Blockade in Non–Small Cell Lung Cancer. Science. 2015;348:124–128. doi: 10.1126/science.aaa1348. PubMed DOI PMC

Liu D., Yang X., Wu X. Tumor Immune Microenvironment Characterization Identifies Prognosis and Immunotherapy-Related Gene Signatures in Melanoma. Front. Immunol. 2021;12:663495. doi: 10.3389/fimmu.2021.663495. PubMed DOI PMC

Murciano-Goroff Y.R., Warner A.B., Wolchok J.D. The Future of Cancer Immunotherapy: Microenvironment-Targeting Combinations. Cell Res. 2020;30:507–519. doi: 10.1038/s41422-020-0337-2. PubMed DOI PMC

Gainor J.F., Shaw A.T., Sequist L.V., Fu X., Azzoli C.G., Piotrowska Z., Huynh T., Zhao L., Fulton L., Schultz K.R., et al. EGFR Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non-Small Cell Lung Cancer (NSCLC): A Retrospective Analysis. Clin. Cancer Res. 2016;22:4585–4593. doi: 10.1158/1078-0432.CCR-15-3101. PubMed DOI PMC

Han J., Khatwani N., Searles T.G., Turk M.J., Angeles C.V. Memory CD8+ T Cell Responses to Cancer. Semin. Immunol. 2020;49:101435. doi: 10.1016/j.smim.2020.101435. PubMed DOI PMC

Tumeh P.C., Harview C.L., Yearley J.H., Shintaku I.P., Taylor E.J.M., 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. PubMed DOI PMC

Zhang J., Li S., Liu F., Yang K. Role of CD68 in Tumor Immunity and Prognosis Prediction in Pan-Cancer. Sci. Rep. 2022;12:7844. doi: 10.1038/s41598-022-11503-2. PubMed DOI PMC

Griss J., Bauer W., Wagner C., Simon M., Chen M., Grabmeier-Pfistershammer K., Maurer-Granofszky M., Roka F., Penz T., Bock C., et al. B Cells Sustain Inflammation and Predict Response to Immune Checkpoint Blockade in Human Melanoma. Nat. Commun. 2019;10:4186. doi: 10.1038/s41467-019-12160-2. PubMed DOI PMC

Willsmore Z.N., Harris R.J., Crescioli S., Hussein K., Kakkassery H., Thapa D., Cheung A., Chauhan J., Bax H.J., Chenoweth A., et al. B Cells in Patients With Melanoma: Implications for Treatment With Checkpoint Inhibitor Antibodies. Front. Immunol. 2021;11:622442. doi: 10.3389/fimmu.2020.622442. PubMed DOI PMC

Koyama S., Akbay E.A., Li Y.Y., Herter-Sprie G.S., Buczkowski K.A., Richards W.G., Gandhi L., Redig A.J., Rodig S.J., Asahina H., et al. Adaptive Resistance to Therapeutic PD-1 Blockade Is Associated with Upregulation of Alternative Immune Checkpoints. Nat. Commun. 2016;7:10501. doi: 10.1038/ncomms10501. PubMed DOI PMC

Acharya N., Sabatos-Peyton C., Anderson A.C. Tim-3 Finds Its Place in the Cancer Immunotherapy Landscape. J. Immunother. Cancer. 2020;8:e000911. doi: 10.1136/jitc-2020-000911. PubMed DOI PMC

Liu M., Wang X., Wang L., Ma X., Gong Z., Zhang S., Li Y. Targeting the IDO1 Pathway in Cancer: From Bench to Bedside. J. Hematol. Oncol. 2018;11:100. doi: 10.1186/s13045-018-0644-y. PubMed DOI PMC

Tawbi H.A., Schadendorf D., Lipson E.J., Ascierto P.A., Matamala L., Castillo Gutiérrez E., Rutkowski P., Gogas H.J., Lao C.D., De Menezes J.J., et al. Relatlimab and Nivolumab versus Nivolumab in Untreated Advanced Melanoma. N. Engl. J. Med. 2022;386:24–34. doi: 10.1056/NEJMoa2109970. PubMed DOI PMC

Long G.V., Dummer R., Hamid O., Gajewski T.F., Caglevic C., Dalle S., Arance A., Carlino M.S., Grob J.-J., Kim T.M., et al. Epacadostat plus Pembrolizumab versus Placebo plus Pembrolizumab in Patients with Unresectable or Metastatic Melanoma (ECHO-301/KEYNOTE-252): A Phase 3, Randomised, Double-Blind Study. Lancet Oncol. 2019;20:1083–1097. doi: 10.1016/S1470-2045(19)30274-8. PubMed DOI

Mariathasan S., Turley S.J., Nickles D., Castiglioni A., Yuen K., Wang Y., Kadel III E.E., Koeppen H., Astarita J.L., Cubas R., et al. TGFβ Attenuates Tumour Response to PD-L1 Blockade by Contributing to Exclusion of T Cells. Nature. 2018;554:544–548. doi: 10.1038/nature25501. PubMed DOI PMC

Allan S.E., Alstad A.N., Merindol N., Crellin N.K., Amendola M., Bacchetta R., Naldini L., Roncarolo M.G., Soudeyns H., Levings M.K. Generation of Potent and Stable Human CD4+ T Regulatory Cells by Activation-Independent Expression of FOXP3. Mol. Ther. 2008;16:194–202. doi: 10.1038/sj.mt.6300341. PubMed DOI

González-Navajas J.M., Fan D.D., Yang S., Yang F.M., Lozano-Ruiz B., Shen L., Lee J. The Impact of Tregs on the Anticancer Immunity and the Efficacy of Immune Checkpoint Inhibitor Therapies. Front. Immunol. 2021;12:625783. doi: 10.3389/fimmu.2021.625783. PubMed DOI PMC

Ohue Y., Nishikawa H. Regulatory T (Treg) Cells in Cancer: Can Treg Cells Be a New Therapeutic Target? Cancer Sci. 2019;110:2080–2089. doi: 10.1111/cas.14069. PubMed DOI PMC

Zuo T., Wang L., Morrison C., Chang X., Zhang H., Li W., Liu Y., Wang Y., Liu X., Chan M.W.Y., et al. FOXP3 Is an X-Linked Breast Cancer Suppressor Gene and an Important Repressor of the HER-2/ErbB2 Oncogene. Cell. 2007;129:1275–1286. doi: 10.1016/j.cell.2007.04.034. PubMed DOI PMC

Wang L., Liu R., Li W., Chen C., Katoh H., Chen G.-Y., McNally B., Lin L., Zhou P., Zuo T., et al. Somatic Single-Hits Inactivate the X-Linked Tumor Suppressor FOXP3 in the Prostate. Cancer Cell. 2009;16:336–346. doi: 10.1016/j.ccr.2009.08.016. PubMed DOI PMC

Hinz S., Pagerols-Raluy L., Oberg H.-H., Ammerpohl O., Grüssel S., Sipos B., Grützmann R., Pilarsky C., Ungefroren H., Saeger H.-D., et al. Foxp3 Expression in Pancreatic Carcinoma Cells as a Novel Mechanism of Immune Evasion in Cancer. Cancer Res. 2007;67:8344–8350. doi: 10.1158/0008-5472.CAN-06-3304. PubMed DOI

Ebert L.M., Tan B.S., Browning J., Svobodova S., Russell S.E., Kirkpatrick N., Gedye C., Moss D., Ng S.P., MacGregor D., et al. The Regulatory T Cell-Associated Transcription Factor FoxP3 Is Expressed by Tumor Cells. Cancer Res. 2008;68:3001–3009. doi: 10.1158/0008-5472.CAN-07-5664. PubMed DOI

Shang B., Liu Y., Jiang S., 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. PubMed DOI PMC

Ma G.-F., Miao Q., Liu Y.-M., Gao H., Lian J.-J., Wang Y.-N., Zeng X.-Q., Luo T.-C., Ma L.-L., Shen Z.-B., et al. High FoxP3 Expression in Tumour Cells Predicts Better Survival in Gastric Cancer and Its Role in Tumour Microenvironment. Br. J. Cancer. 2014;110:1552–1560. doi: 10.1038/bjc.2014.47. PubMed DOI PMC

Hao Q., Zhang C., Gao Y., Wang S., Li J., Li M., Xue X., Li W., Zhang W., Zhang Y. FOXP3 Inhibits NF-ΚB Activity and Hence COX2 Expression in Gastric Cancer Cells. Cell. Signal. 2014;26:564–569. doi: 10.1016/j.cellsig.2013.11.030. PubMed DOI

Cioplea M., Nichita L., Georgescu D., Sticlaru L., Cioroianu A., Nedelcu R., Turcu G., Rauta A., Mogodici C., Zurac S., et al. FOXP3 in Melanoma with Regression: Between Tumoral Expression and Regulatory T Cell Upregulation. J. Immunol. Res. 2020;2020:5416843. doi: 10.1155/2020/5416843. PubMed DOI PMC

Yang S., Liu Y., Li M.-Y., Ng C.S.H., Yang S., Wang S., Zou C., Dong Y., Du J., Long X., et al. FOXP3 Promotes Tumor Growth and Metastasis by Activating Wnt/β-Catenin Signaling Pathway and EMT in Non-Small Cell Lung Cancer. Mol. Cancer. 2017;16:124. doi: 10.1186/s12943-017-0700-1. PubMed DOI PMC

Tan C.L., Kuchroo J.R., Sage P.T., Liang D., Francisco L.M., Buck J., Thaker Y.R., Zhang Q., McArdel S.L., Juneja V.R., et al. PD-1 Restraint of Regulatory T Cell Suppressive Activity Is Critical for Immune Tolerance. J. Exp. Med. 2020;218:e20182232. doi: 10.1084/jem.20182232. PubMed DOI PMC

Kamada T., Togashi Y., Tay C., Ha D., Sasaki A., Nakamura Y., Sato E., Fukuoka S., Tada Y., Tanaka A., et al. PD-1+ Regulatory T Cells Amplified by PD-1 Blockade Promote Hyperprogression of Cancer. Proc. Natl. Acad. Sci. USA. 2019;116:9999–10008. doi: 10.1073/pnas.1822001116. PubMed DOI PMC

Revenko A., Carnevalli L.S., Sinclair C., Johnson B., Peter A., Taylor M., Hettrick L., Chapman M., Klein S., Solanki A., et al. Direct Targeting of FOXP3 in Tregs with AZD8701, a Novel Antisense Oligonucleotide to Relieve Immunosuppression in Cancer. J. Immunother. Cancer. 2022;10:e003892. doi: 10.1136/jitc-2021-003892. PubMed DOI PMC

Selby M.J., Engelhardt J.J., Quigley M., Henning K.A., Chen T., Srinivasan M., Korman A.J. Anti-CTLA-4 Antibodies of IgG2a Isotype Enhance Antitumor Activity through Reduction of Intratumoral Regulatory T Cells. Cancer Immunol. Res. 2013;1:32–42. doi: 10.1158/2326-6066.CIR-13-0013. PubMed DOI

Hodi F.S., Butler M., Oble D.A., Seiden M.V., Haluska F.G., Kruse A., MacRae S., Nelson M., Canning C., Lowy I., et al. Immunologic and Clinical Effects of Antibody Blockade of Cytotoxic T Lymphocyte-Associated Antigen 4 in Previously Vaccinated Cancer Patients. Proc. Natl. Acad. Sci. USA. 2008;105:3005–3010. doi: 10.1073/pnas.0712237105. PubMed DOI PMC

Salmi S., Siiskonen H., Sironen R., Tyynelä-Korhonen K., Hirschovits-Gerz B., Valkonen M., Auvinen P., Pasonen-Seppänen S. The Number and Localization of CD68+ and CD163+ Macrophages in Different Stages of Cutaneous Melanoma. Melanoma Res. 2019;29:237–247. doi: 10.1097/CMR.0000000000000522. PubMed DOI PMC

Van Dalen F., van Stevendaal M., Fennemann F., Verdoes M., Ilina O. Molecular Repolarisation of Tumour-Associated Macrophages. Molecules. 2018;24:9. doi: 10.3390/molecules24010009. PubMed DOI PMC

Arlauckas S.P., Garris C.S., Kohler R.H., Kitaoka M., Cuccarese M.F., Yang K.S., Miller M.A., Carlson J.C., Freeman G.J., Anthony R.M., et al. In Vivo Imaging Reveals a Tumor-Associated Macrophage Mediated Resistance Pathway in Anti-PD-1 Therapy. Sci. Transl. Med. 2017;9:eaal3604. doi: 10.1126/scitranslmed.aal3604. PubMed DOI PMC

Gunnarsson U., Strigård K., Edin S., Gkekas I., Mustonen H., Kaprio T., Böckelman C., Hagström J., Palmqvist R., Haglund C. Association between Local Immune Cell Infiltration, Mismatch Repair Status and Systemic Inflammatory Response in Colorectal Cancer. J. Transl. Med. 2020;18:178. doi: 10.1186/s12967-020-02336-6. PubMed DOI PMC

Morrison C., Pabla S., Conroy J.M., Nesline M.K., Glenn S.T., Dressman D., Papanicolau-Sengos A., Burgher B., Andreas J., Giamo V., et al. Predicting Response to Checkpoint Inhibitors in Melanoma beyond PD-L1 and Mutational Burden. J. Immunother. Cancer. 2018;6:32. doi: 10.1186/s40425-018-0344-8. PubMed DOI PMC

Larkin J., Chiarion-Sileni V., Gonzalez R., Grob J.J., Cowey C.L., Lao C.D., Schadendorf D., Dummer R., Smylie M., Rutkowski P., et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N. Engl. J. Med. 2015;373:23–34. doi: 10.1056/NEJMoa1504030. PubMed DOI PMC

Yang J., Dong M., Shui Y., Zhang Y., Zhang Z., Mi Y., Zuo X., Jiang L., Liu K., Liu Z., et al. A Pooled Analysis of the Prognostic Value of PD-L1 in Melanoma: Evidence from 1062 Patients. Cancer Cell Int. 2020;20:96. doi: 10.1186/s12935-020-01187-x. PubMed DOI PMC