BACKGROUND: In a prospective study with long-term follow-up, we analyzed circulating T cell subsets in patients with metastatic colorectal cancer (mCRC) in the context of primary tumor sidedness, KRAS status, and clinical outcome. Our primary goal was to investigate whether baseline levels of circulating T cell subsets serve as a potential biomarker of clinical outcome of mCRC patients treated with an anti-VEGF-based regimen. METHODS: The study group consisted of 36 patients with colorectal adenocarcinoma who started first-line chemotherapy with bevacizumab for metastatic disease. We quantified T cell subsets including Tregs and CD8+ T cells in the peripheral blood prior to therapy initiation. Clinical outcome was evaluated as progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). RESULTS: 1) mCRC patients with KRAS wt tumors had higher proportions of circulating CD8+ cytotoxic T cells among all T cells but also higher measures of T regulatory (Treg) cells such as absolute count and a higher proportion of Tregs in the CD4+ subset. 2) A low proportion of circulating Tregs among CD4+ cells, and a high CD8:Treg ratio at initiation of VEGF-targeting therapy, were associated with favorable clinical outcome. 3) In a subset of patients with primarily right-sided mCRC, superior PFS and OS were observed when the CD8:Treg ratio was high. CONCLUSIONS: The baseline level of circulating immune cells predicts clinical outcome of 1st-line treatment with the anti-VEGF angio/immunomodulatory agent bevacizumab. Circulating immune biomarkers, namely the CD8:Treg ratio, identified patients in the right-sided mCRC subgroup with favorable outcome following treatment with 1st-line anti-VEGF treatment.

Department of Comprehensive Cancer Care Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Laboratory Medicine Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Oncological and Experimental pathology Masaryk Memorial Cancer Institute Brno Czech Republic

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno Czech Republic

See more in PubMed

Titu LV, Monson JR, Greenman J. The role of CD8(+) T cells in immune responses to colorectal cancer. Cancer Immunol Immunother. 2002;51(5):235–247. doi: 10.1007/s00262-002-0276-4. PubMed DOI PMC

Chiba T, Ohtani H, Mizoi T, Naito Y, Sato E, Nagura H, Ohuchi A, Ohuchi K, Shiiba K, Kurokawa Y, et al. Intraepithelial CD8+ T-cell-count becomes a prognostic factor after a longer follow-up period in human colorectal carcinoma: possible association with suppression of micrometastasis. Brit J Cancer. 2004;91(9):1711–1717. doi: 10.1038/sj.bjc.6602201. PubMed DOI PMC

Naito Y, Saito K, Shiiba K, Ohuchi A, Saigenji K, Nagura H, Ohtani H. CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer Res. 1998;58(16):3491–3494. PubMed

Oberg A, Samii S, Stenling R, Lindmark G. Different occurrence of CD8+, CD45R0+, and CD68+ immune cells in regional lymph node metastases from colorectal cancer as potential prognostic predictors. Int J Color Dis. 2002;17(1):25–29. doi: 10.1007/s003840100337. PubMed DOI

Ohtani H. Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human colorectal cancer. Cancer Immun. 2007;7:4. PubMed PMC

Mlecnik B, Tosolini M, Kirilovsky A, Berger A, Bindea G, Meatchi T, Bruneval P, Trajanoski Z, Fridman WH, 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(6):610–618. doi: 10.1200/JCO.2010.30.5425. PubMed DOI

Galon J, Mlecnik B, Bindea G, Angell HK, 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(2):199–209. doi: 10.1002/path.4287. PubMed DOI PMC

Taube JM, Klein A, Brahmer JR, Xu H, Pan X, Kim JH, Chen L, Pardoll DM, Topalian SL, Anders RA. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. Clin Cancer Res. 2014;20(19):5064–5074. doi: 10.1158/1078-0432.CCR-13-3271. PubMed DOI PMC

Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol. 2010;10(7):490–500. doi: 10.1038/nri2785. PubMed DOI

Whiteside TL. What are regulatory T cells (Treg) regulating in cancer and why? Semin Cancer Biol. 2012;22(4):327–334. doi: 10.1016/j.semcancer.2012.03.004. PubMed DOI PMC

Langier S, Sade K, Kivity S. Regulatory T cells: the suppressor arm of the immune system. Autoimmun Rev. 2010;10(2):112–115. doi: 10.1016/j.autrev.2010.08.013. PubMed DOI

Ling KL, Pratap SE, Bates GJ, Singh B, Mortensen NJ, George BD, Warren BF, Piris J, Roncador G, Fox SB, et al. Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients. Cancer Immun. 2007;7:7. PubMed PMC

Sundstrom P, Stenstad H, Langenes V, Ahlmanner F, Theander L, Ndah TG, Fredin K, Borjesson L, Gustavsson B, Bastid J, et al. Regulatory T cells from Colon Cancer patients inhibit effector T-cell migration through an adenosine-dependent mechanism. Cancer Immunol Res. 2016;4(3):183–193. doi: 10.1158/2326-6066.CIR-15-0050. PubMed DOI

Ward-Hartstonge KA, Kemp RA. Regulatory T-cell heterogeneity and the cancer immune response. Clin Transl Immunol. 2017;6(9):e154. doi: 10.1038/cti.2017.43. PubMed DOI PMC

Luu M, Steinhoff U, Visekruna A. Functional heterogeneity of gut-resident regulatory T cells. Clin Transl Immunol. 2017;6(9):e156. doi: 10.1038/cti.2017.39. PubMed DOI PMC

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(2):186–192. doi: 10.1200/JCO.2008.18.7229. PubMed DOI

Zhuo C, Xu Y, Ying M, Li Q, Huang L, Li D, Cai S, Li B. FOXP3+ Tregs: heterogeneous phenotypes and conflicting impacts on survival outcomes in patients with colorectal cancer. Immunol Res. 2015;61(3):338–347. doi: 10.1007/s12026-014-8616-y. PubMed DOI

Tosolini M, Kirilovsky A, Mlecnik B, Fredriksen T, Mauger S, Bindea G, Berger A, Bruneval P, Fridman WH, Pages F, et al. Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) in patients with colorectal cancer. Cancer Res. 2011;71(4):1263–1271. doi: 10.1158/0008-5472.CAN-10-2907. PubMed DOI

Verma C, Eremin JM, Robins A, Bennett AJ, Cowley GP, El-Sheemy MA, Jibril JA, Eremin O. Abnormal T regulatory cells (Tregs: FOXP3+, CTLA-4+), myeloid-derived suppressor cells (MDSCs: monocytic, granulocytic) and polarised T helper cell profiles (Th1, Th2, Th17) in women with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC) and surgery: failure of abolition of abnormal treg profile with treatment and correlation of treg levels with pathological response to NAC. J Transl Med. 2013;11:16. doi: 10.1186/1479-5876-11-16. PubMed DOI PMC

Yamamoto T, Yanagimoto H, Satoi S, Toyokawa H, Hirooka S, Yamaki S, Yui R, Yamao J, Kim S, Kwon AH. Circulating CD4+CD25+ regulatory T cells in patients with pancreatic cancer. Pancreas. 2012;41(3):409–415. doi: 10.1097/MPA.0b013e3182373a66. PubMed DOI

Ihara F, Sakurai D, Horinaka A, Makita Y, Fujikawa A, Sakurai T, Yamasaki K, Kunii N, Motohashi S, Nakayama T, et al. CD45RA(−)Foxp3(high) regulatory T cells have a negative impact on the clinical outcome of head and neck squamous cell carcinoma. Cancer Immunol Immunother. 2017;66(10):1275–1285. doi: 10.1007/s00262-017-2021-z. PubMed DOI PMC

Jass JR. Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology. 2007;50(1):113–130. doi: 10.1111/j.1365-2559.2006.02549.x. PubMed DOI

Budinska E, Popovici V, Tejpar S, D'Ario G, Lapique N, Sikora KO, Di Narzo AF, Yan P, Hodgson JG, Weinrich S, et al. Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer. J Pathol. 2013;231(1):63–76. doi: 10.1002/path.4212. PubMed DOI PMC

Sadanandam A, Lyssiotis CA, Homicsko K, Collisson EA, Gibb WJ, Wullschleger S, Ostos LC, Lannon WA, Grotzinger C, Del Rio M, et al. A colorectal cancer classification system that associates cellular phenotype and responses to therapy. Nat Med. 2013;19(5):619–625. doi: 10.1038/nm.3175. PubMed DOI PMC

Dienstmann R, Vermeulen L, Guinney J, Kopetz S, Tejpar S, Tabernero J. Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer. Nat Rev Cancer. 2017;17(2):79–92. doi: 10.1038/nrc.2016.126. PubMed DOI

Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol. 2005;23(3):609–618. doi: 10.1200/JCO.2005.01.086. PubMed DOI

Deschoolmeester V, Baay M, Lardon F, Pauwels P, Peeters M. Immune cells in colorectal Cancer: prognostic relevance and role of MSI. Cancer Microenviron. 2011;4(3):377–392. doi: 10.1007/s12307-011-0068-5. PubMed DOI PMC

Boissiere-Michot F, Lazennec G, Frugier H, Jarlier M, Roca L, Duffour J, Du Paty E, Laune D, Blanchard F, Le Pessot F, et al. Characterization of an adaptive immune response in microsatellite-instable colorectal cancer. Oncoimmunology. 2014;3:e29256. doi: 10.4161/onci.29256. PubMed DOI PMC

Bencsikova B, Bortlicek Z, Halamkova J, Ostrizkova L, Kiss I, Melichar B, Pavlik T, Dusek L, Valik D, Vyzula R, et al. Efficacy of bevacizumab and chemotherapy in the first-line treatment of metastatic colorectal cancer: broadening KRAS-focused clinical view. BMC Gastroenterol. 2015;15:37. doi: 10.1186/s12876-015-0266-6. PubMed DOI PMC

Alfaro C, Suarez N, Gonzalez A, Solano S, Erro L, Dubrot J, Palazon A, Hervas-Stubbs S, Gurpide A, Lopez-Picazo JM, et al. Influence of bevacizumab, sunitinib and sorafenib as single agents or in combination on the inhibitory effects of VEGF on human dendritic cell differentiation from monocytes. British J Cancer. 2009;100(7):1111–1119. doi: 10.1038/sj.bjc.6604965. PubMed DOI PMC

Osada T, Chong G, Tansik R, Hong T, Spector N, Kumar R, Hurwitz HI, Dev I, Nixon AB, Lyerly HK, et al. The effect of anti-VEGF therapy on immature myeloid cell and dendritic cells in cancer patients. Cancer Immunol Immunother. 2008;57(8):1115–1124. doi: 10.1007/s00262-007-0441-x. PubMed DOI PMC

Wada J, Suzuki H, Fuchino R, Yamasaki A, Nagai S, Yanai K, Koga K, Nakamura M, Tanaka M, Morisaki T, et al. The contribution of vascular endothelial growth factor to the induction of regulatory T-cells in malignant effusions. Anticancer Res. 2009;29(3):881–888. PubMed

Terme M, Tartour E, Taieb J. VEGFA/VEGFR2-targeted therapies prevent the VEGFA-induced proliferation of regulatory T cells in cancer. Oncoimmunology. 2013;2(8):e25156. doi: 10.4161/onci.25156. PubMed DOI PMC

Manzoni M, Rovati B, Ronzoni M, Loupakis F, Mariucci S, Ricci V, Gattoni E, Salvatore L, Tinelli C, Villa E, et al. Immunological effects of bevacizumab-based treatment in metastatic colorectal cancer. Oncology. 2010;79(3–4):187–196. doi: 10.1159/000320609. PubMed DOI

Selingerova I, Dolezelova H, Horova I, Katina S, Zelinka J. Survival of patients with primary brain tumors: comparison of two statistical approaches. PLoS One. 2016;11(2):e0148733. doi: 10.1371/journal.pone.0148733. PubMed DOI PMC

Roselli M, Formica V, Cereda V, Jochems C, Richards J, Grenga I, Orlandi A, Ferroni P, Guadagni F, Schlom J. The association of clinical outcome and peripheral T-cell subsets in metastatic colorectal cancer patients receiving first-line FOLFIRI plus bevacizumab therapy. Oncoimmunology. 2016;5(7):e1188243. doi: 10.1080/2162402X.2016.1188243. PubMed DOI PMC

Ostrizkova L, Petruzelka L, Hejduk K, Zdrazilova-Dubska L, Vocka M, Brancikova D, Bencsikova B, Vyzula R, Obermannova R. Right-sided colon cancer is associated with increased frequency of KRAS mutation and with a poor outcome in patients with metastatic disease treated in the first line with bevacizumab and chemotherapy. Annals Oncol. 2016;27(Suppl 2):114–115. doi: 10.1093/annonc/mdw200.33. DOI

Obermannova R, Ostrizkova L, Hejduk K, Zdrazilova-Dubska L, Vocka M, Vyzula R, Bencsikova B, Petruzelka L. Right-sided versus left-sided primary tumor location in patients with KRASmut metastatic colorectal cancer (mCRC) treated with 1st-line anti-VEGF plus chemotherapy (CTx) - data from the National Czech Registry. Annals Oncol. 2016;27(Suppl 9):168O.

Loree JM, Pereira AAL, Lam M, Willauer AN, Raghav K, Dasari A, Morris VK, Advani S, Menter DG, Eng C, et al. Classifying colorectal Cancer by tumor location rather than sidedness highlights a continuum in mutation profiles and consensus molecular subtypes. Clin Cancer Res. 2018;24(5):1062–1072. doi: 10.1158/1078-0432.CCR-17-2484. PubMed DOI PMC

Lal N, White BS, Goussous G, Pickles O, Mason MJ, Beggs AD, Taniere P, Willcox BE, Guinney J, Middleton GW. KRAS mutation and consensus molecular subtypes 2 and 3 are independently associated with reduced immune infiltration and reactivity in colorectal Cancer. Clin Cancer Res. 2018;24(1):224–233. doi: 10.1158/1078-0432.CCR-17-1090. PubMed DOI PMC

Cremolini C, Loupakis F, Antoniotti C, Lupi C, Sensi E, Lonardi S, Mezi S, Tomasello G, Ronzoni M, Zaniboni A, et al. FOLFOXIRI plus bevacizumab versus FOLFIRI plus bevacizumab as first-line treatment of patients with metastatic colorectal cancer: updated overall survival and molecular subgroup analyses of the open-label, phase 3 TRIBE study. Lancet Oncol. 2015;16(13):1306–1315. doi: 10.1016/S1470-2045(15)00122-9. PubMed DOI