Original Article| Volume 166, ISSUE 6, P721-732.e1, December 2015

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

      Preclinical studies have suggested that cytotoxic agents and radiation may partly deliver their antitumor activities by activating antitumor immune response. However, the alterations of tumor immune microenvironment including immunosuppressive molecules during chemoradiotherapy and their associations with clinical features and prognosis in rectal cancer have not been thoroughly investigated. Therefore, we investigate the densities of cluster of differentiation 8 (CD8) positive tumor-infiltrating lymphocytes (TILs), CD4+TILs, natural killer cell (NK)-TILs, myeloid-derived suppressor cells (MDSCs), transcription factor forkhead box P3 (FOXP3)+TILs, programmed death ligand-1 (PD-L1), and cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) before and after neoadjuvant chemoradiotherapy (nCRT) in rectal cancer patients to determine their predictive and prognostic effects. We screen 62 rectal cancer patients who underwent nCRT followed by radical surgery. Pretreatment biopsy specimens and posttreatment surgically resected specimens of all patients are retrieved to perform the immunohistochemistry of CD8, CD4, CD56, FOXP3, CD33, CD11b, PD-L1, and CTLA-4. The CD8+TILs and CD4+TILs in post-nCRT resected specimens are significantly higher than that in pre-nCRT biopsy specimens (P = 0.004 and 0.005, respectively). Expressions of MDSC, FOXP3+TILs, and CTLA-4 are relative stable after nCRT. Tumors with high density of CD8+TILs, CD4+TILs, and low MDSC-TILs are more sensitive to nCRT (P = 0.022, 0.022 and 0.005, respectively). High pretreatment CD8+TILs are associated with better disease-free survival and overall survival (P = 0.016 and 0.022, respectively). NK-TILs are detected only in 6 of 62 rectal cancer specimens evaluated. Cell surface PD-L1 positive by tumor cells (1 of 62) and stroma cells (3 of 62) are very low. We may conclude that tumor immunity is activated after nCRT by increased infiltrating CD8+ and CD4+ T cells and relative stable numbers MDSC-TILs, FOXP3+TILs, and coinhibitory molecules. Pre-nCRT CD8+TILs, CD4+TILs, and MDSC-TILs are sensitive predictive marker for response to CRT, and high CD8+TILs are associated with better prognosis.


      CD8 (cluster of differentiation 8), NK (natural killer cell), MDSCs (myeloid-derived suppressor cells), TILs (tumor-infiltrating lymphocytes), FOXP3 (transcription factor forkhead box P3), PD-L1 (programmed death ligand-1), CTLA-4 (cytotoxic T lymphocyte-associated antigen-4), nCRT (neoadjuvant chemoradiotherapy), LARC (locally advanced rectal cancer), TRG (tumor regression grade), HPF (high-power fields), DFS (disease-free survival), OS (overall survival), CRC (colorectal cancer), DAPI (4′,6-diamidino-2-phenylindole), PD-1 (programmed death 1), IHC (immunohistochemistry), LN (lymph node), HR (hazard ratio)
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        • Jemal A.
        • Bray F.
        • Center M.M.
        • et al.
        Global cancer statistics.
        CA Cancer J Clin. 2011; 61: 69-90
        • Galon J.
        • Costes A.
        • Sanchez-Cabo F.
        • et al.
        Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.
        Science. 2006; 313: 1960-1964
        • Gooden M.J.
        • de Bock G.H.
        • Leffers N.
        • Daemen T.
        • Nijman H.W.
        The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis.
        Br J Cancer. 2011; 105: 93-103
        • Aliper A.M.
        • Frieden-Korovkina V.P.
        • Buzdin A.
        • Roumiantsev S.A.
        • Zhavoronkov A.
        Interactome analysis of myeloid-derived suppressor cells in murine models of colon and breast cancer.
        Oncotarget. 2014; 5: 11345-11353
        • Di Meo S.
        • Airoldi I.
        • Sorrentino C.
        • et al.
        Interleukin-30 expression in prostate cancer and its draining lymph nodes correlates with advanced grade and stage.
        Clin Cancer Res. 2014; 20: 585-594
        • Sakaguchi S.
        • Sakaguchi N.
        • Asano M.
        • Itoh M.
        • Toda M.
        Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.
        J Immunol. 1995; 155: 1151-1164
        • Zou W.
        Regulatory T cells, tumour immunity and immunotherapy.
        Nat Rev Immunol. 2006; 6: 295-307
        • Erfani N.
        • Mehrabadi S.M.
        • Ghayumi M.A.
        • et al.
        Increase of regulatory T cells in metastatic stage and CTLA-4 over expression in lymphocytes of patients with non-small cell lung cancer (NSCLC).
        Lung Cancer. 2012; 77: 306-311
        • Frey D.M.
        • Droeser R.A.
        • Viehl C.T.
        • et al.
        High frequency of tumor-infiltrating FOXP3(+) regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients.
        Int J Cancer. 2010; 126: 2635-2643
        • Huang Y.
        • Liao H.
        • Zhang Y.
        • et al.
        Prognostic value of tumor-infiltrating FoxP3+ T cells in gastrointestinal cancers: a meta analysis.
        PLoS One. 2014; 9: e94376
        • Bloch O.
        • Crane C.A.
        • Kaur R.
        • et al.
        Gliomas promote immunosuppression through induction of B7-H1 expression in tumor-associated macrophages.
        Clin Cancer Res. 2013; 19: 3165-3175
        • Lu B.
        • Chen L.
        • Liu L.
        • et al.
        T-cell-mediated tumor immune surveillance and expression of B7 co-inhibitory molecules in cancers of the upper gastrointestinal tract.
        Immunol Res. 2011; 50: 269-275
        • Zou W.
        • Chen L.
        Inhibitory B7-family molecules in the tumour microenvironment.
        Nat Rev Immunol. 2008; 8: 467-477
        • Keir M.E.
        • Butte M.J.
        • Freeman G.J.
        • Sharpe A.H.
        PD-1 and its ligands in tolerance and immunity.
        Annu Rev Immunol. 2008; 26: 677-704
        • Ghebeh H.
        • Mohammed S.
        • Al-Omair A.
        • et al.
        The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors.
        Neoplasia. 2006; 8: 190-198
        • Thompson R.H.
        • Gillett M.D.
        • Cheville J.C.
        • et al.
        Costimulatory molecule B7-H1 in primary and metastatic clear cell renal cell carcinoma.
        Cancer. 2005; 104: 2084-2091
        • Tsushima F.
        • Tanaka K.
        • Otsuki N.
        • et al.
        Predominant expression of B7-H1 and its immunoregulatory roles in oral squamous cell carcinoma.
        Oral Oncol. 2006; 42: 268-274
        • Brunner M.C.
        • Chambers C.A.
        • Chan F.K.
        • et al.
        CTLA-4-Mediated inhibition of early events of T cell proliferation.
        J Immunol. 1999; 162: 5813-5820
        • Salvi S.
        • Fontana V.
        • Boccardo S.
        • et al.
        Evaluation of CTLA-4 expression and relevance as a novel prognostic factor in patients with non-small cell lung cancer.
        Cancer Immunol Immunother. 2012; 61: 1463-1472
        • Topalian S.L.
        • Hodi F.S.
        • Brahmer J.R.
        • et al.
        Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.
        N Engl J Med. 2012; 366: 2443-2454
        • Robert C.
        • Thomas L.
        • Bondarenko I.
        • et al.
        Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.
        N Engl J Med. 2011; 364: 2517-2526
        • Shi S.J.
        • Wang L.J.
        • Wang G.D.
        • et al.
        B7-H1 expression is associated with poor prognosis in colorectal carcinoma and regulates the proliferation and invasion of HCT116 colorectal cancer cells.
        PLoS One. 2013; 8: e76012
        • Dilmec F.
        • Ozgonul A.
        • Uzunkoy A.
        • Akkafa F.
        Investigation of CTLA-4 and CD28 gene polymorphisms in a group of Turkish patients with colorectal cancer.
        Int J Immunogenet. 2008; 35: 317-321
        • Rodel C.
        • Liersch T.
        • Becker H.
        • et al.
        Preoperative chemoradiotherapy and postoperative chemotherapy with fluorouracil and oxaliplatin versus fluorouracil alone in locally advanced rectal cancer: initial results of the German CAO/ARO/AIO-04 randomised phase 3 trial.
        Lancet Oncol. 2012; 13: 679-687
        • Burnette B.
        • Fu Y.X.
        • Weichselbaum R.R.
        The confluence of radiotherapy and immunotherapy.
        Front Oncol. 2012; 2: 143
        • Dieci M.V.
        • Criscitiello C.
        • Goubar A.
        • et al.
        Prognostic value of tumor-infiltrating lymphocytes on residual disease after primary chemotherapy for triple-negative breast cancer: a retrospective multicenter study.
        Ann Oncol. 2014; 25: 611-618
        • Demaria S.
        • Formenti S.C.
        Radiation as an immunological adjuvant: current evidence on dose and fractionation.
        Frontiers in Oncology. 2012; 2: 153
        • Shinto E.
        • Hase K.
        • Hashiguchi Y.
        • et al.
        CD8+ and FOXP3+ tumor-infiltrating T cells before and after chemoradiotherapy for rectal cancer.
        Ann Surg Oncol. 2014; 21: S414-S421
        • Meng X.
        • Wang R.
        • Huang Z.
        • et al.
        Human epidermal growth factor receptor-2 expression in locally advanced rectal cancer: association with response to neoadjuvant therapy and prognosis.
        Cancer Sci. 2014; 105: 818-824
        • Rathore A.S.
        • Goel M.M.
        • Makker A.
        • Kumar S.
        • Srivastava A.N.
        Is the tumor infiltrating natural killer cell (NK-TILs) count in infiltrating ductal carcinoma of breast prognostically significant?.
        Asian Pac J Cancer Prev. 2014; 15: 3757-3761
        • Sconocchia G.
        • Eppenberger S.
        • Spagnoli G.C.
        • et al.
        NK cells and T cells cooperate during the clinical course of colorectal cancer.
        Oncoimmunology. 2014; 3: e952197
        • Kalbasi A.
        • June C.H.
        • Haas N.
        • Vapiwala N.
        Radiation and immunotherapy: a synergistic combination.
        J Clin Invest. 2013; 123: 2756-2763
        • Frey B.
        • Rubner Y.
        • Kulzer L.
        • et al.
        Antitumor immune responses induced by ionizing irradiation and further immune stimulation.
        Cancer Immunol Immunother. 2014; 63: 29-36
        • Yasuda K.
        • Nirei T.
        • Sunami E.
        • Nagama H.
        • Kitayama J.
        Density of CD4(+) and CD8(+) T lymphocytes in biopsy samples can be a predictor of pathological response to chemoradiotherapy (CRT) for rectal cancer.
        Radiat Oncol. 2011; 6: 49
        • Deng L.
        • Liang H.
        • Burnette B.
        • et al.
        Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice.
        J Clin Invest. 2014; 124: 687-695
        • Postow M.A.
        • Callahan M.K.
        • Barker C.A.
        • et al.
        Immunologic correlates of the abscopal effect in a patient with melanoma.
        N Engl J Med. 2012; 366: 925-931
        • Golden E.B.
        • Demaria S.
        • Schiff P.B.
        • Chachoua A.
        • Formenti S.C.
        An abscopal response to radiation and ipilimumab in a patient with metastatic non-small cell lung cancer.
        Cancer Immunol Res. 2013; 1: 365-372
        • Son C.H.
        • Bae J.H.
        • Shin D.Y.
        • et al.
        CTLA-4 blockade enhances antitumor immunity of intratumoral injection of immature dendritic cells into irradiated tumor in a mouse colon cancer model.
        J Immunother. 2014; 37: 1-7
        • Halama N.
        • Braun M.
        • Kahlert C.
        • et al.
        Natural killer cells are scarce in colorectal carcinoma tissue despite high levels of chemokines and cytokines.
        Clin Cancer Res. 2011; 17: 678-689
        • Taube J.M.
        • Klein A.
        • Brahmer J.R.
        • et al.
        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: 5064-5074
        • Droeser R.A.
        • Hirt C.
        • Viehl C.T.
        • et al.
        Clinical impact of programmed cell death ligand 1 expression in colorectal cancer.
        Eur J Cancer. 2013; 49: 2233-2242
        • Zhao L.W.
        • Li C.
        • Zhang R.L.
        • et al.
        B7-H1 and B7-H4 expression in colorectal carcinoma: correlation with tumor FOXP3(+) regulatory T-cell infiltration.
        Acta Histochem. 2014; 116: 1163-1168
        • Stone H.B.
        • Peters L.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
        • Plotnikov A.
        • Niego B.
        • Ophir R.
        • Korenstein R.
        • Keisari Y.
        Effective treatment of mouse metastatic prostate cancer by low electric field enhanced chemotherapy.
        Prostate. 2006; 66: 1620-1630
        • Galon J.
        • Fridman W.H.
        • Pages F.
        The adaptive immunologic microenvironment in colorectal cancer: a novel perspective.
        Cancer Res. 2007; 67: 1883-1886
        • Richards C.H.
        • Roxburgh C.S.
        • Powell A.G.
        • et al.
        The clinical utility of the local inflammatory response in colorectal cancer.
        Eur J Cancer. 2014; 50: 309-319
        • Lin Y.C.
        • Mahalingam J.
        • Chiang J.M.
        • et al.
        Activated but not resting regulatory T cells accumulated in tumor microenvironment and correlated with tumor progression in patients with colorectal cancer.
        Int J Cancer. 2013; 132: 1341-1350
        • Callahan M.K.
        • Wolchok J.D.
        At the bedside: CTLA-4- and PD-1-blocking antibodies in cancer immunotherapy.
        J Leukoc Biol. 2013; 94: 41-53
        • Nielsen H.J.
        • Hansen U.
        • Christensen I.J.
        • et al.
        Independent prognostic value of eosinophil and mast cell infiltration in colorectal cancer tissue.
        J Pathol. 1999; 189: 487-495