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The Role of Tumor-Infiltrating Lymphocytes in Development, Progression, and Prognosis of Non–Small Cell Lung Cancer

Open ArchivePublished:February 01, 2016DOI:https://doi.org/10.1016/j.jtho.2016.01.015

      Abstract

      A malignant tumor is not merely an accumulation of neoplastic cells, but constitutes a microenvironment containing endothelial cells, fibroblasts, structural components, and infiltrating immune cells that impact tumor development, invasion, metastasis, and outcome. Hence, the evolution of cancers reflects intricate cellular and molecular interactions between tumor cells and constituents of the tumor microenvironment. Recent studies have shed new light on this complex interaction between tumor and host immune cells and the resulting immune response. The composition of the immune microenvironment differs across patients as well as in cancers of the same type, including various populations of T cells, B cells, dendritic cells, natural killer cells, myeloid-derived suppressor cells, neutrophils, and macrophages. The type, density, location, and organization of immune cells within solid tumors define the immune contexture, which has proved to be a major determinant of tumor characteristics and patient outcome. Lung cancer consists mostly of non–small cell lung cancer (85%); it is our most deadly malignant disease, with the 5-year survival rate being merely 15%. This review focuses on the immune contexture; the tumor-suppressing roles of tumor-infiltrating lymphocytes; and the relevance of this immune contexture for cancer diagnostics, prognostication, and treatment allocation, with an emphasis on non–small cell lung cancer.

      Keywords

      Introduction

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      Cancer Immunosurveillance, Immunoediting, and Immune Contexture

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      Figure thumbnail gr1
      Figure 1Immunosurveillance and immunoediting. There is an integration of immunoediting and oncogenesis during cancer progression. Oncogenesis leads to transformed cells, which are attacked by immune cells owing to neoantigen presentation. During immunosurveillance, cancer immunoediting incorporates three phases: elimination, equilibrium, and escape. In the elimination phase the cancer immunosurveillance successfully eradicates cancer cells of the developing tumor. During the equilibrium phase the tumor cells that have survived the immunosurveillance and the host immune system are in balance. In the escape phase the tumor cells have evaded detection and elimination by the host immune system. The immunosurveillance imposes a selection of transformed cells that acquire tactics to escape control. Their genetic instability facilitates evolution of strategies for immune evasion or suppression, which may tilt the tumor microenvironment from hostile to supportive for the transformed cells. At some point, a state of equilibrium may be achieved, corresponding to a clinically occult dormant disease. Further iteration of evasion mechanisms may ultimately drive immune suppression beyond the local microenvironment, accomplishing immune escape and in this manner licensing invasive and metastatic behavior. CD8+, cytotoxic T cells; CD4+, T helper cells; NK, natural killer cells; NKT, natural killer T cells; DC, mature dendritic cells; Treg, regulatory T cells; MDSC, myeloid-derived suppressor cells; γδ, gamma delta T cells.
      Adapted from Mittal et al.,
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      Immunosurveillance and Tertiary Lymphoid Structures

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      Figure thumbnail gr2
      Figure 2The immune contexture in non–small cell lung cancer includes the tumor core, invasive margin, tertiary lymphoid structures (TLSs), and tumor microenvironment, as well as the distribution of various immune cells. The immune microenvironment in lung tumors comprises T cells (T), B cells (B), natural killer cells (NK), mature (mDCs) and immature dendritic cells (follicular dendritic cells [FDCs]), tumor-associated macrophages [TAMs, type 2 (M2)], myeloid-derived suppressor cells (MDSCs), neutrophils (N1, antitumorigenic; N2, protumorigenic), and mast cells (M). The great majority of immune cells are found at the interface between the tumor and the normal tissue (invasive margin), and some are organized into tertiary TLSs. The latter are considered a gateway for the entrance of immune cells from the blood to the tumor (by means of peripheral node addressin–expressing high endothelial venules [HEVs]). This process is highly regulated through chemokine/chemokine receptors, interleukins, integrins, and adhesion molecule expression or secretion. Ab, antibody; TAAs, tumor-associated antigens; mB, memory B cell; PC, plasma cells; TFH, follicular helper T cell; AT2, alveolar type II cells; MA, alveolar macrophage.
      Adapted from Fridman et al.
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      Prognostic Immune Markers in NSCLC

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      CD4+ T cells in cancer stroma, not CD8+ T cells in cancer cell nests, are associated with favorable prognosis in human non-small cell lung cancers.
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      • Sautes-Fridman C.
      • et al.
      The immune contexture in human tumours: impact on clinical outcome.
      Judging by these data, TILs appeared to be important players for in situ immunity of cancers.
      On the other hand, it should be noted that lung cancers may progress despite the presence of TILs, as effector immune cells may be anergic with reduced functions in the lung cancer microenvironment,
      • Platonova S.
      • Cherfils-Vicini J.
      • Damotte D.
      • et al.
      Profound coordinated alterations of intratumoral NK cell phenotype and function in lung carcinoma.
      • Prado-Garcia H.
      • Romero-Garcia S.
      • Aguilar-Cazares D.
      • et al.
      Tumor-induced CD8+ T-cell dysfunction in lung cancer patients.
      • Gupta P.K.
      • Godec J.
      • Wolski D.
      • et al.
      CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.
      • Pauken K.E.
      • Wherry E.J.
      Overcoming T cell exhaustion in infection and cancer.
      or the tumor cells may induce a loss or down-regulation of HLA class I molecules during tumor progression, modulating the susceptibility of tumor cells to lysis by cytotoxic CD8+ T lymphocytes and NK cells.
      • So T.
      • Takenoyama M.
      • Mizukami M.
      • et al.
      Haplotype loss of HLA class I antigen as an escape mechanism from immune attack in lung cancer.
      In addition, it has been observed that lung cancer cells are able to produce immunosuppressive factors in the tumor microenvironment (soluble forms of major histocompatibility class I chain-related molecule A, cytokines, and chemokines), impairing CD8+ T-cell–mediated tumor cell lysis or promoting tumor cell progression or survival.
      • Salih H.R.
      • Rammensee H.G.
      • Steinle A.
      Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding.
      • Itakura E.
      • Huang R.R.
      • Wen D.R.
      • et al.
      IL-10 expression by primary tumor cells correlates with melanoma progression from radial to vertical growth phase and development of metastatic competence.
      • Kirshberg S.
      • Izhar U.
      • Amir G.
      • et al.
      Involvement of CCR6/CCL20/IL-17 axis in NSCLC disease progression.
      • Massague J.
      TGFbeta in Cancer.
      • Su L.
      • Zhang J.
      • Xu H.
      • et al.
      Differential expression of CXCR4 is associated with the metastatic potential of human non-small cell lung cancer cells.
      • Wald O.
      • Shapira O.M.
      • Izhar U.
      CXCR4/CXCL12 axis in non small cell lung cancer (NSCLC) pathologic roles and therapeutic potential.
      • Zhu Y.M.
      • Webster S.J.
      • Flower D.
      • et al.
      Interleukin-8/CXCL8 is a growth factor for human lung cancer cells.

      NSCLC and TILs

      In the literature, there are 17 larger studies (sample size N > 200) investigating the prognostic impact of TILS in NSCLC.
      • Johnson S.K.
      • Kerr K.M.
      • Chapman A.D.
      • et al.
      Immune cell infiltrates and prognosis in primary carcinoma of the lung.
      • Al-Shibli K.I.
      • Donnem T.
      • Al-Saad S.
      • et al.
      Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer.
      • Goc J.
      • Germain C.
      • Vo-Bourgais T.K.
      • et al.
      Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8+ T cells.
      • Al-Shibli K.
      • Al-Saad S.
      • Andersen S.
      • et al.
      The prognostic value of intraepithelial and stromal CD3-, CD117- and CD138-positive cells in non-small cell lung carcinoma.
      • Alifano M.
      • Mansuet-Lupo A.
      • Lococo F.
      • et al.
      Systemic inflammation, nutritional status and tumor immune microenvironment determine outcome of resected non-small cell lung cancer.
      • Djenidi F.
      • Adam J.
      • Goubar A.
      • et al.
      CD8+CD103+ tumor-infiltrating lymphocytes are tumor-specific tissue-resident memory T cells and a prognostic factor for survival in lung cancer patients.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      • Horne Z.D.
      • Jack R.
      • Gray Z.T.
      • et al.
      Increased levels of tumor-infiltrating lymphocytes are associated with improved recurrence-free survival in stage 1A non-small-cell lung cancer.
      • Ilie M.
      • Hofman V.
      • Ortholan C.
      • et al.
      Predictive clinical outcome of the intratumoral CD66b-positive neutrophil-to-CD8-positive T-cell ratio in patients with resectable nonsmall cell lung cancer.
      • Kayser G.
      • Schulte-Uentrop L.
      • Sienel W.
      • et al.
      Stromal CD4/CD25 positive T-cells are a strong and independent prognostic factor in non-small cell lung cancer patients, especially with adenocarcinomas.
      • Kilic A.
      • Landreneau R.J.
      • Luketich J.D.
      • et al.
      Density of tumor-infiltrating lymphocytes correlates with disease recurrence and survival in patients with large non-small-cell lung cancer tumors.
      • Kim M.Y.
      • Koh J.
      • Kim S.
      • et al.
      Clinicopathological analysis of PD-L1 and PD-L2 expression in pulmonary squamous cell carcinoma: comparison with tumor-infiltrating T cells and the status of oncogenic drivers.
      • Ruffini E.
      • Asioli S.
      • Filosso P.L.
      • et al.
      Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms.
      • Schalper K.A.
      • Brown J.
      • Carvajal-Hausdorf D.
      • et al.
      Objective measurement and clinical significance of TILs in non-small cell lung cancer.
      • Sterlacci W.
      • Wolf D.
      • Savic S.
      • et al.
      High transforming growth factor beta expression represents an important prognostic parameter for surgically resected non-small cell lung cancer.
      • Suzuki K.
      • Kadota K.
      • Sima C.S.
      • et al.
      Clinical impact of immune microenvironment in stage I lung adenocarcinoma: tumor interleukin-12 receptor beta2 (IL-12Rbeta2), IL-7R, and stromal FoxP3/CD3 ratio are independent predictors of recurrence.
      • Koh J.
      • Go H.
      • Keam B.
      • et al.
      Clinicopathologic analysis of programmed cell death-1 and programmed cell death-ligand 1 and 2 expressions in pulmonary adenocarcinoma: comparison with histology and driver oncogenic alteration status.
      Sixteen and 13 of these studies have been published since 2008 and 2011, respectively, clearly demonstrating the rapidly increasing interest within this topic. For details on study cohort size, included cell type, pathological TNM stage (pStage), and assessed tissue compartment and immune cells (markers), see Table 1. CD8+ TILs were examined in 14 of 17 studies, whereas CD3+ and CD4+ TILs where assessed in six and five studies, respectively. In two of the included studies, TILs were not specified because assessments were based on hematoxylin and eosin staining alone. Three of the studies included validation cohorts.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      • Schalper K.A.
      • Brown J.
      • Carvajal-Hausdorf D.
      • et al.
      Objective measurement and clinical significance of TILs in non-small cell lung cancer.
      • Suzuki K.
      • Kadota K.
      • Sima C.S.
      • et al.
      Clinical impact of immune microenvironment in stage I lung adenocarcinoma: tumor interleukin-12 receptor beta2 (IL-12Rbeta2), IL-7R, and stromal FoxP3/CD3 ratio are independent predictors of recurrence.
      Table 1Prognostic Impact of Immune Cells in Larger (N > 200) NSCLC Studies
      StudyNHistolologic DiagnosisP StageMarkersTissue CompartmentTMA vs. WSPrognostic Impact (PI) by TILs?PI within pStage?
      Johnson et al. (2000)
      • Johnson S.K.
      • Kerr K.M.
      • Chapman A.D.
      • et al.
      Immune cell infiltrates and prognosis in primary carcinoma of the lung.
      710NOSI–IIICD3, CD8, CD57, CD68Intraepithelial and stromalWSTotal cohort (HE), TILs no significant PI. IHC analysis was performed (N = 95). Epithelial CD3 pos PI for OS (univariate)No
      Al-Shibli et al. (2008)
      • Al-Shibli K.I.
      • Donnem T.
      • Al-Saad S.
      • et al.
      Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer.
      335ADC, SCC, NOSI–IIIACD4, CD8, CD20Intraepithelial and stromalTMAStromal and epithelial CD4 and CD8 correlate with improved DSS. Both are independent PIs in stromaNo
      Ruffini et al. (2009)
      • Ruffini E.
      • Asioli S.
      • Filosso P.L.
      • et al.
      Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms.
      1290ADC, SCC, NET, SCLCI–IIIATILs (CD3, CD4, CD8, CD20)IntraepithelialWSPI assessed for total TILs. Independent PI overall and for SCCPartly
      Al-Shibli et al. (2010)
      • Al-Shibli K.
      • Al-Saad S.
      • Andersen S.
      • et al.
      The prognostic value of intraepithelial and stromal CD3-, CD117- and CD138-positive cells in non-small cell lung carcinoma.
      335ADC, SCC, NOSI–IIIACD3, CD117, CD138Intraepithelial and stromalTMAStromal + epithelial CD3 correlates with improved DSS. Stromal CD3 has independent pos PIPartly
      Kilic et al. (2011)
      • Kilic A.
      • Landreneau R.J.
      • Luketich J.D.
      • et al.
      Density of tumor-infiltrating lymphocytes correlates with disease recurrence and survival in patients with large non-small-cell lung cancer tumors.
      219ADC, SCC, NOSITILs (NOS)Intraepithelial and stromalWSTILs (total) correlate with reduced recurrence rate and prolonged DFS (univariate)Only stage I
      Horne et al. (2011)
      • Horne Z.D.
      • Jack R.
      • Gray Z.T.
      • et al.
      Increased levels of tumor-infiltrating lymphocytes are associated with improved recurrence-free survival in stage 1A non-small-cell lung cancer.
      273ADC, SCC, NOSIATILs (NOS)Intraepithelial and stromalWSTotal TILs show pos PI (RFS, univariate)Only stage I
      Sterlacci et al. (2012)
      • Sterlacci W.
      • Wolf D.
      • Savic S.
      • et al.
      High transforming growth factor beta expression represents an important prognostic parameter for surgically resected non-small cell lung cancer.
      383ADC, SCC, NOSI–IVCD4, CD8IntraepithelialTMALow CD4/CD8 ratio pos PI for ADC in stage I (univariate)Partly
      Ilie et al. (2012)
      • Ilie M.
      • Hofman V.
      • Ortholan C.
      • et al.
      Predictive clinical outcome of the intratumoral CD66b-positive neutrophil-to-CD8-positive T-cell ratio in patients with resectable nonsmall cell lung cancer.
      632ADC, SCC, NOSI–IIICD8, CD66bIntraepithelial (central tumor)TMACD8 not pos PI. Low CD66b/CD8 ratio has pos independent PINo
      Kayser et al. (2012)
      • Kayser G.
      • Schulte-Uentrop L.
      • Sienel W.
      • et al.
      Stromal CD4/CD25 positive T-cells are a strong and independent prognostic factor in non-small cell lung cancer patients, especially with adenocarcinomas.
      232ADC, SCC, NOSI–IVCD3, CD8, CD4/CD25Intraepithelial and stromalTMAHigh stromal CD3 and CD4/CD25 ratio favor better prognosis (univariate). High stromal CD4/CD25 ratio has independent PINo
      Suzuki et al. (2013)
      • Suzuki K.
      • Kadota K.
      • Sima C.S.
      • et al.
      Clinical impact of immune microenvironment in stage I lung adenocarcinoma: tumor interleukin-12 receptor beta2 (IL-12Rbeta2), IL-7R, and stromal FoxP3/CD3 ratio are independent predictors of recurrence.
      956ADCIFOXP3, CD3, CD4, CD8, CD45ROIntraepithelial and stromalTMAHigh FOXP3/CD3 ratio has negative PI. FOXP3-mediated protumor environment, but is overcome by high stromal CD3Only stage I
      Goc et al. (2014)
      • Goc J.
      • Fridman W.H.
      • Hammond S.A.
      • et al.
      Tertiary lymphoid structures in human lung cancers, a new driver of antitumor immune responses.
      458ADC, SCC, NOSI–IVCD8, mDCIntraepithelial and stromalWSIn TLS, mDC density correlates with CD8 activation, Th phenotype, and cytotoxic orientationNo
      Alifano et al. (2014)
      • Alifano M.
      • Mansuet-Lupo A.
      • Lococo F.
      • et al.
      Systemic inflammation, nutritional status and tumor immune microenvironment determine outcome of resected non-small cell lung cancer.
      303ADC, SCC, NOSI–IVCD8, CD1AIntraepithelial and stromalWSHigh CD8 cell count shows reduced RR = 0.37. CD8 T-cell density has independent pos PIPartly
      Schalper et al. (2015)
      • Schalper K.A.
      • Brown J.
      • Carvajal-Hausdorf D.
      • et al.
      Objective measurement and clinical significance of TILs in non-small cell lung cancer.
      552ADC, SCC, NOSI–IVCD3, CD8, CD20Intraepithelial, stromal, totalTMATotal CD8 has independent pos PI in both cohorts. Total CD3 has independent pos PI in one cohortNo
      Kim et al. (2015)
      • Kim M.Y.
      • Koh J.
      • Kim S.
      • et al.
      Clinicopathological analysis of PD-L1 and PD-L2 expression in pulmonary squamous cell carcinoma: comparison with tumor-infiltrating T cells and the status of oncogenic drivers.
      331SCCI–IIICD8, PD-1, PD-L1IntraepithelialTMACases with high PD-L1 had also high CD8 cell infiltration. Cases with high CD8 and PD-1 TILs had longer DFSNo
      Donnem et al. (2015)
      • Donnem T.
      • Kilvaer T.K.
      • Andersen S.
      • et al.
      Strategies to implement immunoscore in resected non-small cell lung cancer into the clinic.
      797ADC, SCC, NOSI–IIIACD8StromalTMACD8 and pStage has independent. pos PI in whole cohort, all end points. CD8 has significant PI within each pStage (univariate)Yes
      Koh et al. (2015)
      • Koh J.
      • Go H.
      • Keam B.
      • et al.
      Clinicopathologic analysis of programmed cell death-1 and programmed cell death-ligand 1 and 2 expressions in pulmonary adenocarcinoma: comparison with histology and driver oncogenic alteration status.
      497ADCI–IIICD8Intraepithelial and stromalTMACD8 TILs have pos PI. High CD8 count is significantly associated with increased DFSNo
      Djenidi et al. (2015)
      • Djenidi F.
      • Adam J.
      • Goubar A.
      • et al.
      CD8+CD103+ tumor-infiltrating lymphocytes are tumor-specific tissue-resident memory T cells and a prognostic factor for survival in lung cancer patients.
      101ADC, SCC, NOSICD3, CD8, CD103Intraepithelial and stromalWSCD103 TILs have pos PI, promoting CTL. CD8CD103 TILs lead to activation-induced cell death.No
      ADC, adenocarcinoma; CD, cluster of differentiation; CTL, cytotoxic lymphocyte; DFS, disease-free survival; DSS, disease-specific survival; HE, hematoxylin and eosin staining; IHC, immunohistochemical; mDC, mature dendritic cells; NET, neuroendocrine tumors; NOS, not otherwise specified; NSCLC, non–small cell lung cancer; OS, overall survival; PI, prognostic impact; PD-1, programmed death receptor 1; PD-L1, programmed death ligand 1; pos, positive; pStage, pathological stage; RFS, relapse-free survival; RR, Relative risk; SCC, squamous cell carcinoma; SCLC, small cell lung cancer; Th, TIL helper cell; TILs, tumor-infiltrating lymphocytes; TLS, tertiary lymphoid structures; TMA, tissue micro array; WS, whole slide.
      As CD8+ T cells also constitute most of the CD3+ category, these cells have consistently been associated with a beneficial prognostic impact in all studies based on CD markers (see Table 1). In half of the studies, CD8+ T cells in fact mediated an independent positive prognostic impact. It should be noted that in the study by Ruffini et al., tumor specimens were stained for CD3+, CD8+, CD4+, and CD20 but the analyses were performed for all TILs and not according to T cell type.
      • Ruffini E.
      • Asioli S.
      • Filosso P.L.
      • et al.
      Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms.
      In the study by Donnem et al., one training set and three validation cohorts with a total of 797 patients were examined.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      In that study, density of CD8+ T cells was assessed in stroma only and showed a strong independent positive prognostic impact on disease-free survival, disease-specific survival, and overall survival. The method used for assessing the CD8+ cell density in stromal compartments is briefly presented in Figure 3. Rapidly accumulating research data strongly document that high levels of tumor-infiltrating cytotoxic CD8+ and CD3+ T cells assessed in the stromal or tumor epithelial compartment of NSCLC are associated with significant beneficial patient outcomes mediated by immunity toward the tumor.
      • Geng Y.
      • Shao Y.
      • He W.
      • et al.
      Prognostic role of tumor-infiltrating lymphocytes in lung cancer: a meta-analysis.
      Furthermore, stromal assessments of TILs appear to have a superior prognostic impact when compared with epithelial assessments, which has recently been supported by a meta-analysis of 29 small and large studies of the prognostic role of various TILs in lung cancer.
      • Geng Y.
      • Shao Y.
      • He W.
      • et al.
      Prognostic role of tumor-infiltrating lymphocytes in lung cancer: a meta-analysis.
      Figure thumbnail gr3
      Figure 3Representative examples of CD8 immunostaining in non–small cell lung cancer tissue microarray (TMA) cores. (A and B) In the large majority of non–small cell lung cancer tissues, histologic examination will show a mixture of epithelial tumor tissue (red) and tumor-related stroma (green). (CF): Within each TMA core, the percentages of CD8+ lymphocytes (stained brown) in comparison with the total amount of nucleated cells in the stromal compartments (green) can be estimated. In our experience with TMAs, the determination of CD8+ appears to be most reliable when assessed in the stromal (green) compartment. The magnified panels constitute examples of a negative CD8+ score (C), low-density score (≤25% CD8+ cells [D]), intermediate-density score (>25% to ≤50% CD8+ cells [E]), and high-density score (>50% CD8+ cells [F]) in tumor stroma at ×400 magnification.

      Development of Immunoscore and TNM-Immune in Cancer

      Since the host immune system appeared to mediate a significant prognostic role in cancer, Galon et al. have led the translational research development within this field in colorectal cancer.
      • Mlecnik B.
      • Tosolini M.
      • Kirilovsky A.
      • et al.
      Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction.
      • Galon J.
      • Pages F.
      • Marincola F.M.
      • et al.
      Cancer classification using the Immunoscore: a worldwide task force.
      • Galon J.
      • Angell H.K.
      • Bedognetti D.
      • et al.
      The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures.
      • Galon J.
      • Mlecnik B.
      • Bindea G.
      • et al.
      Towards the introduction of the ‘Immunoscore' in the classification of malignant tumours.
      They have developed a remarkable diagnostic tool that uses assessments of CD8+ and CD3+ TILs in colorectal cancer tissue to create an “Immunoscore” as an essential prognostic and predictive supplement to TNM classification; it is designated TNM-Immune (TNM-I). Their findings suggest that the Immunoscore classification provides prognostic significance superior to that of the Dutch Association of Comprehensive Cancer Centers/Union for International Cancer Control TNM classification system, as a multivariate Cox analysis revealed the immune criteria to remain highly significant whereas the TNM variables and tumor differentiation did not.
      • Mlecnik B.
      • Tosolini M.
      • Kirilovsky A.
      • et al.
      Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction.
      This publication led to an editorial in Journal of Clinical Oncology with the title “TNM Staging in Colorectal Cancer: T is for T cell and M is for Memory.”
      • Broussard E.K.
      • Disis M.L.
      TNM staging in colorectal cancer: T is for T cell and M is for memory.
      In NSCLC, several groups have evaluated the prognostic impact of TILs, but only our group has focused on an Immunoscore assessment (low, intermediate, or high density of stromal CD8+ T cells) and TNM-I.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      Using principally the same strategy as the French research group used for colorectal cancer,
      • Mlecnik B.
      • Tosolini M.
      • Kirilovsky A.
      • et al.
      Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction.
      we were able to demonstrate a solid prognostic impact of the Immunoscore within each examined NSCLC pStage (IA, IB, IIA, IIB, and IIIA [Fig. 4]).
      • Donnem T.
      • Kilvaer T.K.
      • Andersen S.
      • et al.
      Strategies to implement immunoscore in resected non-small cell lung cancer into the clinic.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      In the multivariate analysis of this study, CD8+ T cell density had a prognostic impact equal to that of NSCLC pStage, which significantly enhanced the discriminatory prognostic prediction. Presently, there is a national initiative in Norway for a prospective study validating an Immunoscore in NSCLC.
      • Donnem T.
      • Kilvaer T.K.
      • Andersen S.
      • et al.
      Strategies to implement immunoscore in resected non-small cell lung cancer into the clinic.
      However, there are still several challenges regarding establishment of an Immunoscore, such as standardizing (1) immunohistochemical staining procedures, (2) the scoring system, and (3) the localization for scoring. According to our experience and results from the literature (see Table 1), stromal scoring of CD8+ T cells appears superior to scoring in the tumor epithelial compartment.
      • Geng Y.
      • Shao Y.
      • He W.
      • et al.
      Prognostic role of tumor-infiltrating lymphocytes in lung cancer: a meta-analysis.
      But where in the tumor stromal areas should the CD8+ cell assessment be performed (in the stroma in general or at the invasive margin)? Recent data from Donnem et al. indicate that the strongest prognostic impact of stromal scoring was at the invasive tumor margin, although verification is warranted.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      Figure thumbnail gr4
      Figure 4From tumor, node, and metastasis (TNM) to TNM-Immunoscore (TNM-I) in non–small cell lung cancer? The American Joint Committee of Cancer and the Union for International Cancer Control define and regularly update the TNM classification systems. The current seventh edition of the lung cancer staging system, which was based on an initiative by the International Association for the Study of Lung Cancer, was first published in 2009 and took effect in January 2010.
      • Sobin L.G.M.
      • Wittekind C.
      TNM Classification of Malignant Tumors.
      When Donnem et al. assessed the association between Immunoscore (low, intermediate, and high density of stromal CD8+ T cells) and pathological stage (pStage) (IA, IB, IIA, IIB, and IIIA), Immunoscore distinctly discriminated patients in two or three prognostic groups within each TNM pStage.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      Within the respective pStages IIB and IIIA, in particular, there were substantial differences in prognosis when assessed by Immunoscore.
      Data from Donnem et al.
      • Donnem T.
      • Kilvaer T.K.
      • Andersen S.
      • et al.
      Strategies to implement immunoscore in resected non-small cell lung cancer into the clinic.
      and Donnem et al.
      • Donnem T.
      • Hald S.M.
      • Paulsen E.E.
      • et al.
      Stromal CD8+ T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.
      Currently, there are national and international initiatives to include assessments of TIL infiltration in colorectal cancer, breast cancer, and NSCLC as an adjunct to the TNM classification to improve patients’ diagnoses, therapy, and prognosis.
      • Donnem T.
      • Kilvaer T.K.
      • Andersen S.
      • et al.
      Strategies to implement immunoscore in resected non-small cell lung cancer into the clinic.
      • Galon J.
      • Pages F.
      • Marincola F.M.
      • et al.
      Cancer classification using the Immunoscore: a worldwide task force.
      • Salgado R.
      • Denkert C.
      • Demaria S.
      • et al.
      The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.

      Immunoscore and Immunotherapies

      The host immune system may contribute to both the development of cancer and the efficacy of cancer therapies, in particular where the induced tumor cell death is considered “immunogenic.” The field of immunotherapy has had a renaissance in recent years, especially owing to the remarkable clinical efficacy observed after therapy with immune checkpoint inhibitors against various cancers, including NSCLC.
      • Ansell S.M.
      • Lesokhin A.M.
      • Borrello I.
      • et al.
      PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma.
      • Herbst R.S.
      • Soria J.C.
      • Kowanetz M.
      • et al.
      Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.
      • Hodi F.S.
      • O'Day S.J.
      • McDermott D.F.
      • et al.
      Improved survival with ipilimumab in patients with metastatic melanoma.
      • Powles T.
      • Eder J.P.
      • Fine G.D.
      • et al.
      MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer.
      • Topalian S.L.
      • Hodi F.S.
      • Brahmer J.R.
      • et al.
      Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.
      Recently, Kim et al. demonstrated that expression of programmed death ligand 1 (PD-L1) on pulmonary squamous cell carcinoma cells was significantly associated with increased CD8+ TILs, which again was correlated with increased programmed death 1–positive (PD-1+) TILs in pulmonary squamous carcinoma.
      • Kim M.Y.
      • Koh J.
      • Kim S.
      • et al.
      Clinicopathological analysis of PD-L1 and PD-L2 expression in pulmonary squamous cell carcinoma: comparison with tumor-infiltrating T cells and the status of oncogenic drivers.
      It has further been shown, that the efficacy of immune checkpoint blockade depends on TILs infiltrating and acting in the tumor tissues. It has been proposed that future strategies must be aimed at increasing CD8+ TILs in number and functionality while minimizing their impairment by the immunosuppressive environment.
      • Aerts J.G.
      • Hegmans J.P.
      Tumor-specific cytotoxic T cells are crucial for efficacy of immunomodulatory antibodies in patients with lung cancer.
      There are currently efforts to predict which patients will respond to therapy with checkpoint blockade involving, in particular, anti–PD-1 and anti–PD-L1.
      • Anagnostou V.K.
      • Brahmer J.R.
      Cancer immunotherapy: a future paradigm shift in the treatment of non-small cell lung cancer.
      Here the immune contexture and the Immunoscore may allow prediction of response to immune-related therapies.
      • Sznol M.
      • Chen L.
      Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer—response.
      • Taube J.M.
      • Anders R.A.
      • Young G.D.
      • et al.
      Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape.
      • Teng M.W.
      • Ngiow S.F.
      • Ribas A.
      • et al.
      Classifying Cancers Based on T-cell Infiltration and PD-L1.
      • Angell H.
      • Galon J.
      From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer.

      Conclusion

      In lung cancer development, neoplastic cells interact with their microenvironment, in which local cancer antigen-specific immune responses decide the destiny of the cancer cells. In recent years, there has been an increasing awareness of the immune contexture of NSCLC. This is defined according to immune cell type, density, and location, as well as according to the functional orientation of the involved immune cell populations in situ in the NSCLC microenvironment. The immune contexture represents, in fact, a major player in the development and fate of malignant disease. Elements of this immunogenic microenvironment and, in particular, the presence of TLSs correlate with survival. The possibility of assessing and evaluating a tumor’s immune contexture will be a pivotal instrument to identify subsets of patients at high risk for relapse in order to modify therapy or allocate patients for adjuvant treatment. An established standardized immunopathological assessment, such as an Immunoscore added to the TNM classification (TNM-I), may be fundamental to offering a highly improved prognostic, and most possibly, also a predictive tool. A TNM-I for NSCLC will need to be evaluated and validated in large prospective studies before implementation. There are already proposals for revision of the forthcoming eighth edition of the TNM classification for lung cancer.
      • Rami-Porta R.
      • Bolejack V.
      • Crowley J.
      • et al.
      The IASLC Lung Cancer Staging Project: proposals for the revisions of the T descriptors in the forthcoming eighth edition of the TNM classification for lung cancer.
      The immune contexture should be incorporated into future editions of the TNM classification, as research has demonstrated substantial room for improvements regarding prognostic and possibly also predictive value. As outlined earlier in this article, the Immunoscore can delineate profound variety in NSCLC survival within each pStage. In this regard, Immunoscore should be further investigated in NSCLC and definitely evaluated for future TNM-I classifications. A score involving assessment of in situ immunity will also be fundamental for optimization of novel therapies such as immune checkpoint blockade, as it is essential to select patients who will respond to this treatment modality.
      • Teng M.W.
      • Ngiow S.F.
      • Ribas A.
      • et al.
      Classifying Cancers Based on T-cell Infiltration and PD-L1.

      Acknowledgments

      We are grateful for the financial support from the Norwegian Cancer Society and the Northern Norway Health Authorities.

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      • Expanding Tumor Lymphocytic Infiltration as a Prognostic Tool to Patients with NSCLC Who Are Treated with Radiotherapy?
        Journal of Thoracic OncologyVol. 11Issue 11
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          We commend Bremnes et al.1 for their thoughtful review on “The role of tumor-infiltrating lymphocytes in the development, progression and prognosis of non-small cell lung cancer.” Their review did not, however, comment on a recent study by Brambilla et al.,2 which assessed the impact of tumor lymphocytic infiltration (TLI) on survival in a homogeneous population of patients with resected NSCLC.2 In this study, among patients treated on prospective trials of cisplatin-based adjuvant chemotherapy, TLI was associated with improved overall survival in a discovery set, and again in a validation set.
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