Prognostic Immune Markers in Non-Small Cell Lung Cancer

Author Manuscript Published OnlineFirst on June 9, 2011; DOI: 10.1158/1078-0432.CCR-10-2805 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Kei Suzuki1, Stefan S Kachala1, Kyuichi Kadota1,2, Ronglai Shen3, Quincy Mo3, David G Beer4, Valerie W Rusch1, William D Travis5, Prasad S Adusumilli1, 6

1Division of Thoracic Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York. 2Department of Pathology, Kagawa University, Japan. 3Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York. 4Department of Surgery, University of Michigan, Ann Arbor, Michigan. 5Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York. 6Center of Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York, New York.

Keywords: immune response, NSCLC, lung adenocarcinoma, tumor immunology, tumor stroma

Please send direct correspondence to: Prasad S Adusumilli MD 1275 York Avenue Memorial Sloan-Kettering Cancer Center New York, NY 10065 Phones: 212-639-8093 Fax: 646-422-2340 Email: [email protected]

Supported in part by American Association of Cancer Research (AACR) Lung Cancer Translational Research Award, American Association for Thoracic Surgery (AATS) -Third Edward D. Churchill Research Scholarship, IASLC – International Association for the Study of Lung Cancer Young Investigator Award, National Lung Cancer Partnership / LUNGevity Foundation Research Grant, William H. Goodwin and Alice Goodwin and the Commonwealth Foundation for Cancer Research and the Experimental Therapeutics Center, New York State Empire Clinical Research Investigator Program (ECRIP), and Stony Wold-Herbert Fund

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 9, 2011; DOI: 10.1158/1078-0432.CCR-10-2805 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Statement of Translational Relevance

Recurrence in lung cancer continues to pose a major clinical challenge, with rates of

recurrence as high as 40% among early stage patients who undergo resection with curative

intent. This highlights the need for additional prognostic markers and therapeutic targets. The

host immune response to tumor is a critical component in tumor progression and a prognostic

marker in colorectal and ovarian cancers. In this first comprehensive review of immune

markers of non-small cell lung cancer (NSCLC) in (a) the tumor microenvironment, (b)

peripheral blood, and (c) gene profiling studies, we identify those immune markers that are

prognostic. This review provides a foundation for future studies investigating tumor

immunology in NSCLC.

Abstract

Tumor-associated immune responses have polarized effects in regulating tumor growth.

While a clear association has been shown between the tumor immune response and clinical

outcome in colorectal and ovarian cancers, the role of immune markers for stratifying

prognosis in non-small cell lung cancer (NSCLC) is less defined. Herein we review the

prognostic significance of published immune markers in the tumor microenvironment as well

as peripheral blood of NSCLC patients. To identify prognostic immune genes, we reviewed

all published gene profiling studies in NSCLC and delineated the significance of immune

genes by performing subanalysis on the microarray database of the NIH Director’s Challenge

study. This first comprehensive review of prognostic immune markers provides a foundation

for further investigating immune responses in NSCLC.

Introduction

Immune responses within the tumor microenvironment are increasingly implicated as

determining factor in tumor progression and aggressiveness. Understanding these responses

has allowed investigators to use immune markers to stratify prognosis of colorectal and

ovarian cancer patients (1, 2). High levels of intratumoral effector memory cells in colorectal

cancer and CD3+ T cells in ovarian cancer are associated with prolonged survival.

Published studies investigating immune markers as prognosticators in non-small cell

lung cancer (NSCLC) are heterogeneous in histology (adenocarcinoma, squamous cell, and

large cell) and stage (I-IV). In this review, we have identified prognostic immune markers in

the tumor microenvironment and peripheral blood, as well as tumor expression of immune

genes. The immune genes, from 17 published NSCLC gene profiling studies, were evaluated

for prognostic significance in the NIH Director’s Challenge microarray (3), the largest

multi-center gene profiling study to date.

Prognostic Immune Cells

Tumor-Infiltrating Lymphocytes (TILs)

The interaction between tumor and immune cells in the tumor microenvironment is

influenced by the type of immune cells (CD8+, CD4+, CD20+, and FoxP3+ - see Table 1),

their density (as counted microscopically), and location (tumor nest and stroma) (4). Among

T lymphocytes, which comprise 80% of TILs in NSCLC (5), CD8+ cytotoxic lymphocytes

form the effector arm of adaptive immunity and are thought to have protective roles against

tumors. However, five investigations in NSCLC show no correlation between CD8+ TIL

infiltration and survival (6-10). Pertinent to NSCLC patients is the fact that CD8+ T-cell

predominance is a characteristic of smoking-related chronic obstructive pulmonary disease

(11), and they are thought to drive the progression of emphysema (12). In mouse models,

activation of CD8+ T cells are impaired in the presence of cigarette smoke (13, 14), perhaps

explaining this finding in NSCLC patients. In fact, studies that report correlation between

CD8+ TIL and prognosis contradict each other (15, 16). Ruffini et al. demonstrate in their

investigation of 1290 NSCLC tumors that TILs, mostly CD8+, were associated with

prolonged survival but only in a subset of squamous cell carcinomas (n=549) (15). This is

contrasted by Wakabayashi et al., who correlate tumoral CD8+ and shorter overall survival in

NSCLC, especially in adenocarcinoma (16). Examining the functional significance of CD8+

T-cells, Trojan et al. analyzed mRNA ratio of IFN-γ to CD8, where IFN-γ is an effector

cytokine secreted by cytotoxic T cells, specifically in peri-tumoral areas and within tumor

nests. While the number of peritumoral CD8+ T cells correlated with the IFN-γ/CD8 ratio,

this association was not observed intra-tumorally (8). This suggests that while the CD8+

T-cells are able to infiltrate the tumor, they are not able to mount a robust anti-tumor response

once within the tumor nest.

In contrast to CD8+ lymphocytes, stromal CD4+ (16, 17), CD20+ (17, 18), and

co-localization of stromal CD8+ and CD4+ cells (9) have all shown association with

improved survival. The significance of immune cell co-localization is highlighted in a study

by Dieu-Nosjean et al., who demonstrate the presence of tertiary de novo lymphoid structure

in the tumor microenvironment, which they named the tumor-induced bronchus-associated

lymphoid tissue (Ti-BALT) (19). In their investigation of 74 stage I-IIA NSCLC, the presence

of mature dendritic cells (Lamp+, lysosome-associated membrane protein) in Ti-BALT

correlate with higher density of T and B lymphocytes and more importantly prolonged overall

(OS) and disease-free survival (DFS) (4-year DFS of 51% vs 88%). The investigators

postulate that effector immune cell co-localization within Ti-BALT could play a role in

activating an anti-tumor immune response.

Regulatory T-cells (Tregs)

Regulatory T cells (Tregs) suppress the host immune response and are thought to

promote tumor growth. The pro-tumor association of Treg tumor infiltration was examined

by Shimizu et al. in a study of stage I-III 100 NSCLC tumors, demonstrating that

tumor-infiltrating FoxP3+ Tregs correlate with cyclo-oxygenase-2 (COX-2) expression and

increased tumor recurrence (20). This is supported by preclinical findings from Sharma et al.,

who demonstrated that tumor-derived COX-2 and its product prostaglandin E2 (PGE2)

induced in vitro lymphocyte expression of FoxP3. In vivo, inhibition of COX-2 reduced Treg

activity, attenuated FoxP3 expression in TILs, and decreased tumor burden (21). Furthermore,

urinary PGE-M, the major metabolite of PGE2, was proposed as a biomarker to predict

response to COX-2 inhibitors in NSCLC patients (22). Petersen et al. demonstrated that

Treg/TIL Combination Risk Index (the ratio of FoxP3+ to CD3+) correlates with

disease-specific survival (DSS) in patients with stage I NSCLC (23). Patients with high-risk

tumors (30% of tumors) experienced worse DSS (median 53 months) when compared to

patients with intermediate (63 months) and low-risk tumors (>72 months), which had low

FoxP3 and high CD3.

Tumor-Associated Macrophages (TAMs)

TAMs demonstrate both pro- and anti-tumor effects in the tumor microenvironment

(24). Anti-tumor TAMs in NSCLC are of the M1 phenotype and accumulate intra-tumorally

while pro-tumor TAMs of the M2 phenotype accumulate in the stroma and express IL-8,

IL-10, and triggering receptor expressed on myeloid cells (TREM-1). IL-8 is an angiogenic

factor, and TAM’s angiogenic role in NSCLC has been shown by correlating macrophage

density with intra-tumor microvessel counts and poor patient outcomes (25). By performing

RT-PCR for IL-8 in resected specimens, the study concludes that TAMs lead to poor patient

outcome by their angiogenic role through IL-8 production. IL-10 is an immunosuppressive

cytokine, and its expression by TAM has been observed more commonly in stages II/III/IV,

thus correlating with decreased OS (26). TAM expression of TREM-1, which can initiate and

amplify an inflammatory response, is increased in malignant pleural effusions of NSCLC

patients (27). Furthermore, in 68 stage I-III NSCLC patients, increased level of TREM-1 high

TAMs in resected specimen was an independent predictor of shorter OS.

In contrast to these findings, TAMs have also been associated with prolonged survival

in a study of 144 stage I-IV NSCLC (28). Recent studies have shed light on these

contradictory findings, where TAMs have been classified according to their location and

phenotype. In an investigation 175 stage I-III NSCLC tumors, Welsh et al. found that CD68+

macrophages in the stroma are associated with poor prognosis (5-year OS of 27% vs 35%)

while intra-tumoral macrophages are associated with increased survival (5-year OS of 53% vs

8%) (29).

Phenotypically, TAMs are comprised of 2 distinct types: M1, pro-inflammatory with

anti-tumor activity, and M2, which are immunosuppressive, angiogenic, and pro-tumor (30,

31). In a nested case-control study, 20 long-survival patients (median 93 months) had high

tumor infiltration of the M1 phenotype compared to 20 poor-survival patients (median 8

months). M1 was characterized by the expression of human leukocyte antigen (HLA)-DR,

inducible nitric oxide synthase (iNOS), myeloid related protein (MRP) 8/14, and tumor

necrosis factor (TNF)-α (32). Contrasting the anti-tumor association of M1 TAMs, high

numbers of CD204+ M2 TAMs in stroma have been shown to correlate with decreased OS

(33). CD204+ TAMs are also associated with increased tumor expression of IL-10 and

monocyte chemoattractant protein (MCP)-1, two molecules which are known to attract

macrophages.

Tumor-Associated Neutrophils (TANs)

Similar to TAMs, TANs also have polarized functions. Although no published studies

investigate the role of TAN in human NSCLC, recent preclinical work by Fridlender et al.

showed that in mouse models of lung cancer, TGF-β induces a population of TAN with

pro-tumor function (N2) while TGF-β blockade results in anti-tumor neutrophils (N1) (34).

Depletion of these N1 neutrophils resulted in increased tumor growth.

Peripheral Blood Lymphocytes

The universal availability of peripheral blood lymphocyte count has led to its

investigation as a prognostic marker in NSCLC. In resected NSCLC patients (n=177; 72%

Stages I and II), increasing total lymphocyte counts were associated with lower hazard ratios

for death (HR 0.62, CI=0.43-0.9; p=0.012) (35). The same study further identified neutrophil

to lymphocyte ratio (NLR) as a superior predictor of survival compared to pathologic stage

(p=0.001) (35). Specifically, an NLR of >3.81 was found to be a significant predictor of

survival in patients with stage I NSCLC. As for the tumor-promoting Tregs, they have been

observed at an increased level in the peripheral blood of NSCLC patients compared to normal

healthy volunteers (36, 37). These reports correlate with elevated serum and plasma levels of

TGF-β and IL-10, both known promoters of Treg development, in NSCLC patients compared

to healthy controls (38, 39). Peripheral blood levels of Tregs, TGF-β, and IL-10, however,

have not been investigated in predicting clinical outcome of NSCLC patients.

Myeloid-Derived Suppressor Cells (MDSCs):

MDSCs represent a heterogeneous population of myeloid cells comprising immature

macrophages, granulocytes, and DCs at early stages of differentiation that have pro-tumor

effects. MDSCs are mobilized from bone marrow into the peripheral blood by tumor-derived

factors and accumulate in the tumor microenvironment, where they exert their pro-tumor

effect by inhibiting T cell proliferation and activation (40). Similar to Tregs, increased levels

of MDSCs, marked by CD11b+/CD14-/CD15+/ CD33+, were observed in the peripheral

blood of advanced stage NSCLC patients (n = 87) compared to healthy controls (41). In

addition, high levels of MDSCs were associated with decreased levels of CD8+ T cells,

further supporting the pro-tumor effect of MDSCs. No study to date, however, has

investigated the prognostic significance of MDSCs in the tumor microenvironment of patient

samples.

Summary of Prognostic Tumor-Infiltrating Immune Cells

Stromal CD4+ lymphocytes, especially co-localized with CD8+ lymphocytes, stromal

CD20+ B lymphocytes, Ti-BALT, and intra-tumoral macrophages (M1) are associated with

prolonged survival in NSCLC. In contrast, FoxP3+ Tregs, stromal macrophages (M2), and

their associated cytokines – IL-8, IL-10, and TREM-1 – are associated with poor prognosis.

Tumor expression and secretion of COX-2 and IL-10, respectively, lead to shorter survival.

These prognostically significant findings are summarized in Figure 1.

Prognostic Immune Genes

In the large, multi-site, blinded validation study examining lung adenocarcinoma

(NIH Director’s Challenge), one of the four prognostic gene clusters contained genes related

to immunologic function (3). Further pathway analysis demonstrated immune function to be

important in stratifying lung adenocarcinomas into prognostic subgroups (42). In addition to

the NIH Director’s Challenge study, we reviewed all published genomic studies performed on

lung cancer between 2000-2010 that utilized mRNA microarray or RT-PCR. From the 17

studies reviewed (total of 1,615 patients, Supplementary Table 1) (3, 43-58), we identified

the prognostic genes related to immune/inflammatory response (search/selection criteria

shown in Supplementary Figure 1), resulting in a list of 84 genes (Supplementary Table

2).

As shown in Figure 2, there are seven overlapping immune genes from the 17

reviewed studies, and two genes are from a well-investigated chemokine axis in NSCLC –

CCL19/CCR7. CCR7 (C-C chemokine receptor type 7) is a chemokine receptor expressed on

naïve T cells, dendritic cells, natural killer cells, and B cells, and its interaction with its

ligands, CCL19 (C-C motif chemokine 19) and CCL21 (C-C motif chemokine 21), play a

central role in lymphocyte trafficking and homing to lymph nodes (59, 60). In lung cancer,

CCR7 expression on tumor cells by mRNA has been demonstrated as an independent

predictor of lymph node metastasis in an investigation of 71 NSCLC patients (61). It has been

hypothesized that when expressed on tumors cells, the homing mechanism of CCR7

contributes to tumor’s increased potential for lymphatic metastasis.

While CCR7 may increase the metastatic potential of tumor cells, investigators have

attempted to exploit the effect of this chemokine axis on the immune cells to promote tumor

regression. CCL19, or Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC),

expressed in the T cell zone of lymph nodes and dendritic cells, is known to attract immune

cells which express CCR7. While no clinical study has investigated the role of CCL19 in

NSCLC, its ability to promote tumor infiltration of T cells and dendritic cells (DCs) has been

shown in preclinical models to decrease tumor burden. Intra-tumoral injection of CCL19 lead

to increased influx of CD4+ and CD8+ T cells and DCs, increase in anti-tumor factors such

as IFN-γ and IL-12, and a decrease in the immunosuppressive molecule TGF-β (62, 63).

CCL21, the other ligand of CCR7, has been investigated as a potential adjunct to

DC-based therapy due to its ability in promoting co-localized tumor infiltration of DCs and

lymphocyte effector cells (64). Intratumoral injection of DCs transduced to express CCL21

reduced tumor burden in a mouse model of spontaneous bronchoalveolar carcinoma. Based

on these findings, a phase I clinical trial is currently investigating the intratumoral injection

of CCL-21-expressing DCs in stage IIIb/IV NSCLC patients refractory to standard therapy

(65).

Significance of Prognostic Immune Genes in the NIH Director’s Challenge study

Cox proportional hazards model was used to evaluate the association between DFS

and the expression profiles of the 84 identified prognostic immune genes in the Director’s

Challenge dataset (3). This analysis revealed 17 genes which were significantly associated

with recurrence (Table 2). We employed the Ingenuity Pathway Analysis to examine the

networks shared by the 17 genes (Figure 3). STAT3 was a common signal transduction

pathway highlighted in this analysis, which has been implicated in cancer inflammation and

immunity (66).

Although prognostic immune genes are identified in NSCLC, the cells of origin

(tumor cells, tumor-infiltrating immune cells, or both) of these genes remain to be explored.

One of the 17 prognostic genes, IL-7R, is expressed by both the tumor and immune cells.

When expressed on the tumor, IL-7R expression leads to up-regulation of VEGF-D in vitro

and also shorter OS in NSCLC patients (67). On the other hand, it has been demonstrated in

vitro that the expression of IL-7R on CD8+ T cells signifies a long-lived memory phenotype

(68). How the expression of these genes modulates the tumor immune microenvironment is

future area of investigation. CXCR4 is the receptor for stromal-derived factor-1alpha

(CXCL12). In lung adenocarcinoma, tumor expression of CXCL12 has been shown to

correlate with accumulation of CXCR4-expressing immune cells, 30% of which were

pro-tumor regulatory T cells (69).

Conclusion

The immune microenvironment surrounding the tumor is influenced by interactions

among tumor, immune cells (TILs, TAMs, TANs, and MSDCs), and cytokines that shift the

environment to pro- or anti-tumor. In NSCLC, stromal CD4+ T lymphocytes, especially

co-localized with CD8+ T cells, stromal CD20+ B lymphocytes, Ti-BALT, and intra-tumoral

M1 macrophages predicted prolonged patient survival. FoxP3+ Tregs and stromal M2

macrophages are associated with poor clinical outcome. We examined gene expression

profiling studies for insights into the NSCLC tumor microenvironment. In reviewing 17 gene

profile studies, we identified 17 prognostic immune genes by comparing their expression to

tumor recurrence in lung adenocarcinoma patients. Further studies utilizing RT-PCR and

immunohistochemical analysis are necessary to determine the source cell (tumor versus

immune) and how they influence the tumor immune microenvironment. Future studies

investigating a large patient series uniform in histology and stage and assessing

tumor-immune interaction in both tumor nest and tumor-associated stroma may yield

significant information.

Acknowledgements

We thank Eduardo Morales (figure) and Erlin Daley (editorial) for their assistance in

preparation of the manuscript.

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Figure Legends:

Figure 1. Prognostic immune markers in NSCLC. T cells and B cells are associated with

longer survival when found in the stroma along with Ti-BALT which contains Lamp+

dendritic cells. In contrast, FoxP3+ Tregs in the tumor are associated with shorter survival.

Anti-tumor M1 macrophages are characterized by HLA-DR, iNOS, MRP, and TNF-α.

Pro-tumor M2 macrophages express CD204. M2 expression of IL-8, IL-10, and TREM-1

(delineated by arrows) has been shown to correlate with shorter survival. Tumoral expression

of COX-2 recruits FoxP3+ Tregs cells while expression of IL-10 and MCP-1 recruits M2

macrophages. In the peripheral blood, immune suppression is associated with poor clinical

outcomes revealed by low TLCs and elevated NLRs.

COX, cyclooxygenase; DC, dendritic cells; FoxP3, forkhead box P3; IL, interleukn; iNOS,

inducible nitric oxide synthase; LAMP, lysosome-associated membrane protein; MCP,

monocyte chemoattractant protein; MRP, myeloid-related protein; NLR, neutrophil to

lymphocyte ratio; Ti-BALT, tumor-induced bronchus-associated lymphoid tissue; TLC, total

lymphocyte count; TNF, tumor necrosis factor; TREM, triggering receptor expressed on

myeloid cells; Treg, regulatory T cells.

Figure 2. Prognostic genes associated with immunity and inflammation. Prognostic

immune/inflammatory genes from published gene profile studies on lung cancer, with

overlapping immune genes shown in boxes.

Figure 3. Ingenuity Pathway Analysis of 17 significant immune genes. The data set

containing gene identifiers was uploaded into in the application. Each identifier was mapped

to its corresponding object in Ingenuity's Knowledge Base. A P value cutoff of 0.05 was set

to identify molecules whose expression was significantly differentially regulated. These

molecules, called Network Eligible molecules, were overlaid onto a global molecular

network developed from information contained in Ingenuity’s Knowledge Base. Networks of

Network Eligible Molecules were then algorithmically generated based on their connectivity.

Supplementary Figures:

Supplementary Figure 1. Selection criteria for reviewed studies on gene expression

profiles and approach to identification of prognostic immune genes.

Initial search for this review was performed by searches of PubMed and references from

relevant articles from using the search terms “gene expression profiling” and “lung cancer”.

Table 1 Summary of TIL and TAM role in lung cancer with respect to their phenotype and location *

Author # pts stages Observation/Conclusion Survival Advantage I – 54 (57%) Johnson7 95 II – 17 (18%) High CD3+ and S100+ in tumor correlated with longer OS III 20 (21%) Hiraoka9 109 I -67 (61%) Concurrent high CD4+ and CD8+ in stroma correlated with II-III - 42 (39%) longer survival Kikuchi10 161 I – 95 (59%) HLA class I expression correlates with longer OS in stage I II-IV – 66 (41%) HLA class I expression correlated with CD8+ cells I - 714 (55%) Ruffini15 1290 II -265 (21%) TIL (mostly CD8+) in tumor correlated with better OS IIIA -214 (17%) I -107 (60%) High CD4+ in stroma correlated with longer OS 5-yr OS 64% vs 43% TIL Wakabayashi16 178 II -23 (13%) High CD8+ in tumor correlated with shorter OS 5-yr OS 47% vs 60% IIIA -48 (27%) I -212 (63%) High CD4+ in stroma correlated with longer DSS 5-yr DSS 63% vs 42% Al-Shibli17 335 II - 91 (27%) High CD8+ in stroma correlated with longer DSS 5-yr DSS 75% vs 53% IIIA - 32(10%) High CD20+ in stroma correlated with longer DSS 5-yr DSS 61% vs 32% I -66 (58%) Pelletier18 113 II - 20 (18%) Peritumoral CD20+ correlated with longer survival III -29 (26%)

Ti-BALT Dieu-Nosjean19 74 I - 62 (84%) High mature dendritic cells in tertiary lymphoid structures 4-yr DFS 88% vs 51% IIA - 12 (16%) correlated with longer survival I – 68 (68%) High FoxP3+ correlated with shorter time to recurrence Shimizu20 100 II – 14 (14%) COX-2 expression correlated with shorter time to recurrence Treg III – 18 (18%) COX-2 expression correlated with FoxP3+ infiltration Petersen23 64 I - 64 High proportion of FoxP3+ among TIL in tumor median DSS of 53mo, 63mo, and >72 mo correlated with shorter DFS for high, intermediate, and low risk group I - 14 (40%) TAM in stroma correlated with shorter OS Chen25 35 II - 4 (11%) TAM-tumor interaction upregulated IL-8 mRNA expression median OS 16 mo vs 45 mo III - 17 (49%) Zeni26 47 I – 24 (51%) IL-10 high TAMs associated with shorter OS II/III/IV – 23 (49%) IL-10 high TAMs associated with advanced stage I – 24 (35%) Ho27 68 II – 15 (22%) Increased high TREM-1 macrophages correlated Median DFS 22 mo vs not reached TAM III – 29 (43%) with shorter DFS and OS Median OS 29 mo vs not reached I - 79 (55%) Kim28 144 II - 25 (17%) TAM in tumor correlated with longer OS 5-yr OS 64% vs 39% III - 38 (26%) I - 79 (45%) Welsh29 175 II - 44 (25%) High TAM in tumor correlated with longer OS 5-yr OS of 53% vs 8% IIIA - 34 (19%) I – 26 (65%) Ohri32 40 II – 8 (20%) Increased M1 in long survivors III – 6 (15%) Ohtaki33 170 IA – 95 (56%) High stromal CD204+ (M2) associated with shorter survival 5-yr OS of 61% vs 89% IB-IIIA – 75 (44%)

* Abbreviations: TIL – tumor-infiltrating lymphocytes, Ti-BALT – tumor-induced bronchus-associated lymphoid tissue, Treg – regulatory T-cells, TAM – tumor-associated macrophages, OS – overall survival, DSS – disease-specific survival, DFS – disease-free survival Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2011 American Association for Cancer Research. Downloaded from Table 2 Prognostic immune genes of statistical significance following computational analysis and their functions

Gene symbol p.value (HR) Gene Name Function Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonJune9,2011;DOI:10.1158/1078-0432.CCR-10-2805 LIME1 (20q13.3) 0.0007 (1.0036) LCK interacting transmembrane adaptor 1 Involved in BCR (B-cell antigen receptor)-mediated signaling in B-cells Involved in TCR (T-cell antigen receptor)-mediated T-cell signaling in T-cells IL7R (5p13) 0.0012 (0.9993) interleukin-7 receptor subunit alpha; CD127 Receptor for interleukin-7 clincancerres.aacrjournals.org Expressed at high levels on naïve T cells CD59 (11p13) 0.0034 (0.9997) CD59 glycoprotein Potent inhibitor of the complement membrane attack complex (MAC) action membrane attack complex inhibitory protein RHOH (4p13) 0.0102 (0.9977) Rho-related GTP-binding protein RhoH Negative regulator of hematopoietic progenitor cell proliferation, survival and migration CYFIP2 (5q33.3) 0.0109 (0.9989) cytoplasmic FMR1-interacting protein 2 Involved in T-cell adhesion Stimulates a migratory response in CD4+ lymphocytes, monocytes, and eosinophils IL16 (15q26.3) 0.0128 (0.9880) interleukin-16 Primes CD4+ T-cells for IL-2 and IL-15 responsiveness lymphocyte chemoattractant factor Induces T-lymphocyte expression of interleukin 2 receptor on September 30, 2021. © 2011American Association for Cancer IFI35 (17q21) 0.0184 (1.0007) interferon-induced 35 kDA protein Unknown Research. ENPP2 (8q24.1) 0.0192 (0.9996) Ectonucleotide pyrophosphatase family member 2 Involved in several motility-related processes autotaxin CXCR4 (2q21) 0.0247 (0.9997) C-X-C chemokine receptor type 4 Receptor for CXCL12 stromal cell-derived factor 1 receptor BMP7 (20q13) 0.0292 (1.0061) bone morphogenic protein 7 Induces cartilage and bone formation CCR7 (17q12-21.2) 0.0311 (0.9986) C-C chemokine receptor type 7 Receptor for MIP-3β (macrophage inflammatory protein 3 beta) MS4A1 (11q12) 0.0351 (0.9993) B-lymphocyte antigen CD20 Involved in the regulation of B-cell activation and proliferation CCR6 (6q27) 0.0405 (0.9907) C-C chemokine recptor type 6 Receptor for MIP-3α (macrophage inflammatory protein 3 alpha) CCL19 (9p13) 0.0432 (0.9996) CCL19 protein Plays a role in inflammatory and immunological responses and also in normal lymphocyte recirculation and homing Plays a role in trafficking of T-cells in thymus, and T-cell and B-cell migration to secondary lymphoid organs CCL8 (17q11.2) 0.0446 (1.0009) C-C motif chemokine 8 Chemotactic factor that attracts monocytes, lymphocytes, basophils and eosinophils monocyte chemotactic protein 2 CCR2 (3p21.31) 0.0446 (0.9975) C-C chemokine receptor type 2 Receptor for the MCP(monocyte chemoattractant protein)-1, 3 and 4 monocyte chemoattractant protein 1 receptor BST2 (19p13.1) 0.0468 (1.0002) bone marrow stromal antigen 2 Involved in pre-B-cell growth tetherin; CD317

Author Manuscript Published OnlineFirst on June 9, 2011; DOI: 10.1158/1078-0432.CCR-10-2805 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

TREM-1 [27] IL-8 [25] MDSC M2 [41] CD68 [29] CD204 [33] NLR >3.81[35] IL-10 [26,33] MCP-1 [33] Treg N

FoxP3/CD3 [23]

COX-2 [20] Stroma Tumor Peripheral Blood

Ti-BALT [19] M1

CD68 [28, 29] DC TLC HLA-DR, iNOS >1500/mm3 [35] MRP, TNF-α [32] LAMP [19] T

CD20 [17, 18] T CD4 [16, 17] CD4 + CD8 [9]

RHOH Boutros Lau Raz Chen (4) (1) CCR2 CCR7 (8) (3) Roapman (10) LCK Director’s Borczuk Lu Challenge (2) (6) ENPP2 (43)

Hou Hayes Beer Larsen CCL5 (2) (7) (4) (3) Lee (3) Aviel- Tomida Bianchi CCL19 Ronen (1) (1) (1)

CCL19 CCR6 CHEMOKINE CCR7 CCL8 CXCR4* STAT5B SPI1 STAT5A

JAK3 MS4A1* CCR2 RELA STAT3 FOXP3

IFI16 IFI35 IL7R GATA3 GABPB1 CASP3 ZAP70 PTK2B RHOH TP53 NEDD4

Prognostic Immune Markers in Non-Small Cell Lung Cancer

Kei Suzuki, Stefan S Kachala, Kyuichi Kadota, et al.

Clin Cancer Res Published OnlineFirst June 9, 2011.

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