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Prognostic Effect of Epithelial and Stromal Lymphocyte Infiltrationin Non^Small Cell Lung CancerKhalid I. Al-Shibli,1,2 TomDonnem,3,4 Samer Al-Saad,2,5 Magnus Persson,2,5

RoyM. Bremnes,3,4 and Lill-Tove Busund2,5

Abstract Purpose:Themajor value of prognostic markers in potentially curable non-small cell lung cancer(NSCLC) should be to guide therapy after surgical resection. In this regard, the patients’ immunestatus at the time of resection may be important and also measurable. The immune system hasparadoxical roles during cancer development. However, the prognostic significance of tumor-infiltrating lymphocytes is controversial.The aim of this study is to elucidate the prognostic signif-icance of epithelial and stromal lymphocyte infiltration in NSCLC.Experimental Design:Tissue microarrays from 335 resected NSCLC, stage I to IIIAwere con-structed from duplicate cores of viable and representative neoplastic epithelial and stromal areas.Immunohistochemistry was used to evaluate the epithelial and stromal CD4+, CD8+, and CD20+

lymphocytes.Results: Inunivariate analyses, increasingnumbers of epithelial CD8+ (P =0.023), stromal CD8+

(P = 0.002), epithelial CD20+ (P = 0.023), stromal CD20+ (P < 0.001), and stromal CD4+

(P < 0.001) lymphocytes correlated significantly with an improved disease-specific survival.No such relation was noted for epithelial CD4+ cells. Furthermore, a low level of stromal CD8+

lymphocyte infiltration was associated with an increased incidence of angiolymphatic invasion(P = 0.032). In multivariate analyses, a high number of stromal CD8+ (P = 0.043) and CD4+

(P = 0.002) cells were independent positive prognostic factors for disease-specific survival.Conclusions: High densities of CD4+ and CD8+ lymphocytes in the stroma are independentpositive prognostic indicators for resected NSCLC patients.This may suggest that these cells aremediating a strong antitumor immune response in NSCLC.

Despite medical advances, lung cancer remains the leadingcause of cancer death mainly due to delayed diagnosis resultingin the low resection rate of 20% (1, 2). The major histologicsubtypes of lung cancer are non-small cell lung cancer (NSCLC;80%) and small cell lung cancer (20%; ref. 2). The only chancefor cure is surgical treatment in early stages of disease. However,as much as 60% of stages IB to IIIA patients relapse after surgeryand die of metastasis (2).

Activation of the adaptive immune system may suppressmalignant cells, whereas activation of various types of innateimmune cells may promote tumor growth (3). The adaptiveimmunity is orchestrated by antigen-specific T and B lympho-cytes and inhibits tumor growth through direct killing by CTLsas well as a combination of cytokine- and antibody-mediated

tumor cell lysis (3). Recently, it became clear that analysis of thetumor stroma is paramount, as many of these antitumor effectsact mainly on the tumor stroma (4), and cancer infiltration bytumor-reactive T lymphocytes is required for efficient tumoreradication (5). However, cancer cells can escape the immunesurveillance by many mechanisms such as suppression ofcytotoxic T cells by regulatory T cells and by accumulation ofmyeloid suppressor cells (5–7).

Tumor-infiltrating lymphocytes can be divided into threegroups: (a) lymphocytes within cancer cell nests (epitheliallymphocytes), (b) lymphocytes in the central cancer stroma(stromal lymphocytes), and (c) lymphocytes present along theinvasive margins (peritumoral lymphocytes; ref. 8). In NSCLC,about two-thirds of inflammatory cells are lymphocytes, amongthese 80% are T cells (9), expressing various activation antigens(10). CD8+ cells in malignant tumors have been associated witha better survival in small cell lung carcinoma, carcinomas of theendometrium, bile duct, colon, esophagus, and urothelium, aswell as uveal melanoma and follicular lymphoma (11–18). Ithas been shown that NSCLC contain higher proportions ofCD8+ T lymphocytes than their corresponding peripheral bloodsamples (10), and a higher number of tumor-infiltratingCD8+ lymphocytes is associated with tumor cell apoptosis inNSCLC (19).

The role of CD8+ lymphocytes in NSCLC is, however,controversial and most of the studies contain either relativelyfew cases and/or neglect the stromal component. In addition,

Imaging, Diagnosis, Prognosis

Authors’Affiliations: 1Department of Pathology, NordlandCentral Hospital, Bod8,Norway; Institutes of 2Medical Biologyand 3ClinicalMedicine, UniversityofTroms8;and Departments of 4Oncology and 5Pathology, University Hospital of NorthernNorway,Troms8, NorwayReceived1/18/08; revised 4/24/08; accepted 4/24/08.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Khalid I. Al-Shibli, Department of Pathology, NordlandCentral Hospital, 8092 Bod8, Norway. Phone: 47-7553-84-38/47-9282-87-19;Fax: 47-7553-4074; E-mail: [email protected].

F2008 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-08-0133

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CD4+ T and B lymphocytes are double-edged swords in tumorimmunity, and their role is controversial in many cancersincluding NSCLC. Pelletier et al. (20) found peritumoral B, andnot T, lymphocytes to be related to a better survival.Wakabayashi et al. (8) found stromal CD4+ cells to be a goodprognostic indicator. Hiraoka et al. (21) found only theconcurrent infiltration by CD8+ and CD4+ T lymphocytes inthe tumor epithelial cells to be associated with a better survival,whereas Nakamura et al. (22) showed no prognostic signifi-cance, neither for the total intraepithelial T cells nor for itshelper and cytotoxic subtypes, in NSCLC.

To elucidate the prognostic significance of lymphocyteinfiltrate in NSCLC, we analyzed the degree of infiltration ofCD4+, CD8+, and CD20+ lymphocytes in 335 resected NSCLCin both epithelial and stromal compartments and studied theirrelations to other clinicopathologic variables as well as theproportion of lymphocytes compared to CD138+ and CD68+

cells.

Patients andMethods

Patients and clinical data. Patients diagnosed with NSCLC, patho-logic stage I to IIIA at the University Hospital of Northern Norway andNordland Central Hospital from 1990 to 2004 were used in thisretrospective study. In total, 371 patients were obtained from thehospitals’ database. Of these, 36 patients were excluded from the studydue to (a) radiotherapy or chemotherapy before surgery (n = 10), (b)other malignancy within 5 years before NSCLC diagnosis (n = 13), and

(c) adequate paraffin-embedded fixed tissue blocks not available(n = 13). Thus, 335 patients were included in this study.

The median follow-up was 96 (range, 10-179) months. Twopathologists (S.A.S. and K.A.S.) reviewed all the cases; the diagnosisof carcinoma, histologic type, angiolymphatic invasion, and pathologicstage were confirmed before including any case in the study. The tumorswere staged according to the International Union Against Cancer’stumor-node-metastasis classification (23). Stage IA and IB patients weregrouped together as stage I, and stage IIA and IIB patients were groupedas stage II. Histologic classification was done according to the WHOguidelines (24).

The National Data Inspection Board and the Regional Committee forResearch Ethics in Northern Norway approved this study.

Microarray construction. Tissue microarrays were constructed asreported previously (25, 26). Studies suggest that punching multiple0.6 mm cores from different regions captures the heterogeneity of thetumors more accurately than single 2 to 4 mm core (25). Hence, wechose using four 0.6-mm cores that were selected to be as representativeas possible, after reviewing all the original sections of the tumor andtaking the fact of heterogeneity in consideration. In addition, all thesurface area of the four cores was counted. Two areas of viable invasivecarcinoma tissue (neoplastic epithelium) and two from the surround-ing, central tumor stroma were selected and marked on the donorblocks. The tissue microarrays were obtained using a tissue-arrayinginstrument (Beecher Instruments) from two epithelial areas and twostromal areas and transferred to the recipient blocks. Lung tissuewithout signs of pathologic changes localized distant from the primarytumor as well as lung tissue samples from 20 normal lungs were used ascontrol. To include all the 1,340 cores plus the control cores, eighttissue array blocks were constructed. Multiple 5 Am sections were cut

Fig. 1. Immunohistochemical microscopicpictures of tissue microarray of NSCLCrepresenting different scores forepithelial and stromal CD8+ lymphocytes.A, epithelial CD8 low score. B, epithelialCD8 high score. C, stromal CD8 lowscore.D, stromal CD8 high score. Originalmagnification, �200.

Prognostic Effect of Lymphocyte Infiltration in NSCLC

www.aacrjournals.org Clin Cancer Res 2008;14(16) August15, 20085221



with a Micron microtome (HM355S) and stained by specific antibodiesfor immunohistochemistry analysis.

In most NSCLC cases, the malignant cells proliferate along thealveolar walls of the lung and lack stroma at the invasive margin (8); inthis study, we analyzed infiltration of CD4+, CD8+, and CD20+

lymphocytes in the neoplastic epithelial areas and in the centralstromal areas.

Immunohistochemistry. Ventana Benchmark, XT automated slidestainer (Ventana Medical System) was used for immunohistochemistry.Sections were deparaffinized with xylene and rehydrated with ethanol.Antigen retrieval was done by placing the specimens in 0.01 mol/L

citrate buffer (pH 6.0) and exposed to two repeated microwave heatingof 10 min at 450 W. The DAKO Envision+ System-HRP (diaminobenzi-dine) kit was used as endogen peroxidase blocking. As negative stainingcontrols, the primary antibodies were replaced with the primary antibodydiluents. Primary mouse monoclonal antibodies were incubated for16 min (CD20, clone L26 Ventana), 20 min (CD4, clone 1F6 Novocastra,dilution 1:5), 32 min (CD8, clone 1A5 Ventana), 16 min (CD68, cloneKP1, Ventana), and 32 min (CD138, clone B-A38, Ventana) in roomtemperature. The Ventana antibodies were prediluted from the manu-facturer. As secondary antibodies, biotinylated goat anti-mouse IgG andmouse anti-rabbit IgM were used. The diaminobenzidine was used to

Table 1. Prognostic clinicopathologic variables as predictors for DSS in 335 NSCLS (univariate analysis,log-rank test)

Characteristics Patients [n (%)] Median survival (mo) 5-y survival (%) P

Age (y)V65 156 (47) 104 57 0.62>65 179 (53) NR 58

SexFemale 82 (25) 127 65 0.19Male 253 (75) 84 55

SmokingNever 15 (5) 19 43 0.13Present 215 (64) NR 60Previous 105 (31) 84 54

Performance statusNormal 197 (59) NR 62 0.04Slightly reduced 120 (36) 61 52In bed <50% 18 (5) 36 40

Weight loss<10% 303 (90) 127 57 0.92>10% 32 (10) NR 57

HistologySCC 191 (57) NR 65 0.30Adenocarcinoma 95 (28) 52 44Bronchioloalveolar carcinoma 18 (5) NR 67Large cell carcinoma 31 (9) 84 54

DifferentiationPoor 138 (41) 48 48 0.001Moderate 144 (43) NR 64Well 53 (16) NR 65

Surgical procedureLobectomy + wedge* 243 (73) NR 61 0.0009Pneumonectomy 92 (27) 35 46

StageI 212 (63) NR 68 <0.00001II 91 (27) 41 46IIIA 32 (10) 18 22

Tumor status1 90 (27) NR 75 0.0022 218 (65) 84 523 27 (8) 42 43

Nodal status0 232 (69) NR 66 <0.000011 76 (23) 37 432 27 (8) 18 20

Surgical marginsFree 307 (92) 127 58 0.34Not free 28 (8) 64 51

Vascular infiltrationNo 284 (85) NR 61 0.0005Yes 51 (15) 25 35

Postoperative radiotherapyNo 276 (82) NR 61 0.002Yes 59 (18) 41 42

Abbreviation: NR, not reached.*Wedge (n = 10).





visualize the antigens. This was followed by application of liquiddiaminobenzidine and substrate-chromogen, yielding a brown reactionproduct at the site of the target antigen. Finally, slides were counter-stained with hematoxylin to visualize the nuclei. For each antibody,including controls, staining were done in a single experiment.

Scoring of immunohistochemistry. By light microscopy, the tissuesections were scored for the degree of infiltration of CD4+, CD8+, andCD20+ lymphocytes. The percentages of lymphocytes compared withthe total amount of nucleated cells in the epithelial and stromalcompartments were assessed. Arbitrary cutoff points at 1%, 5%, 25%, or50% for each cell/compartment according to the degree of cell densitieswere used, as these percentages are easy to follow and reproduce indaily practice.

For CD8+ cells, the epithelial infiltrate was scored as low V5% orhigh >5% in the whole surface area of the two epithelial cores andwas scored as low V50% or high >50% of the total nucleated cells inthe whole surface area of the two stromal cores. CD20+ (B) cells werevery sparse and its infiltrate was scored as low if they represent <1% ofthe nucleated cells or if absent, or high otherwise, in both epithelialand stromal compartments. CD4+ cells were scored as high if z5% orz25% of the total nucleated cells in the epithelial and stromal cores,respectively. The interstitial tissue of the nonneoplastic/normalcontrols show no or sparsely scattered CD20+ cells (0 to <1% of thetotal nucleated cells) and few CD4+ and CD8+ cells (0 to <5% of thetotal nucleated cells).

All samples were anonymized and independently scored by twopathologists (S.A.S. and K.A.S.). In case of disagreement, the slides werere-examined and the observers reached a consensus. When assessingone marker in a given core, both observers were blinded to the scoresof other markers as well as to the patient’s outcome. The interobserverscoring agreement between the two pathologists was tested on thecurrent material in a previous report (27). The mean correlationcoefficient (r) was 0.95 (range, 0.93-0.98). Examples of stromal andepithelial cores stained for CD8 lymphocytes are shown in Fig. 1.

Statistical analysis. All statistical analyses were done using thestatistical package SPSS version 15. The m2 test and Fisher’s exact test

were used to examine the association between the density of thelymphocyte infiltrates and various clinicopathologic variables. Univar-iate analyses were done by using the Kaplan-Meier method, andstatistically significant differences between survival curves were assessedby the log-rank test. Disease-specific survival (DSS) was determinedfrom the date of surgery to the time of lung cancer death. To assess theindependent value of different pretreatment variables on survival in thepresence of all other variables, multivariate analysis was carried outusing the Cox proportional hazards model. Only variables with asignificant P value from the univariate analysis were entered into theCox regression analysis. Probability for stepwise entry and removalwas set at 0.05 and 0.10, respectively. The significance level was set atP < 0.05.

Results

Clinicopathologic variables. Demographic, clinical, and his-topathologic variables are shown in Table 1. Patients’ ageranged between 28 and 85 years (median, 67 years), and 75%of the patients were males. The NSCLC tumors comprised 191squamous cell carcinomas (SCC), 95 adenocarcinomas, 31large cell carcinomas, and 18 bronchioloalveolar carcinomas.Due to nodal metastasis and/or nonradical surgical margins,59 patients received postoperative radiotherapy. There were232 lymph node–negative and 103 positive cases (76 N1 and27 N2).

Univariate analysis. Performance status, pathologic stage,T status, N status, differentiation, surgical procedure, vascularinfiltration, and postoperative radiotherapy were all significantindicators for disease-free survival in univariate analysis.

Lymphocyte infiltration. Tumor-infiltrating lymphocyteswere observed in both epithelial and stromal compartmentsand were generally more abundant in the stroma. Tumor inflam-matory cells were mainly lymphocytes (f65%) compared with

Table 2. Intraepithelial and stromal lymphocyte subsets and their prediction for DSS in NSCLC patients(univariate analysis; log-rank test)

Marker expression Patients, n (%) Mean survival (mo) 5-y survival (%) P

CD4Tumor

Low 252 (75) 107 56 0.46High 78 (24) 113 60Missing 3 (1)

StromaLow 82 (25) 70 42 <0.001High 251 (74.5) 121 63Missing 2 (0.5)

CD8Tumor

Low 243 (42) 102 54 0.023High 85 (58) 125 68

StromaLow 268 (80) 99 53 0.002High 61 (18) 143 75Missing 6 (2)

CD20Tumor

Low 263 (78.5) 104 55 0.023High 68 (20.5) 115 67Missing 4 (1)

StromaLow 42 (16.5) 56 32 <0.001High 292 (83) 115 61Missing 1 (0.5)





20% to 25% CD68+ cells and 5% to 10% plasma cells. Plasmacells were more abundantly found in the stroma of SCCscompared with other histologic types (high expression; SCCs61%, adenocarcinomas 35%, bronchioloalveolar carcinomas29%, and large cell carcinomas 50%; P < 0.001). Similar resultswere obtained for the epithelial compartment.

In univariate analyses, the group of patients with highepithelial CD8+ lymphocytes had a significantly better DSSthan those with low CD8+ cells (P = 0.023; Table 2; Fig. 2). Bysubgroup analysis, this significance was seen only in patientswithout lymph node metastasis (N0; P = 0.018), whereaspatients with lymph node metastasis showed a tendency forbetter DSS but did not reached the significant level. Furtherstratifying the cases based on histology revealed that thesignificance was limited to SCCs (P = 0.011). Epithelial CD20+

lymphocytes showed a similar significance (P = 0.023). Thissignificance was limited to SCCs (P = 0.030) and mostsignificant in T2 cases (P = 0.017). Epithelial CD4+ cellsshowed no significant correlation with DSS even after analysisof the cases in subgroups.

Regarding stromal lymphocytes, a high number of CD8+ cellswas significantly associated with an improved DSS (P = 0.002;Table 2; Fig. 2). This was limited to large cell carcinomas andSCCs. Stromal CD4+ lymphocytes showed a highly significantcorrelation with DSS (P < 0.001), strongest in N1 cases.Similarly, CD20+ cells in the stroma showed a significantassociation with DSS (P < 0.001). Both stromal CD20+ andCD4+ significance were limited to SCCs. Concurrent infiltratesof epithelial CD4+ and CD8+ cells failed to show prognosticsignificance, whereas concurrent infiltrates of stromal CD4+

and CD8+ cells was a significant and strong positive indicator(P < 0.001; Fig. 3). Furthermore, a high level of stromal CD8+

lymphocytes was associated with a lower incidence ofangiolymphatic invasion (P = 0.032). However, neitherepithelial nor stromal CD4+, CD8+, or CD20+ lymphocytesshowed any correlation with smoking, performance status, sex,or tumor differentiation. In addition, there were significantcorrelations between the amounts of CD4+ and CD8+ cells inthe stroma (r = 0.18; P = 0.001) and in the tumor epithelium(r = 0.274; P < 0.001).

Fig. 2. DSS curves for stromal and epithelial CD4+, CD8+, and CD20+ lymphocytes in NSCLC.





Multivariate analyses. All significant demographic, clinico-pathologic, and lymphocyte infiltrate variables from theunivariate analyses were entered into the multivariate Coxregression analysis. These data are presented in Table 3. Ahigh number of stromal CD4+ (P = 0.002) and stromal CD8+

(P = 0.043) lymphocytes were independent prognostic factorsfor DSS.

Discussion

Tumor-infiltrating lymphocytes are considered to be anindication of the host immune reaction to tumor antigens(28). In this study, we evaluated whether there is an associationbetween the amount of epithelial and stromal CD4+, CD8+,and CD20+ lymphocytes and the clinical course in 335 primaryresected NSCLC. Our data suggest that high intensities ofstromal CD4+ cells as well as epithelial and stromal CD8+ andCD20+ cells are significantly associated with an improvedsurvival in NSCLC. In fact, the strong association betweensurvival and the stromal intensities of CD4+ and CD8+ wereindependent of the other clinicopathologic prognostic factors.Furthermore, a high stromal infiltration of CD8+ lymphocyteswas associated with a lower incidence of angiolymphaticinvasion in our cohort. As expected, the patients’ survival washighly linked to stage of disease.

The fact that our patients were diagnosed over a 15-yearperiod (1990-2004) may have introduced a bias due to altereddiagnostic techniques (e.g., introduction of spiral computedtomographic scan and positron emission tomographic scan).However, the cited disease stage is based on the pathologictumor-node-metastasis (examination of the surgical specimen),which should be minimally altered during the period. Adjuvantchemotherapy was not introduced in Norway during thisperiod.

Our findings regarding CD8+ cells suggest that these cells canmodify tumor stroma and/or epithelium in a way that reducesthe disease progression and metastatic potential. In studies onadaptive immunotherapy and tumor vaccination in colorectalcarcinoma, the outcome was superior among patients whoseprimary tumor contained high level of CD8+ lymphocytes (29).Tormanen-Napankangas et al. (19) found that a higher numberof epithelial CD8+ lymphocytes is associated with tumor cellapoptosis in NSCLC, but this finding did not translate to a

survival benefit, whereas Trojan et al. (30) could not show anyinfluence of epithelial CD8+ cells and IFN-g/CD8 ratio onoverall survival in their study of 31 NSCLC patients. However,in a laser microscopic study on 16 randomly selected stage INSCLC, Verdegaal et al. (31) found a vigorous antitumorimmune response mediated by CD8+ T cells despite HLAhaplotype loss in 69% of the tumors. They also found 90% ofthe CD8+ cells to be activated cytotoxic cells. The positiveprognostic effect of CD8+ cells in NSCLC despite their tendencyto loose HLA antigens may be explained by Kikuchi et al. (32)in a study of 161 NSCLC tumors. A negative HLA was observedin 54, weak in 57, and strong in 50 cases. The density of cancer-infiltrating CD8+ cells in HLA-I-negative tumors was signifi-cantly decreased compared with that in HLA-I strong tumors(P < 0.01).

Epithelial and stromal CD8+ cells, in our study, correlatedstrongly with outcome in patients without lymph nodemetastases, a finding that may indicate a key role of these cellsin tumors restricted to the lung tissue. In addition, the detectionof a significant association between increased stromal CD8+

cells and low incidence of angiolymphatic invasion mayindicate a protective role of these cells in preventing tumorcell infiltration into the vascular spaces. This may suggest thatthe positive prognostic effect of CD8+ cells may be mediated, atleast in part, by suppressing micrometastases, which is a majorcause of cancer-related death following surgical radical resec-tion. A similar suggestion was made by Chiba et al. (33) in theirstudy on colon cancer.

Moreover, our data showed that stromal CD4+ T cells areassociated with a favorable DSS in NSCLC. A similar result has

Table 3. Results of Cox regression analysissummarizing significant independent prognosticfactors

Factor Hazard ratio(95% confidence ratio)

P

Tumor status 0.004*1 1.0002 2,248 (1.351-3.740) 0.0023 2,623 (1.311-5.248) 0.006

Nodal status <0.001*0 1.0001 2.286 (1.492-3.501) <0.0012 3.110 (1.783-5.424) <0.001

Performance status 0.025*Normal 1.000Slightly reduced 1.564 (1.056-2.316) 0.026In bed <50% 2.342 (0.967-5.676) 0.059

Differentiation 0.048*Poor 1.000Moderate 0.611 (0.407-0.916) 0.017Well 0.670 (0.363-1.238) 0.20

Vascular infiltrationNo 1.000Yes 1.961 (1.207-3.186) 0.007

CD4+ cells, stromaHigh 1.000Low 2.624 (1746-3.945) <0.001

CD8+ cells, stromaHigh 1.000Low 1.893 (1.021-3.511) 0.043

*Overall significance as a prognostic factor.

Fig. 3. DSS analysis for combined stromal CD4+ and CD8+ lymphocytes inNSCLC.





been reported by Wakabayashi et al. (8) who studied epithelialand stromal CD4+ and CD8+ cells in 178 NSCLC patients andobserved only CD4+ in cancer stroma to be associated with abetter prognosis. This finding points to the fact that CD4+

lymphocytes may be needed for initiating and maintaininganticancer immune responses. In the absence of CD4+ cell help,specific CD8+ lymphocytes can become lethargic (34) or bedeleted (35). In fact, CD4+ cells are essential for CD8+

transformation into long-lived functional effector cells (36).This positive interaction between CD4+ and CD8+ lymphocytesin our cohort is shown by the observation of a significantcorrelation between the degree of infiltration of the two celltypes and by the finding of an improved DSS in cases withconcurrent high stromal CD4+ and CD8+ cell counts whencompared with heterogeneous high/low or low/high andhomogenous low/low counts. In addition, CD4+ cells caninhibit tumor growth in the absence of CD8+ cells by directlyses or recruiting other cells (3). However, CD4+ cells are amixture of CD4+/CD25+Foxp3+ (regulatory T cells) and CD4+/Foxp3- cells. The first subtype is known to suppress tumorimmunity and enhance cancer growth and metastases (7, 29).In animal models, removal of CD4+/CD25+ T cells improvedimmune-mediated tumor clearance and enhanced the responseto immunotherapy (37, 38). Our results suggest that in NSCLCthe net effect of CD4+ cells is toward supporting cancereradication. Badoual et al. (39) found that even CD4+

regulatory T cells were, paradoxically, associated with a superiorlocoregional control (but not survival) in head and neck SCC.This may also explain our results; more studies involving CD4+

subtypes in NSCLC are needed to test this hypothesis.Epithelial CD4+ cells failed to show a prognostic significance.

Hiraoka et al. (21) studied epithelial CD8+ and CD4+ cells in109 NSCLC patients and found only the concurrent high levelsof epithelial CD8+ and CD4+ cells to be a significantindependent indicator for a better survival. A similar analysisin our cases failed to reveal a significant prognostic relevance.

In our cohort, the independently positive prognostic effect ofCD4+ and CD8+ cells in the stroma points to the significance ofthe stromal component in modulating cancer cells. Antitumorcytokines contributing to tumor suppression act mainly in thestroma, whereas killing mediated by molecules such as perforinor Fas ligand acts mainly on tumor cells and to a lesser extenton the stroma (4). Similarly, in an adoptive T-cell transfer

model, Spiotto et al. (40) showed that tumor rejection requiresantigen presentation to CD8+ cells by cancer cells and cross-presentation by bone marrow–derived and non-bone mar-row–derived stromal cells. In fact, T-cell-produced cytotoxicmolecules and cytokines destroy the stromal cells, therebywithdrawing essential resources and leading to tumor infarctionand subsequent T-cell-mediated elimination of residual tumorcells (41). Treating well-established tumors expressing lowlevels of antigens with local irradiation or chemotherapy causessufficient release of antigens to sensitize stromal cells fordestruction by cytotoxic CD8+ cells (42). In addition, theantitumor effect of cytotoxic lymphocytes may be theoreticallystrengthened by strategies inhibiting both the differentiationblock of CD8+ cells and the early Fas expression that leads toapoptosis of lymphoid cells.

Studying several prognostic factors in 113 NSCLC tumors,including ‘‘peritumoral’’ B (CD20+) and T (CD43+) lympho-cytes, Pelletier et al. (20) found B cells to be associated with abetter survival (P = 0.04). Our study showed a similar effect forstromal, as well as epithelial, B cells in univariate analysis. B cellsare precursors of antibody-mediated immunity, which maycontribute in tumor eradication (43). Additionally, the presenceof tumor-specific B cells may be a mirror of the host’s overallimmunity in NSCLC (20). On the other hand, the precipitationof immunoglobulin in the tissue may have a stimulatory effecton the innate immune cells, which may enhance tumor growthby secreting mediators stimulating angiogenesis (3). In fact, deVisser et al. (44) showed in an animal model that B lymphocytesare required for establishing chronic inflammatory states thatpromote de novo carcinogenesis.

In conclusion, a high number of stromal CD4+ and CD8+

cells are independent positive prognostic indicators for resectedNSCLC patients. It may be hypothesized that the majority ofCD8+ lymphocytes in NSCLC tumors are activated cytotoxiccells mediating a vigorous antitumor immune response andthat CD4+ cells play a central role in this immune response.Before these findings can be used for novel targeted therapystrategies, the underlying mechanisms need to be furthercharacterized.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.



References1. GoyaT, Asamura H,Yoshimura H, et al. Prognosis of6644 resected non-small cell lung cancers in Japan:a Japanese lung cancer registry study. Lung Cancer2005;50:227^34.2. Parkin DM, Muir CS. Cancer incidence in five conti-nents. Comparability and quality of data. IARC SciPubl 1992;120:45^173.3. de Visser KE, Eichten A, Coussens LM. Paradoxicalroles of the immune system during cancer develop-ment. Nat Rev Cancer 2006;6:24^37.4. BlankensteinT. The role of tumor stroma in the inter-action between tumor and immune system. Curr OpinImmunol 2005;17:180^6.5.Mukai S, KjaergaardJ, Shu S, Plautz GE. Infiltration oftumors by systemically transferred tumor-reactive Tlymphocytes is required for antitumor efficacy. CancerRes1999;59:5245^9.6. Costello RT, Gastaut JA, Olive D.Tumor escape fromimmune surveillance. Arch ImmunolTher Exp (Warsz)1999;47:83^8.

7. Enarsson K, Lundin BS, Johnsson E, Brezicka T,Quiding-Jarbrink M. CD4+ CD25high regulatoryTcellsreduce T cell transendothelial migration in cancerpatients. EurJ Immunol 2007;37:282^91.8.Wakabayashi O,Yamazaki K, Oizumi S, et al. CD4+ Tcells in cancer stroma, not CD8+ Tcells in cancer cellnests, are associated with favorable prognosis in hu-man non-small cell lung cancers. Cancer Sci 2003;94:1003^9.9. Kataki A, Scheid P, Piet M, et al. Tumor infiltratinglymphocytes and macrophages have a potential dualrole in lung cancer by supporting both host-defenseand tumor progression. J Lab Clin Med 2002;140:320^8.10. Kuo SH, Chang DB, Lee YC, Lee YT, Luh KT.Tumour-infiltrating lymphocytes in non-small cell lungcancer are activated T lymphocytes. Respirology1998;3:55^9.11. Eerola AK, Soini Y, Paakko P. A high number of tu-mor-infiltrating lymphocytes are associated with a

small tumor size, low tumor stage, and a favorableprognosis in operated small cell lung carcinoma. ClinCancer Res 2000;6:1875^81.12. Kondratiev S, Sabo E,Yakirevich E, Lavie O, ResnickMB. Intratumoral CD8+ T lymphocytes as a prognos-tic factor of survival in endometrial carcinoma. ClinCancer Res 2004;10:4450^6.13.OshikiriT,MiyamotoM, ShichinoheT, et al. Prognos-tic value of intratumoral CD8+ T lymphocyte in extra-hepatic bile duct carcinoma as essential immuneresponse. J Surg Oncol 2003;84:224^8.14. Schumacher K, HaenschW, Roefzaad C, SchlagPM. Prognostic significance of activated CD8(+)Tcell infiltrations within esophageal carcinomas. Can-cer Res 2001;61:3932^6.15. Sharma P, ShenY,Wen S, et al. CD8 tumor-infiltrat-ing lymphocytes are predictive of survival in muscle-invasive urothelial carcinoma. Proc Natl Acad SciUS A 2007;104:3967^72.16. Staibano S, Mascolo M, Tranfa F, et al. Tumor





infiltrating lymphocytes in uveal melanoma: a linkwith clinical behavior? Int J Immunopathol Pharmacol2006;19:171^9.17.Wahlin BE, Sander B, Christensson B, Kimby E.CD8+ T-cell content in diagnostic lymph nodesmeasured by flow cytometry is a predictor of sur-vival in follicular lymphoma. Clin Cancer Res 2007;13:388^97.18. NaitoY, Saito K, Shiiba K, et al. CD8+ Tcells infiltrat-ed within cancer cell nests as a prognostic factor inhuman colorectal cancer. Cancer Res 1998;58:3491^4.19. Tormanen-Napankangas U, Soini Y, Paakko P. Highnumberof tumour-infiltrating lymphocytes is associat-ed with apoptosis in non-small cell lung carcinoma.APMIS 2001;109:525^32.20. Pelletier MP, Edwardes MD, Michel RP, Halwani F,Morin JE. Prognostic markers in resectable non-smallcell lung cancer: a multivariate analysis. Can J Surg2001;44:180^8.21. Hiraoka K, Miyamoto M, ChoY, et al. Concurrent in-filtration by CD8+ Tcells and CD4+ Tcells is a favour-able prognostic factor in non-small-cell lungcarcinoma. BrJCancer 2006;94:275^80.22. Nakamura H, Saji H, Ogata A, et al. Immunologicparameters as significant prognostic factors in lungcancer. Lung Cancer 2002;37:161^9.23. SihoeAD,Yim AP. Lung cancer staging. J Surg Res2004;117:92^106.24. Travis WD. Pathology of lung cancer. Clin ChestMed 2002;23:65^81.25. Kallioniemi OP,Wagner U, KononenJ, Sauter G. Tis-sue microarray technology for high-throughput mo-lecular profiling of cancer. Hum Mol Genet 2001;10:657^62.

26. Bremnes RM, Camps C, Sirera R. Angiogenesis innon-small cell lung cancer: the prognostic impact ofneoangiogenesis and the cytokines VEGF and bFGFin tumours andblood. LungCancer 2006;51:143^58.27. DonnemT, Al-Saad S, Al-Shibli K, et al. Inverseprognostic impact of angiogenic marker expression intumor cells versus stromal cells in non small cell lungcancer. Clin Cancer Res 2007;13:6649^57.28. Dunn GP, BruceAT, Ikeda H, Old LJ, Schreiber RD.Cancer immunoediting: from immunosurveillance totumor escape. Nat Immunol 2002;3:991^8.29. Parmiani G.Tumor-infiltratingTcellsLfriendor foe ofneoplastic cells? NEngl JMed 2005;353:2640^1.30.Trojan A, UrosevicM, Dummer R, Giger R,Weder W,Stahel RA. Immune activation status of CD8+ Tcellsinfiltrating non-small cell lung cancer. Lung Cancer2004;44:143^7.31. Verdegaal EM, Hoogstraten C, Sandel MH, et al.Functional CD8+ T cells infiltrate into nonsmall celllung carcinoma. Cancer Immunol Immunother 2007;56:587^600.32. Kikuchi E,Yamazaki K, TorigoeT, et al. HLA class Iantigen expression is associated with a favorableprognosis in early stage non-small cell lung cancer.Cancer Sci 2007;98:1424^30.33. ChibaT, Ohtani H, MizoiT, et al. Intraepithelial CD8+

T-cell-count becomes a prognostic factor after a lon-ger follow-up period in human colorectal carcinoma:possible association with suppression of micrometa-stasis. BrJCancer 2004;91:1711^7.34. Bourgeois C,Veiga-Fernandes H, Joret AM, RochaB, Tanchot C. CD8 lethargy in the absence of CD4help. EurJ Immunol 2002;32:2199^207.35. Kurts C, Carbone FR, Barnden M, et al. CD4+ Tcellhelp impairs CD8+ T cell deletion induced by cross-

presentationof self-antigens and favors autoimmunity.JExp Med1997;186:2057^62.36. Bevan MJ. Helping the CD8(+) T-cell response.Nat Rev Immunol 2004;4:595^602.37. Ghiringhelli F, Larmonier N, Schmitt E, et al.CD4+CD25+ regulatoryTcells suppress tumor immu-nity but are sensitive to cyclophosphamide whichallows immunotherapyof established tumors to be cu-rative. EurJ Immunol 2004;34:336^44.38. Sutmuller RP, van Duivenvoorde LM, van EA, et al.Synergism of cytotoxicT lymphocyte-associated anti-gen 4 blockade and depletion of CD25(+) regulatoryT cells in antitumor therapy reveals alternative path-ways for suppression of autoreactive cytotoxicT lym-phocyte responses. JExp Med 2001;194:823^32.39. Badoual C, Hans S, Rodriguez J, et al. Prognosticvalue of tumor-infiltrating CD4+ T-cell subpopulationsin head and neck cancers. Clin Cancer Res 2006;12:465^72.40. Spiotto MT, Schreiber H. Rapid destruction of thetumor microenvironment by CTLs recognizing can-cer-specific antigens cross-presented by stromal cells.Cancer Immun 2005;5:8.41. KammertoensT, SchulerT, BlankensteinT. Immuno-therapy: target the stroma to hit the tumor.TrendsMolMed 2005;11:225^31.42. Zhang B, Bowerman NA, SalamaJK, et al. Inducedsensitization of tumor stroma leads to eradication ofestablished cancer byT cells. J Exp Med 2007;204:49^55.43. Gilboa E. The promise of cancer vaccines. Nat RevCancer 2004;4:401^11.44. deVisser KE, Korets LV, Coussens LM.Denovo car-cinogenesis promoted by chronic inflammation is Blymphocyte dependent. Cancer Cell 2005;7:411^23.



2008;14:5220-5227. Clin Cancer Res Khalid I. Al-Shibli, Tom Donnem, Samer Al-Saad, et al.

Small Cell Lung Cancer−Infiltration in Non Prognostic Effect of Epithelial and Stromal Lymphocyte

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