Intratumoral T-Cell Infiltrates and MHC Class I Expression

in Patients with Stage IV Melanoma

Salah-Eddin Al-Batran,1Mohammad-Reza Rafiyan,

1Akin Atmaca,

1Antje Neumann,

1

Julia Karbach,1Armin Bender,

1Eckhart Weidmann,

1Hans-Michael Altmannsberger,

2

Alexander Knuth,3and Elke Jager

1

1II. Medizinische Klinik-Onkologie and 2Pathologisches Institut, Krankenhaus Nordwest, Frankfurt, Germany and 3UniversitatsspitalZurich, Zurich, Switzerland

Abstract

The infiltration of tumors by T cells has been shown tocorrelate with prolonged patients’ survival. However, itremains unclear why only some tumors are infiltrated withT cells. This study was designed to investigate possiblecorrelations between intratumoral T-cell infiltrates and theexpression of cancer-associated antigens and MHC class Iand II molecules in patients with melanoma. Fresh frozensamples from 124 stage IV melanoma patients wereanalyzed by immunohistochemistry for the expression ofMelan-A/MART-1, tyrosinase, gp100, NY-ESO-1, and MHCclass I and II. Intratumoral T-cell and B-cell infiltrates weredetected by staining with anti-CD4, anti-CD8, anti-CD3, andL26 antibodies. The NY-ESO-1 serum antibody status wasassessed by Western blot analysis. Intratumoral CD8+ andCD4+ T cells were detected in 63.9% and 71.3% of patients,respectively. We observed a significant heterogeneity of theexpression of the melanocyte differentiation antigens, NY-ESO-1, and MHC class I and II molecules. The onlysignificant correlation was found between the expressionof MHC class I and the presence of CD4+ and CD8+ T cells(P < 0.0001). There was a strong association between thesetwo variables with respect to the density and distribution ofinfiltrating T cells and the pattern of MHC class I expression( focal versus homogenous). Intratumoral T-cell infiltrationis closely correlated with the MHC class I expression but notwith the expression of differentiation antigens, cancer-associated antigens, or MHC class II molecules. Theseresults may have implications for the definition of prog-nostic variables and for the identification of patients whomay benefit from antigen-specific cancer immunotherapy.(Cancer Res 2005; 65(9): 3937-41)

Introduction

The infiltration of tumors by T cells has been shown tocorrelate with the clinical outcome of patients with several typesof cancer, including melanoma, renal cell, prostate, breast,colorectal, and esophageal cancers (1–6). Most recently, a clearassociation between the presence of intratumoral T cells andprolonged patients’ progression-free and overall survival inovarian cancer was established (7). However, the pattern of T-cellinfiltration of tumors is heterogeneous and it remains unclear why

only some tumors are infiltrated with T cells. The present study wasdesigned to investigate this issue by evaluating the expression ofcancer-associated antigens, MHC class I and II molecules, andintratumoral T and B cells in samples of advanced melanoma.

Materials and Methods

Patients and samples. We evaluated fresh frozen samples from 124

stage IV melanoma patients between January 2001 and December 2002. All

patients had metastatic disease and had failed at least one palliativechemotherapy regimen. All tumor samples were obtained from metastatic

lesions. Informed consent was obtained from all patients.

Immunohistochemistry. Cryosections (5 Am thickness) were fixed in

acetone and stained with H&E or immunostained by indirect immuno-peroxidase method (DAKO, Glostrup, Denmark) as recommended by the

manufacturer. Monoclonal antibodies against the T-cell markers CD3,

CD4, and CD8, against B-cell marker (L26), against the MHC class Icomplex (W6/32; ref. 8), and against the HLA-DR of MHC class II complex

(L243), the melanocyte differentiation antigens Melan-A/MART-1 (A103;

ref. 9), tyrosinase (T311; ref. 10), and gp100 (HMB45; ref. 11), and the

cancer testis antigen NY-ESO-1 (E978; ref. 12) were used. The sectionswere examined by a pathologist and two independent additional

investigators trained in the histopathology of melanoma. Tumor-

infiltrating T cells were defined as T cells detected within the tumor

cell nests (intratumoral T cells) and were evaluated by semiquantitativescoring on a scale of 1+ to 3+ (scattered or mild 1+, moderate 2+, and

strong 3+). Tumor-associated antigens and MHC molecules were graded

as 1+, 2+, or 3+ (5-24%, 25-50%, or >50% positive tumor cells, respectively).

The overall distribution of stained cells was graded as focal (one or fewtumor cell nests were positive or infiltrated by T cells) or diffuse.

Heterogeneous staining (1+ to 3+) was considered as diffuse and graded

according to the area of the highest level of antigen expression.Tumor typing for NY-ESO-1 mRNA. Expression of NY-ESO-1 mRNA in

tumor specimens was assessed by reverse transcription-PCR (RT-PCR) using

primers described previously (13).

Detection of NY-ESO-1 serum antibody. NY-ESO-1 serum antibodieswere assayed by Western blot analysis using NY-ESO-1 recombinant protein

purified from Escherichia coli (14) as described.

Results

Intratumoral T-cell infiltration. Intratumoral CD8+ and CD4+

T cells were detected in 78 of 122 (63.9%) and 87 of 122 (71.3%)patients, respectively. In most cases, the observed T-cell infiltrationwas mild to moderate (Table 1). The results for CD8+ and CD4+ cellswere closely correlated. In the majority of cases (109 of 122),intratumoral CD8+ and CD4+ cells were either both detectable orboth absent. Total intratumoral (CD3+) lymphocytes were evaluatedin a smaller patient population and found to be positive in 43 of 62(67.7%) patients, correlating well with the results of intratumoralCD8+ and CD4+ cells. Positive immunoreactivity of the B-cell markerL26 was detected in 90 of 116 (77.6%) cases.

Note: S-E. Al-Batran and M-R. Rafiyan contributed equally to this work.Requests for reprints: Salah-Eddin Al-Batran, II. Medizinische Klinik-Onkologie,

Krankenhaus Nordwest, 60488 Frankfurt, Germany. Phone: 49-69-76013340; Fax: 49-69-76013655; E-mail: albatran@aol.com.

I2005 American Association for Cancer Research.

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NY-ESO-1 expression and NY-ESO-1 antibody. Tumors from48 of 124 (38.7%) patients with stage IV melanoma expressed NY-ESO-1 as detected by either immunohistochemistry (28 of 60;46.6%) or RT-PCR (20 of 64; 31.3%). The staining pattern for NY-ESO-1 was heterogeneous. In most cases, 5% to 50% of the tumorcells were positive, corresponding to 1+ to 2+ in our grading system(Table 1). No positive correlation between NY-ESO-1 expressionand the presence of intratumoral CD4+ or CD8+ T cells could befound (Ps = 0.21 and 0.11, respectively, Fisher’s exact test). Therewere no correlations between both variables with respect to theintensity or the overall distribution of immunoreactivity in thetumor. Eight of 45 (17.8%) patients had detectable NY-ESO-1 serumantibody, and in seven of eight patients, tumor samples expressedNY-ESO-1 as detected by RT-PCR.Expression of the melanocyte differentiation antigens. The

expression proportions are shown in Table 1. In the cases shownhere, the number of positive cells was comparatively high (3+ in38% of patients), staining was more homogenous, and focalexpression was less frequent. No statistically significant correla-tions between the expression of any antigen and the T-cellinfiltration were found.MHC class I and II expression. Eighty-nine of 123 (72.4%)

samples were positive for MHC class I (W6/32) and 53 of 55(96.4%) samples tested were positive for MHC class II (HB55). Thestaining pattern for MHC class I was more heterogeneouscompared with MHC class II. Focal expression was observed in17 of 89 (19.2%) cases (Table 1).Relation between MHC class I expression and T-cell

infiltration. Positive W6/32 immunoreactivity was stronglyassociated with the presence of CD8+ and CD4+ T cells. Inaddition, there was a clear correlation between W6/32 immu-noreactivity and CD8+/CD4+ infiltration with respect to thenumber and distribution of positive cells and the overall patternof MHC class I expression ( focal versus homogenous). Forinstance, focal MHC class I staining was observed in 17 cases, 13of which exhibited focal CD8+ lymphocyte infiltration (Fig. 1Aand B). Only one of these cases showed a homogenous but weak(+1) infiltration, and the remaining three were negative.Conversely, negative staining with W6/32 was found in 34 cases,

30 of which were also negative for CD8+ T cells (Fig. 1E and F), 3showed a weak positive infiltration, corresponding to 1+ in ourgrading system, and 1 was not evaluable for CD8+ T-cellinfiltration. Tables 2, 3, and 4 show the strong statisticalassociations between the intensity and distribution of T-cellinfiltrates and MHC class I expression.

Discussion

Growing evidence has supported the view that the immunesystem may be able to interact with determinants of tumor cellsin vivo (15, 16). Mainly the presence of tumor-infiltrating T cells hasbeen shown to correlate with improved patients’ clinical outcome(1–7). In some cases, the specificity of infiltrating CD8+ T cells wasdirected against cancer-associated antigens expressed by thetumor sample. However, it remains unclear why only some tumorsare infiltrated with T cells, and little is known about the relationbetween tumor-infiltrating T cells and the expression of tumor-associated antigens or MHC molecules.Previous studies on limited patient populations described a

decrease of expression of the melanocyte differentiation antigensalong with more advanced clinical stages (Table 5; refs. 11,17, 18). The expression of MHC molecules has also been shownto be reduced in advanced tumor stages (19–21). In single cases,

Table 1. Intratumoral T-cell and B-cell infiltrates and the expression of cancer-associated antigens and MHC class I and IImolecules

Antigen Patients Positive, n (%) Scoring Focal n (%)

1+ 2+ 3+

CD4 122 87 (71.3) 50 (57.5) 33 (37.9) 4 (04.6) 15 (12.3)CD8 122 78 (63.9) 42 (53.8) 26 (33.3) 10 (12.8) 14 (11.5)

CD3 62 42 (67.7) 8 (19.0) 22 (52.4) 12 (28.6) 3 (07.1)

L26 116 90 (77.6) 42 (46.6) 33 (36.7) 15 (16.7) 10 (11.1)

NY-ESO-1 124 48 (38.7)* 16 (57.1)c

11 (39.3)c

1 (03.6)c

4 (14.3)c

Melan-A/MART-1 121 106 (87.6) 20 (18.9) 46 (43.4) 40 (37.7) 7 (06.7)

Tyrosinase 121 103 (85.1) 36 (35.0) 37 (35.9) 30 (29.1) 5 (04.8)

Gp100 121 95 (78.5) 32 (33.7) 29 (30.5) 34 (35.8) 13 (13.7)

MHC I 123 89 (72.4) 39 (43.8) 38 (42.7) 12 (13.5) 17 (19.2)MHC II 55 53 (96.4) 12 (22.6) 39 (73.6) 2 (03.8) 7 (13.2)

*As detected by either immunohistochemistry (28 of 60; 46.6%) or RT-PCR (20 of 64; 31.3%).cThe scoring is based on the results of immunohistochemistry.

Table 2. Correlation between MHC class I expression andCD8+ T-cell infiltration with respect to the variables:positive/negative

n = 122* CD8+ cells

present

CD8+ cells

absent

P

W6/32 negative (n = 33) 3 30 <0.0001c

W6/32 positive (n = 89) 75 14

*Number of samples evaluable for W6/32 and CD8+ T cells.cFisher’s exact test.

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a loss of expression of MHC class I molecules was observed insingle growing tumors of patients with a mixed response topeptide vaccination therapy, representing a potential mechanismof immune escape from antigen-specific T-cell recognition (22–24).Our results confirm these observations, showing that in most casesof advanced melanoma MHC class I molecules were expressed in<50% of the tumor cells or were absent. Interestingly, MHC class IImolecules were regularly expressed in the majority of samplesevaluated.We compared in our samples the presence of tumor-infiltrating T

cells with the expression status for MHC molecules and melanocytedifferentiation antigens and found that the immunohistochemicaldetection of MHC class I molecules by positive staining with W6/32was significantly associated with tumor-infiltrating CD4+ and CD8+

T cells. On the other hand, no significant correlations were foundbetween detectable tumor-infiltrating T cells and the expression ofmelanocyte differentiation antigens or MHC class II molecules.The presence of tumor-infiltrating T cells did not correlate eitherwith the expression of the highly immunogenic cancer testisantigen NY-ESO-1 (13, 14) or with the status of NY-ESO-1 serumantibody.

Both the loss of tumor-associated antigens and the down-

regulation or alteration of MHC class I and II molecules have been

considered to be potential means of tumor escape from immunerecognition (25–28). Our analysis of tumor samples of patients

Table 3. Correlation between MHC class I expression andCD8+ T-cell infiltration with respect to the variables: focal/homogenous

n = 75* Focal CD8+

cell infiltration

Homogenous

CD8+ cell

infiltration

P

W6/32 focal

expression (n = 14)

13 1 <0.0001c

W6/32 homogenous

expression (n = 61)

1 60

*Number of samples evaluable for W6/32 and CD8+ T cells.cFisher’s exact test.

Figure 1. MHC class I expression andCD8+ T-cell infiltration in three differentcases of metastatic melanoma. A and B,a sample with focal expression of MHCclass I (W6/32) scored as 2+ (A ) and focalCD8+ T-cell infiltration (B) withcorresponding areas of strong ("""),moderate (""), and negative (")MHC class I expression/CD8+ T-cellinfiltration. C and D, a sample withhomogeneous MHC class I expression,3+ (C) and CD8+ T-cell infiltration,3+ (D ). E and F, a sample with a total lossof MHC class I expression (E ) and nointratumoral CD8+ T-cell infiltrates (F ).Original magnification, �100 (A and B),�600 (C and D), and �400 (E and F ).

T-Cell Infiltrates and MHC Class I in Stage IV Melanoma

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with metastatic melanoma by immunohistochemistry showed asignificant heterogeneity of the expression of the melanocytedifferentiation antigens Melan-A/MART-1, tyrosinase, and gp100,the cancer testis antigen NY-ESO-1, and MHC class I and II mole-cules and the levels of tumor-infiltrating CD4+ and CD8+ T cells.However, tumor-infiltrating CD4+ and CD8+ Tcells were restricted totumors that were MHC class I positive. In a recent report, clonallyexpanded tumor-infiltrating lymphocytes in melanoma lesions didnot correlate with the heterogeneous expression of melanocytedifferentiation antigens, whereas MHC class I molecules were notevaluated (29). A positive correlation between tumor-infiltratinglymphocytes and MHC class I molecules was found in a series of 18head and neck squamous cell carcinoma biopsies (30). Our resultsadd to these observations and indicate that MHC class I–expressingtumor cells may trigger intratumoral T-cell infiltrates in vivo . Lowlevels of MHC expression may lead to reduced antigen presentation,which might prevent sufficient immunologic recognition and

subsequently the intratumoral accumulation of CD4+ and CD8+ Tcells. The expression of MHC class I molecules in metastatic diseasemay represent a major prognostic factor for the clinical benefitresulting from spontaneous and specific vaccine-induced antitumorimmunity. The targeting of MHC class II–restricted epitopes, theadministration of immunotherapy in adjuvant settings or in earlystages of cancer, the use of drugs that enhance the expression ofMHC class I molecules, or the selection of patients with tumors thathomogeneously express MHC class I molecules for clinical vaccinetrials may represent effective strategies for making antigen-specificcancer vaccines more effective.

Acknowledgments

Received 12/28/2004; revised 2/10/2005; accepted 2/21/2005.Grant support: Cancer Research Institute, New York and Krebforschung Rhein

Main e.V.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Table 4. Correlation between MHC class I expression andCD8+ T-cell infiltration as graded by the scoring system(1+ to 3+)

MHC class I expression (n = 75)* CD8+ T-cell infiltration

1+ 2+ 3+

1+ 27 27 0 0

2+ 36 11 23 2

3+ 12 1 3 8

*Number of samples positive for W6/32 and CD8+ T cells.

Table 5. Inverse correlation of melanocyte differentiationantigen expression and clinical stage in patients withmelanoma (%)

Antigen Stage I Stage II Stage II Stage IV

Melan-A/MART-1

(n = 57; ref. 17)

100 88 90 75

Tyrosinase

(n = 50; ref. 18)

100 100 86 86

NOTE: Adapted from Hofbauer et al. (17, 18).

References1. Vesalainen S, Lipponen P, Talja M, et al. Histologicalgrade, perineural infiltration, tumour-infiltrating lym-phocytes and apoptosis as determinants of long-termprognosis in prostatic adenocarcinoma. Eur J Cancer1994;30:1797–803.

2. Halpern AC, Schuchter LM. Prognostic models inmelanoma. Semin Oncol 1997;24:2–7.

3. Marrogi AJ, Munshi A, Merogi AJ, et al. Study of tumorinfiltrating lymphocytes and transforming growthfactor-h as prognostic factors in breast carcinoma. IntJ Cancer 1997;74:492–501.

4. Naito Y, Saito K, Shiiba K, et al. CD8+ T cells infiltratedwithin cancer cell nests as a prognostic factor in humancolorectal cancer. Cancer Res 1998;58:3491–4.

5. Nakano O, Sato M, Naito Y, et al. Proliferative activityof intratumoral CD8(+) T-lymphocytes as a prognosticfactor in human renal cell carcinoma: clinicopathologicdemonstration of antitumor immunity. Cancer Res2001;61:5132–6.

6. Schumacher K, Haensch W, Roefzaad C, et al.Prognostic significance of activated CD8+ T cellinfiltrations within esophageal carcinomas. CancerRes 2001;61:3932–6.

7. Zhang L, Conejo-Garcia JR, Katsaros D, et al. Intra-tumoral T cells, recurrence, and survival in epithelialovarian cancer. N Engl J Med 2003;16:203–13.

8. Barbatis C, Woods J, Morton JA, et al. Immunohisto-chemical analysis of HLA (A, B, C) antigens in liverdisease using amonoclonal antibody. Gut 1981;22:985–91.

9. Chen YT, Stockert E, Jungbluth A, et al. Serological

analysis of Melan-A(MART-1), a melanocyte-specificprotein homogeneously expressed in human melano-mas. Proc Natl Acad Sci U S A 1996;93:5915–9.

10. Jungbluth AA, Iversen K, Coplan K, et al. T311—ananti-tyrosinase monoclonal antibody for the detectionof melanocytic lesions in paraffin embedded tissues.Pathol Res Pract 2000;196:235–42.

11. Marincola FM, Hijazi YM, Fetsch P, et al. Analysis ofexpression of the melanoma-associated antigensMART-1 and gp100 in metastatic melanoma cell linesand in in situ lesions. J Immunother Emphasis TumorImmunol 1996;19:192–205.

12. Jungbluth AA, Chen YT, Stockert E, et al. Immuno-histochemical analysis of NY-ESO-1 antigen expressionin normal and malignant human tissues. Int J Cancer2001;92:856–60.

13. Chen YT, Scanlan MJ, Sahin U, et al. A testicularantigen aberrantly expressed in human cancersdetected by autologous antibody screening. Proc NatlAcad Sci U S A 1997;94:1914–8.

14. Stockert E, Jager E, Chen YT, et al. A survey of thehumoral immune response of cancer patients to apanel of human tumor antigens. J Exp Med 1998;187:1349–54.

15. Old LJ. Immunotherapy for cancer. Sci Am1996;275:136–43.

16. Rosenberg SA. A new era of cancer immunotherapy:converting theory to performance. CA Cancer J Clin1999;49:70–3.

17. Hofbauer GF, Kamarashev J, Geertsen R, et al.Melan A/MART-1 immunoreactivity in formalin-fixedparaffin-embedded primary and metastatic melanoma:

frequency and distribution. Melanoma Res 1998;8:337–43.

18. Hofbauer GF, Kamarashev J, Geertsen R, et al.Tyrosinase immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma:frequency and distribution. J Cutan Pathol 1998;25:204–9.

19. Munzarova M, Zemanova D, Rejthar A, et al. HLA-DRantigen expression on melanoma metastases and thecourse of the disease. Cancer Immunol Immunother1989;30:185–9.

20. Taramelli D, Fossati G, Mazzocchi A, et al. Classes Iand II HLA and melanoma-associated antigen expres-sion and modulation on melanoma cells isolated fromprimary and metastatic lesions. Cancer Res 1986;46:433–9.

21. Geertsen RC, Hofbauer GF, Yue F, et al. Higherfrequency of selective losses of HLAA and B allospeci-ficities in metastasis than in primary melanoma lesions.J Invest Dermatol 1998;111:497–502.

22. Mendez R, Serrano A, Jager E, et al. Analysis ofHLA class I expression in different metastases fromtwo melanoma patients undergoing peptide immuno-therapy. Tissue Antigens 2001;57:508–19.

23. Jager E, Ringhoffer M, Altmannsberger M, et al.Immunoselection in vivo : independent loss of MHCclass I and melanocyte differentiation antigen expres-sion in metastatic melanoma. Int J Cancer 1997;10:142–7.

24. Khong HT, Wang QJ, Rosenberg SA. Identification ofmultiple antigens recognized by tumor-infiltratinglymphocytes from a single patient: tumor escape by

Cancer Research

Cancer Res 2005; 65: (9). May 1, 2005 3940 www.aacrjournals.org

Research. on September 5, 2020. © 2005 American Association for Cancercancerres.aacrjournals.org Downloaded from

antigen loss and loss of MHC expression. J Immunother2004;27:184–90.

25. Maeurer MJ, Gollin SM, Storkus WJ, et al. Tumorescape from immune recognition: loss of HLA-A2melanoma cell surface expression is associated with acomplex rearrangement of the short arm of chromo-some 6. Clin Cancer Res 1996;2:641–52.

26. Garcia-Lora A, Algarra I, Garrido F. MHC class Iantigens, immune surveillance, and tumor immuneescape. J Cell Physiol 2003;195:346–55.

27. Ahmad M, Rees RC, Ali SA. Escape from immuno-therapy: possible mechanisms that influence tumorregression/progression. Cancer Immunol Immunother2004;53:844–54.

28. Atkins D, Ferrone S, Schmahl GE, et al. Down-regulation of HLA class I antigen processing molecules:an immune escape mechanism of renal cell carcinoma?J Urol 2004;171:885–9.

29. Bernsen MR, Diepstra JH, van Mil P, et al. Presenceand localization of T-cell subsets in relation to

melanocyte differentiation antigen expression andtumour regression as assessed by immunohistochemis-try and molecular analysis of microdissected T cells.J Pathol 2004;202:70–9.

30. Hald J, Rasmussen N, Claesson MH. In vivoinfiltration of mononuclear cells in squamous cellcarcinoma of the head and neck correlates with theability to expand tumour-infiltrating T cells in vitro andwith the expression of MHC class I antigens on tumourcells. Cancer Immunol Immunother 1994;39:383–90.

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2005;65:3937-3941. Cancer Res   Salah-Eddin Al-Batran, Mohammad-Reza Rafiyan, Akin Atmaca, et al.   Patients with Stage IV MelanomaIntratumoral T-Cell Infiltrates and MHC Class I Expression in

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