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Autoimmunity Resulting From Cytokine Treatment Predicts Long-Term Survival in Patients With Metastatic Renal Cell Cancer

From the Departments of Hematology and Oncology, and Endocrinology, Medizinische Hochschule Hannover, Hannover, and Department of Cell Biology and Immunobiology, National Center for Biotechnology (GBF), Braunschweig, Germany; Ludwig Cancer Institute, Brussels, Belgium; and Institute Necker Enfants Malades, Paris, France.

Address reprint requests to Jens Atzpodien, Medizinische Hochschule Hannover, Department of Hematology and Oncology, Carl-Neuberg Str, D-30623 Hannover, Germany.

	ABSTRACT

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PURPOSE: In patients undergoing cytokine therapy, systemically applied interleukin-2 (IL-2) and/or interferon-alpha (IFN-) have been reported to induce thyroid dysfunction as well as thyroid autoantibodies. We analyzed the correlation of thyroid autoimmunity with HLA phenotype, various other autoimmune parameters, and patient survival.

PATIENTS AND METHODS: For this purpose, antithyroglobulin autoantibodies, antimicrosomal thyroid autoantibodies, thyroglobulin receptor autoantibodies, thyroid dysfunction, and multiple clinical parameters were determined in 329 unselected patients with metastatic renal cell cancer before and after systemic IL-2 and IFN-2 therapy. For statistical analysis, we used both univariate and multivariate Cox proportional hazards models and the two-tailed Fisher's exact test.

RESULTS: Antithyroglobulin autoantibodies and antimicrosomal thyroid autoantibodies were detected in 60 patients (18%); positive autoantibody titers of various other autoimmune parameters were statistically unrelated. The presence of thyroid autoantibodies was correlated with prolonged survival (P < .0001). There was a statistically significant difference in frequencies of HLA-Cw7 expression between thyroid autoantibody-positive and -negative patients (P .05), and the Cw7 expression was associated with prolonged overall survival (P = .009).

CONCLUSION: The evaluation of thyroid autoantibodies during cytokine therapy could be a useful prognostic marker for patients with renal cell carcinoma who benefit from cytokine treatment. IL-2– and IFN-2-induced tumor control and prolonged survival may require breaking of immunologic tolerance against self-antigens.

	INTRODUCTION

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IN METASTATIC RENAL CELL cancer treatment with recombinant cytokines, interleukin-2 (IL-2) and interferon-alpha (IFN-) 2 have shown promising results.^1-3 Reversible thyroid dysfunction and the transient induction of thyroid autoantibodies occur in up to 60% of metastatic cancer patients treated with immunotherapy consisting of IL-2 alone or in combination with either IFN- or lymphokine-activated killer cells.^4,5 Cytokine-induced hypothyroidism has been suggested to correlate with a favorable tumor response.^4-7

We assessed the prognostic significance of thyroid autoantibodies in correlation with the HLA phenotype, various other autoimmune parameters, and overall survival in a retrospective analysis of 329 unselected metastatic renal cell cancer patients treated at our institution with subcutaneous IL-2 and IFN-2.

	PATIENTS AND METHODS

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Patients and Collection of Samples
This study was approved by the institutional review board of the Medizinische Hochschule Hannover. The study cohort consisted of 329 unselected patients treated at our institution since May 1989 with histologically confirmed metastatic renal cell cancer and clinically progressive disease; all patients had a Karnofsky performance status of at least 70%. After giving written informed consent, patients received 8-week cycles of subcutaneous IL-2 and IFN-2 with (n = 247) or without (n = 82) intravenous fluorouracil: IL-2 was administered at 20.0 x 10⁶ IU/m² three times per week in weeks 1 and 4 and at 5 x 10⁶ IU/m² three times per week in weeks 2 and 3. In addition, patients received recombinant IFN-2 at 6 x 10⁶ U/m² once per week in weeks 1 and 4 and three times per week in weeks 2 and 3, and at 10 x 10⁶ U/m² three times per week in weeks 5 through 8.⁸ None of the patients received agents with antithyroid activity. Patients' characteristics are listed in Table 1.

Serologic Assays
Peripheral-blood samples were obtained from all patients both before and after 8 weeks of therapy. Sera were frozen at -70°C until analysis. The following thyroid parameters were tested: triiodothyronine and thyroxine (Corning RIA, Fernwald, Germany), thyrotropin (Corning IRMA), thyroglobulin autoantibodies (ATA) and antimicrosomal autoantibodies (AMA) (both by enzyme-linked immunosorbent assay from Pharmacia, Freiburg, Germany), and thyroid receptor antibodies (Brahms radioligand assay; Brahms, Berlin, Germany). In addition, the following autoimmune parameters were determined in 125 patients (30 with thyroid autoantibodies and 95 without thyroid autoantibodies), using standard enzyme-linked immunosorbent assay and histologic immunofluorescence procedures: rheumatoid factor, antibodies against mitochondria, skeletal muscle, smooth muscle, heart muscle, gastric parietal cells, nuclear antigens, cytoplasmic antigens on neutrophilic granulocytes (cANCA), and perinuclear antigens on neutrophilic granulocytes (pANCA). Patients were selected randomly for testing of additional autoimmune parameters. The following clinical pretreatment parameters were evaluated: erythrocyte sedimentation rate, lactate dehydrogenase, hemoglobin, neutrophil count, lung and bone metastases, time of progress since tumor diagnosis, age, and sex.

Complete HLA phenotyping was performed for 70 samples; standard microlymphocytotoxicity assays were used on peripheral-blood mononuclear cells. Patients chosen for HLA phenotyping were selected retrospectively on the basis of survival status.

Statistical Analysis
The statistical end point in our analysis was overall survival from time of entry onto the study. We calculated univariate hazard ratios with 95% confidence intervals, using the Cox proportional hazards model.⁹ The simultaneous prognostic effect of various factors was determined in a multivariate analysis using the Cox proportional hazards model (forward selection of variables). The probability of overall survival was plotted over time according to the method of Kaplan and Meier.¹⁰ Differences between groups in overall survival were tested with log-rank statistics. The results of the multivariate analysis are summarized in Table 2. The frequency of HLA antigens was determined retrospectively in 70 patients; each HLA allele was treated as a binary characteristic, either present or absent. The distribution of each HLA antigen was related to the thyroid autoimmune status by use of a two-tailed Fisher's exact test. Associations with a P value equal to or less than .05 were considered worthy of further study. Independent confirmation of these apparent associations was obtained by a study of the relationship between HLA phenotype and patients' overall survival, using the univariate Cox proportional hazards model.

	RESULTS

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Thyroid Autoimmunity and Thyroid Dysfunction
The thyroid autoantibody status and thyroid function of 329 unselected, IL-2– and IFN-2–treated patients with metastatic renal cell cancer are described in Table 1. After 8 weeks of therapy, thyroid autoantibodies were detected in 60 patients (18%). There was a strong statistical correlation between thyroid autoantibody formation and development of thyroid dysfunction (P < .0001) diagnosed in 65% of autoantibody-positive patients, whereas 68% of the autoantibody-negative patients remained free of thyroid dysfunction. Subclinical hyperthyroidism was the most common abnormality, occurring in 62 patients (18.8%) (Table 1). Of the 60 thyroid autoantibody-positive patients, 25 patients had thyroid autoantibodies before treatment. In the overwhelming majority of these 25 patients, we detected higher autoantibody titers after immunotherapy (mean, 3.9-fold increase; SD, 3.47).

Survival Analysis
The median survival time of all 329 patients was 22 months. By univariate analysis, there were significant differences in overall survival (P < .0001) between thyroid autoantibody-positive (median not reached; 5-year survival estimate, 54%) and thyroid autoantibody-negative patients (median, 20 months; 5-year survival probability, 15%) (Fig 1, top); there was no significant difference in overall survival (P = .19) between patients with cytokine-induced thyroid autoantibodies (n = 35) and patients with preexisting (n = 25) thyroid autoimmunity (Fig 1, bottom).

View larger version (14K): [in this window] [in a new window]   Fig 1. The top panel shows the thyroid autoimmunity and survival in 329 metastatic renal cell carcinoma patients treated with subcutaneous IL-2 and IFN-2. Survival curves (Kaplan-Meier estimate) were determined for all patients (I; n = 329) and patients with (II; n = 60) or without (III; n = 269) thyroid autoimmunity. The P value was determined by log-rank test comparing II and III; tick marks represent patients whose data were censored. The bottom panel shows the survival of metastatic renal cell carcinoma patients with pre-existing versus treatment-induced thyroid autoantibodies. Survival curves (Kaplan-Meier estimate) were determined for thyroid autoantibody-positive patients with preexisting (A; n = 25) and treatment-induced (B; n = 35) thyroid autoantibodies. The P value was determined by log-rank test; tick marks represent patients whose data were censored.

To assess the multivariate prognostic significance of thyroid autoimmunity, we established a Cox proportional hazards model containing known clinical predictors of survival.⁸ Notably, the presence of thyroid autoantibodies (P < .0001) was rendered statistically independent along with other predictors (absence of lung metastases, neutrophil count > 6,000/µL, hemoglobin < 100 g/L, and lactate dehydrogenase > 240 U/L).

HLA Phenotype Analysis
The HLA phenotype analysis in a subgroup of 70 patients showed statistically significant (P < .05) correlation of HLA phenotype and thyroid autoantibody status. The A23, A25, Cw6, and Cw7 alleles were more frequent in ATA/AMA-positive patients, whereas the A3, B27, DR16, and DQ8 alleles were more frequent in ATA/AMA-negative patients (Table 3). Expression of class I antigen Cw7 was associated with prolonged survival (P = .009).

Lack of Association With Other Autoimmune Phenomena
A subgroup of 125 patients (30 patients with and 95 patients without thyroid autoantibodies) was evaluated for various other autoimmune parameters before and after treatment with IL-2 and IFN-2. All parameters tested were statistically independent of thyroid autoimmunity. The presence of the following autoimmune parameters was not associated with prolonged survival, as evaluated by the log-rank test: Rheumafactor (P < .53), antimitochondrial antibody (P < .6), antiskeletal antibody (P < .16), anti–smooth muscle antibody (P < .17), antiheart antibody (P < .08), antistomach antibody (P < .75), antinuclear antibody (P < .92), cANCA (not assessable; one positive patient in 125 tested patients), and pANCA (P < .44). Also, in this subgroup of patients, thyroid autoantibodies were rendered independent prognostic factors for overall survival (P < .017).

	DISCUSSION

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Several authors have described the frequent development of reversible thyroid dysfunction in association with transient thyroid autoantibody formation after IL-2– and/or IFN-–based therapy in advanced malignancies. Thyroid autoantibody–related hypothyroidism has been suggested to be associated with a favorable tumor response.^4-7 The current data provide compelling evidence for this hypothesis by showing that a positive thyroid autoantibody titer represents a highly significant, independent predictor of survival in renal cell cancer patients undergoing cytokine therapy. Thus, in metastatic renal cell cancer patients receiving systemic IL-2 and IFN-2, the 5-year survival estimate was 54% in thyroid autoantibody-positive patients compared with 15% in thyroid autoantibody-negative patients. This correlation was statistically independent of thyroid autoantibody subtype (ATA and/or AMA) and of the time of thyroid autoantibody formation (preexisting versus induced autoantibodies). Notably, all other preexisting or therapy-induced autoimmune phenomena were clinically and statistically unrelated to the development of thyroid autoantibodies and showed no correlation with a prolonged survival.

Cytokine-induced thyroid dysfunction might be discussed in the context of autoimmune thyroid disease (AITD), with the clinical spectrum ranging from hyperthyroidism in Graves' disease to hypothyroidism in Hashimoto's thyroiditis. Data from animal models and human disease indicate that T cell–mediated autoimmunity to the thyroid gland plays a major role in the pathogenesis of AITD^11,12 and is often associated with thyroid autoantibodies to thyroglobulin and/or thyroid microsomal antigen.¹³ T-lymphocyte infiltration of the thyroid gland is predominantly of the CD8⁺ phenotype.¹⁴ These autoreactive T cells could be specifically cytotoxic for thyrocytes. Another relevant observation may be the aberrant expression of HLA-DR found on thyroid epithelial cells in AITD.¹⁵ Recombinant IFN-gamma can induce the expression of HLA-DR antigens on cultured thyroid epithelial cells¹⁶ and thus permit enhanced presentation of thyroid autoantigens to T cells of the CD4⁺ subset. Furthermore, susceptibility and resistance to autoimmune thyroiditis have been found to be related to certain HLA class II antigens.^17-19 Several authors have previously discussed these results in the context of cytokine-induced thyroid dysfunction and thyroid autoantibody formation in cancer patients; activation of preexisting autoreactive CD8⁺ cytotoxic and CD4⁺ helper T-cell clones and the secondary release of other cytokines have been suggested to induce and potentially exacerbate preexisting AITD while compromising self-tolerance.^15-19

Our investigations support the hypothesis of cytokine-induced AITD and extend previous observations in that IL-2– and IFN-–based immunotherapy may induce pleiotropic autoimmunity, including the induction of de novo and the reactivation of former autoimmune responses. The correlation of the thyroid autoantibodies with the prolonged survival in the present cohort of advanced renal cell carcinoma patients is not fully understood. One possibility is that the cytokines enhance the immune response to certain autoantigens as well as to antigens present on tumor cells. Furthermore, the immune responses to thyroid tissue and tumor tissue might be similarly regulated, as it has been reported that immune responses to antigen presented by nonprofessional antigen-presenting cells benefit from cytokines such as IL-2.²⁰ Thus, cytokine-induced autoimmunity might be caused by mechanisms of peripheral immunoregulation similar to those involved in controlling tumor growth. Also, a specific cross-reaction of thyroid autoimmune responses caused by shared antigens between renal cell carcinoma cells and thyroid cells might be possible. A specific cross-reactivity could explain why all the other determined immune responses besides thyroid autoimmunity are rendered less relevant for effective, long-lasting tumor control. This hypothesis might be underscored by the higher incidence of thyroid autoantibodies in a total of 329 renal cell carcinoma patients before treatment (7.6%) compared with approximately 3% to 4% in the general population.²¹

The present association between thyroid autoimmunity and expression of certain HLA class I antigens suggests a role for HLA in conferring an enhanced ability to present self-antigens to the MHC-restricted cellular immune system. HLA-Cw7, which has been a marker of prolonged survival in our study, is more frequent in thyroid autoantibody-positive patients and shows a higher frequency than expected in whites.²² HLA-Cw7 has been previously reported to favor autoimmune diseases other than AITD.^23,24 However, it should be noted that a prospective controlled study in a larger cohort of patients will be necessary to confirm our observation concerning HLA-dependent thyroid autoimmunity.

In the present study, we suggest that IL-2 and IFN-2 treatment may alter immune responsiveness and self-tolerance in a genetically predisposed subgroup of patients and result in effective antitumor immunity. Future research will focus on the dissection of these mechanisms.

	ACKNOWLEDGMENTS

 
We are grateful to Harald von Boehmer for critical review of the manuscript.

	REFERENCES

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1. Rosenberg SA, Lotze MT, Muul LM, et al: A progress report on treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 316:889-897, 1987[Abstract]

2. Atzpodien J, Lopez H änninen E, Kirchner H, et al: Multiinstitutional home-therapy trial of recombinant human interleukin-2 and interferon alpha-2 in progressive metastatic renal cell carcinoma. J Clin Oncol 13:497-501, 1995[Abstract/Free Full Text]

3. Facendola G, Locatelli MC, Pizzocaro G, et al: Subcutaneous administration of interleukin-2 and interferon-alpha-2b in advanced renal cell carcinoma: A confirmatory study. Br J Cancer 72:1531-1535, 1995[Medline]

4. Atkins MB, Mier JW, Parkinson DP, et al: Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells. N Engl J Med 318:1557-1562, 1988[Abstract]

5. Weijl NI, Van Der Harst D, Brand A, et al: Hypothyroidism during immunotherapy with interleukin-2 is associated with antithyroid antibodies and response to treatment. J Clin Oncol 11:1376-1383, 1993[Abstract/Free Full Text]

6. Scalzo S, Gengaro A, Boccoli G, et al: Primary hypothyroidism associated with interleukin-2 and interferon alpha-2 therapy of melanoma and renal carcinoma. Eur J Cancer 26:1152-1156, 1990

7. Sauter NP, Sauter MD, Atkins MB, et al: Transient thyrotoxicosis and persistent hypothyroidism due to acute autoimmune thyroiditis after interleukin-2 and interferon-alpha therapy for metastatic carcinoma: A case report. Am J Med 92:441-444, 1992[Medline]

8. Lopez H änninen E, Kirchner H, Atzpodien J: Interleukin-2 based home therapy of metastatic renal cell carcinoma: Risk and benefits in 215 consecutive single institution patients. J Urol 155:19-25, 1995

9. Cox DR: Regression models and life-tables. J R Stat Soc (B) 34:187-220, 1972

10. Kaplan EL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958

11. Strakosh CR, Wenzel BE, Row VV, et al: Immunology of autoimmune thyroid diseases. N Engl J Med 307:1499-1507, 1982[Medline]

12. Canonica GW, Cosulich ME, Croci R, et al: Thyreoglobulin-induced T cell in vitro proliferation in Hashimoto's thyroiditis: Identification of the responsive subset and effect of monoclonal antibodies directed to Ia antigens. Clin Immunol Immunopathol 32:142-151, 1984[Medline]

13. Volpe R: Immunology of human thyroid disease, Volpe R (ed):Autoimmunity in Endocrine Disease73-98, 1986

14. Margolick JB, Hsu SM, Volkman DJ, et al: Immunohistochemical characterization of intrathyroid lymphocytes in Graves' disease: Interstitial and intraepithelial populations. Am J Med 76:815-821, 1984[Medline]

15. Bottazzo GF, Pujol-Borrell R, Hanafusa T, et al: Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity. Lancet 2:1115-1119, 1983[Medline]

16. Todd I, Pujol-Borrell R, Hammond LJ, et al: HLA-DR expression by thyroid epithelium. Clin Exp Immunol 61:265-273, 1985[Medline]

17. Honda K, Tamai H, Morita T, et al: Hashimoto's thyroiditis and HLA in Japanese. J Clin Endocrinol Metab 69:1268-1273, 1989[Abstract/Free Full Text]

18. Tamai H, Uno H, Hirota Y, et al: Immunogenetics of Hashimoto's and Graves' disease. J Clin Endocrinol Metab 60:62-66, 1985[Abstract/Free Full Text]

19. Uno H, Sasazuki T, Tamai H, et al: Two major genes, linked to HLA and Gm, control susceptibility to Graves' disease. Nature 292:768-770, 1981[Medline]

20. Heath WR, Allison J, Hoffmann MW, et al: Autoimmune diabetes as a consequence of locally produced IL-2. Nature 359:547-549, 1992[Medline]

21. Hashimoto's thyroiditis: A disease of autoimmunity, in DeGroot LJ, Larsen PR, Refetott S, et al (eds): The Thyroid and Its Diseases (ed 5). New York, NY, John Wiley Publications, 1984

22. Baur MP: Population analysis on the basis of deduced haplotypes from random families, in Albert ED, Baur MP, Mayr WR (eds): Histocompatibility Testing 1984. New York, NY, Springer Verlag, 1984

23. Tiwari JL, Terasaki PI (eds): HLA and Disease Associations. New York, NY, Springer Verlag, 1985

24. Leidenius MH, Koskimies SA, Kellokumpu IH, et al: HLA antigens in ulcerative colitis and primary sclerosing cholangitis. APMIS 103:519-524, 1995[Medline]

Submitted July 8, 1998; accepted October 13, 1998.

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