[CANCER RESEARCH 53, 5654-5662, December 1, 1993] Evidence for Local and Systemic Activation of Immune Cells by Peritumoral Injections of Interleukin 2 in Patients with Advanced Squamous Cell Carcinoma of the Head and Neck 1 Theresa L. Whiteside, 2 Eric Letessier, Hideki Hirabayashi, Domenico Vitolo, John Bryant, Leon Barnes, Carl Snyderman, Jonas T. Johnson, Eugene Myers, Ronald B. Herberman, John Rubin, John M. Kirkwood, and Daniel R. Viock 3 Departments of Pathology [T. L. W., E. L., D. V, L. B., R. B. H.], Otolaryngology [1t. H., C. S., J. T. J., E. M.], and Medicine JR. B. H., J. M. K., D. R. V], University of Pittsburgh School of Medicine, Pittsburgh Cancer Institute [T. L. W., E. L., H. H., D. V., J. B., C. S., J. T. J., E. M., R. B. H., J. M. K., D. R. V.], and Eye and Ear Institute [H. H., C. S., J. T. J., E. M.], Pittsburgh, Pennsylvania 15213; and Department of Otolaryngology, Albert Einstein School of Medicine, New York, New York 10708 [J. R.] ABSTRACT Interleukin 2 (IL2) was injected peritumorally and intranodally in 36 patients with unresectable squamous cell carcinoma of the head and neck enrolled in an Eastern Cooperative Oncology Group-sponsored phase Ib trial (EST P-Z388). Groups of 6 patients received escalating doses(200, 2 x liP, 2 • 104, 2 • 105, 2 • 106, and 4 • 106 units) of IL2 daily 5 times/week for 2 weeks. Tumor biopsies were obtained before and after IL2 therapy. Tumor tissue was provided for histology, and the remaining fresh tissue was divided for snap-freezing in -75~ and for separation of tumor-infiltrating lymphocytes (TIL) and tumor cells. Immunophenotyp. ing of TIL performed on cryostat sections of paired pre- and post-IL2 biopsy tissues showed increases after IL2 therapy in the number of T-cells (P = 0.005), natural killer (NK; CD16 § cells (P = 0.0001), CD25 § cells (P = 0.004), and HLA-DR + cells (P = 0.001) accumulating in the tumor stroma. In the tumor parenchyma, NK cells (P = 0.0001) and HLA-DR § cells (P = 0.003) were increased after IL2 therapy. The T:NK cell ratios in the tumor stroma and parenchyma were decreased after therapy, suggest- ing selective accumulation of NK cells. By flow cytometry, TIL recovered from post-IL2 biopsy tissues were enriched (P < 0.05) in CD3-CD56 § (NK) cells. In situ hybridization with [3ss] cDNA probes for cytokines and IL2 receptors indicated that the numbers of cells expressing mRNA for IL2, tumor necrosis factor a, ILl-t, v-interferon, transforming growth factor 1~, and IL2 receptor p55 or p70 were increased in post-IL2 biopsy tissues as compared to pre-IL2 tissues. Cytolytic activity of TIL isolated from post-IL2 tissues was also increased, as determined in 4-h S~Cr release assays against K562 targets (12 +_ 3 mean iytic units/107 cells _+ SEM pre-IL2 versus 46 -+ 13 post-IL2; n - 16) and against autologous tumor (13 _+8 versus 68 _+26; n -- 9). Fresh TIL of one clinical responder showed relatively high levels (195 lytic units) of autotumor cytotoxicity after IL2 therapy versus no activity prior to therapy. In the blood, NK and lym- phokine-activated killer cell activity, and percentages of CD3-CD56 § NK cells and of activated (CD25 § T-lymphocytes were increased for all doses of IL2. A significant dose-response effect was observed for the percentage of CD3-CD56 § NK cells and lymphokine activated killer cell cytotoxicity against autologous tumor, with the highest values observed at the two highest doses of IL2. Our data indicate that local as well as systemic activation of antitumor effector cells occurred during local ad- ministration of IL2 in patients with squamous cell carcinoma of the head and neck. Received 4/19/93; accepted 9/22/93. The costs of publication of this article were defrayedin part by the paymentof page charges. This article must thereforebe hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 1The clinical trial described was sponsored by the Eastern Cooperative Oncology Group. This study was supported in part by the Pathology Education and Research Foundation, University of Pittsburgh, and by the Mary Hillman Jennings Foundation Grant to the Eye and Ear Institute. 2 To whom requests for reprints should be addressed, at Pittsburgh Cancer Institute, W1041 Biomedical Science Tower, DeSoto at O'Hara Street, 10th Floor West Wing, Pittsburgh, PA 15213. 3 Present address: Hematology/Oncology Division, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. INTRODUCTION IL24 therapy has been used in a number of clinical trials in patients with advanced malignancies (1-4). In the vast majority of these trials, IL2 has been delivered systemically as either a bolus injection (5) or 24-h continuous infusion (6). Relatively few trials have evaluated locoregional delivery of IL2 (7-9), although both clinical and experi- mental studies indicate that locally injected IL2 can modulate host immunoreactivity and mediate antitumor effects (10, 11). SCCHN represent a logical choice for locoregional IL2 therapy because they tend to grow and recur locally, are generally well infiltrated by MNC, are surrounded by extensive lymph node network, and are well vas- cularized and accessible to local delivery of therapeutic agents. Pa- tients with SCCHN, especially those with inoperable disease, often have cellular immune defects (12, 13), and IL2-mediated up-regula- tion of local antitumor responses might be expected to be beneficial in such individuals. Additionally, the majority of patients with advanced disease fail to respond to surgery and/or radiotherapy, and chemo- therapy has not been shown to have a significant impact on overall survival (14). Novel therapeutic approaches, including locoregional administration of cytokines, offer an opportunity for improving sur- vival in this group of patients, and their effectiveness deserves to be evaluated in clinical trials. Local administration of low doses of natural IL2 was reported to result in a complete or partial response in 30% and a minimal response in 40% of 20 patients with recurrent SCCHN (15, 16). In a pilot study of four patients with primary SCCHN who received preoperative local therapy with IL2, a decrease or disappearance of neoplastic lesions was documented clinically and histologically (17). More recently in a new series of clinical trials with recombinant IL2, these early prom- ising results have not been reproduced (18). However, neither the optimal IL2 dose nor the exact protocol for its optimal perilesional administration has been determined, and differences in these param- eters as well as the clinical status of the patients treated have limited the assessment of the value of this form of biological therapy. A Phase Ib clinical trial of the effects of peritumoral and intranodal injections of IL2 in patients with advanced SCCHN has been spon- sored by the Eastern Cooperative Oncology Group (19) and this provided us with an opportunity to monitor both local and systemic immune responses of patients treated with escalating doses of rlL2. We present here results of this study, which provided evidence for local activation of TIL in post-IL2 tissues and also for systemic activation of immune effector cells after local therapy with rlL2. Our 4 The abbreviations used are: IL2, interleukin2; rlL2, recombinantIL2; IL2R, inter- leukin 2 receptor;TIL, tumor-infiltrating lymphocytes; AuTu, autologoustumor; SCCHN, squamous cell carcinomaof the head and neck; MNC, mononuclear cell; LU, lytic units; PBS, phosphate-bufferedsaline; MAbs, monoclonalantibodies; HPF, high power field; DEP, diethyl pyrocarbonate;SSC, saline-sodiumcitrate buffer (1 • = 0.15 M NaC1/0.015 M sodium citrate); cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; NK, natural killer; LAK, lymphokine-activated killer. 5654 Research. on June 25, 2015. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from