Short-term autologous tumor cell lines for the active specific immunotherapy of patients with metastatic melanoma

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Abstract

We established short-term cell lines for 108/170 (64%) patients with metastatic melanoma. Tumor cell numbers were expanded to 108, then cells were irradiated, aliquoted, and cryopreserved for clinical use. Vaccines have been used to treat 69 patients with clinical follow up for 33 who had measurable metastatic disease at the time vaccine therapy was initiated (METS), and 33 who had no evidence of disease (NED) at the time of vaccine therapy following surgical resection of metastases. The protocol called for a baseline test of delayed tumor hypersensitivity (DTH), three weekly injections, a repeat of the DTH test, then monthly injections for an additional 5 months. Objective tumor responses were noted in 3/26 (12%) patients who received a minimum of three vaccinations, one complete, and two partial, with survivals of 36, 46+, and 78+ months. Only 6/64 (9.4%) had a positive DTH (>10 mm) at baseline, including three METS, all of whom progressed within 4 months and died within a year, and three who are still NED after more than 5 years. Conversion of DTH from negative to positive was documented in 18/44 (41%) patients who were tested at week 0 and 4. At a median follow up of greater than 5 years, the median overall survival (OS) was 40 months for ‘NED’ with a 5-year survival rate of 39%, and 8.6 months with a 5-year survival rate of 10% for ‘METS’. The 18 patients who had conversion of their DTH had a median event-free survival (EFS) of 15.8 months and 5-year EFS of 32% compared to 4.2 months and 9% for the 26 non-converters (P=0.012, two-tailed, log-rank test). Among patients who were NED when treatment started, the 12 patients whose DTH converted had a median overall survival of 61.4 months with 5-year survival of 63% compared to 9.7 months and 0% for the 13 non-converters (P=0.0026). This treatment approach is feasible, produces minimal toxicity, and is associated with long-term survival in a significant subset of patients.

Introduction

Patients with melanoma have been the focus of vaccine therapy and active specific immunotherapy for over 30 years [1]. Promising results of vaccine therapies have been reported for patients at high risk of recurrence because of the finding of lymph node metastases at the time of initial diagnosis, [2], [3], [4], [5], [6] or who had in transit metastases [7] and for selected patients who had distant metastatic disease [8], [9], [10], [11]. The sources of tumor antigen in these studies have included fresh cells from autologous tumor samples (a mixture of normal and malignant cells), irradiated allogeneic tumor cell lines, lysates from tumor cell lines, and purified antigens. There is tremendous interest in the role that viral vectors, antigen-presenting cells and non-specific and cytokine adjuvants may play in vaccine strategies. However, the only published prospective, randomized, placebo-controlled trial of a melanoma vaccine, a lysate of allogeneic melanoma cells delivered with vaccinia virus, failed to show a progression-free survival or overall survival advantage for treated patents [12], [13].

For several years we have been trying to establish short-term cultures of autologous tumor cells for clinical use as active specific immunotherapy in a variety of tumor types [14], [15], [16], [17], [18], [19]. Theoretical advantages of such an autologous cell line approach include: (1) the presence of the patient's own histocompatibility antigens; (2) the presence of tumor-associated antigens that are relevant for that individual patient; (3) the presentation of antigens that are expressed by tumor cells that have proliferative capacity; (4) the presentation of antigens in the context of the tumor cell membrane, where it would have to be recognized by the immune system for an effective cellular and/or humoral immune response; (5) the absence of non-malignant, cellular elements such as blood cells, fibroblasts, and other normal tissue elements; and (6) the availability of autologous tumor cells as targets for in vitro testing for the presence of antibody-specific and lymphocyte-specific activity induced by vaccination. Limitations of this approach include: (1) restriction to patients who have sufficient quantities of tumor accessible for surgical resection, which usually means gross metastatic disease; (2) the inability to establish a cell line for all patients; (3) a variable delay of 2–4 months from the time that vaccine therapy is considered to the time that a therapeutic product can be made available; (4) the costs associated with a patient-specific cell culture approach; and (5) the simultaneous presentation of normal antigens, which is a theoretical concern for any cell or cell-lysate vaccine strategy.

We previously demonstrated that short-term cell lines of melanoma, free of contaminating normal fibroblasts, could be established from fresh tumors [14], [15], [16]. We have consistently achieved success rate of greater than 60% in establishing short-term tumor cell lines from patients with melanoma [19]. In this report we describe the results of efforts to grow autologous melanoma cells from resected metastatic specimens, and the clinical results associated with the use of such cells in combination with various adjuvants in patients with metastatic melanoma. This report expands on a previous publication that described preliminary results associated with the first 52 patients who had received an autologous vaccine at a median follow up of 3.5 years [11]. This update describes 66 evaluable patients with a median follow up of greater than 5 years from initiation of vaccine therapy.

Section snippets

Objectives

The primary objective of this research was to establish tumor cell lines from resected metastatic melanoma tissues, so that these cells might be used in the preparation of autologous tumor vaccines for the patient from whom the sample was obtained. The secondary objective was to prepare vaccines, treat melanoma patients with the vaccines that were prepared from these cell lines, and determine the immune effects and anticancer effects of the vaccines.

Establishment of tumor cell lines

Portions of resected metastatic melanoma

Cell lines and vaccines

Between October 1990 and December 1999, the Hoag Cancer Center cell biology laboratory received 181 tumor samples from 170 patients with metastatic melanoma. Short-term melanoma cell lines were successfully established for 108/170 patients (64%). Ultimately 69/170 (40%) of all patients for whom tumor was submitted, actually received vaccine therapy; 69/108 (64%) of patients for whom a cell line was successfully established received vaccine therapy; and so far 69/80 (86%) of all patients for

Feasibility

Even though there are many theoretical advantages for an autologous tumor cell line as a source of tumor-associated antigen for vaccination, most investigators have shied away from this approach for a variety of technical and practical reasons. From a commercial standpoint, the necessity to establish a new cell line for every single patient is a major deterrent as is the inability to establish a cell line for every patient. We were able to grow cell lines from 60% of resected metastatic

Acknowledgements

This work was supported in part by philanthropic gifts from the Circle 1000 of Hoag Hospital and is dedicated to Shankar K. Nayak, Ph.D., who died in 1998 after devoting his scientific career to the in vitro growth of human cancers. The content of this article was presented in part in abstract form at the International Conference on Advances in Cancer Immunotherapy that was held in Princeton, New Jersey in March 2000. We would like to recognize Hoag medical oncologists Khosrow Mahdavi and Frank

Dr Dillman is Medical Director and Director of Clinical and Laboratory Cancer Research at the Hoag Cancer Center in Newport Beach, California, Chairman of the Cancer Biotherapy Research Group (1990–2002), and President of the Society for Biological Therapy (2000–2002).

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    Dr Dillman is Medical Director and Director of Clinical and Laboratory Cancer Research at the Hoag Cancer Center in Newport Beach, California, Chairman of the Cancer Biotherapy Research Group (1990–2002), and President of the Society for Biological Therapy (2000–2002).

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