Human xenograft-nude mouse model of adoptive immunotherapy with human melanoma-specific cytotoxic T-cells.

We investigated the efficacy of human melanoma-specific cytotoxic T-cells (CTLs) in treating experimental human melanoma metastases in a nude mouse model of adoptive immunotherapy. Hepatic metastases were generated by the intrasplenic injection of 1.5 x 10(6) human melanoma cells. Animals were then randomized to receive saline, interleukin-2 only, or CTLs and interleukin-2. CTLs were effective when administered 3 or 7 days after generation of hepatic metastases, with 96 and 88% of animals disease-free, respectively, when examined at one month. Interleukin-2 alone was not effective. In addition, CTLs were effective when as few as 2.5 x 10(6) T-cells were adoptively transferred. Only 33% of the animals were tumor-free when CTLs were administered on day 10, and CTLs were not effective when given at day 14. Human CTLs that were not cytotoxic for the tumor line used in vivo, when tested in a 51Cr assay, were also not effective in the model of immunotherapy. This suggests that the tumor-specific CTLs maintain their specificity in vivo, and eliminates a nonspecific inflammation directed against the human CTLs as a possible cause of the antitumor effect. These studies lay the foundation for clinical trials of CTLs in the adoptive immunotherapy of patients with metastatic melanoma.

Generation of human autologous melanoma-specific cytotoxic T-cells using HLA-A2-matched allogeneic melanomas.

Autologous tumor-specific cytotoxic T-lymphocytes (CTLs), generated by repeated stimulation with autologous melanoma and expanded in interleukin 2, are major histocompatibility complex restricted. These CTLs recognize a common tumor-associated antigen in the presence of HLA class I determinants, suggesting that allogeneic melanomas which express the restricting HLA-A region antigen could substitute for the autologous tumor in the generation of CTLs. This was investigated in the HLA-A2 system. Four T-cell lines were established by stimulation of lymphocytes with either autologous tumor or an HLA-A2-matched allogeneic melanoma. Allogeneic stimulated CTLs specifically lysed the autologous tumor and demonstrated an identical pattern of HLA-A2 restriction, when compared to the autologous stimulated CTLs. Lysis by the allogeneic stimulated CTLs was blocked by a monoclonal antibody to HLA class I antigens; lysis was also inhibited by both autologous tumor or HLA-A2 allogeneic melanomas when evaluated in cold target competition studies. The allogeneic stimulated CTLs proliferated in response to both autologous tumor and HLA-A2 melanomas, but not in response to HLA-A2 nonmelanomas. By phenotypic analysis these CTLs were CD3+ and predominantly CD8+ cells. We conclude that autologous tumor-specific CTLs can be generated using HLA-A region-matched allogeneic melanomas for stimulation. Since established, HLA-typed melanoma tumor lines can be used in the absence of autologous tumor; this procedure can be applied clinically to a broad patient population and may prove useful in the adoptive immunotherapy of melanoma.

Relationship between disease-free interval and survival in patients with recurrent melanoma.

A total of 2468 patients with recurrent melanoma were subdivided on the basis of disease-free interval: group 1 had recurrences within 1 year (n = 810), group 2 at years 1 to 3 (n = 1001), group 3 at years 3 to 5 (n = 363), group 4 at years 5 to 10 (n = 329), and group 5 after 10 years (n = 145). Ten-year survivals were 21%, 23%, 25%, 28%, and 35%, respectively. Patients who had recurrences within 1 year had a decreased median survival compared with those who had later recurrences, although the differences were not clinically significant (only 6 to 8 months). Survival was improved for the few patients who had recurrences longer than 10 years from diagnosis. However, for the majority of patients, who had recurrences between 1 and 10 years, the disease-free interval did not predict subsequent survival. The data support the hypothesis that malignant cells can exist in a state of relative quiescence for extended periods. Once disease reactivation occurs, however, the subsequent survival is relatively predictable and is independent of the initial period of tumor dormancy.

Treatment of human melanoma hepatic metastases in nude mice with human cytotoxic T lymphocytes.

We investigated the effects of human melanoma-specific cytotoxic T lymphocytes in treating experimental human melanoma hepatic metastases in a nude mouse model of adoptive immunotherapy. Hepatic metastases were generated by intrasplenic injection of 1.5 x 10(6) human melanoma cells. Three days after injection, animals received salt solution and interleukin 2 or interleukin 2 and cytotoxic T lymphocytes. Twenty-four of 25 control animals had developed multiple tumor nodules in the liver; 11 of 13 animals receiving only interleukin 2 also had significant tumor burdens. In striking contrast, 17 of 18 animals receiving cytotoxic T lymphocytes and interleukin 2 had no gross or histologic evidence of tumors. The remaining animal had a 2-mm nodule. Human tumor-specific cytotoxic T lymphocytes are effective in vivo in a model of adoptive immunotherapy and may prove useful in adoptive immunotherapy of humans with metastatic melanoma.

Malignant melanoma in black Americans. A trend toward improved survival.

A search of patients seen at the Duke University Melanoma Clinic (Durham, NC) identified 79 black patients. Five- and 10-year survival was 35% and 26%, respectively. Patients presenting in the recent decade had less advanced disease, resulting in an improved 5-year survival of 49%. Five-year survival for white patients was 74%. Black patients had more negative prognostic indicators when compared with white patients. In a multivariate analysis, however, race was not a prognostic indicator, suggesting melanoma is not inherently more aggressive in black patients. The trend during the past decade has been for black patients to present with less advanced disease, with improved survival. This may be related to better appreciation of the disease by patients and physicians alike. Continued emphasis on education and early diagnosis may result in further improvement for black patients with melanoma.

Possibilities of immunotherapy and gene therapy for malignant melanoma.

Over the last decade, both immunotherapy and gene therapy have emerged as exciting new modalities in the treatment of malignant melanoma. The fact that many melanoma patients mount cellular and humoral responses against their tumors, and that melanomas express both HLA antigens and tumor-associated antigens (TAA), has led to an increased interest in the treatment of melanoma by manipulation of the immune system. Advances have occurred in several areas, including a) the use of monoclonal antibodies, alone or in combination with cytokines, b) tumor vaccines, using whole cell preparations or cloned melanoma antigens, c) adoptive immunotherapy, with tumor-infiltrating lymphocytes (TILs) and cytotoxic T lymphocytes (CTLs), and d) gene therapy, designed to increase the immunogenicity of the tumor, increase the effectiveness of the TILs, or alter the basic mechanisms of tumor cell growth and regulation. Some of the advances in these areas are discussed.

Late recurrence of malignant melanoma. Analysis of 168 patients.

Analysis of 7104 patients with melanoma seen at Duke University identified 168 who experienced their first recurrence 10 or more years after diagnosis, for an incidence of 2.4%. This included patients with all stages of disease. There was no sex, age, or primary site predominance. The mean disease-free interval for cutaneous melanomas was 14.3 years versus 22.3 years for ocular primary melanomas. The prognosis following relapse was related to the site of recurrence. Survival after local or regional node recurrence was often prolonged; survival after distant metastases was usually limited. Patients with ocular primaries had the highest incidence of distant metastases, and the shortest subsequent survival. An additional 483 patients were identified who survived 10 or more years without evidence of recurrence; of these 651 patients with long disease-free intervals, 25% (168 of 651) developed recurrent disease. This demonstrates that a 10-year disease-free interval cannot be considered a cure, and emphasizes the importance of continued annual follow-up.

The role of elective lymph node dissection in the management of patients with thick cutaneous melanoma.

A retrospective search of patients seen at the Duke Melanoma Clinic from 1970 to 1986 identified 308 clinically Stage I patients, with 4.0 to 10.0 mm cutaneous melanomas. Five-year and ten-year survival was 56% and 43%, respectively. Elective lymph node dissection (ELND) was done in 116 patients (37.7%); there was no difference in disease-free interval (DFI) or survival between these patients versus patients treated with wide excision only (P = 0.9). Thirty-two patients (27.6%) had pathologically positive nodes on ELND. These patients had a shorter DFI (P = 0.05) and survival (P = 0.03) compared with patients with negative node dissections. When further divided by Breslow's thickness, this difference persisted in patients with 4.0 to 6.0 mm lesions (P = 0.01). However, for thicker lesions (greater than 6.0 mm), there was no difference in survival between the node-negative and node-positive groups (P = 0.9). The mean follow-up was 7.1 years. Elective lymph node dissection was not done in 192 patients; 78 of these recurred first in the regional nodes. These 78 patients were compared with the 32 patients who had pathologically positive nodes by ELND to see if patient survival was improved by early removal of nodal disease. There was no difference in DFI (P = 0.5) or survival (P = 0.3) between these two groups. It is concluded that ELND may provide prognostic information for patients with thick cutaneous melanomas. However, there was no change in DFI or ultimate survival when patients were followed, and nodes removed when clinically positive. The authors do not recommend ELND for patients with thick melanomas because the risk of distant metastases outweighs any benefit of regional node dissection.

Cell binding and tumor inhibiting functions of a new antihuman melanoma murine monoclonal antibody.

Murine, antihuman melanoma cell monoclonal antibody (mAb) 16.C8 was generated by fusing the murine myeloma cell line P3X63/Ag8.653 with splenocytes from a nude mouse bearing a human melanoma xenograft, after reconstitution with splenocytes from syngeneic immunocompetent BALB/c mice. The antibody reacted strongly with fresh human melanoma cells and exhibited preferential reactivity with established human melanoma and neuroectodermal tumor cell lines. Electrophoresis and Western blotting experiments indicated that 16.C8 is directed against a sialoglycoprotein antigen with a molecular weight of 110-120 kDa. mAb 16.C8 mediated lysis of melanoma cells in vitro in antibody-dependent cellular cytotoxicity assays using human mononuclear effector cells isolated from normal volunteers or malignant melanoma patients. In addition, the administration of mAb 16.C8 to nude mice bearing established human melanoma lung and liver metastases resulted in significant inhibition of tumor growth as shown by gross and histologic examination. In contrast, animals treated with Hanks' balanced salt solution or nonspecific immunoglobulin exhibited a large tumor burden. These results suggest that mAb 16.C8 may be of value in treatment of metastatic melanoma in humans.

MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.

Modulation of in vitro autologous melanoma-specific cytotoxic T-cell responses by phorbol dibutyrate and ionomycin.

Human melanoma-specific, HLA restricted, cytotoxic T-cell lines can be generated by in vitro stimulation and culturing of peripheral lymphocytes, or lymph node cells, with autologous or HLA-A region matched melanomas in the presence of a low concentration (5 U/ml) of IL-2. Stimulation is followed by a period of clonal expansion and differentiation into cytotoxic T-cells specific for melanoma. We investigated the effect of the PKC modulating drug phorbol dibutyrate combined with the calcium ionophore Ionomycin on growth and differentiation of the cell lines. The growth of the T-cell lines was substantially augmented in the presence of the drugs with increases of 10-fold or more in clonal expansion by 3 weeks of culture. The cell lines were IL-2 dependent for growth in the presence or absence of the drugs and the phenotypic distribution remained predominantly CD3+ T-cells of mixed CD4 and CD8 phenotypes. In spite of the increased rate of growth in the presence of the drugs, autologous melanoma-specific cytotoxicity was almost completely abrogated in those cultures. The cells were, however, nonspecifically lytic in the presence of concanavalin A. The melanoma-specific cytotoxic response was completely restored following culture with IL-2 alone. The results suggest that the human tumor-specific cytotoxic T-cell response can be induced and amplified in the presence of immune modulating drugs.

Inhibition of the growth of human melanoma xenografts in nude mice by human tumor-specific cytotoxic T-cells.

Melanoma-specific T-cells (CTLs) are specifically cytotoxic for autologous tumor, when assayed in vitro. To examine their effectiveness in vivo, we tested the ability of these human T-cells to inhibit growth of human melanoma xenografts by using a Winn assay. Nude mice receiving specific CTLs (n = 10) demonstrated a dramatic inhibition of tumor growth. All treated mice were tumor-free at day 50 and nine remained tumor-free at day 65, vs. control mice (n = 10) with average tumor volumes of 321 mm3 and 808 mm3, respectively. To control for the possibility that a non-specific response to the human T-cells could inhibit tumor growth, an additional group received allospecific CTLs. There was no inhibition of tumor growth in this group (n = 8), with the average tumor volume of 2,768 mm3 at day 40 vs. 1,882 mm3 in the control group (n = 10). We conclude that these tumor-specific CTLs can inhibit tumor growth in vivo and may prove useful in the adoptive immunotherapy of melanoma.