Immunohistochemical determination of HER-2/neu overexpression in malignant melanoma reveals no prognostic value, while c-Kit (CD117) overexpression exhibits potential therapeutic implications

BACKGROUND: HER-2/neu and c-kit (CD117) onco-protein are increasingly being recognized as targets for therapy in solid tumors, but data on their role in malignant melanoma is currently limited. We studied the prevalence of overexpression of HER-2/neu and c-Kit in 202 patients with malignant melanoma to evaluate a possible prognostic value of these molecular targets in malignant melanoma. METHODS: Overexpression of HER-2/neu and c-Kit was evaluated using immunohistochemical assays in 202 archival tissue specimens. RESULTS: Between 1991 and 2001, 202 subjects (109 males; 54% and 93 females; 46%) with malignant melanoma were studied with a mean age of 57 years (age range: 15-101 years). The most common histologic type was amelanotic melanoma (n = 62; 30.7%) followed by superficial spreading melanoma (n = 54; 26.7%). The depth of penetration of melanoma (Breslow thickness, pT Stage) ranged from 0.4 mm (stage pT1) to 8.0 mm (stage pT4A). Mean thickness was 2.6 mm (stage pT3A). The ECOG performance scores ranged from 0 to 3. Only 2 patients (0.9%) revealed HER-2/neu overexpression, whereas 46 (22.8%) revealed c-Kit overexpression. Multivariate analysis performed did not show a significant difference in survival between c-Kit positive and negative groups (p = 0.36). Interestingly, not only was c-Kit more likely to be overexpressed in the superficial spreading type, a preliminary association between the presence or absence of c-Kit overexpression and the existence of another second primary tumor was also observed. CONCLUSIONS: The results of our large study indicate that the HER-2/neu onco-protein neither has a role in melanogenesis nor is a potential target for clinical trials with monoclonal antibody therapy. This indicates there is no role for its testing in patients with malignant melanoma. Although c-Kit, expressed preferentially in the superficial spreading type, may not have prognostic value, it does have significant therapeutic implications as a molecular target warranting further investigation.     

T lymphocyte subsets in patients with malignant melanoma

Summary T lymphocyte subset profiles were determined by monoclonal antibodies on cryopreserved peripheral blood lymphocytes from 57 patients with malignant melanoma and 19 healthy controls. Quantitation of percentages of total T cells (OKT3.PAN), helper (OKT4.IND) or suppressor (OKT8.SUP) cells, and the ratio of helper/suppressor subsets revealed no correlation of these markers with stage of disease or clinical outcome. A sequential study of these markers on peripheral blood lymphocytes from three stage I melanoma patients with subsequent recurrent disease showed no fluctuations that could be correlated to tumor progression. This study indicates that there is no systemic imbalance in T cell subsets in malignant melanoma and that quantitation of these subsets cannot predict the clinical course of this disease.     

Speed of growth of melanoma: statistical analysis of the average ages of patient groups

Based on data from 1,657 cases, the average age of patients with melanoma of the same type, level, resp. thickness was calculated. It was shown that the average age of the melanoma patients increased with progressive tumor growth, i.e., with increasing level and thickness. The time span for thin (less than 0.75 mm thickness), superficial spreading melanoma (SSM) to reach the level of a far-advanced tumor with a thickness of more than 3.00 mm was 7.8 years. For nodular melanomas (NM), the corresponding period was 9.5 years. Longer intervals were determined by analysis of the data according to tumor levels. The difference in the average age of patients with early invasive SSM (level II) as compared with patients with level V tumors was 11.4 years. Patients with level V NM were on the average 10.0 years older than patients with level III NM. Differences in the average age of patients with SSM or NM from one level to the other ranged between 3 and 4 years (median: 3.9 years). The differences in the thickness of the groups were similar (median: 3.1 years).     

Identification of specific cytolytic immune responses against autologous tumor in humans bearing malignant melanoma

Tumor-infiltrating lymphocytes from six patients with metastatic malignant melanoma were expanded by culture in recombinant interleukin 2. Three of the preparations were highly cytotoxic against autologous fresh melanoma tumor cells, but not against autologous fresh normal cells or allogeneic fresh tumor targets. The other three were highly cytotoxic against autologous fresh melanoma tumor cells and also had a limited capacity to kill allogeneic fresh tumor targets. The tumor-associated specific killer cells could be expanded from threefold to 95,652-fold with maintenance of specific antitumor lysis. The expanded tumor-infiltrating cells were Leu-4+ T cells, and in five of six patients the majority were Leu-3+. These studies demonstrate that the melanoma-bearing patient raises an immune response against autologous tumor and presents a method for the generation of human lymphocytes with antitumor reactivity that may be useful in the adoptive immunotherapy of tumors.     

Leukocyte infiltration and tumor cell plasticity are parameters of aggressiveness in primary cutaneous melanoma

Various clinical and experimental observations detected an immunological host defense in cutaneous melanoma. In order to investigate the prognostic value of leukocyte effector mechanisms, we examined the presence of different subsets of leukocytes in tumor samples of 58 patients diagnosed with primary cutaneous melanoma. The presence of T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, CD16+ cells and macrophages was correlated to Breslow depth. A significantly higher amount of several subsets of leukocytes was found in samples with a more progressed tumor stage and survival analysis demonstrated that a higher amount of T lymphocytes and CD16+ cells was associated with a short survival. The amount of FOXP3+ regulatory T lymphocytes did not correlate with survival, nevertheless, it correlated with the amount of total infiltrate. In contrast, analysis of the expression of CD69, a marker for activated lymphocytes, demonstrated that patients with a higher amount of CD69+ lymphocytes had a better survival. In addition, a new parameter for aggressiveness of melanoma, tumor cell plasticity [i.e., the presence of periodic acid Schiff’s (PAS) reagent positive loops], also predicted short survival and a trend of a higher amount of tumor infiltrating leukocytes in tumors with PAS positive loops was observed. These findings demonstrate that leukocyte infiltration and the presence of PAS loops is a sign of tumor aggressiveness and may have prognostic value.     

Disease-related lymphocyte cytotoxicity in vitro in patients with transitional cell carcinoma of the urinary bladder. Comparisons with other malignancies,acute cystitis,and healthy donors

Summary Blood lymphocytes from 100 patients with transitional cell carcinoma of the urinary bladder (TCC-bladder) were studied for their cytotoxicity in vitro against a panel of allogeneic tissue culture cell lines. Of the TCC-bladder patients, 45 were untreated for their disease, while 55 had been treated with local radiotherapy up to 12 years before testing. Control lymphocytes were obtained from (1) 45 untreated, age- and sex-matched patients with other neoplastic diseases, mainly urogenital cancers; (2) 19 patients with acute cystitis; and (3) 45 healthy donors. Lymphocytes from individual donors within all five groups were frequently cytotoxic to any one of the target cells. However, the lymphocytes from each of the two TCC-bladder groups were markedly more cytotoxic to two different bladder tumor targets than to control targets derived from normal bladder epithelium, from colon carcinoma, or from malignant melanoma. Similar comparisons made within each of the three control donor groups did not show this. The results indicate that the two bladder tumor targets were not more susceptible to lymphocyte-mediated lysis than the control targets. The mean cytotoxicity displayed by the lymphocytes from both TCC-bladder groups to the bladder tumor targets was significantly higher than that of the cancer control group and that of the healthy donors. No such elevation was seen when the cancer control group or the cystitis patients were compared with healthy donors. Although untreated TCC-patients with a larger tumor burden (stages T3–T4) appeared to be slightly less cytotoxic to all target cells than those with a smaller tumor burden (T1–T2), these differences were not statistically significant. On the other hand, among the treated TCC-patients, in the main those tested more than 1 year and up to 5 years after therapy exhibited a significantly elevated mean cytotoxicity to the bladder tumor targets. Within all five donor groups, the overall cytotoxicity to the bladder tumor targets and the normal bladder targets showed a statistically highly significant correlation. However, while there was no correlation for the untreated TCC-bladder patients and the clinical controls between cytotoxicity to the bladder tumor targets on one hand and non-bladder targets on the other, the cytotoxicity to the bladder tumor targets of the treated TCC-bladder patients was also correlated with that to the colon carcinoma and the melanoma targets. The results indicate that cytotoxicity in both TCC patients and controls reflects recognition by the lymphocytes of a variety of antigens, shared to different degrees by different groups of target cells. Furthermore, in TCC-bladder patients there is a superimposed cytotoxicity, which is related to their disease and which probably reflects reactions against one or several tumor-associated antigens.     

Rapid expansion of tumor-reactive cells from HLA-matched siblings for adoptive immunotherapy of melanoma

BACKGROUND: Administration of expanded tumor-infiltrating lymphocytes in association with lymphodepleting chemotherapy is effective in some patients with advanced malignant melanoma. However, obtaining lymphocytes and subsequent expansion is labor intensive, making it impractical for broad clinical application. Allogeneic transplantation may have anti-melanoma efficacy because of a graft vs. tumor effect. The disappointing tumor control observed post-transplant suggests that adoptive immunotherapy using melanoma-reactive cells will be essential for sustained responses. METHODS: Melanoma cell lines were grown from two patients with advanced disease. High-level CD80 and CD86 expression was obtained in the tumor lines using a retroviral vector for gene transfer. Transduced melanoma and controls were cultured with mononuclear cells from HLA-identical sibling donors. RESULTS: Expression of CD80 and CD86, particularly the former, promoted marked expansion of lymphocytes from HLA-matched sibling donors. Proliferation of up to 300-fold after 4 weeks of culture was observed. Lymphocytes from cultures stimulated with CD80 demonstrated cytotoxicity against recipient-untransfected melanoma (45-75% specific lysis at an E:T ratio of 50:1). Although expanded lymphocytes were predominantly CD4(+), cytotoxicity was greatest in the numerically smaller CD8(+) subpopulation. Both CD4(+) and CD8(+) cells secreted IFN-gamma (but not IL-4) on exposure to untransduced stimulator melanoma cells. DISCUSSION: Our strategy generates a large number of melanoma-reactive lymphocytes from HLA-identical siblings using a 4-week culture strategy. Lymphocytes expanded in this way offer an alternative to tumor-infiltrating lymphocytes for allogeneic cellular immunotherapy after stem cell transplantation in young patients.     

Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.     

Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines.

Activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP is believed to play a critical role during tumor progression and metastasis. In this study we present evidence that MT1-MMP plays a key role for the in vitro invasiveness of malignant melanoma. Melanoma cell lines secreted latent MMP-2 when cultured on plastic. However, when cells were grown in floating type I collagen lattices, only high invasive melanoma cells activated proMMP-2. Activation could be inhibited by antibodies against MT1-MMP, by addition of recombinant tissue inhibitor of metalloproteinases (TIMP)-2 and by inhibition of MT1-MMP cleavage. MT1-MMP protein was detected as an inactive protein in all cell lines cultured as monolayers, whereas in collagen gels, active MT1-MMP protein was detected in the membranes of both high and low invasive melanoma cells. Production of TIMP-2 was about 10-fold higher in low invasive cells as compared with high invasive melanoma cells and was further increased in the low invasive cells upon contact to collagen. Thus, in melanoma cells TIMP-2 expression levels might regulate MT1-MMP-mediated activation of proMMP-2. High invasive melanoma cells displayed increased in vitro invasiveness, which was inhibited by TIMP-2. These data indicate the importance of these enzymes for the invasion processes and support a role for MT1-MMP as an activator of proMMP-2 in malignant melanoma.     

Selective growth, in vitro and in vivo, of individual T cell clones from tumor-infiltrating lymphocytes obtained from patients with melanoma

In recent clinical trials in patients with metastatic melanoma, adoptive transfer of tumor-reactive lymphocytes mediated the regression of metastatic tumor deposits. To better understand the role of individual T cell clones in mediating tumor regression, a 5' RACE technique was used to determine the distribution of TCR beta-chain V region sequences expressed in the transferred cells as well as in tumor samples and circulating lymphocytes from melanoma patients following adoptive cell transfer. We found that dominant T cell clones were present in the in vitro-expanded and transferred tumor-infiltrating lymphocyte samples and certain T cell clones including the dominant T cell clones persisted at relatively high levels in the peripheral blood of the patients that demonstrated clinical responses to adoptive immunotherapy. However, these dominant clones were either undetected or present at a very low level in the resected tumor samples used for tumor-infiltrating lymphocyte generation. These data demonstrated that there was selective growth and survival, both in vitro and in vivo, of individual T cell clones from a relatively small number of T cells in the original tumor samples. These results suggest that the persistent T cell clones played an active role in mediating tumor regression and that 5' RACE analysis may provide an important tool for the analysis of the role of individual T cell clones in mediating tumor regression. A similar analysis may also be useful for monitoring autoimmune responses.     

Flow cytometry assessment of residual melanoma cells in tumor-infiltrating lymphocyte cultures

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.     

Relationship between regulatory and type 1 T cells in dogs with oral malignant melanoma

Recent data suggest a decreased prevalence of IFN‐γ‐producing T lymphocytes (Type 1 T cells) in tumor‐bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti‐tumor responses.     

Lentigo Maligna and Malignant Melanoma

Lentigo maligna, a precancerous lesion, is a brown-black irregularly pigmented freckle, usually occurring on the face of the elderly subject. In a series of 99 patients with malignant melanomas, lentigo maligna was the pre-existing lesion in 21. The clinical and histological findings, and previous publications on the subject are reviewed. Lentigo maligna itself is not a superficial malignant melanoma. After the development of malignant melanoma from lentigo maligna, eight of 21 patients developed metastatic disease. This seems to indicate that once malignant melanoma has developed (whether de novo from the junctional portion of a pre-existing nevus, or from a lentigo maligna), the outlook is the same. During the development of malignant melanoma from lentigo maligna there is an indefinite period when it is virtually impossible to determine histologically whether malignant melanoma is present. Naturally, the inclusion of these indefinite cases will greatly influence reported results of treatment.     

Epidemiological and clinical characteristics of malignant melanoma in area of West Herzegovina from 1997 to 2010

Incidence rate of cutaneous malignant melanoma (MM), one of the most aggressive skin tumours, is increasing nowadays. Etiology of MM has not been fully understood. Various etiological factors are of relevance for the occurrence of the disease. The solar radiation as well as long term exposure to ultraviolet radiation has the greatest impact on development of this skin tumour. Melanoma risk factors have different associations with melanoma on body sites. This study investigates the epidemiological and clinical characteristics of MM such as age, gender, distribution of MM on the body and type of melanoma in the area of West Herzegovina, on the sample of 205 patients. It presents the occurrence of MM in the period from 1997. to 2010. Both, females and males have increased the risk of melanoma on the trunk (45.9%). Different body sites receive various amounts of sun exposure, yet melanomas occur on all parts of the body. This may represent different pathways in the etiology of melanoma based on body location. The most frequent type of MM was superficial spreading melanoma (SSM) 47.8%. According to our investigation incidence rate was 18.6% (per 1000 patients).     

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.     

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.     

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dentritic cell/melanoma cell hybridomas

Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70 +/- 9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63+/-8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32+/-6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.     

Surgical considerations in the management of malignant melanoma of the ear

Malignant melanomas of the external ear are rare and are difficult lesions to treat because of the cosmetic importance and the reconstructive difficulty of their location. The literature suggests that these lesions have a worse prognosis than melanomas occurring elsewhere and that radical resection is the "correct" treatment. To clarify this issue, we examined 21 consecutive patients (19 male, 2 female) with malignant melanoma of the ear seen at the Yale-New Haven Hospital over the last 10 years. Nineteen patients had a diagnosis of primary malignant melanoma of the ear, one had a local recurrence, and one had an in-transit melanoma from an unknown primary site. The mean thickness of the lesions was 2.7 mm. Two patients had palpable nodes, which in both cases turned out to be histologically positive for tumor. All patients underwent local excision and reconstruction using chondrocutaneous or fasciocutaneous flaps or skin grafts. There was one local recurrence (0.5 mm original thickness); there were two patients with regional recurrences, both of whom died within a year with disseminated disease. Forty-three percent have been followed for 5 or more years and all are alive and free of disease. This suggests that malignant melanoma of the ear may be safely treated by conservative excision and reconstruction.     

Dendritic cell-based combined immunotherapy with autologous tumor-pulsed dendritic cell vaccine and activated T cells for cancer patients: rationale, current progress, and perspectives

Effective adoptive cancer immunotherapy depends on an ability to generate tumor-antigen-presenting cells and tumor-reactive effector lymphocytes and to deliver these effector cells to the tumor. Dendritic cells (DCs) are the most potent antigen-presenting cells, capable of sensitizing T cells to new and recall antigens. Many studies have shown that tumors express unique proteins that can be loaded on DCs to trigger an immune response. The current experimental and clinical statuses of adoptive transfer of tumor antigen-pulsed DCs and vaccine-primed activated T cells are summarized herein. Clinical trials of antigen-pulsed DCs have been conducted in patients with various types of cancer, including non-Hodgkin lymphoma, multiple myeloma, prostate cancer, renal cell carcinoma, malignant melanoma, colorectal cancer, and non-small cell lung cancer. These studies have shown that antigen-loaded DC vaccination is safe and promising for the treatment of cancer. In addition, tumor vaccine-primed T cells have been shown to induce antitumor activity in vivo. Several clinical studies are being conducted on the use of vaccine-primed T cells such as tumor-drainage lymph node. It is reasonable to consider using both tumor antigen-pulsed DCs and vaccine-primed lymphocytes as adjuvants. We are now investigating the use of autologous whole tumor antigen-pulsed DCs and the DC vaccine-primed activated lymphocytes in patients with multiple metastasis of solid tumors.     

Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.