Andrew N. Cornforth

Intralesional Lymphokine-activated Killer Cells as Adjuvant Therapy for Primary Glioblastoma

Despite recent advances, median survival for patients with resectable glioblastoma multiforme (GBM) is only 12 to 15 months. We previously observed minimal toxicity and a 9.0-month median survival after treatment with intralesional autologous lymphokine-activated killer (LAK) cells in 40 patients with recurrent GBM. In this study, GBM patients were treated with adjuvant intralesional LAK cells. Eligible patients had completed primary therapy for GBM without disease progression. LAK cells were produced by incubating autologous peripheral blood mononuclear cells with interleukin-2 for 3 to 7 days and then placed into the surgically exposed tumor cavity by a neurosurgeon. The 19 men and 14 women had a median age of 57 years. Prior therapy included surgical resection (97%), partial brain irradiation (97%), γ knife radiosurgery (97%), and temozolomide chemotherapy (70%). Median time from diagnosis to LAK cell therapy was 5.3 months (range: 3.0 to 11.1u2009mo). LAK cell treatment was well tolerated; average length of hospitalization was 3 days. At the time of this analysis, 27 patients have died; the median survival from the date of original diagnosis is 20.5 months with a 1-year survival rate of 75%. In subset analyses, superior survival was observed for patients who received higher numbers of CD3+/CD16+/CD56+ (T-LAK) cells in the cell products, which was associated with not taking corticosteroids in the month before leukopheresis. Intralesional LAK cell therapy is safe and the survival sufficiently encouraging to warrant further evaluation in a randomized phase 2 trial of intralesional therapies with LAK or carmustine-impregnated wafers.

Phase II trial of dendritic cells loaded with antigens from self-renewing, proliferating autologous tumor cells as patient-specific antitumor vaccines in patients with metastatic melanoma: final report.

UNLABELLEDnBetween January 2001 and September 2007, we treated 54 metastatic melanoma patients with patient-specific tumor cell vaccines consisting of dendritic cells (DCS), derived from their peripheral blood cells that were cultured in interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor (GM-CSF), which had phagocytosed irradiated autologous tumor cells from a continuously proliferating, self-renewing, autologus tumor cell (TC) culture. The loaded DCs were injected subcutaneously in 500 microg of GM-CSF weekly x three, and then monthly for 5 months, for a total of up to 8 injections. The 34 men and 20 women had a median age of 50.5 years; 32 had M1c (visceral metastases and/or elevated lactate dehydrogenase) as their most advanced disease stage. Overall, 83% had received other systemic therapies, including interferon-alpha (n = 20), biochemotherapy (n = 19), GM-CSF (n = 19), chemotherapy (n = 16), IL-2 (n = 13), and other investigational vaccines (n = 7). Patients received an average of 7.4 vaccinations. Treatment was well-tolerated, with most patients experiencing only mild local pruritus and/or erythema. A positive delayed-type hypersensitivity reaction to purified autologous tumor cells was observed at baseline in only 1 of 54 patients, compared to 12 of 54 following vaccination (p = 0.001). The projected 5-year survival rate is an impressive 54% at a median follow-up of 4.5 years (range, 2.4-7.4) for the 30 surviving patients. This survival was superior to that observed following vaccination with irradiated TC in 48 melanoma patients in a previous trial (64 versus 31 months, p = 0.016). This patient-specific vaccine warrants further investigation, based on its safety and encouraging survival rates. (nnnCLINICAL TRIAL NUMBERnNCI-V01-1646).

Features Associated with Survival in Metastatic Melanoma Patients Treated with Patient-Specific Dendritic Cell Vaccines

Previously, a 54% 5-year survival was reported for metastatic melanoma patients treated with patient-specific vaccines consisting of autologous dendritic cells loaded with antigens from autologous proliferating tumor cells. This study attempted to determine which clinical and laboratory factors best explained long-term survival in this group of patients. Univariate analyses were used to identify factors associated with continuous survival after initiating vaccine therapy. Multivariate logistic regression was used to identify independent factors to classify survival at 3.5 years. Survivors were followed a minimum of 3.7 years (median: 5.7). Univariate analyses identified eight features associated with improved survival: Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0, no measurable disease at study entry, receiving 8 vaccinations, age <50 years, normal baseline lactate dehydrogenase, no history of visceral metastases, anergy to standard skin tests, and failure of interferon-gamma (IFN-γ) to induce apoptosis in autologous tumor cells. After examining 54 variables for which complete information was available over all patients, the best multivariate regression for survival at 3.5 years utilized six features: prior radiation therapy, younger age, male gender, ECOG PS 0, higher numbers of cells administered during the first 3 injections, and lower numbers of viable cells administered during the first 3 injections. This model correctly classified survival for 28 of 32 patients (87%) and death for 20 of 22 (91%). When features with incomplete information were included in the analysis, addition of IFN-γ-induced apoptosis (n=49) improved predictive accuracy to 27 of 29 (93%) for survival and 19 of 20 (95%) for death. Dependencies between variables were common, but these multivariate linear models yielded high classification accuracy for survival at 3.5 years and identified two features of the vaccine itself as being of independent significance.

Cancer stem cell antigen-based vaccines: the preferred strategy for active specific immunotherapy of metastatic melanoma?

Introduction: There are now two chemotherapy agents, one tyrosine kinase inhibitor and three immunotherapy products approved for the treatment of metastatic melanoma, but an unmet need persists because these options are toxic and of limited therapeutic benefit. Active specific immunotherapy with therapeutic vaccines could be a useful addition to the therapeutic armamentarium, especially in patients whose tumor burden has been reduced by other treatment modalities. Areas covered: This article reviews various sources of melanoma antigens, such as peptides, gangliosides, autologous tumor and cancer stem cells including allogeneic and autologous cell lines. The advantages and disadvantages of various antigen sources and allogeneic and autologous approaches are discussed with an emphasis on the theoretical benefits of immunizing against cancer stem cells. The results from published randomized trials testing the benefit of various vaccine approaches are summarized, as well as promising results from three Phase II trials (one randomized) of patient-specific stem cell antigen-based products. Expert opinion: Immune responses directed toward the unique neoantigens and stem cell antigens expressed on continuously proliferating, self-renewing, autologous tumor cells could potentially overcome the limitations inherent in these other antigen-based approaches, that to date, have yielded disappointing results in randomized trials.

Durable Complete Response of Refractory, Progressing Metastatic Melanoma After Treatment with a Patient-Specific Vaccine

A patient with metastatic melanoma who experienced a durable complete response after treatment with a patient-specific vaccine has been described in this article. This 59-year-old woman presented with cervical spine metastases and, within the year, had experienced local disease progression and, despite various therapies, metastases to the axilla, rectum, gall bladder, and multiple soft-tissue sites. She had previously received radiation therapy, combination chemotherapy, interleukin-2 plus interferon biotherapy, and gamma knife radiosurgery, and undergone multiple surgical resections. At the time vaccine therapy was initiated, she had multiple, new, measurable, soft-tissue metastases that were increasing in size. She was treated with a vaccine consisting of autologous dendritic cells incubated with irradiated tumor cells from an autologous tumor cell line and suspended in granulocyte-macrophage colony stimulating factor (GM-CSF), with subcutaneous injections once a week for 3 weeks and monthly for 5 months. There was evidence of disease regression by the completion of therapy. A few months later a complete response was documented by radiologic scans, and subsequently reconfirmed at 6-month intervals. She remains in complete remission >2.5 years after starting the vaccine, and >2 years after completing the vaccine, and survives >4 years after her initial presentation with bone, bowel, and lymph node metastases. This is the first time she has been in a complete remission since her initial diagnosis. Patient-specific vaccines can sometimes induce durable complete regression of progressing soft-tissue melanoma metastases.

Increases in Serum TARC/CCL17 Levels Are Associated with Progression-Free Survival in Advanced Melanoma Patients in Response to Dendritic Cell-Based Immunotherapy

IntroductionChanges in the levels of serum cytokines and growth factors are associated with response to therapy. We examined cytokine, chemokine, and growth factor levels in serum collected from normal volunteers or metastatic melanoma patients receiving dendritic cell-based immunotherapy.Materials and MethodsUsing an array for 42 cytokines, chemokines, or growth factors, sera from normal controls and metastatic melanoma patients at baseline and week 4 were analyzed for qualitative changes. Quantitative determination of the levels of the chemokine thymus and activation-regulated chemokine (TARC/CCL17) was determined by enzyme-linked immunosorbent assay (ELISA).ResultsSignificant qualitative differences were noted in serum cytokine, chemokine, and growth factor levels of metastatic melanoma patients versus the normal controls at baseline. The results also demonstrated a significant decrease in the level of angiogenin (Pu2009=u20090.026) and a significant increase in TARC/CCLl7 (Pu2009=u20090.008) from week 0 to week 4 which was associated with improved overall survival (Pu2009=u20090.059). Higher TARC/CCL17 levels were observed by ELISA at week 4 and a log-rank comparison revealed a significant association between high serum TARC/CCL17 levels at week 4 and progression-free survival (Pu2009=u20090.005). Receiver–operator characteristic analysis revealed that week 4 serum TARC/CCL17 levels were predictive of progression-free and overall survival, indicating that serum TARC/CCL17 might be of predictive value of response to dendritic cell-based anti-melanoma immunotherapy.

Characterization of cancer cell lines established from two human metastatic breast cancers

SummaryCell lines are valuable resources for the study of the malignancy and potential therapy of human breast cancer. A major problem with adapting fresh breast tumor specimens to grow in vitro is contamination by fibroblasts. Previously, we have reported a technique to overcome this problem (Nayak, S. K; Dillman, R. O. Clin. Biotechnol. 3:237–242; 1991). We have recently established two new breast cancer cell lines, HH315 and HH375, that were derived from abdominal and supraclavicular lymph node metastases from two patients. They were characterized by (1) growth kinetics; (2) staining with monoclonal antibodies (MoAbs) to cytokeratin-19, epithelial membrane antigen (EMA), anticarcinoembryonic antigen (CEA), breast cancer antigen 1 (BRST-1), breast cancer antigen 2 (BRST-2), Her2/neu, and p53; (3) expression of domains of urinary plasminogen activator (uPA), neural cell adhesion molecule (NCAM), and haptoglobin (Hp) (Harvey et al., 1997); and (4) karyotypic analysis. Growth kinetic studies showed that doubling times for both lines ranged from 48 to 96 h. These two cell lines were found to have characteristics of the metastatic breast cancer cells. Both lines stained positive with MoAbs to cytokeratin-19 and EMA, thus confirming their epithelial origin. They also strongly reacted with the pan-breast carcinoma MoAbs BRST-1 and BRST-2, and carcinoembryonic CEA MoAb. Both cell lines overexpressed the oncogene proteins Her2/neu and p53. The tumor cells were negative for estrogen and progesterone receptors. HH315 cells were poorly differentiated, whereas the HH375 cells exhibited adenocarcinoma morphology. Both cell lines showed intense cell surface and some cytoplasmic staining for uPA, NCAM, and Hp domains, which is a characteristic of malignant neoplasms (Harvey et al., 1997). The HH375 cell line showed two cell types, of which 60% were hyperdiploids with 60–70 chromosomes and 5–10 marker chromosomes. The remaining cells were polyploid with more than 200 chromosomes. Cell line HH315 consisted of only a polyploid population. These cell lines may be useful in breast cancer research.

Characterization of interferon-γ-treated melanoma tumor cells for use in dendritic cell-based immunotherapy.

Efficient delivery of tumor-associated antigens to professional antigen-presenting cells is important for inducing a response in patients receiving cancer immunotherapy. Interferon-gamma (IFN-γ) is used by the immune system to combat viral and fungal infections by restricting cell proliferation and, in some cases, inducing apoptosis. Using IFN-γ to activate target tumor cells prior to antigen loading of dendritic cells (DCs) may enhance the beneficial qualities of whole-cell tumor vaccines. The incubation of melanoma cell cultures with IFN-γ resulted in an increase in the expression of major histocompatibility complex molecules and ICAM-1 but generally decreased the expression of melanoma-associated tumor antigens. Additionally, important immune-stimulating molecules (heat-shock proteins, high-mobility group box-1 protein, and calreticulin) were also present but differentially regulated by IFN-γ. Loading of DCs with IFN-γ-treated tumor cells resulted in a small but significant increase in the expression of CD83-positive DCs, indicating the initiation of DC maturation (p=0.019). IFN-γ treatment of melanoma cell lines prior to antigen loading of DCs may aid in antigen processing and presentation.

Phase III randomized, double-blinded, placebo-controlled multicenter trial of melapuldencil-t: autologous dendritic cells loaded with irradiated autologous tumor cells (dc-tc) in gm-csf in patients with metastatic melanoma

Background Genomic analyses have shown that melanoma patients have hundreds to thousands of non-synonymous mutations including unique tumor associated antigens (TAA) that can be recognized by their immune systems. The efficacy of anti-checkpoint monoclonal antibodies provides further proof that host recognition of TAA exists, but many patients do not benefit from such therapy, perhaps because they lack sufficient TAA recognition. One way to enhance TAA recognition is immunization. The best source of TAA may be autologous tumor cells that self-renew and proliferate in tissue culture (patient specific tumor stem cells). Sequential Phase II trials in metastatic melanoma patients tested the clinical benefit of repeated s.c. injections of autologous dendritic cells loaded with antigens from irradiated tumor cells derived from autologous melanoma cell lines (DC-TC), and suspended in GM-CSF. In a single arm trial 5-year survival was 50% for 54 patients treated with DC-TC. In a randomized Phase II trial, 2-year survival was 72% for 18 DC-TC patients compared to 31% in the control arm (HR = 0.27, p = 0.007). Toxicities associated with DCTC were minimal (n = 72). Improvements in manufacturing have increased the probability of establishing a cell line and decreased the time needed to produce the product. Melapuldencel-T (DC-TC) has shown sufficient promise to receive special product assessment (SPA) and fast track designation in association with approval of this pivotal Phase III trial.

Phase I trial of active specific immunotherapy with autologous dendritic cells pulsed with autologous irradiated tumor stem cells in hepatitis B positive patients with hepatocellular carcinoma

Background Active or chronic infections may be barriers to successful vaccines, and patients with hepatitis typically are excluded from clinical trials testing therapeutic anticancer vaccines. One concern is vaccines may exacerbate the underlying infection. Hepatocellular carcinoma (HCC) often arises in the context of viral hepatitis B (HBV) or hepatitis C (HBC), with or without cirrhosis. HCC is seldom cured by standard therapy because of undetectable micrometastatic disease that is present at diagnosis. Effective nontoxic adjunctive treatments might improve outcomes for HCC patients who are at high risk for recurrence and death despite surgical resection of their primary hepatoma. Active specific immunotherapy (ASI) with autologous dendritic cells loaded with antigens from autologous tumor stem cell lines has been associated with promising long-term survival in metastatic melanoma, but patients with HBV or HCV were ineligible. Therefore a Phase I safety trial in HCC patients with HBV and/or HIV was warranted. Patients and methods Patients with previously untreated HCC who were candidates for surgical resection, but not curable by resection or liver transplant were enrolled in Shanghai, China. Patients had a solitary lesion >5 cm in diameter, or 3 lesions with at least one > 3 cm, but no regional or distant metastatic disease. Patients had a good performance status, adequate blood counts and organ function. An autologous tumor cell (TC) line was established from the resected tumor. Irradiated autologous TC were incubated with autologous dendritic cells (DC) to create a patient-specific DC-TC product which was cryopreserved. Approximately eight weeks after one course of transarterial chemoembolization therapy (TACE), patients received three weekly subcutaneous injections of DC-TC suspended in 500 micrograms of sargramostim. Patients were monitored for toxicity for 8 weeks from the start of treatment.