Richard Heller

Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis

Non-receptor and receptor tyrosine kinases, such as Src and EGF receptor (EGFR), are major inducers of vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis. While tyrosine kinases signal through multiple pathways, signal transducer and activation of transcription 3 (Stat3) is a point of convergence for many of these and is constitutively activated with high frequency in a wide range of cancer cells. Here, we show that VEGF expression correlates with Stat3 activity in diverse human cancer cell lines. An activated Stat3 mutant (Stat3C) up-regulates VEGF expression and stimulates tumor angiogenesis. Stat3C-induced VEGF up-regulation is abrogated when a Stat3-binding site in the VEGF promoter is mutated. Furthermore, interrupting Stat3 signaling with dominant-negative Stat3 protein or Stat3 antisense oligonucleotide in tumor cells down-regulates VEGF expression. Consistent with an important role of Stat3 in VEGF up-regulation induced by various oncogenic tyrosine kinases, v-Src-mediated VEGF expression is inhibited when Stat3 signaling is blocked. Moreover, chromatin immunoprecipitation assays indicate that Stat3 protein binds to the VEGF promoter in vivo and mutation of a Stat3-binding site in the VEGF promoter abrogates v-Src-induced VEGF promoter activity. These studies provide evidence that the VEGF gene is regulated directly by Stat3 protein, and indicate that Stat3 represents a common molecular target for blocking angiogenesis induced by multiple signaling pathways in human cancers.

Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells.

Although tumor progression involves processes such as tissue invasion that can activate inflammatory responses, the immune system largely ignores or tolerates disseminated cancers. The mechanisms that block initiation of immune responses during cancer development are poorly understood. We report here that constitutive activation of Stat-3, a common oncogenic signaling pathway, suppresses tumor expression of proinflammatory mediators. Blocking Stat-3 in tumor cells increases expression of proinflammatory cytokines and chemokines that activate innate immunity and dendritic cells, leading to tumor-specific T-cell responses. In addition, constitutive Stat-3 activity induces production of pleiotropic factors that inhibit dendritic cell functional maturation. Tumor-derived factors inhibit dendritic cell maturation through Stat-3 activation in progenitor cells. Thus, inhibition of antitumor immunity involves a cascade of Stat-3 activation propagating from tumor to dendritic cells. We propose that tumor Stat-3 activity can mediate immune evasion by blocking both the production and sensing of inflammatory signals by multiple components of the immune system.

The orderly progression of melanoma nodal metastases

ObjectiveThe aim of this study was to determine the order of melanoma nodal metastases. Summary Background DataMost solid tumors are thought to demonstrate a random nodal metastatic pattern. The incidence of skip nodal metastases precluded the use of sampling procedures of first station nodal basins to achieve adequate pathological staging. Malignant melanoma may be different from other malignancies in that the cutaneous lymphatic flow is better defined and can be mapped accurately. The concept of an orderly progression of nodal metastases is radically different than what is thought to occur in the natural history of metastases from most other solid malignancies. MethodsThe investigators performed preoperative and intraoperative mapping of the cutaneous lymphatics from the primary melanoma in an attempt to identify the “sentinel” lymph node in the regional basin. All patients had primary melanomas with tumor thicknesses > 0.76 mm and were considered candidates for elective lymph node dissection. The sentinel lymph node was defined as the first node in the basin from which the primary site drained. The sentinel lymph node was harvested and submitted separately to pathology, followed by a complete node dissection. The null hypothesis tested was whether nodal metastases from malignant melanoma occurred in equal proportions among sentinel and nonsentinel nodes. ResultsForty-two patients met the criteria of the protocol based on prognostic factors of their primary melanoma. Thirty-four patients had histologically negative sentinel nodes, with the rest of the nodes in the basin also being negative. Thus, there were no skip metastases documented. Eight patients had positive sentinel nodes, with seven of the eight having the sentinel node as the only site of disease. In these seven patients, the frequency of sentinel nodal metastases was 92%, whereas none of the higher nodes had documented metastatic disease. Nodal involvement was compared between the sentinel and nonsentinel nodal groups, based on the binomial distribution. Under the null hypothesis of equality in distribution of nodal metastases, the probability that all seven unpaired observations would demonstrate that involvement of the sentinel node is 0.008.

Phase I Trial of Interleukin-12 Plasmid Electroporation in Patients With Metastatic Melanoma

PURPOSE Gene-based immunotherapy for cancer is limited by the lack of safe, efficient, reproducible, and titratable delivery methods. Direct injection of DNA into tissue, although safer than viral vectors, suffers from low gene transfer efficiency. In vivo electroporation, in preclinical models, significantly enhances gene transfer efficiency while retaining the safety advantages of plasmid DNA. PATIENTS AND METHODS A phase I dose escalation trial of plasmid interleukin (IL)-12 electroporation was carried out in patients with metastatic melanoma. Patients received electroporation on days 1, 5, and 8 during a single 39-day cycle, into metastatic melanoma lesions with six 100-mus pulses at a 1,300-V/cm electric field through a penetrating six-electrode array immediately after DNA injection. Pre- and post-treatment biopsies were obtained at defined time points for detailed histologic evaluation and determination of IL-12 protein levels. RESULTS Twenty-four patients were treated at seven dose levels, with minimal systemic toxicity. Transient pain after electroporation was the major adverse effect. Post-treatment biopsies showed plasmid dose proportional increases in IL-12 protein levels as well as marked tumor necrosis and lymphocytic infiltrate. Two (10%) of 19 patients with nonelectroporated distant lesions and no other systemic therapy showed complete regression of all metastases, whereas eight additional patients (42%) showed disease stabilization or partial response. CONCLUSION This report describes the first human trial, to our knowledge, of gene transfer utilizing in vivo DNA electroporation. The results indicated this modality to be safe, effective, reproducible, and titratable.

Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy.

Electrochemotherapy (ECT) enhances the effectiveness of chemotherapeutic agents by administering the drug in combination with short intense electric pulses. ECT is effective because electric pulses permeabilize tumour cell membranes and allow non-permeant drugs, such as bleomycin, to enter the cells. The aim of this study was to demonstrate the anti-tumour effectiveness of ECT with bleomycin on cutaneous and subcutaneous tumours. This article summarizes results obtained in independent clinical trials performed by five cancer centres. A total of 291 cutaneous or subcutaneous tumours of basal cell carcinoma (32), malignant melanoma (142), adenocarcinoma (30) and head and neck squamous cell carcinoma (87) were treated in 50 patients. Short and intense electric pulses were applied to tumours percutaneously after intravenous or intratumour administration of bleomycin. The tumours were measured and the response to the treatment evaluated 30 days after the treatment. Objective responses were obtained in 233 (85.3%) of the 273 evaluable tumours that were treated with ECT. Clinical complete responses were achieved in 154 (56.4%) tumours, and partial responses were observed in 79 (28.9%) tumours. The application of electric pulses to the patients was safe and well tolerated. An instantaneous contraction of the underlying muscles was noticed. Minimal adverse side-effects were observed. ECT was shown to be an effective local treatment. ECT was effective regardless of the histological type of the tumour. Therefore, ECT offers an approach to the treatment of cutaneous and subcutaneous tumours in patients with minimal adverse side-effects and with a high response rate.

In vivo gene electroinjection and expression in rat liver

In vivo targeted gene transfer by non‐viral vectors is subjected to anatomical constraints depending on the route of administration. Transfection efficiency and gene expression in vivo using non‐viral vectors is also relatively low. We report that in vivo electropermeabilization of the liver tissue of rats in the presence of genes encoding luciferase or β‐galactosidase resulted in the strong expression of these genetic markers in rat liver cells. About 30–40% of the rat liver cells electroporated expressed the β‐galactosidase genetic marker 48 h after electroporation. The marker expression was also detected at least 21 days after transfection at about 5% of the level 48 h after electroporation. The results indicate that gene transfer by electroporation in vivo may avoid anatomical constraints and low transfection efficiency.

Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy

Electroporation is a process that causes a transient increase in the permeability of cell membranes. It can be used to increase the intracellular concentration of chemotherapeutic agents in tumor cells (electrochemotherapy; ECT). A clinical study was initiated to determine if this mode of treatment would be effective against certain primary and metastatic cutaneous malignancies. A group of six patients, three with malignant melanoma, two with basal cell carcinoma, and one with metastatic adenocarcinoma, were enrolled in the study. The treatment was administered in a two‐step process.

Detection of Submicroscopic Lymph Node Metastases with Polymerase Chain Reaction in Patients with Malignant Melanoma

BackgroundThe presence or absence of lymph node metastases in patients with malignant melanoma is the most powerful prognostic factor for predicting survival. If regional nodal metastases are found, the 5-year survival for the patient decreases approximately 50%. If the presence or absence of regional nodal metastases will determine which patients receive formal dissections or which patients enter adjuvant trials, then a technique is needed to accurately screen lymph node samples for occult disease. Routine histopathologic examination routinely underestimates the number of patients with metastases. This study was initiated to develop a highly sensitive clinically applicable method to detect micrometastases by examining lymph nodes for the presence of tyrosinase messenger RNA (mRNA). The hypothesis was that if mRNA for tyrosinase is found in the lymph node preparation, that finding is good evidence that metastatic melanoma cells are present. MethodsThe assay is accomplished using the combination of reverse transcription and double-round polymerase chain reaction (RT-PCR). The amplified samples are examined on a 2% agarose gel and tyrosinase cDNA is seen as a 207 base pair fragment. Lymph node preparations from 29 patients who were clinically stage I and II and undergoing elective node dissections were analyzed both by standard pathologic staining and RT-PCR. ResultsEleven of 29 lymph node (38%) samples from 29 patients with intermediate thickness melanoma were pathologically positive. Nineteen of the 29 lymph node preparations (66%) were RT-PCR-positive, and these included all of the pathologically positive samples, so that the false-negative rate was 0. In a spiking experiment, one SK-Mel-28 melanoma cell in a background of one million normal lymphocytes could be detected, thus indicating the sensitivity of this method. In addition, analysis by restriction enzyme mapping showed that the amplified 207-bp PCR product produced is part of the tyrosinase gene sequence.

Treatment of cutaneous and subcutaneous tumors with electrochemotherapy using intralesional bleomycin

Electrochemotherapy (ECT) is performed by locally administering a chemotherapeutic agent in combination with electric pulses. Previous clinical studies have demonstrated the effectiveness of ECT. In these initial trials, the drug was administered intravenously, followed by administration of electric pulses directly to the tumor. This study was initiated to determine whether an intralesional injection of the drug in combination with electric pulses could provide an improved result. A group of 34 patients was studied.

Novel electrode designs for electrochemotherapy

Direct current pulses for electrochemotherapy treatment are typically administered using two parallel plate electrodes that are placed on either side of the tumor. This simple design has produced high response rates (70 to 85%) in animal studies and in clinical trials. However, parallel plate electrodes are not suitable for all situations. This study describes five novel electrode designs and compares their effectiveness to a parallel plate design for treating melanoma tumors in mice. Results for the 2 x 2 needle array design showed 50% increases in doubling time and in complete response rate compared to the standard parallel plate electrode.