Sergiusz Nawrocki

Expression of interleukin‐6, interleukin‐6 receptor, and glycoprotein 130 correlates with good prognoses for patients with breast carcinoma

Interleukin‐6 (IL‐6) is secreted by normal epithelial breast cells but not by oncogene‐transformed cells. Interleukin‐6 is able to inhibit growth of breast carcinoma cells in culture. Interleukin‐6 exerts its activity via two receptor subunits, IL‐6R and glycoprotein 130 (gp130). The expression of these receptor subunits in breast tumors has been studied, but there are no previous reports of their prognostic significance, to the authors knowledge.

Genetically modified tumour vaccines (GMTV) in melanoma clinical trials

Since melanoma is a model immunogenic malignancy incurable in the disseminated phase of its natural course different immunotherapeutic approaches are tested in clinical trials. A number of tumour vaccines genetically modified (GMTV), with various immunostimulatory factors, are tested in phase I/II clinical trials. These factors include cytokines, tumour antigens (TA), costimulatory molecules or HLA antigens. We have designed a novel, mixed auto/allogeneic cellular melanoma vaccine modified with the IL-6 and the sIL-6R genes. Preclinical studies in a mouse model demonstrated that the IL-6/sIL-6R based vaccine is able to elicit efficient anti-tumour responses, mediated by CD8+ and NK cells, which resulted in inhibition of the tumour growth, metastases formation and prolonged survival of the animals treated. Irradiation of vaccine cells does not only lead to their sterilisation but also causes increased secretion of exogenous IL-6 and sIL-6R. Since January 1996 we have vaccinated more than one hundred metastatic melanoma patients. Promising clinical results (22% CR+PR, 32% SD) and the evidence of immune responses in the vaccinated patients have prompted us to design a phase III clinical trial which is to be open in 2000.

Genetically modified tumour vaccines: an obstacle race to break host tolerance to cancer.

The development of genetically modified tumour vaccines (GMTV) has been prompted by a better understanding of antitumour immune responses and genetic engineering technologies, as well as the identification of numerous tumour antigens (TA) in several malignancies which occasionally induce spontaneous tumour regressions. Cellular vaccines are based on autologous or allogeneic tumour cells genetically engineered to secrete different cytokines, co-stimulatory molecules, or allogeneic HLA molecules in order to provide a strong stimulatory signal together with the presented TA. Another promising approach that is targeted towards breaking immune tolerance to TA, exploits dendritic cells (DC) loaded or genetically modified with TA (and sometimes cytokines). Effective nonviral and viral gene delivery systems have been constructed including a third generation of adenoviral, lentiviral and hybrid vectors. Studies in mice demonstrated that therapeutic, curative immune responses might be elicited by GMTV. Promising results from animal studies are rarely seen in human trials. Several reasons, such as numerous escape mechanisms of slowly evolving spontaneous tumours and immune incompetence of advanced patients, are major concerns. Improved monitoring of immune responses to GMTV is essential to distinguish between responders and non-responders in order to tailor immune therapy strategy to the individual patient.

Clinical trials of active cancer immunotherapy

Active immunotherapy of cancer needs its own clinical trials methodology. The standard methodology paradigm for clinical trials in oncology was developed for cytotoxic drugs, which differ dramatically from cancer vaccines in their mode of action and toxicity profile. To minimize the risk of overlooking benefits for patients, Mackiewicz and Nawrocki invite to open discussion on vaccine trials methodology. Our point of view is based on several Phase I and II trials with hundreds of melanoma patients treated with allogenic cellular vaccine genetically modified with cytokine genes. We feel that a simplified two-stage clinical trial design without a separate Phase I is justified. In the first stage, preliminary efficacy together with proof-of-principle and feasibility issues could be addressed. For real efficacy assessment, careful consideration of end points is necessary. Immunologic responses and objective clinical responses are not the best measures of vaccine efficacy for many patients who benefit from treatment. Randomized single institution studies with time-to-event end points are probably well suited for such combined Phase I/II studies. In the second stage trial (Phase III), the final efficacy analysis with comparator arm is needed.

Humoral responses to melanoma vaccine, genetically modified with interleukin 6 and soluble interleukin 6 receptor.

Production of antibodies directed against the tumour antigens is well recognised in cancer patients. However, role of antibodies in antitumour immune responses and their clinical significance is not clear, especially when specific immune therapies aim boosting of cellular responses to tumour cells1-3. Moreover, antagonisms between cellular and humoral responses were observed mainly in animal models4-5. Recently, so called immune enhancement, in human colon cancer patients was also demonstrated6in which depletion of B lymphocytes correlated with tumour regression. On the other hand, several reports suggested that humoral responses are associated with positive clinical outcomes.

Immunogene Therapy of Human Melanoma

IL-6 is a multifunctional cytokine that exerts its activity via a receptor complex (IL-6R) composed of two subunits, an IL-6-binding α chain (gp80) and a signal transducing β chain (gp130) (1). The soluble form of the α subunit (sIL-6R) acts agonistically with IL-6 to enhance its activity both in vitro and in vivo (2,3). Moreover, the IL-6/sIL-6R complex displays a broader range of biological activities than IL-6 alone since it is capable of activating cells which possess gp130 but lack gp80 (2). In our pre-clinical studies, introduction of the IL-6 and sIL-6R genes into a weakly immunogenic murine melanoma cell line (B-78-H1) inhibited tumor growth potential and ability to metastasize concomitant with the stimulation of a potent, specific and long-lasting anti-melanoma immune response (3,4). The IL-6/sIL-6R complex was significantly better at inducing anti-melanoma activity than IL-6 alone. Analysis of tumor infiltrates demonstrated that IL-6/sIL-6R-secreting melanoma cells attracted CD8+ T cells and NK cells but not CD4+ T cells or dendritic cells (unpublished results).

Flow cytometric cytotoxicity assay with GFP gene modified target cells

The cytotoxicity assays have been one of the essential tools in studies of effector functions of the immune system. In order to avoid use of radioactive materials many groups have developed alternative ways to quantitate cell mediated lysis. These methods include release of target cell cytoplasmic enzymes (1), dye inclusion or vital dye uptake reduction (2-4). In most of the assays target cells have to be labeled prior analysis which albeit easy to perform is unnecessarily time consuming Here we describe the measurement by flow cytometry killing activities of human NK cells against K562 cells stably transduced to express enhanced green fluorescent protein (EGFP). After short incubation with effector cells killed EGFP positive K562 cells can be later easily identified by propidium iodide (PI) staining. Since EGFP is expressed in every target cell it requires no further cell labelling, cell purification, or extra washing steps thus, it is more time efficient than current assay methods.

Molecular profiling of tumours for precision oncology – high hopes versus reality

Treatment based on molecular profiling of tumor is advertised however there are very limited clinical data supporting this approach so far. Only one, relatively small, randomized clinical trial (SHIVA) have not met its primary endpoint - prolongation of PFS. Some other unpublished series were reported during ASCO 2017 and are discussed in this review. There are many issues to be resolved before the tumor profiling will enter the clinical practice with significant benefit for patients, eg. spatial and temporal heterogeneity of tumor cells in individual patient, wide access to targeted therapies, toxicity of combined targeted therapies.

The outcomes of Polish patients with advanced BRAF-positive melanoma treated with vemurafenib in a safety clinical trial.

Aim of the study The BRAF inhibitor vemurafenib has improved progression-free survival and overall survival in patients with BRAFV600-mutation-positive metastatic melanoma. Here we present the results of an open-label safety study with vemurafenib in patients with metastatic melanoma enrolled in Polish oncological centres. Material and methods Patients with untreated or previously treated Stage IIIC/IV BRAFV600 mutation-positive melanoma were treated with oral vemurafenib in an initial dose of 960 mg twice daily. Assessments for safety and efficacy were made every 28 days. For the survival analysis the Kaplan-Meier estimator was used with the log-rank tests for bivariate comparisons. Results In total, 75 Polish patients were enrolled in the safety study across four centres. At data cut-off, 28 patients died (37%), mainly (26) due to disease progression; 33 (44%) patients continued vemurafenib after disease progression. The objective response rate was 46%, including two patients with a complete response and 29 with a partial response. Median progression-free survival was 7.4 months. The one-year overall survival rate was 61.9% (median overall survival was not reached). Seventy-three (97.3%) patients reported adverse events (AEs), and grade 3–5 toxicity was reported in 49.4% (37) patients. The most common AEs were: skin lesions (including rash and photosensitivity), arthralgia, and fatigue. Conclusions The overall safety profile and response rate of vemurafenib were comparable to those reported in previous studies of this drug. Our study confirmed the value of well-established prognostic features for overall survival, such as initial LDH (lactate dehydrogenase) level and AJCC staging.

The 14th European Immunology Meeting - EFIS 2000

The 14th European Immunology Meeting - EFIS 2000, held in Poznan, Poland on 23 - 27 September 2000, was the last major meeting of European immunolgists in the second millennium. This conference was intended to summarise past achievements and to present future prospects in immunology. The philosophy of the scientific program was to fuse fundamental and clinical immunology and give a chance for basic scientists and clinicians to discuss mutual topics in a general view. There were eight state-of-art lectures, 12 ‘meet an expert’ sessions, 20 plenary sessions and 46 workshops. More than 900 works were presented. Significant interest was focused on several aspects of cancer immunology and immunotherapy. EFIS 2000 was accompanied by six pre-congress satellite symposia held in various Polish cities. The topics were, ‘Heat shock proteins: immune, stress response and apoptosis’ (Gdansk), ‘Infectious immunity and vaccines’ (Kazimierz Dolny), ‘Mononuclear phagocytes in basic and clinical immunology’ (Cracow), ‘Immunology of reproduction’ (Poznan), ‘Primary immunodeficiencies’ (Warsaw) and ‘Glycoimmunology’ (Wroclaw).