First Eastern European experience of isolated limb infusion for in-transit metastatic melanoma confined to the limb: Is it still an effective treatment option in the modern era?

BACKGROUND: Isolated limb infusion (ILI) with cytotoxic agents is a simple and effective treatment option for patients with melanoma in-transit metastases (ITMs) confined to an extremity. Data for ILIs performed in Europe are sparse and to date no Eastern European ILI experience has been reported. The aim of the current study was to evaluate the efficacy of ILI in Estonia. PATIENTS AND METHODS: Data for twenty-one patients were collected and analysed. All patients had melanoma ITMs and underwent an ILI between January 2012 and May 2018. The cytotoxic drug combination of melphalan and actinomycin-D was used. Drug circulation times were 20-30 min under mildly hyperthermic conditions (38-39 °C). Primary outcome measures were treatment response and overall survival. RESULTS: Nineteen lower limb and two upper limb ILIs were performed. The female to male ratio was 18:3. The overall response rate (complete + partial response) was 76% (n = 16), with a complete response in 38% (n = 8). The overall long-term limb salvage rate was 90% (n = 19). During follow-up, eight patients (38%) died, two due to metastatic melanoma. Five-year overall survival was 57%. CONCLUSION: This first Eastern European report of ILI for melanoma ITMs shows results comparable to those from other parts of the world. In this era of effective targeted and immune therapies, ILI remains a useful treatment option, with a high overall response rate and durable responses in patients with melanoma ITMs confined to a limb.

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4.

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.

Soft Tissue Cancer Management: Isolated Limb Infusion for Sarcoma.

BACKGROUND: Sarcoma is a heterogeneous group of malignancies comprising almost 80 subtypes of bone and soft tissue cancers. Previously, all subtypes were managed identically. Advancements in biological and genetic studies have revealed that sarcoma subtypes display varying characteristics and therefore require tailored treatments. Locally advanced soft tissue malignancies of both the trunk and the extremities can present significant challenges for treatment. At present, a negative surgical resection margin is the only definitive treatment despite attempts to use neoadjuvant and adjuvant therapies. In patients with locally advanced non-resectable soft tissue sarcoma (STS), the current practice would advocate amputation. However, studies suggest that limb salvage may be possible with radiotherapy or regional chemotherapy using isolated limb perfusion or isolated limb infusion (ILI). An ideal treatment modality for non-resectable STS would strive for preservation of anatomy and functionality as well as improve quality of life. The aim of the study was to investigate the efficacy of isolated limb infusion as an alternative treatment modality for non-resectable locally advanced STS. METHODS: The efficacy of ILI was retrospectively investigated in 10 patients with STS. All patients received ILI with melphalan and actinomycin at the North Estonia Medical Centre Foundation, Tallinn, Estonia from September 1, 2014 to May 31, 2018. The procedures were performed in a lower extremity in 8 patients and in an upper extremity in 2 patients. The 6-month overall response rate was 78% and the overall limb salvage rate was 100%. The distant metastatis-free survival was longer for responders than for non-responders. RESULTS AND CONCLUSIONS: ILI is an alternative treatment modality for regional disease control and limb preservation in patients with cutaneous and soft tissue malignant neoplasms of the extremities. The short-term response rates are encouraging and the median overall survival shows good results in this highly complex patient population.

Porcine circovirus type 2 ORF3 protein induces apoptosis in melanoma cells.

BACKGROUND: The current treatment of malignant melanoma is limited by the lack of effective therapeutic approaches, and alternative treatments are needed. Proliferative diseases such as melanoma and other cancers may be treatable by virally-encoded apoptotic proteins that are targeted to rapidly multiplying cells. Caspase-dependent apoptosis, that is frequently used in chemotherapy, can boost the cell proliferation that caspase-independent cell death does not. METHODS: In the current study, the porcine circovirus type 2 (PCV2), proapoptotic protein ORF3 was expressed in mouse and human cancer cell lines, and its apoptotic activity was assessed. RESULTS: Quantitative assessment of the apoptotic cells by flow cytometry showed that apoptotic cell death was significantly increased in ORF3-expressing malignant cells, compared to ORF3 non-expressing cells. Our data show that PCV2 ORF3 induces apoptosis in a caspase-3 and -8 independent manner. ORF3 expression seems to cause an increase in abnormal mitosis in B16F10 melanoma cells by interacting with centrosomes and thereby disrupting the formation of the mitotic spindle. In addition, we show that ORF3 of PCV2 also exhibits significant anti-tumor effects in vivo. Although the expression of Regulator of G protein Signaling (RGS)-16 by recipient mice inhibited the development of grafted melanoma in vivo, it was not required for the antitumoral activity of ORF3. CONCLUSION: PCV2 ORF3 causes abnormal mitosis in rapidly dividing cells and increases the apoptosis of cancer cells. Apoptin might, therefore, be considered to develop future antitumoral strategies.

Antibody response against cancer-testis antigens MAGEA4 and MAGEA10 in patients with melanoma.

Melanoma-associated antigen A (MAGEA) represent a class of tumor antigens that are expressed in a variety of malignant tumors, however, their expression in healthy normal tissues is restricted to germ cells of testis, fetal ovary and placenta. The restricted expression and immunogenicity of these antigens make them ideal targets for immunotherapy in human cancer. In the present study the presence of naturally occurring antibodies against two MAGEA subfamily proteins, MAGEA4 and MAGEA10, was analyzed in patients with melanoma at different stages of disease. Results indicated that the anti-MAGEA4/MAGEA10 immune response in melanoma patients was heterogeneous, with only ~8% of patients having a strong response. Comparing the number of strongly responding patients between different stages of disease revealed that the highest number of strong responses was detected among stage II melanoma patients. These findings support the model that the immune system is involved in the control of melanoma in the early stages of disease.