Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4 T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.

The contactless high intensity pulsed electromagnetic field (HI-PEMF)-induced increase of cell membrane permeability is similar to conventional electroporation, with the important difference of inducing an electric field non-invasively by exposing a treated tissue to a time-varying magnetic field. Due to the limited number of studies in the field of electroporation induced by HI-PEMF, we designed experiments to explore the feasibility of such a contactless delivery technique for the gene electrotransfer of nucleic acids in tissues in vivo. By using HI-PEMF for gene electrotransfer, we silenced enhanced green fluorescent protein (EGFP) with siRNA molecules against EGFP in B16F10-EGFP tumors. Six days after the transfer, the fluorescent tumor area decreased by up to 39% as determined by fluorescence imaging in vivo. In addition, the silencing of EGFP to the same extent was confirmed at the mRNA and protein level. The results obtained in the in vivo mouse model demonstrate the potential use of HI-PEMF-induced cell permeabilization for gene therapy and DNA vaccination. Further studies are thus warranted to improve the equipment, optimize the protocols for gene transfer and the HI-PEMF parameters, and demonstrate the effects of HI-PEMF on a broader range of different normal and tumor tissues.

Background: The Ki-67 proliferative index (PI) is part of the diagnosis of nodal B-cell lymphoma (nBCL), but its determination in cytological samples is not standardized. We aimed to establish an approach for the accurate determination of the Ki-67 PI in cytological slides to differentiate between indolent and aggressive nBCLs. Methods: Patients diagnosed with nBCL by fine-needle aspiration biopsy and subsequent excision biopsy were included. Cell suspensions were prepared from biopsy samples for CD3/Ki-67 double immunocytochemical staining and flow-cytometric verification of lymphoma B-cell counts. The Ki-67 PI was assessed by manual counting and eyeballing in cytology and eyeballing in histology. The cut-off values for the differentiation between aggressive and indolent lymphomas were determined for each method. Results: A strong correlation between manual and flow-cytometric counting of lymphoma B cells was confirmed (interclass correlation coefficient (IC coef.) = 0.78). The correlation of the Ki-67 PI determined in cytological and histological slides was also strong (IC coef. > 0.80). Histologically, 55 cases were classified as indolent and 31 as aggressive nBCLs. KI-67 PI cut-off values of 28.5%, 27.5%, and 35.5% were established for manual counting and eyeballing in cytology and eyeballing in histology, respectively, with high sensitivity and specificity. Conclusions: The Ki-67 PI, assessed by manual counting and eyeballing in cytological samples, accurately differentiates between indolent and aggressive nBCLs.

Flow cytometry is helpful in differentiating between B-cell lymphoma (BCL) and reactive lymphocytic proliferation (RLP) in FNA biopsies. However; the presence of inconclusive surface immunoglobulin light chains (sIg LC) poses a problem. We investigated the usefulness of additional tests; namely Bcl-2 expression and expression of cytoplasmic Ig LC (cIg LC), mainly on samples with inconclusive sIg LC. Both tests were performed on 232 FNA samples from lymph nodes. Bcl-2 alone was determined qualitatively and quantitatively on 315 samples. The quantitative test was correctly positive in 76% of cases and falsely negative in 24%. The correctly positive results of the qualitative test were 11% points lower. cIg LC correctly identified 65% of BCL with dual positive sIg LC; 36% of BCL with difficult to interpret sIg LC and only 7% of BCL with negative sIg LC. The best results in differentiating between BCL and RLP were obtained when all three tests were used together. In samples with inconclusive sIg LC and additional monoclonal or polyclonal populations the κ:λ ratios did not differentiate between RLP and BCL. We propose that in case of inconclusive sIg LC Bcl-2 test is used first. The addition of cIg LC test is sensible only in cases with dual positive and difficult to interpret sIg LC.

Flow cytometric (FC) immunophenotyping and T-cell receptor (TCR) gene rearrangement studies are essential ancillary methods for the characterisation of T-cell lymphomas. Traditional manual gating and polymerase chain reaction (PCR)-based analyses can be labour-intensive, operator-dependent, and have limitations in terms of sensitivity and specificity. The objective of our study was to investigate the efficacy of the Phenograph and t-SNE algorithms together with an antibody specific for the TCR β-chain constant region 1 (TRBC1) to identify monoclonal T-cell populations. FC- and PCR-based clonality analyses were performed on 275 samples of T-cell lymphomas, B-cell lymphomas, and reactive lymphocytic proliferations. Monotypic T-cell populations were identified in 65.1% of samples by manual gating and 72.4% by algorithm-driven analysis, while PCR-based analysis detected clonal T cells in 68.0%. Of the 262 monotypic populations identified, 46.6% were classified as T-cell lymphomas and 53.4% as T-cell populations of uncertain significance (T-CUS). Algorithm-driven gating identified monotypic populations that were overlooked by manual gating or PCR-based methods. The study highlights the difficulty in distinguishing monotypic populations as T-cell lymphoma or T-CUS. Further research is needed to establish criteria for distinguishing between these populations and to improve FC diagnostic accuracy.

Interleukin-12 (IL-12) has demonstrated potent antitumor effects by activating natural killer (NK) and T cells, but its systemic administration poses challenges due to toxicity. This study investigated gene electrotransfer (GET) as a localized delivery method for IL-12 plasmids in B16F10 and CT26 tumor cells and its effect on the expression of damage-associated molecular patterns (DAMPs) and several cytokines. We observed the activation of cytosolic sensors and cytokine production, with the expression of IL-6 and TNF-α upregulated only after IL-12 GET, suggesting the involvement of inducible damage-associated molecular patterns (iDAMPs) in the immune response to IL-12 gene therapy. These findings highlight the multifaceted immune activation triggered by GET, offering insights for improving IL-12-based cancer therapies.