The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia.

BACKGROUND: Flow cytometry plays is important in the diagnosis of acute lymphoblastic leukaemia (ALL) and when antigen-specific immunotherapy is indicated. We have investigated the effects of prednisolone, vincristine, daunorubicin, asparaginase and methotrexate on the antigen expression on blast cells that could influence the planning of antigen-specific therapy as well as risk-based treatment assignment. PATIENTS AND METHODS: Patients aged ≤ 17 years with de novo B-cell ALL (B-ALL) were enrolled in the study. Blast cells were isolated and exposed in vitro to 5 individual cytotoxic drugs in logarithmically increasing concentrations. Then, the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c and CD137 antigens was determined by quantitative flow cytometry. RESULTS: Cytotoxic drugs caused dose-dependent or dose-independent modulation of antigen expression. Daunorubicin caused a dose-dependent down-modulation of CD10, CD19, CD34, CD45 and CD58 and an up-modulation of CD137. Vincristine caused a dose-dependent down-modulation of CD19 and CD58 and an up-modulation of CD45. Daunorubicin also caused dose-independent down-modulation of CD27 and prednisolone down-modulation of CD10, CD19, CD27, CD34 and CD58. Down-modulation of CD20 was detected only in relation to the specific dose of daunorubicin. CONCLUSIONS: The results of the study have shown that cytotoxic drugs can alter the expression of antigens that are important for immunotherapy. Importantly, daunorubicin, prednisolone and vincristine caused down-modulation of CD19 and CD58, suggesting that these drugs are better avoided during bridging therapy prior to bispecific antibodies or CAR-T cell therapy. In addition, immunophenotypic changes on blast cells induced by different drugs could also influence risk-based treatment assignment.

The prognostic significance of tumor-immune microenvironment in ascites of patients with high-grade serous carcinoma.

BACKGROUND: High-grade serous carcinoma (HGSC) is often associated with ascites at presentation. Our objective was to quantify immune cells (ICs) in ascites prior to any treatment was given and evaluate their impact on progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: Forty-seven patients with primary HGSC and ascites were included. Flow-cytometric analysis was performed to detect percentages of CD3+ T cells (CD4+, CD8+, Tregs, and NKT cells), B cells, NK cells (CD56brightCD16- and CD56dimCD16+ subsets), macrophages and dendritic cells (DCs). Furthermore, CD103 expression was analyzed on T cells and their subsets, while PD-1 and PD-L1 expression on all ICs. Cut-off of low and high percentages of ICs was determined by the median of variables, and correlation with PFS and OS was calculated. RESULTS: CD3+ cells were the predominant ICs (median 51%), while the presence of other ICs was much lower (median ≤10%). CD103+ expression was mostly present on CD8+, and not CD4+ cells. PD-1 was mainly expressed on CD3+ T cells (median 20%), lower expression was observed on other ICs (median ≤10%). PD-L1 expression was not detected. High percentages of CD103+CD3+ T cells, PD-1+ Tregs, CD56brightCD16- NK cells, and DCs correlated with prolonged PFS and OS, while high percentages of CD8+ cells, macrophages, and PD-1+CD56brightCD16- NK cells, along with low percentages of CD4+ cells, correlated with better OS only. DCs were the only independent prognostic marker among all ICs. CONCLUSIONS: Our results highlight the potential of ascites tumor-immune microenvironment to provide additional prognostic information for HGSC patients. However, a larger patient cohort and longer follow-up are needed to confirm our findings.

Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune status.

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.

Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity.

Background Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.

Electrotransfer of siRNA to Silence Enhanced Green Fluorescent Protein in Tumor Mediated by a High Intensity Pulsed Electromagnetic Field.

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.

Usefulness of Bcl-2 Expression and the Expression of Cytoplasmic Immunoglobulin Light Chains in the Differentiation Between B-Cell Lymphoma and Reactive Lymphocytic Proliferations in FNA.

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.

Comparable effectiveness and immunomodulatory actions of oxaliplatin and cisplatin in electrochemotherapy of murine melanoma.

Interest in platinum-based chemotherapeutics such as oxaliplatin (OXA) and cisplatin (CDDP) has been reinvigorated by their newly described impacts on tumor-specific immune responses. In addition to CDDP, OXA is frequently used to treat cancers. Based on the characteristics of OXA, which are similar to those of CDDP, and the presumably more pronounced immunomodulatory effect of OXA, OXA is a candidate for electrochemotherapy (ECT). We compared the effectiveness of intratumoral ECT with OXA to that of ECT with CDDP in murine B16F10 melanoma to determine the equieffective dose. Special attention was given to the elicitation of immunogenic cell death and local immune response. Based on the in vitro and in vivo results pertaining to effectiveness and drug uptake in cells and tumors, ECT with OXA is as effective as ECT with CDDP when the OXA dose is increased 1.6-fold. Exposure of melanoma cells to ECT induces immunogenic cell death when either OXA or CDDP is used, which correlates with a comparable increase in lymphocyte infiltration into tumors after ECT with either OXA or CDDP. Based on these results, OXA is a valid platinum-based drug for use with ECT, and the effectiveness of ECT with OXA is comparable to that of the well-established ECT with CDDP. Furthermore, both drugs display equal and specific immune responses following ECT.