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.

IntroductionIn addition to its direct cytotoxic effects, ablative therapies as electrochemotherapy (ECT) can elicit indirect antitumor effects by triggering immune system responses. Here, we comprehensively analyzed this dual effectiveness of intratumoral ECT with chemotherapeutic drugs bleomycin (BLM), oxaliplatin (OXA), and cisplatin (CDDP). Our aim was to determine if ECT can act as in situ vaccination and thereby induce an abscopal effect. By evaluating ECT’s potential for in situ vaccination, our goal was to pave the way for future advancements for its combination with emerging (immuno)therapies, leading to enhanced responses and outcomes.MethodsWe employed two mouse tumor models, the immunologically cold B16F10 melanoma and 4T1 mammary carcinoma, to explore both local and systemic (i.e., abscopal) antitumor effects following equieffective intratumoral ECT with BLM, OXA, and CDDP. Through histological analyses and the use of immunodeficient and metastatic (for abscopal effect) mouse models, we identified and compared both the cytotoxic and immunological components of ECT’s antitumor efficiency, such as immunologically recognizable cell deaths (immunogenic cell death and necrosis) and immune infiltrate (CD11+, CD4+, CD8+, GrB+).ResultsDifferences in immunological involvement after equieffective intratumoral ECT were highlighted by variable kinetics of immunologically recognizable cell deaths and immune infiltrate across the studied tumor models. Particularly, the 4T1 tumor model exhibited a more pronounced involvement of the immune component compared to the B16F10 tumor model. Variances in the antitumor (immune) response were also detected based on the chemotherapeutic drug used in ECT. Collectively, ECT demonstrated effectiveness in inducing in situ vaccination in both tumor models; however, an abscopal effect was observed in the 4T1 tumor model only.ConclusionsThis is the first preclinical study systematically comparing the immune involvement in intratumoral ECT’s efficiency using three distinct chemotherapeutic drugs in mouse tumor models. The demonstrated variability in immune response to ECT across different tumor models and chemotherapeutic drugs provides a basis for future investigations aimed at enhancing the effectiveness of combined treatments.

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.

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.

Introduction/Background High-grade serous cancer is often associated with ascites at presentation. Our objective was to quantify immune cells in the ascites before treatment and evaluate their impact on patient survival. Methodology Forty-seven patients with primary disease and ascites were included in the study. Flow cytometry 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. CD103 epithelial marker was further analyzed on T cells, and PD-1 and PD-L1 immune checkpoint molecules were analyzed on all immune cells. Cut-off of low and high percentages of immune cells was determined by the median of the variables, and the correlation with progression-free survival and overall survival was calculated. Results CD3+ T cells were the predominant cells in the ascites (median 51%), while the presence of other immune cells was much lower (median ≤10%). PD-1 was mainly expressed on CD3+ T cells (median 20%), lower expression was observed on macrophages (median 10%), dendritic cells (median <10%), NK cells, and B cells (median <5%). PD-L1 expression was not detected. Progression-free survival and overall survival were significantly better in patients with high percentages of CD103+CD3+ T cells, PD-1+Tregs, CD56brightCD16- NK cells, and dendritic cells. High percentages of CD8+ T cells, macrophages, and PD-1+CD56brightCD16- NK cells, and low percentages of CD4+ also indicated significantly better overall survival. Conclusion Our results highlight the potential of the ascites tumor immune microenvironment to provide novel prognostic markers for patients diagnosed with primary high-grade serous cancer. Disclosures The authors made no disclosures. The study was funded by the research program P3–0289 of the Slovenian Research Agency.

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 progres-sion-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 (CD56(bright)CD16- and CD56(dim)CD16(+) 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 percent-ages 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 (me-dian <= 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, CD56(bright)CD16- NK cells, and DCs correlated with prolonged PFS and OS, while high percentages of CD8(+) cells, macrophages, and PD-1(+)CD56(bright)CD16- 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

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.

The Physics of Cancer, pp. 145-164 (2020) No AccessChapter 7: Computational Modeling and Experimental Investigations to Enhance the Successful Response of Anti-PD-1 Cancer ImmunotherapiesDamijan Valentinuzzi, Katja Uršič, Matea Maruna, Simon Buček, Urban Simončič, Martina Vrankar, Maja Čemažar, Gregor Serša, and Robert JerajDamijan ValentinuzziJožef Stefan Institute, Ljubljana, SloveniaFaculty of Mathematics and Physics, University of Ljubljana, Ljubljana, Slovenia, Katja UršičInstitute of Oncology Ljubljana, Ljubljana, Slovenia, Matea MarunaInstitute of Oncology Ljubljana, Ljubljana, Slovenia, Simon BučekInstitute of Oncology Ljubljana, Ljubljana, Slovenia, Urban SimončičJožef Stefan Institute, Ljubljana, SloveniaFaculty of Mathematics and Physics, University of Ljubljana, Ljubljana, Slovenia, Martina VrankarInstitute of Oncology Ljubljana, Ljubljana, Slovenia, Maja ČemažarInstitute of Oncology Ljubljana, Ljubljana, Slovenia, Gregor SeršaInstitute of Oncology Ljubljana, Ljubljana, Slovenia, and Robert JerajJožef Stefan Institute, Ljubljana, SloveniaFaculty of Mathematics and Physics, University of Ljubljana, Ljubljana, SloveniaDepartment of Medical Physics, University of Wisconsin, Madison, WI, USAhttps://doi.org/10.1142/9789811223495_0007Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Immunotherapies with anti-programmed death-1 antibodies (anti-PD-1) have revolutionized cancer treatment. However, the majority of patients still fail to respond and the reasons remain largely unknown. In order to identify tumor characteristics associated with treatment failure, we developed a computational model capable of simulating tumor response to anti-PD-1 immunotherapy, validated with on-site in vitro and in vivo experiments. The experiments were conducted using non-responsive and responsive murine cell lines, 4T1 mammary carcinoma and CT26 colon carcinoma, respectively. Tumors were induced in athymic nude mice, wild-type mice, and wild-type mice receiving anti-PD-1 immunotherapy to: (1) assess Gompertzian parameters describing intrinsic tumor growth in the absence of T cells, (2) assess the immune-related parameters, and (3) validate model's predictive ability, respectively. Flow-cytometric analyses of major histocompatibility complex (MHC) class I and PD-1 ligand (PD-L1) expression were performed to assess initial heterogeneity in tumor cell subpopulations. Model parameter sensitivity studies were performed to establish importance of different tumor characteristics on the observed tumor response to anti-PD-1 immunotherapy. The model correctly predicted the observed trend of response to anti-PD-1 in 4T1, while the predictions in CT26, where dichotomous responses were observed experimentally, were less accurate. Sensitivity studies of measured model parameters and initial conditions suggested that complete responses to anti-PD-1 immunotherapy might be possible only in the case of homogeneous MHC class I positive tumors. On the other hand, heterogeneous tumors containing MHC class I negative tumor cell subpopulations were associated with resistance to anti-PD-1 therapy. Keywords: Computational modelmathematical modeltumor modelinganti-PD-1immunotherapyMHC class IPD-L14T1 mammary carcinomaCT26 colon carcinomatumor biologyT cells FiguresReferencesRelatedDetails The Physics of CancerMetrics History KeywordsComputational modelmathematical modeltumor modelinganti-PD-1immunotherapyMHC class IPD-L14T1 mammary carcinomaCT26 colon carcinomatumor biologyT cellsPDF download

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.

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.

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.