Aliya®Pulsed Electric Field (PEF) technology is an emerging strategy in the field of cancer treatment, offering a novel approach to ablation therapy that does not rely on thermal mechanisms. By employing a multi-stage experimental setup, including potato tuber, porcine liver, and murine breast cancer models, we explored the dose-response relationship on ablation and immune modulation by varying the pulse packets delivered from 20 to 100. The biologic response observed with 60 packets represented a minimum effective dose yielding reproducible ablation parameters, immune response, and efficacy which could be augmented with immune checkpoint blockade. This pre-clinical analysis provides a first step toward understanding the therapeutic index for PEF technology beyond ablation, a consideration that will require robust clinical validation in well-designed prospective studies.

Background: The AFFINITY trial (NCT05890872) is a prospective, non-randomized, open-label, single-arm study evaluating the safety, immunological impact, and preliminary efficacy of Aliya pulsed electric field ablation in patients with solid tumors. Thirty-one patients were enrolled; thirty received lung lesion ablation prior to continuation on standard-of-care treatment. This manuscript reports six-month local control outcomes and immunological response characteristics. Radiological outcomes were assessed using a modified RECIST 1.1, and immunological impact was evaluated via changes in peripheral blood immunocyte populations and detection of immunoglobulins (Ig) to tumor-associated antigens in serum post-ablation. Methods: Twenty-eight patients underwent radiological assessment of ablated lesions at approximately 1-, 3-, and 6-month post-ablation to evaluate local control. Peripheral blood was collected for immune monitoring using flow cytometry and to detect IgG responses to biopsy-specific and tumor-associated antigens. Results: At 6 months, two cohorts emerged: 12 received ablation only, and 16 received ablation plus systemic and/or focal therapies (radiotherapy or second ablation). In the ablation-only group, imaging showed local control in all ablated lesions (8/12 SD, 4/12 PR), suggesting local efficacy without systemic therapy in those patients. Immunophenotyping showed dynamic changes in circulating immune cells, including T and B cell activation. A subset also exhibited modulation of tumor antigen-specific IgG, indicating a systemic humoral response. Conclusions: This analysis provides preliminary evidence that this form of ablation may promote local tumor control and modulate systemic immune function. These findings support the immunogenic potential of this specialized energy and warrant further investigation. Extended 12-month data for the full cohort will be reported in a future manuscript.

Intoduction:Non-thermal ablation, including irreversible electroporation (IRE) and Aliya®, an advanced biphasic pulsed electric field (aPEF) technology, have emerged as effective tumor ablation approaches, particularly in sensitive anatomical locations. These methods not only ablate tumors but also may stimulate immune responses. Methods:This study compares the immunological impact of biphasic aPEF and IRE in a murine breast cancer model. Equal-sized tumor ablations were performed using both technologies, followed by analysis of cytokine profiles, immune cell populations, tumor growth, and overall survival. Results:aPEF induced a differentiated tumor microenvironment four days post-ablation compared to IRE, with greater intratumoral infiltration of T-cells, B-cells, increased M1 macrophages, and decreased myeloid-derived suppressor cells. Analysis of systemic circulating immunocytes 14 days post-ablation showed elevated levels of B-cells, CD4 and CD8 T-cells (including memory subpopulations) in the aPEF-ablated groups. aPEF also resulted in better control of ablated and contralateral tumor growth, leading to improved median survival. Discussion:This study demonstrates that the specific biphasic aPEF system evaluated here induces a stronger immunostimulatory effect and superior tumor control compared to IRE, supporting the notion that not all non-thermal ablation is equal, and each may be better suited to different objectives. Further clinical investigations into the potential for better clinical outcomes from this specific advanced pulsed electric field technology is warranted.

e20503 Background: Tumor ablation is generally restricted to control focal disease. There have been rare reports of off-target tumor response (abscopal effect), presumably through the upregulation of tumor-targeting adaptive immunity. Aliya Pulsed Electric Field (PEF) ablation has demonstrated this preclinically, with corresponding increases in antigen-specific CD8 T-cells and tumor-specific antibodies. This report evaluates the immunostimulatory effects of Aliya PEF in patients, focusing on immune cell population dynamics and tumor-specific antibody levels. Methods: In AFFINITY (NCT05890872), patients with stage-IV NSCLC or metastatic cancers to the lungs underwent Aliya PEF ablation followed by first-line standard-of-care (SOC) therapy. Tumor biopsies and blood samples were collected at baseline (pre-PEF) and approximately 3-, 10-, 30-, and 90-days post-PEF; only patients with adequate samples were included in the analysis. ELISA quantified tumor-specific IgG antibodies in patient serum at days 10, 30, 90 bound to tumor biopsy lysate proteins or common tumor antigens (NY-ESO-1, MAGE-A3, MAGE-A4 and WT1). Antibody levels were normalized to day 0, with responders defined as having at least one timepoint with a >15% antibody increase relative to baseline. Flow cytometry (FC) analyzed immune cell populations (days 3, 10, 30), and population changes were evaluated via paired-sample t-test. Results: Tumor-specific IgG analysis of the 19 evaluable patients revealed that 58% (11/19) exhibited increased antibody levels post-PEF. Separately, antibodies to common tumor antigens showed increases in antibodies to NY-ESO-1 (n=5), MAGE-A3 (n=5), MAGE-A4 (n=6), and WT1 (n=4). FC analysis of the 24 evaluable patients revealed immunocyte changes at each timepoint (Table 1). Notably, plasma blasts increased in 71% of patients, peaking at day 10, while plasma cells increased in 59% of patients, peaking at day 30. A strong correlation (R= 0.80) was noted between increased tumor-specific antibodies and plasma cell populations. Conclusions: This data suggests Aliya PEF may induce tumor-specific immunostimulation across diverse cancer histologies, with significant shifts in multiple adaptive immune cell populations and subpopulations as well as increased circulating tumor-specific IgG antibodies in most patients. Future analyses will incorporate SOC therapy’s role and whether PEF-induced changes may invoke systemic disease control and augment systemic therapies. Clinical trial information: NCT05890872 . Mean fold-change vs. baseline (p < 0.05) in circulating immune populations following Aliya ablation (approximate days). Population Day 3 Day 10 Day 30 Activated CD4+ 7.25 9.25 3.1 T regs CD4+ 0.6 0.6 Activated CD8+ 3.9 3.01 3.4 Activated Memory B-cells 5.9 5.3 Switched Memory B-cells 4.3 Plasma blast 6.6 6.1 Plasma cell 7.7

Aliya Pulsed Electric Fields (PEF) is an ablation modality that does not rely on thermal processes to kill cells, providing a means to destroy tissue without damaging the extracellular matrix and potentially promoting an immune response. The Aliya PEF system uses an advanced waveform design compared with irreversible electroporation (IRE) that makes it is simpler to deliver and generates less mechanical trauma than IRE waveforms. This study evaluates whether these improvements to PEF technology provide differentially favorable immune response. Female Balb/c mice were orthotopically inoculated in the mammary fat pad with 200,000 cells from the EMT6 murine breast cancer cell line. When the tumors reached 5-7 mm in size (10 days), the mice were randomly assigned to Sham treatment (n=4), IRE (n=6), or Aliya PEF (n=6), where the ablation technologies were titrated to target matched-size ablations for approximately 80% of the tumor volume. Four days later, the mice were euthanized to collect the treated tumors as well as peripheral blood. The tumor samples were lysed to extract total proteins to quantify 32 cytokines by multiplex laser bead technology. Flow cytometry on blood samples was used to enumerate dendritic and natural killer cells, T and B lymphocytes, macrophage (M1 and M2), and myeloid derived suppressor cells (mMDSC). The intratumoral cytokine profiles revealed unique tumor microenvironments, where Aliya PEF-treated tumors had higher concentration of LIX, MIP2, and MIP1A and lower concentrations of IL-13, Il-2, Eotaxin, Il-12, IL-7, RANTES and GM-CSF compared to both Sham and IRE treatment. These cytokines were unchanged between Sham and IRE. Flow cytometric analyses revealed that Aliya PEF-treated tumors four days post-ablation had a higher percentage of T-cells (CD3+), B-cells, dendritic, and natural killer cells compared to Sham and IRE. Moreover, Aliya PEF-treated tumors had fewer immunosuppressive mMDSC and M2 macrophages, while the level of M1 was unchanged between groups. The IRE treatment did not promote any statistical change in the recruitment of these immune populations. The proprietary Aliya PEF ablation modality resulted in important differences in post-ablation intratumoral cytokines that indicate distinct effects in the tumor microenvironment between the ablation technologies. This resulted in increased intratumoral immune cell populations for Aliya PEF relative to Sham or IRE. These data suggest that Aliya PEF generates a uniquely favorable immunostimulatory profile versus IRE in a murine model.

e14543 Background: Pulsed electric field (PEF) tissue destruction has demonstrated meaningful systemic immune responses in mice that are superior to thermal ablation, and synergize effectively with systemic chemotherapy and checkpoint blockade (CPB) immunotherapy. Immune cell populations are increased by PEF, including tumor specific activated CD8 T-cells and plasma B-cells. Antitumor immunity can include the presence of circulating anti-cancer antibodies. However, the presence and role of circulating tumor specific antibodies induced by PEF have not been explored. Serological antibodies are often detected with the ELISA assay, where tumor-associated antigens are coated to a microtiter plate to attract the corresponding serum antibodies. Here, we quantify IgG tumor specific antibodies after PEF using an in-house ELISA. Methods: Mice bearing immune-warm (EMT6) and immune-cold (4T1) orthotopic murine breast tumors were treated with a biphasic PEF dose comparable to Aliya titrated for mouse tumors. To detect tumor specific circulating IgG1 immunoglobulin in serum, an ELISA plate was coated with EMT6 and 4T1 protein lysate, as well as with a known 4T1-expressed tumor-specific antigen (gp70). Serum draws from the treated, untreated, and naïve mice were exposed to the coated wells for 1 hour. Bound IgG were detected by anti-mouse IgG (HRP tagged) antibody added to the wells and incubated 1 hour. A TMB substrate was added and color development was allowed for 15 minutes. The optical density was quantified using a microtiter plate reader. Results: ELISA quantification of serum IgG at multiple timepoints show that 4T1 tumor-specific IgG antibodies were increased 14.3- and 2.6-fold by day 20 in the PEF-treated and untreated group survivors relative to Naïve mice, respectively, (OD=0.86 and 0.15 v. 0.06). Furthermore, IgG antibodies specific to the gp70 antigen of the 4T1 cell line were increased by 44.9- and 2.7-fold in the PEF-treated and untreated groups relative to naïve mice (OD=3.14 and 0.19 v. 0.07). In the EMT6 model, at 3 months post-treatment, only the PEF-ablated group mice survived, and showed 15.5-fold higher antibodies against EMT6 tumor antigens compared to naive mice (OD=0.62 v. 0.04). Conclusions: The specific form of PEF in this study generated tumor-specific IgG long-lived antibodies in mice for immune-warm and immune-cold tumor models, corroborating previous findings of increased murine B-cell and plasma cell populations. This effect has not been well-reported for other ablation technologies. Increases were evident by Day 20 post-PEF and persisted at least 3 months. These murine data suggest the PEF used may invoke potent, long-lived immune responses and anti-tumor immune surveillance, offering a potential approach for in situ tumor vaccination to improve metastatic disease outcomes. Future studies should determine response durability and its role in attaining distant tumor clearance.

Purpose: To compare the immune response and survival after size -matched radiofrequency (RF) ablation and a proprietary form of pulsed electric field (PEF) ablation in murine tumors. Material and Methods: Orthotopically inoculated EMT6 or 4T1 murine tumors received sham, RF ablation, or PEF ablation. 4T1 tumor ablations included subgroups with intraperitoneal checkpoint inhibition immunotherapy (alpha PD-1). Blood was collected for cytokine profiling and flow cytometry. Tumor size was measured and survival was monitored. Tumor samples were processed for histology, immunohistochemistry, flow cytometry, and cytokine profiling. Lungs were collected from 4T1 -bearing mice for hematoxylin and eosin histology to assess metastatic spread and abscopal effect induced by ablation. Results: PEF elicited distinct immunomodulatory effects, with clear differences in serum and tumor cytokine profiles compared with RF ablation, including intratumoral downregulation of vascular endothelial growth factor, hypoxiainducible factor 1 alpha,c-MET, interleukin-10, Ki67, and tumor necrosis factor-alpha (all P < .05). PEF increased innate immune activation, with enhanced recruitment of dendritic cells, M1 macrophages, and natural killer cells coupled with a reduction in M2 macrophages and myeloid -derived suppressor cells (all P < .05). Concurrently, PEF strengthened adaptive immunity compared with RF ablation, characterized by increased antigen -specific T cells and decreased regulatory T cells (all P < .05). PEF stalled tumor growth and increased survival at the end of the study (>= 4x versus RFA). Finally, PEF promoted an abscopal effect of clearing metastases in the lungs, which was stronger in combination with alpha PD-1 than with PEF alone. Conclusions: The proprietary form of PEF used in this study evoked a preferential immunostimulatory profile versus RF ablation thermal ablation in mice, with implications for enhancing the therapeutic effectiveness of checkpoint inhibition immunotherapy for immunotherapy-unresponsive tumors.

Chemo-immunotherapy uses combined systemic therapies for resectable and unresectable tumors. This approach is gaining clinical momentum, but survival increases leave considerable room for improvement. A novel form of Pulsed Electric Field (PEF) ablation combines focal tissue destruction with immune activation in preclinical settings. The PEFs induce lethal cell damage without requiring thermal processes, leaving cellular proteins intact. This affords PEF a favorable safety profile, improved antigenicity, and significant immunostimulatory damage-associated molecular pattern release compared to other focal therapies. Preclinical investigations demonstrate a combinatorial benefit of PEF with immunostimulation. This study evaluates whether this proprietary PEF therapy induces an immunostimulatory effect sufficient to augment systemic neoadjuvant chemotherapy and immunotherapy to reverse metastatic disease in an immune-cold murine tumor model. To determine whether PEF improves a neoadjuvant chemo-immunotherapy standard-of-care, partial PEF ablation was delivered to orthotopically inoculated 4T1 metastatic tumors in addition to combinations of cisplatin chemotherapy and/or αPD-1 immunotherapy, followed by resection. In addition, to determine whether PEF combined with chemo-immunotherapy improves local and metastatic response in unresectable populations, partial PEF ablation was added to chemo-immunotherapy in mice that did not receive resection. Blood cytokines and flow cytometry evaluated immune response. Partial PEF ablation generates an immunostimulatory tumor microenvironment, increases systemic immune cell populations, slows tumor growth, and prolongs survival relative to neoadjuvant systemic therapies-alone. These data suggest the addition of this proprietary PEF locoregional therapy may synergize with systemic standard-of-care paradigms to improve outcomes with potential or demonstrated metastatic disease in both resectable and unresectable patient cohorts.

Introduction: Pulsed Electric Field (PEF) therapy is a novel approach to treat cancer by providing focal tumor ablation while sparing sensitive structures within the tumor micro-environment. Previous studies have demonstrated that PEF induces immunogenic cell death by activating inflammatory signaling pathways and stimulation of antigen presenting cells withing the tumor micro-environment. PEF ablation provides a tumor focused in situ vaccination strategy that combines with checkpoint blockade. We hypothesized that PEF ablation should synergize with chemotherapy and anti-PD1 in a model where checkpoint inhibitors are inactive. Experimental Design: To test this hypothesis, we utilized a highly aggressive syngeneic orthotopic triple negative breast cancer 4T1 mouse model that is non-responsive to anti-PD1 and only marginally responsive to cisplatin to determine if this combination treatment could prolong survival. Thirty-two Balb/c mice were inoculated in the mammary fat pad with 200,000 4T1 cells. Once tumors were established (5mm in diameter), animals were randomized to the following groups: (A) Sham/IgG control (n=8; needle inserted but no PEF), (B) PEF only (n=8), (C) cisplatin + anti-PD-1 (n=8) and (D) PEF + cisplatin + anti PD-1 (n=8). Tumors in groups B and D were treated once with PEF. Anti-PD1 and isotype matched control IgG was administered once per week (200μg, i.p injections) starting on the day of PEF (Group D) for four weeks. Cisplatin was administered (2mg/kg, i.v) once per week for four weeks starting on the day of PEF (d=0) for four weeks. All the mammary fat pad tumors were resected when they achieved a tumor volume of ~200 mm3 to recapitulate post-resection metastatic relapse (See Schematic 1). Results and Conclusions: All mice in the Sham-control IgG group needed to be euthanized by day 43 due to metastases in multiple organs including lungs, bones, liver and brain. All mice in the cisplatin + anti PD-1 group had to be euthanized by day 74. Interestingly, all groups that were treated with PEF survived much longer. On day 90, 28% of mice in the PEF alone group were alive and 43% were alive in the PEF + anti-PD1 + Cisplatin (see Figure 1). The rank-log p-value for the Kaplan-Meier survival curve was statistically significant. In conclusion, our results demonstrate that PEF treatment in combination with anti-PD1 and Cisplatin significantly prolongs survival in a highly aggressive triple negative breast tumor mouse model that is non-responsive to immunotherapy. These findings support evaluation of PEF in cancer patients that are receiving combination of checkpoint blockade and chemotherapy. Citation Format: Chiara Pastori, Mukta Wagh, Ebtesam Nafie, Fatima Murad, Mohit Trikha, Robert Neal. Combination of pulsed electric field, immunotherapy and cisplatin significantly prolongs survival in an orthotopic breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6392.

Pulsed electric field (PEF) technologies treat many types of tissue. Many systems mandate synchronization to the cardiac cycle to avoid the induction of cardiac arrhythmias. Significant differences between PEF systems make the assessment of cardiac safety from one technology to another challenging. A growing body of evidence suggests that shorter duration biphasic pulses obviate the need for cardiac synchronization, even when delivered in a monopolar fashion. This study theoretically evaluates the risk profile of different PEF parameters. It then tests a monopolar, biphasic, microsecond-scale PEF technology for arrhythmogenic potential. PEF applications of increasing likelihood to induce an arrhythmia were delivered. The energy was delivered throughout the cardiac cycle, including both single and multiple packets, and then with concentrated delivery on the t-wave. There were no sustained changes to the electrocardiogram waveform or to the cardiac rhythm, despite delivering energy during the most vulnerable phase of the cardiac cycle, and delivery of multiple packets of PEF energy across the cardiac cycle. Only isolated premature-atrial contractions (PAC) were observed. This study provides evidence that certain varieties of biphasic, monopolar PEF delivery do not require synchronized energy delivery to prevent harmful arrhythmias.

Patients living with chronic bronchitis (CB) suffer from physical limitations and poor quality of life. In general, treatment options that directly address the mucus hypersecretion component of CB are quite limited. Chronic airway inflammation and the associated hypersecretion and cough that are pathognomonic for CB generally result from long-term exposure to airway irritants such as tobacco use and other environmental insults. This, in turn, results in an increase in the quantity and change in composition of the airway mucosa as a consequence of altered goblet cells, club cells, and submucosal glands. Pulsed electric fields (PEFs) provide a method for eradicating the cellular constituents of tissue with limited impact on the stromal proteins. Preclinical evidence in porcine airways demonstrated that particular PEF waveforms allowed for salutary remodeling of the epithelial and submucosal airway tissue layers and appeared to foster rapid regeneration and recovery of the tissue. Therefore, a therapeutic opportunity might exist whereby the application of a specific form of PEF may result in a reduction of the cellular secretory constituents of the airway while also reducing airway mucosal inflammation. This review discusses the use of such PEF to address the underlying disease processes in CB including challenges around device design, dosing, and appropriate delivery methods. Further, we outline considerations for the transition to human airways along with a brief examination of the initial work treating CB patients, suggesting that the therapy is well tolerated with limited adverse events.

Background Pulsed Electric Field (PEF) ablation kills cells via non-thermal processes, offering improved safety and distinct immunomodulatory effects versus Radiofrequency (RFA) thermal ablation. A proprietary PEF system with an optimized immunostimulatory waveform was modified for murine treatments and previously shown to induce local and systemic anti-tumor immunity in conjunction with aPD-1.1–3 This study compares the immune responses stimulated by this PEF versus RFA, particularly regarding innate and adaptive immune cell populations. Methods PEF or RFA treatments with equivalent ablation volumes were delivered to 4T1 orthotopic mammary murine tumors, followed by tumor resection seven days later (figure 1). Sham mice did not receive energy but underwent resection at the same timepoint. Immune cell populations in the resected tumors, including dendritic cells (DCs), Natural Killer cells, monocytic-derived immune suppressor cells (mMDSCs), neutrophils, M1 macrophages, and M2 macrophages, were assessed using flow cytometry. Flow cytometry was also used to quantify circulating regulatory T-cells and to measure tumor-specific CD8+ T-cell activation using gp70 tetramers in blood samples taken 21 days after treatment. Survival from metastatic burden was monitored. Results PEF-treated mice significantly suppressed tumor growth and increased survival compared to RFA-treated mice despite matched partial ablation volumes (figure 2). Relative to sham controls, PEF ablation induced significant increases in 7-day intratumoral DCs (p=0.009), NK cells (p=0.006), neutrophils (p=0.01), and M1 Macrophages, while decreasing the immunosuppressive mMDSCs (p=0.001) and M2 macrophages (p=0.05) (figure 3). Conversely, RFA did not influence any of these cell populations relative to sham. At 21-days post-treatment, circulating T-cells increased in the PEF group relative to sham and RFA groups (figure 4A). Further, gp70 tetramer assay showed a significant increase in Tet+CD8+ T cells in PEF versus sham and RFA mice (figure 4B). Finally, PEF did not significantly affect regulatory T-cells, while RFA substantially increased this immunosuppressive cell population (figure 4C). Examination of the lungs in mice euthanized for burden confirmed lung metastases, while those surviving to the terminal endpoint confirmed the absence of metastasis, indicating systemic immune anti-cancer protection (figure 5). Conclusions PEF with a proprietary immunostimulatory waveform significantly increased innate and adaptive anti-cancer immune cell populations in both short- and long-term timeframes. In contrast, RFA thermal ablation had minimal impact and even increased certain immunosuppressive cell populations. As a result, PEF treatment enhanced tumor response and improved survival compared to RFA treatment. These findings indicate that the PEF employed in this study may induce a beneficial immunostimulatory profile and better outcomes in cancer patients versus thermal ablation. References C Pastori, M Wagh, E Nafie, F Murad, R Neal. Pulsed Electric Field (PEF) Ablation Invokes Different Immune Cytokine Profile and Tumor Response than Radiofrequency Thermal Ablation for Matched Ablation Volumes in the EMT6 Mouse Model. SIR 2023, Phoenix, AZ. E Nafie, C Pastori, M Wagh, F Murad, M Trikha, R Neal. Pulsed electrical fields in combination with anti-PD1 and survival of mice with TNBC (EMT6) murine breast tumor. Journal of Clinical Oncology 2023;41(16_suppl):540–540 E Nafie, C Pastori, M Wagh, M Trikha, R Neal. Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model. Cancer Research 2023;83(7_Supplement):6638.

BACKGROUND:Effects of contact force (CF) on lesion formation during pulsed field ablation (PFA) have not been well validated. The purpose of this study was to determine the relationship between average CF and lesion size during PFA using a swine-beating heart model. METHODS:A 7F catheter with a 3.5-mm ablation electrode and CF sensor (TactiCath SE, Abbott) was connected to a PFA system (CENTAURI, Galvanize Therapeutics). In 5 closed-chest swine, biphasic PFA current was delivered between the ablation electrode and a skin patch at 40 separate sites in right ventricle (28 Amp) and 55 separate sites in left ventricle (35 Amp) with 4 different levels of CF: (1) low (CF range of 4-13 g; median, 9.5 g); (2) moderate (15-30 g; median, 21.5 g); (3) high (34-55 g; median, 40 g); and (4) no electrode contact, 2 mm away from the endocardium. Swine were sacrificed at 2 hours after ablation, and lesion size was measured using triphenyl tetrazolium chloride staining. In 1 additional swine, COX (cytochrome c oxidase) staining was performed to examine mitochondrial activity to delineate reversible and irreversible lesion boundaries. Histological examination was performed with hematoxylin and eosin and Masson trichrome staining. RESULTS:Ablation lesions were well demarcated with triphenyl tetrazolium chloride staining, showing (1) a dark central zone (contraction band necrosis and hemorrhage); (2) a pale zone (no mitochondrial activity and nuclear pyknosis, indicating apoptosis zone); and a hyperstained zone by triphenyl tetrazolium chloride and COX staining (unaffected normal myocardium with preserved mitochondrial activity, consistent with reversible zone). At constant PFA current intensity, lesion depth increased significantly with increasing CF. There were no detectable lesions resulting from ablation without electrode contact. CONCLUSIONS:Acute PFA ventricular lesions show irreversible and reversible lesion boundaries by triphenyl tetrazolium chloride staining. Electrode-tissue contact is required for effective lesion formation during PFA. At the same PFA dose, lesion depth increases significantly with increasing CF.

Focal tumor ablation may cause secondary immunogenic upregulation that enhances local and distant tumor responses, with profiles that are unique to each modality., Pulsed electric fields (PEFs) use short duration-high voltage electrical pulses to destabilize and kill cells through various biochemical processes. Because it does not rely on thermal effects PEF does not damage interstitial proteins, fostering improved antigen integrity and immune signaling which may lead to lesion resolution. This study compared whether cell death and subsequent interstitial effects caused by PEF and radiofrequency ablation (RFA) result in differentiated primary tumor responses. Thirty female Balb/c mice, divided into sham, PEF, and RFA treatment groups (n = 10/group) were transplanted with EMT6 cells in the left mammary fat pad. Eight days after challenge (d = 0), mice in the PEF and RFA groups received treatment with doses selected to achieve similar subtotal ablation volumes. Three mice from each group were euthanized at day 4 for H&E analysis of ablation zone. The remainder were survived to 10-days post-ablation to monitor tumor growth. Serum was collected on Days -1, +1, +4 and +10 for cytokine analysis. Four-day histology demonstrated comparable subtotal ablation areas between PEF and RFA treatment. While RFA-treated tumors presented immune cells accumulating at the tumor-treatment zone boundary, PEF-treated tumors showed immune cell infiltration throughout the tumor. Further, PEF treatment slowed tumor growth compared with RFA or sham treatments, with day 10 average tumor volumes of 335, 791, and 857 mm3 for PEF, RFA, and sham group, respectively. Ingenuity Pathway Analysis (IPA) of 32 serum cytokines revealed most notable differences between groups on day 4 post-ablation. The PEF-treated group had a significant decrease in the levels of CSF1, CSF2, CSF3, CCL2, IL1β, IL6, IL13, and TNF, and an increase in the levels of IL4. These changes suggest that PEF is associated to 1) decreased accumulation of myeloid derived suppressor cells (MDSCs), 2) decreased the proliferation of tumor-associated macrophages (TAMs), 3) decreased proliferation, survival and invasion of tumor cells. Conversely, these changes were all the opposite for the RFA group. PEF treatment may cause a stronger EMT6 tumor response in mice compared with RFA treatment. These data suggest that PEF may have a greater local and distant tumor response benefit versus RFA thermal ablation. Further studies should explore potential abscopal effects.

Background: Although anti-PD1 therapy is standard of care in multiple tumor types, unmet need remains for those non-responsive to immunotherapy. Pulsed Electric Field (PEF) treatment may alter the tumor micro-environment, converting an immune “cold” tumor into an immune “hot” tumor, triggering an adaptive immune response and abscopal effect. We evaluated PEF in combination with anti-PD1 in a highly aggressive orthotopic triple negative breast tumor mouse model. Methods: Murine mammary 4T1 cells (2 × 105) non-responsive to anti-PD1 therapy were orthotopically implanted in mammary fat of 48 female Balb/C mice. Once tumors were established (5 mm diameter), mice were randomized into six groups: Sham+IgG controls, aPD-1-only, PEF (1X), PEF (2X), aPD-1 + PEF (1X), and aPD-1 + PEF (2X). Groups 4 and 6 received two PEF treatments, five days apart. Anti-PD1 and isotype matched control IgG was administered (200 ug, intraperitoneal) weekly, starting 3 days after PEF treatment. Tumor volumes were recorded 3x/week and standard survival analyses were conducted per IACUC protocol. In the cohort of tumors collected 30 days after PEF, flow cytometry (FC) evaluated T-cell immune infiltrates and associated markers. Blood was collected at different time points to evaluate systemic immunological response. Tumor-specific cytotoxic lymphocytes (CTL) were isolated from tumor-infiltrating and circulating lymphocytes by gp70 MHC tetramer formation assay. Results: Single PEF reduced tumor growth compared to IgG and aPD-1-only groups by 65% and 62%, respectively. Double PEF treatment reduced tumor growth by 74% and prolonged survival. FC demonstrated circulating helper T-cells and CTL frequency was increased two weeks post-PEF compared to the control group. The tetramer detected substantial increases in antigen-specific T-cell populations in both single and double PEF treatment groups. PEF groups had significant tumor and spleen infiltration of CD8 and CD4 T-cells, including increased tetramer levels. Anti-PD1-only animals showed no meaningful reductions in tumor growth, prolonged survival, or significant changes in CD8, CD4 T cells or in the tetramer assay. Conclusions: PEF treatments prior to initiation of anti-PD1 in orthotopic 4T1 mouse model triggers a tumor-specific adaptive immune response in an immune “cold” tumor model, irrespective of anti-PD-1 therapy. PEF increases circulating and tumor-infiltrating T-cells and antigen-specific T-cells, with these effects observed after a single or two treatments with PEF. Collectively, our results demonstrate that PEF treatment prior to initiation of anti-PD1 treatment can reduce tumor growth, prolong survival, and activate an adaptive immune response in an immune “cold” tumor. These findings suggest that PEF treatment merits further evaluation in IO-non-responsive cancer patients. Citation Format: Ebtesam Nafie, Mukta Wagh, Chiara Pastori, Mo Trikha, Robert Neal. Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6638.

BACKGROUND Pulsed electrical field (PEF) ablation may cause tissue heating. These changes are reportedly small, but each PEF system and waveform will have a different behavior, and data are lacking. OBJECTIVES This study sought to compare the temperature profile of focal point, monopolar biphasic PEF ablation versus radiofrequency (RF).METHODS Ablation lesions were performed on perfused thigh muscle of swine. PEF lesions were performed with 3 compatible ablation catheters at the highest (25 amp) energy, and 1 catheter (Tacticath SE) was also used at the 22-and 19-amp levels. Temperature changes in the tissue were measured using fluoroptic temperature probes inserted at the muscle surface, as well as 3 mm and 7 mm below the surface. Temperatures were recorded continuously at baseline, during delivery, and after ablation. Muscle temperatures were compared with those of RF lesions performed with 1 catheter (Tacticath SE) at 30 W for 30 seconds.RESULTS PEF ablation with 3energy settings produced small temperature changes. Maximum average temperature rise for PEF for the maximum (25-amp) energy setting (32 lesions) was 7.6 degrees C, 2.8 degrees C, and 0.9 degrees C at the surface, 3-mm depth, and 7-mm depth, respectively. The temperature rise was dose dependent, with lower energy settings yielding less temperature rise. RF ablations (10 lesions) produced temperature increases of 16.6 degrees C, 39.8 degrees C, and 9.5 degrees C at the surface, 3-mm depth, and 7-mm depth, respectively.CONCLUSIONS PEF caused detectable temperature changes in muscle tissue, which never exceeded 2.8 degrees C at the 3-mm depth versus baseline. By contrast, RF produced substantial temperature rises. These data support that focal monopolar biphasic energy delivered by this PEF technology retains a favorable thermal safety profile.(c) 2023 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

540 Background: Delivery of pulsed electric field (PEF) energy is used to destabilize cells through multiple biochemical processes. We questioned whether a specific PEF treatment type could synergize with anti-PD1 in an orthotopic Triple Negative Breast Cancer (TNBC) EMT6 mouse model. Our previous studies have demonstrated that PEF treatment induces neo-antigen presentation and triggers an innate and adaptive immune response. Anti-PD1 therapy is ineffective at blocking growth of (TNBC) EMT6 tumors that are orthotopically implanted in mice. We evaluated whether a biphasic monopolar form of PEF could synergize with anti-PD1 when it was administered intraperitoneally or intra-tumorally, and whether there was a change in immune response post treatment. Methods: Forty-eight female Balb/c mice were implanted with 200,000 EMT6 cells into the mammary fat pads. Once tumor diameter reached 5 mm they were randomized into 6 groups of 8 animals: 1) Sham/IgG; 2) intra-tumoral (IT) anti-PD-1; 3) intra-peritoneal (IP) anti-PD-1; 4) PEF + anti-PD-1(IP); 5) PEF + anti-PD-1(IT) 6) 2X PEF on day 0 and day 7 + anti-PD-1(IT administered weekly). Sham-PEF mimicked the same procedure but without PEF. Anti-PD1 was administered weekly, IT or IP at 200 µg/mouse and PEF was delivered once on day of randomization. Tumors were measured three times per week, and animal survival was monitored until tumor volume necessitated euthanasia (≥2000mm 3 ). Blood was collected on day 14 for flow cytometric analysis of systemic immune response which included quantifying CD3 positive T cells, B cells and NK cells. Results: Anti-PD1 treatment (IT or IP) did not reduce tumor growth, nor did it prolong survival compared to Sham/IgG treated animals. All anti-PD1 alone (IT or IP) as well as Sham treated animals had to be euthanized by day 35. In contrast, PEF combined with anti-PD1 given IT and IP significantly reduced tumor growth by 83% and 70%, respectively, on Day 14 compared to anti-PD-1 only treated animals (p = 0.03). Interestingly, EMT6 bearing mice treated with double PEF + IT administered anti-PD1 survived longer compared to mice treated with single PEF + IT administered anti-PD1. On day 65, 71% of double PEF + IT administered anti-PD1 were still alive compared to 12.5% of mice treated with PEF + IT administered anti-PD1 (p = 0.002). Flow cytometry analysis on day 14 indicated an increase in circulating adaptive (T- and B-cells) and innate (NK cells) immunocytes in the PEF + anti-PD1 treated animals compared to the control. Conclusions: Collectively, these results indicate that some types of PEF ablation synergize with weekly anti-PD1 in stimulating an increase of CD3+ T cells, B cells and NK cells, reducing tumor growth and prolonging survival of tumor bearing mice. These results suggest the synergistic potential of combining Immunotherapy with some forms of PEF, which warrants further study in patients.

Background Anti-PD1 and systemic chemotherapy, with or without resection, is the standard of care for many tumor types. However, many tumors do not respond to immunotherapy or the combination. Immune 'cold' tumors may become immune 'hot' under the influence of pulsed electric field (PEF) therapy, inducing an adaptive immune response and an abscopal effect. This study determines whether adding PEF to an anti-PD1 and cisplatin regimen improves outcomes in an immune cold tumor model. Methods Twenty female Balb/c mice were inoculated in the mammary fat pad with 200,000 4T1 cells, a highly aggressive syngeneic orthotopic triple negative breast cancer 4T1 mouse model that is non-responsive to anti-PD1 and only marginally responsive to cisplatin for each treatment group. Once tumors were established (5mm diameter), animals were randomized to systemic-only Cisplatin+anti-PD1 (SOC) or PEF+Cisplatin+anti-PD1 (PEF+SOC) treatment groups. Anti-PD1 (200μg, IP) and Cisplatin (2mg/kg, IV) were administered once weekly for eight weeks, starting on the PEF day (figure 1). Primary tumors were measured three times per week, while overall health, metastases, and survival of the mice was monitored until either death by natural causes or until tumor volume necessitated euthanasia (≥2000mm3). Flow cytometry on blood collected at day 14 evaluated the systemic immune response. Results The addition of PEF to the systemic therapy resulted in prolonged survival, with 44.4% of the PEF-inclusive group surviving to Day 55, while all mice in the systemic-only therapy group were euthanized by day 24 due to large tumor size (figure 2A,B). The rank-log p-value for the Kaplan-Meier survival curve was statistically significant, P= 0.0002. Circulating immunocyte analysis at day 14 indicated markedly higher CD3, CD4, and CD8 T cells in the PEF-inclusive group versus the systemic-only therapy group, with increases of 3.2x (figure 3A) and significant increase of central memory t cell (figure 3B,C). CD8 T-cells had increased CD69 MFI 'activated' and reduced PD-1 'exhausted' T-cell markers in the PEF-inclusive group versus systemic-only therapy group (figure 4A,B). Conclusions In a murine model, adding PEF to typical systemic therapy options increases circulating T-cells, with a direct correlation between immunocyte increases and primary tumor response (figure 5A,B). Collectively, our results demonstrate that adding PEF to anti-PD1 and Cisplatin therapy reduces tumor growth, prolongs survival, and activates an adaptive immune response in an immune 'cold' tumor. These results suggest a potential additive benefit to conventional patient care paradigms. Ethics Approval All animal studies were performed in accordance with the protocols and animal care and use guidelines approved by Institutional Animal Care and Use Committee (IACUC) protocol # 2023–05-01.

OBJECTIVES:Pulsed electric field (PEF) therapies employ punctuated energy delivery to kill cells in a volume of tissue through mechanisms that are not dependent on thermal processes. A key component to successful cardiac ablation procedures is ensuring the generation of transmural, contiguous ablation zones, which requires in-depth knowledge regarding treatment sizes for a given therapeutic application.METHODS:In this study, a series of acute treatments were delivered to porcine ventricles, where triphenyl tetrazolium chloride (TTC) vitality stain was used to identify treatment effect sizes for the three focal monopolar CENTAURI PEF cardiac ablation energy settings.RESULTS:Treatment depths were 5.7, 7.2, and 8.2 mm for the 19, 22, and 25 A energy settings, respectively. Gross pathology indicated umbral zones of hemorrhage surrounded by pale avital TTC-negative-negative tissue, which contrasted significantly from radiofrequency ablation (RF) controls. Histologically, treatment zones are identified by regions of contraction band necrosis and cardiomyocytolysis, which contrasted with RF control lesions composed primarily of coagulation necrosis.CONCLUSIONS:Together, these data indicate the ability for focal monopolar PEF treatments to generate deep treatment zones in cardiac ablation without incurring the gross or histological coagulative characteristics of RF thermal lesions.