1137 Background: Triple-negative breast cancer (TNBC) has limited treatment options and poor outcomes, with ~12% 5-year overall survival in stage IV. In early-stage TNBC (II/III), recurrence remains substantial among patients without pathological complete response after chemo-immunotherapy. Current PD-1/PD-L1 inhibitors show limited efficacy in metastatic TNBC (median OS ~23 months) and infrequent durable responses. Novel immune targets are urgently needed to overcome resistance and prevent metastasis. V-domain Ig suppressor of T-cell activation (VISTA) is a next-generation checkpoint with distinct biology, enriched in tumor-infiltrating immune cells in TNBC. Methods: Comprehensive bioinformatic analyses of VSIR (VISTA) and ligands were performed across multi-cohort TNBC datasets spanning early and advanced stages, including pre-treatment, post-treatment, and progression samples. Spatial single-cell profiling of stage III TNBC tumors with recurrence versus non-recurrence was conducted using NanoString CosMx. VISTA expression in tumor tissue and circulating tumor cells (CTCs) was assessed by optimized immunofluorescence and digital pathology. Clinically relevant TNBC mouse models evaluated VISTA-mediated immune modulation alone and combined with chemo- or immunotherapy. Results: Single-cell bioinformatic analyses revealed enrichment of VSIR and ligands SELPLG/LGALS9 in post-treatment and progression-stage TNBC versus pre-treatment, observed in tumors and liquid biopsies. VSIR/SELPLG were enriched in progenitor and terminally exhausted immune subsets, whereas early-stage TNBC showed broader immune distribution. Digital pathology identified VISTA in metastasis-initiating tumor cells and dysfunctional immune cells, with induction following anti–PD-1 therapy in TNBC mouse models. Spatial profiling of recurrence-associated tumors showed co-enrichment of VSIR/SELPLG with inhibitory receptors TIM3, LAG3, and TIGIT across immune niches in recurrence-associated tumors. VISTA targeting with a novel antibody, HMBD-002, enhanced anti-tumor immunity, synergized with chemo- and immunotherapy, and suppressed recurrence and metastatic burden in preclinical models. Conclusions: VISTA is a treatment-emergent checkpoint associated with T-cell exhaustion, recurrence, and metastasis in TNBC. Its enrichment in exhausted T-cell niches, metastasis-initiating tumor cells, and CTCs post–chemo-immunotherapy supports its role in adaptive resistance. VISTA expression in tissue and liquid biopsy CTCs represents a clinically actionable biomarker for patient selection and response monitoring. These data provide strong rationale for early-phase clinical trials of combination VISTA blockade with ICIs in high-risk early-stage TNBC with residual disease and metastatic TNBC after prior ICI exposure.

Allogeneic virus-specific T cell (VST) therapies offer distinct advantages, including scalability, rapid deployment, and manufacturing consistency, and have demonstrated efficacy in multiple clinical trials. However, identifying VST products with high therapeutic potential remains a major hurdle. Here, we present a multidimensional analytical platform that integrates in vitro and in vivo anti-viral reactivity, T cell receptor (TCR) repertoire analysis, gene expression profiling, immunophenotyping, and functional validation in a humanized mouse model. Epstein-Barr virus (EBV)-specific T cells expanded from HLA-diverse healthy donors consistently enriched for TCRs targeting EBV-encoded antigens. Transcriptomic and high-dimensional flow cytometric analyses revealed a distinct effector-associated signature. Importantly, this integrative approach uncovered correlative biomarkers of T cell potency and effector function, validated in an in vivo model of EBV-driven B cell lymphoma. These findings establish a scalable framework for the characterization of allogeneic T cell products and may inform the development of predictive metrics for in vivo efficacy. Allogeneic T cell therapies could be used in therapeutic applications because of their potential for ‘off-the-shelf’ access and standardised production. Here the authors have developed a multidimensional workflow profiling platform for EBV-specific T cell therapy and show that correlative biomarkers of T cell potency and effector function are associated with therapeutic effectiveness in xenogeneic mouse EBV-LCL models.

Supplementary Figure 4. Nuclear p85β expression in Stage III TNBC patients and paxalisib- treated 4T1 TNBC mice.

Supplementary Figure 2. Dose de-escalation of the PI3K-mTOR inhibitor, paxalisib, overcomes toxicity burden in combination treatments.

upplementary Table S2. The 23 antibody CODEX panel.

Supplementary Results to accompany manuscript

Supplementary Figure 1. PI3K-mTOR blockade inhibits proliferation of TNBC cells

Background: The COVID-19 pandemic has significantly impacted people with cancer. Initial vaccine studies excluded patients with malignancy. Immunocompromised individuals remain vulnerable to SARS-CoV-2, necessitating detailed understanding of vaccine response. The epidemiology of COVID-19 in Australia offered unique opportunities to study cancer populations with minimal community exposure to SARS-CoV-2. Methods: SerOzNET prospectively examined previously unvaccinated patients with solid and haematological malignancies receiving up to five COVID-19 vaccine doses. Antibody response was measured by live virus neutralisation assay (neutralising antibody (NAb); positive titre >= 1:20; study primary endpoint) and commercial assay. T cell response was measured by cytometric bead array; positive defined as interferon gamma (IFN-gamma) >= 10 pg/mL in response to Spike antigen. Patient and physician-reported adverse events were secondary endpoints. Outcomes: 395 adults were enrolled prior to receiving mRNA vaccine (BNT162b2 = 347; mRNA-1273 = 1) or viral vector vaccine (ChadOx1-S = 43) for initial two-dose course, plus up to three additional doses. Median age was 58 years (range: 20-85); 60 % were female; 35 % had haematological malignancy, 2/395 (0.5 %) had baseline positive nucleocapsid antibody indicating prior SARS-CoV-2 exposure. NAb response post dose three was demonstrated in 84 % overall; 96 % of patients with solid cancers and 64 % with haematological cancer (p < 0.001). Risk factors for non-response were haematological cancer and anti B-cell therapies. Some patients with haematological cancer seroconverted for the first time after the fourth or fifth dose. IFN-gamma response was seen in many patients with haematological cancer who lacked NAb response. Serious adverse events were rare. COVID19 infection occurred in 29 % with no deaths. Interpretation: COVID-19 vaccination elicits B and T cell responses in patients with solid and haematological cancers, with an acceptable safety profile. A significant proportion of haematological cancer patients require >3 doses to elicit NAb, with many demonstrating T cell response, which may be an alternative pathway of immune protection.

Supplementary Figure 7. Associations between PIK3R2 gene expression and survival probability amongst TNBC patient cohorts.

Supplementary Figure 6. EZH2 co-localizes with NF-κB to activate pro-oncogenic gene expression in TNBC.

Supplementary Figure 3. NanoString nCounter analysis highlighting distinct gene expression profiles in Paxalisib-treated tumors.

Supplementary Table S1. List of the PI3K/mTOR inhibitors

BACKGROUND:Young people undergoing cancer treatment are at increased risk of severe COVID-19 outcomes. Vaccination is recommended; however, data regarding vaccine response are limited. METHODS:A prospective cohort study was conducted of children and adolescents (aged 5-19 years) with current solid or hematological cancer and life expectancy of at least 1 year, eligible for COVID-19 vaccination. Participants received 2 or 3 doses of BNT162b2. Blood was taken at baseline, after dose 1, and then 1 and 3 months after subsequent doses. Safety outcomes and patient-reported adverse events were collected. The proportion with neutralizing antibody (NAb) response after 2 vaccine doses was the primary outcome. Vaccine response was measured by NAb titer (positive ≥1:20), T-cell response (interferon-γ, positive ≥10 pg/mL), and binding antibody titer. RESULTS:Of 113 patients enrolled, 108 (96%) currently or previously received cytotoxic chemotherapy, and most were on current or recent therapy, with 18 (16%) having completed treatment more than 6 months prior to vaccination. Positive NAb response occurred in 52/79 (66%) with samples available after 2 doses and 33/41 (80%) after 3 doses. Interferon-γ response occurred in 44/64 (59%) after 2 doses and 25/34 (74%) after 3 doses. Adverse events were generally mild to moderate, were transient if serious (fever, mucositis, headache), and did not delay cancer treatment. Fever was reported by 12% after doses 1 and 2 and 15% after dose 3. CONCLUSIONS:Most children with cancer respond to BNT162b2 COVID-19 vaccination despite anticancer treatment. Vaccination should not be deferred until treatment completion. These data may have implications for other childhood vaccinations during cancer treatment.

Almost half of patients with triple-negative breast cancer develop distant metastases, heralding unfavorable outcomes. Here, we provide novel insights into the contribution of the PI3K-mTOR pathway to the triple-negative breast cancer phenotypes that promote growth, migration, metastasis, and therapy resistance. Specifically, we demonstrate that dual targeting of PI3K and mTOR but not PI3K alone inhibits cancer cell proliferation and migration in vitro. Dual PI3K-mTOR inhibition with paxalisib not only promotes a favorable mesenchymal-to-epithelial phenotype but also inhibits signatures associated with metastasis-initiating cells, including the highly aggressive cancer stem cell phenotype, persister cancer cell phenotype (p65, FOXQ1, NRF2, and NNMT), and a cancer drug resistance signature (ABCB5, SNAIL, and ALDH1). In vivo, paxalisib overcomes immunotherapy resistance to reduce primary tumor burden, circulating tumor cells, and direct and indirect indicators of metastasis with a favorable toxicity profile. Gene expression and spatial analyses show that paxalisib profoundly affects the immune microenvironment in tumors, reducing adaptive immune phenotypes associated with immunotherapy resistance (exhausted T cells and regulatory T cells) and protumor innate immune populations such as mast cells. PI3K-mTOR blockade acts upstream of EZH2, impacting both the classic repressive catalytic p85β-EZH2-H27ME3 and active EZH2-NF-κB pathways. Our data suggest that dual targeting of the PI3K-mTOR pathway disrupts both the catalytic and noncatalytic axes of EZH2 to inhibit metastasis and enhance cancer immune visibility, potentially increasing the utility of immunotherapy in resistant individuals.

Supplementary Figure 5. PI3K-mTOR inhibition targets the p85β:H3K27Me3 switch.

Supplementary Table S3. Inhibition of human PI3K isoenzymes in vitro by paxalisib in fluorescence polarization biochemical assays.

Adoptive T-cell immunotherapy holds great promise for the treatment of viral complications in immunocompromised patients resistant to standard anti-viral strategies. We present a retrospective analysis of 78 patients from 19 hospitals across Australia and New Zealand, treated over the last 15 years with "off-the-shelf" allogeneic T cells directed to a combination of Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK polyomavirus (BKV), John Cunningham virus (JCV) and/or adenovirus (AdV) under the Australian Therapeutic Goods Administration's Special Access Scheme. Most patients had severe post-transplant viral complications, including drug-resistant end-organ CMV disease, BKV-associated haemorrhagic cystitis and EBV-driven post-transplant lymphoproliferative disorder. Adoptive immunotherapy is well tolerated with few adverse effects. Importantly, 46/71 (65%) patients show definitive clinical improvement including reduction in viral load, clinical symptoms and complete resolution of end-organ disease. In addition, seven high-risk patients remain disease free. Based on this long-term encouraging clinical experience, we propose that a dedicated nationally funded centre for anti-viral cellular therapies should be considered to provide T cell therapies for critically ill patients for compassionate use. Adoptive T-cell immunotherapy offers promise to patients who are resistant to standard anti-viral strategies. Here the authors describe clinical observations in patients with viral complications treated with adoptive immunotherapy over the last 15 years.

Abstract Invasive Lobular Carcinoma (ILC) is the most common special histological subtype of breast cancer. ILC typically present as Oestrogen and Progesterone Receptor positive cancers, without over-expression of HER2 and are defined by their invasive pattern of growth. Despite clinical and biological differences, including diverse sites of metastasis, ILC are managed in the same way as the more commonly diagnosed, Invasive Carcinomas of no special type. Previously, we derived the LobSig lobular specific gene signature in an attempt to prognosticate within an otherwise homogeneous tumour category. We showed that this set of genes could stratify Grade 2 and Nottingham Prognostic Index moderate tumours into high and low risk groups. Herein, we use a nanoString nCounter custom codeset and immunohistochemistry to validate the LobSig signature. Using a CoxBoost analysis we further refined the geneset to 14 genes of interest which we examined using Immunohistochemistry on a large panel of ILC with clinical follow up data. Four targets showed a significant association with breast cancer specific survival, with high levels correlating with the poorest outcomes. Considering the expression data for these 4 candidates together, we performed a Cox Proportional Hazard Regression resulting in a combined prognostic power of (P=0.00034, HR=8.07 (CI 2.58-25.30)), which has superior prognostic power over variables including tumor size and patient age. ‘LobSig4’ represents a readily implementable and informative biomarker set for prognostication in Invasive Lobular Carcinoma. Citation Format: Lauren Kalinowski, Jamie Kutasovic, Sriganesh Srihari, Yufan Feng, Samir Lal, Kaltin Ferguson, Haarika Chittoory, Anna Sokolova, Malcolm Lim, Priyakshi Kalita De Croft, Sunil Lakhani, Peter Simpson, Amy McCart Reed. LobSig4 is a superior and readily implementable ILC-focussed prognostic biomarker set [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-09.