The PI3K/AKT pathway, a key oncogenic signalling pathway, is mutated in 70% of breast cancers (BC). PI3K/AKT coding mutations promote BC progression and are associated with poor survival. However, coding mutations represent <1% of the whole genome. Non-coding mutations (NCMs) can promote oncogenic gene expression in several cancers, including BC. Further study on NCMs can enhance our understanding of BC and help identify novel treatments targets. NCMs surrounding kinome genes were identified from whole genome sequencing of 915 BC tumours. NCMs were prioritised based on recurrence and analysed with the Activity by Contact (ABC) bioinformatic model, which estimates their likelihood to interact with transcription start sites (TSS) and indicates their enhancer potential. Epigenetic data (Hi-C and H3K27ac, ChIP Seq) from BC cell lines (n=12) was screened to identify BC subtype-specific epigenetic changes surrounding the prioritised NCMs. The NCMs’ regulatory effect over gene expression was confirmed by luciferase reporter assays. We identified 3,546 recurrent NCMs surrounding kinome genes in BCs and observed an enrichment of NCMs in gene loci of PI3K/AKT pathway members (p=0.005). We discovered subtype-specific enrichment of NCMs around oncogenes, like AKT2 in HR+ BCs (29/398, 7.2%) or AKT3 for TNBC (21/444, 4.7%). Of the 152 NCMs analysed, 17 (11.2%) reported positive by ABC analysis. 6/17 NCMs were identified in the gene loci of AKT2 or AKT3 (ABC scores 0.023-0.1) and were predicted to interact with genes up to 75kb from the NCM. Epigenetic analysis of ER+ and TNBC cell lines reported peaks of enhancer activity near the AKT2 and AKT3 NCMs, thus supporting the ABC analysis. Luciferase assays confirmed a 1.6-fold increase in luciferase expression for an AKT2 gene loci NCM (AC_A, chr19:41858248) and a 3-fold increase in luciferase expression for an AKT3 gene loci NCM (-_AT, chr1:244065861), whilst we observed a 3-fold decrease for a NCM in AKT2 gene loci (CAG_C, chr19:39686510). We have identified that BCs have an enrichment of NCMs surrounding PI3K/AKT pathway genes. NCMs in the gene loci of AKT2 and 3 were predicted to interact with distal genes and luciferase reporter assays confirmed their regulatory effects in vitro. These results highlight the potential of NCMs to drive BC oncogenesis and potentially act as targets for drug development. Arnau Cuy Saques, Anita White, Kaneez Rabab, Philip Scarry, Francesca Matra, Craig Monger, Adrian Bracken, John Crown, Alexander Eustace. Decoding the noncoding: An insight into the noncoding genome of breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4118.

Carbon dots (CDs) are small-sized, spherical nanoparticles presenting amorphous carbon cores with nanocrystalline regions of a graphitic structure. They show unique properties such as high aqueous solubility, robust chemical inertness, and non-toxicity and can be manufactured at a relatively low cost. They are also well known for outstanding fluorescence tunability and resistance to photobleaching. Together, these properties boost their potential to act as drug delivery systems (DDSs). This work presents a low-cost synthesis of CDs by upcycling spent coffee grounds (SCGs) and transforming them into value-added products. This synthetic route eliminates the use of highly toxic heavy metals, high energy-consuming reactions and long reaction times, which can improve biocompatibility while benefiting the environment. A series of physico-chemical characterisation techniques demonstrated that these SCG-derived CDs are small-sized nanoparticles with tunable fluorescence. In vitro studies with 120 h of incubation of SCG-derived CDs demonstrated their specific antiproliferative effect on the breast cancer CAL-51 cell line, accompanied by increased reactive oxygen species (ROS) production. Importantly, no impact was observed on healthy breast, kidney, and liver cells. Confocal laser scanning microscopy confirmed the intracellular accumulation of SCG-derived CDs. Furthermore, the drug efflux pumps P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP) did not impact CD accumulation in the cancer cells.

Background: NK cells are innate immune cells capable of direct anti-tumour cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC can be mediated by autologous anti-tumour antibodies (aaAbs) produced by the adaptive immune response, or monoclonal antibody (mAb) therapies like trastuzumab that are used to treat HER2+ BC. While the majority of met HER2+ BC pts progress on treatment, higher stromal tumor immune infiltrate has been associated with longer OS. Utilising the “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin, we have identified cohorts of trastuzumab-treated met HER2+ BC pts with OS survival times ranging from 0.3 months (mos) to 223.3 mos. This study examines the tumor and stromal NK cell levels in primary and met samples in the context of OS in this patient population. Methods: Clinico-pathological data was available for formalin-fixed, paraffin-embedded biopsy specimens (n=52 primary, n=28 met biopsies). Immunohistochemical staining was conducted for NK cells using anti-CD56 (Agilent IR62861-2). A DAKO Link 48 Autostainer was utilised for staining. Slide analysis used an Aperio AT2 Digital Slide Scanner (Leica Biosystems), Aperio ImageScope 12.4 software (Leica Biosystems) and QuPath analysis software (University of Edinburgh). Following tumor area annotation, an algorithm identified tumor or stromal NK cells. Data was expressed as number of positively stained cells/mm2 tissue. NK cells were designated as “Present” (≥1 cells/mm2) or “Absent” (0 cells/mm2) in the tumor and stromal compartments. Survival studies utilized the Kaplan Meier method. HR= Hazard ratio, CI=Confidence interval, p,0.05 is significant. Non-parametric statistical tests were utilised for independent (Mann-Whitney test) and paired (Wilcoxon signed rank test) comparisons. Results: NK cell levels were significantly lower in the tumor compartment vs the stromal compartment for both the primary (p<0.001) and met sites (p=0.04). Paired primary and met sample (n=23) NK cell levels were not significantly different within the tumor or stromal compartments. The presence of NK cells displayed a trend towards improved OS in primary tumor (HR 0.57, CI 0.29-1.09, p=0.089)) and stroma (HR 0.50, CI 0.16-1.62, p=0.249) across all pts investigated (n=52). When the pt cohort was divided into long-term responders (>5 year OS) and short-term responders (<5 years OS), the presence of tumor-infiltrating NK cells was significantly associated with better OS in long-term responders (HR 0.21, CI 0.05 – 0.86, p=0.03), but not in short-term responders (HR 0.59, CI 0.05 – 0.28, p=0.19). Conclusions: Our results report a link between OS and tumor NK cell infiltrate in met HER2+ BC pts with long-term responses to trastuzumab. Further expansion of this dataset is warranted. Citation Format: Denis M Collins, Janet McCormack, Laura Ivers, Jose Javier Berenguer-Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex J Eustace, Naomi Walsh, Giuseppe Gullo, Janice Walshe, Aurelie Fabre, John Crown. Tumor/stromal infiltration of Natural Killer (NK) cells and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-05-21.

The EACR-AACR-IACR 2024 Basic and Translational Research Conference, held in Dublin, Ireland, from 27th–29th February, 2024, marked a significant milestone as part of the 60th anniversary celebrations of the Irish Association for Cancer Research (IACR). Organized in collaboration with the European Association for Cancer Research (EACR) and the American Association for Cancer Research (AACR), this prestigious event brought together leading experts in oncology research from around the world. The conference provided a platform for cutting-edge discussions on the latest advancements in immunotherapy, drug combinations, cell-based therapies, liquid biopsies, epigenetics, tumour microenvironment, and novel drug targets. With keynote lectures from esteemed researchers such as Kevan Shokat, Jerome Galon, Suzanne Topalian, and Scott Lowe, the conference facilitated knowledge exchange and fostered international collaboration in the pursuit of improved cancer treatments. The report highlights the key sessions, research breakthroughs, and discussions that shaped this landmark event.

Responses to neoadjuvant chemoradiotherapy for locally advanced rectal cancer are not uniform. The phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways are involved in tumorigenesis and treatment resistance in many cancers; therefore, targeting these pathways could enhance response to chemoradiotherapy.A panel of colorectal cancer (CRC) cell lines (n = 10) with varying PI3K and MAPK mutational backgrounds were treated with combinations of 5-Flourouracil (5-FU), radiation, the PI3K inhibitor copanlisib, and/or the MEK inhibitor refametinib, and their effects on proliferation in vitro were measured. BALB/c SCID mice were implanted with CRC cell lines representative of each mutational background, treated with copanlisib and/or chemoradiotherapy, and monitored for tumor growth.In vitro, PIK3CA mutated cell lines were most sensitive to copanlisib (IC50=28 nM) and KRAS mutated cell lines were most sensitive to refametinib (IC50 = 36 nM), while the combination of copanlisib and refametinib was synergistic in 9/10 cell lines tested. The addition of copanlisib to 5-FU chemoradiotherapy inhibited cell growth compared to 5-FU chemoradiotherapy alone, an effect that was most notable in LS-1034 (KRAS mutated) and Caco-2 (PIK3CA/KRAS wild-type) cell lines. In vivo copanlisib and 5-FU chemoradiotherapy reduced tumor growth in all xenograft models and increased overall survival in LS-1034 and Caco-2 xenografts.Our results suggest that activation of the kinase signalling pathway may modulate PI3K/MEK inhibitor responsiveness in colorectal cancer. Furthermore, the addition of copanlisib to 5-FU chemoradiotherapy resulted in an enhanced anti-proliferative cytotoxic effect compared to 5-FU chemoradiotherapy alone, regardless of the background mutational status, and supports further clinical development of this regimen.

Breast cancer's global prevalence underscores a critical need for novel biomarkers to guide treatment and improve patient outcomes. Biomarker discovery historically focused on mutations in protein coding regions, comprising merely 1% of the genome. However, with advances in whole‐genome sequencing, the functional significance of the noncoding genome—comprising the remaining 99%—has become increasingly evident. Noncoding regions play a vital role in regulating gene expression, and mutations within these regions have been associated with cancer risk, progression, and treatment response. This Review compiles and synthesizes current knowledge on cis‐regulatory alterations (promoters/enhancers) and long noncoding RNAs (lncRNAs) in breast cancer. Key examples include promoter mutations [e.g., rs2279744 (Mouse double minute 2 homolog gene; MDM2 )], enhancer mutations [e.g., rs4784227 (thymocyte selection‐associated high mobility group box family member 3 gene; TOX3 )], and lncRNAs [e.g., HOX transcript antisense intergenic RNA ( HOTAIR )] linked to progression, metastasis, and poor survival. Integrating preclinical ( in vitro , in vivo ) and clinical findings, we emphasize the biomarker and therapeutic potential of these noncoding alterations. This Review also critically identifies the pressing need for more specific functional validation studies to fully elucidate their mechanistic roles. This emerging field offers promising opportunities to advance personalized medicine and refine prognostic/predictive strategies for breast cancer patients.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen, progesterone, and HER2 receptors, and is associated with poor prognosis and limited targeted therapeutic options. TP53 mutations occur in the majority of TNBC cases, disrupting p53’s role in DNA repair and apoptosis. Beyond gene regulation, p53 also influences calcium signalling through store-operated calcium entry (SOCE), a critical pathway for cell survival and death. However, the impact of different TP53 mutation types on calcium signalling remains unclear. Methods: Calcium channel gene expression was analysed using publicly available TNBC datasets. Calcium channel expression and SOCE activity were assessed in TNBC cell lines with different TP53 mutations using quantitative PCR and calcium imaging (Fura-2AM). Cell proliferation was measured using acid phosphatase assays, while apoptosis was evaluated through caspase 3/7 activation using the Incucyte live-cell fluorescent imager. The p53 reactivator COTI-2 was tested for its ability to restore TP53 function and modulate calcium signalling. Results: Analysis revealed significant downregulation of CACNA1D in TP53-mutant TNBCs. TNBC cell lines harbouring frameshift and stop TP53 mutations exhibited reduced SOCE, lower CACNA1D expression, and resistance to thapsigargin-induced apoptosis compared to wild-type cells. In contrast, cells with the TP53 R273H missense mutation demonstrated similar calcium signalling and proliferation to TP53 wild-type cels. COTI-2 treatment restored CACNA1D expression and SOCE in frameshift and stop mutant cells, enhancing apoptotic sensitivity. Combined treatment with COTI-2 and thapsigargin resulted in a synergistic increase in apoptosis. Conclusions: This study identifies a novel link between TP53 mutation type and calcium signalling in TNBC. Reactivating mutant p53 with COTI-2 restores calcium-mediated apoptosis, supporting combination strategies targeting both TP53 dysfunction and calcium signalling.

Background: The docetaxel (T), carboplatin (C) and trastuzumab (H) regimen has been used in the (neo-) adjuvant treatment of HER2+ early stage breast cancer (ESBC). Lapatinib (L) a small molecule HER2 antagonist produces clinical responses following H failure. Methods: We randomly assigned 88 patients with stages Ic–III HER2+ESBC to receive neoadjuvant TCH, TCL or TCHL followed by surgery and 1 year of H. The primary endpoint was pathological complete response (pCR). Secondary objectives were overall and disease-free survival (OS, DFS). Results: The TCL arm was closed following demonstration of inferiority of L in another trial. The pCR rates for TCH and TCHL were 52.8 and 51.6 (p = 1.0). At a median 4.8 years follow-up, TCHL patients had a significantly superior DFS; however, OS was similar. Prophylactic loperamide reduced the frequency of diarrhoea. Serum biomarker analysis identified a link between high tumour T-cell levels and high red blood cell, haematocrit, and haemoglobin following commencement of therapy. Interpretation: The study did not meet its primary endpoint of superior pCR. TCHL produced a significant improvement in DFS. Our study and others suggest a possible role for L in neoadjuvant therapy of HER2+ ESBC. Clinical Trial Registration: NCT01485926.

Advancements in oncology, especially with the era of precision oncology, is resulting in a paradigm shift in cancer care. Indeed, innovative technologies, such as artificial intelligence, are paving the way towards enhanced diagnosis, prevention, and personalised treatments as well as novel drug discoveries. Despite excellent progress, the emergence of resistant cancers has curtailed both the pace and extent to which we can advance. By combining both their understanding of the fundamental biological mechanisms and technological advancements such as artificial intelligence and data science, cancer researchers are now beginning to address this. Together, this will revolutionise cancer care, by enhancing molecular interventions that may aid cancer prevention, inform clinical decision making, and accelerate the development of novel therapeutic drugs. Here, we will discuss the advances and approaches in both artificial intelligence and precision oncology, presented at the 59th Irish Association for Cancer Research annual conference.

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin contains a cohort of met HER2+ BC pts treated with trastuzumab, with OS ranging from short term to durable complete response (never relapsed). While the majority of pts progress on treatment, higher stromal tumor immune infiltrate has been associated with longer OS in met HER2+ BC. Using the SVUH database, we have identified a cohort of met HER2+ BC pts that received trastuzumab and have a broad OS range (0.3 months (mos) to 200.9 mos). This study examines the tumor and stromal levels of pan T cell marker CD3, cytotoxic T cell marker CD8, and immune checkpoints PD-L1 and PD-1 by immunohistochemistry (IHC) in primary and met samples from this cohort. Methods: Clinico-pathological data was available for formalin-fixed, paraffin-embedded biopsy specimens (n=45 primary, n=25 matched met biopsies). IHC staining was conducted for CD3 (Agilent IR50361-2), CD8 (Agilent IR62361-2), PD-L1, (Agilent M365329-2), and PD-1 (Roche 07099029001). A DAKO Link 48 Autostainer was utilised for staining, incorporating positive (tonsil tissue) and negative controls (isotype controls). Slide processing used an Aperio AT2 Digital Slide Scanner (Leica Biosystems), Aperio ImageScope 12.4 software (Leica Biosystems) and QuPath analysis software (University of Edinburgh). Following annotation of tumor areas, an algorithm was trained to identify immune cells and designate them as tumor or stromal. Data was expressed as the number of positively stained cells/mm2 tissue. A median cut-off was applied to denote “high” and “low” expression for CD3 and CD8. For PD-1 and PD-L1, samples with ≥ 1 stained cell/mm2 were designated as positive (pos) and samples with zero stained cells as negative (neg). The Kaplan Meier method was utilised for survival studies. A paired Student’s t test was utilised for primary vs metastatic site comparisons. Results: 51.1% of primary samples displayed high CD3 (23/45) and CD8 (23/45) expression in both the stromal and tumor compartments. Within the tumor compartment, 18.6% (8/43) of samples were pos for PD-L1 expression, and 57.8% (26/45) were pos for PD-1 expression. In the stromal compartment, 30.2% (13/43) of samples were pos for PD-L1 expression, and 68.9% (31/45) were pos for PD-1 expression. High tumor levels of CD3 (median OS CD3 high 113.4 mos vs CD3 low 7.9 mos, HR 0.2938 (95% CI 0.130-0.618), p=0.0012) and CD8 (median OS CD8 high 73.9 mos vs CD8 low 10.8 mos, HR 0.4764 (95% CI 0.238-0.954), p=0.0365), but not stromal levels of these markers, were associated with improved OS. There were no significant differences in CD3, CD8, PD-1 and PD-L1 levels between matched primary and met samples (p >0.05). Tumor, but not stromal, PD-L1 expression was associated with longer OS (HR 0.3120 (95% CI 0.146-0.667), p=0.0027). Interestingly, stromal PD-1 expression, but not tumor PD-1 expression, was also associated with longer OS (median OS PD-1 pos 64.7 mos vs. PD-1 neg 5.6 mos, HR 0.158 (95% CI 0.060-0.411), p=0.0002). Conclusion: Our results report a link between OS and tumor (CD3+, CD8+ and PD-L1+)/stromal (PD-1+) immune cell infiltrate in met HER2+ BC patients treated with trastuzumab. Further expansion of this preliminary dataset is warranted. Citation Format: Denis Collins, Janet McCormack, Laura Ivers, Jose Javier Berenguer-Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC) [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 PO5-06-06.

Cancer, responsible for approximately 10 million lives annually, urgently requires innovative treatments, as well as solutions to mitigate the limitations of traditional chemotherapy, such as long-term adverse side effects and multidrug resistance. This review focuses on Carbon Dots (CDs), an emergent class of nanoparticles (NPs) with remarkable physicochemical and biological properties, and their burgeoning applications in bioimaging and as nanocarriers in drug delivery systems for cancer treatment. The review initiates with an overview of NPs as nanocarriers, followed by an in-depth look into the biological barriers that could affect their distribution, from barriers to administration, to intracellular trafficking. It further explores CDs ' synthesis, including both bottom-up and top-down approaches, and their notable biocompatibility, supported by a selection of in vitro, in vivo, and ex vivo studies. Special attention is given to CDs ' role in bioimaging, highlighting their optical properties. The discussion extends to their emerging significance as drug carriers, particularly in the delivery of doxorubicin and other anticancer agents, underscoring recent advancements and challenges in this field. Finally, we showcase examples of other promising bioapplications of CDs, emergent owing to the NPs flexible design. As research on CDs evolves, we envisage key challenges, as well as the potential of CD-based systems in bioimaging and cancer therapy. This review explores Carbon Dots (CDs) in cancer treatment, highlighting their synthesis, bioimaging applications, and role in drug delivery. The review details potential barriers to drug delivery, and details recent studies and developments, with a focus on the use of CDs as nanocarriers in drug delivery systems designed for the delivery of doxorubicin.+image

Background: Immune checkpoint inhibitors (ICIs) have significantly improved outcomes in various cancers. ICI treatment is associated with the incidence of immune-related adverse events (irAEs) which can affect any organ. Data on irAEs occurrence in relation to sex- differentiation and their association with gender-specific factors are limited. Aims: The primary objective of the G-DEFINER study is to compare the irAEs incidence in female and male patients who undergo ICI treatment. Secondary objectives are: to compare the irAEs incidence in pre- and postmenopausal female patients; to compare the irAEs incidence in female and male patients according to different clinical and gender-related factors (lifestyle, psychosocial, and behavioral factors). Exploratory objectives of the study are to compare and contrast hormonal, gene-expression, SNPs, cytokines, and gut microbiota profiles in relation to irAEs incidence in female and male patients. Methods and Results: The patients are recruited from Fondazione IRCCS Istituto Nazionale dei Tumori, Italy, St Vincent’s University Hospital, Ireland, Oslo University Hospital, Norway, and Karolinska Insitutet/Karolinska University Hospital, Sweden. The inclusion of patients was delayed due to the Covid pandemic, leading to a total of 250 patients recruited versus a planned number of 400 patients. Clinical and translational data will be analyzed. Interpretation: The expected outcomes are to improve the management of cancer patients treated with ICIs, leading to more personalized clinical approaches that consider potential toxicity profiles. The real world nature of the trial makes it highly applicable for timely irAEs diagnosis.

Abstract PIK3CA mutations occur in ~15% of triple-negative breast cancers (TNBCs) and aberrantly activate the PI3K-AKT pathway, resulting in uncontrolled cell proliferation. Alpelisib is approved for hormone receptor-positive (HR+), HER2-negative BC patients with a PIK3CA mutation; however, not all PIK3CA-mutated (MUT), HR+ patients respond to alpelisib and others acquire resistance following exposure, suggesting adaptive mechanisms of PI3K activation. It has been proven that in TNBC, there exists a cohort of mutated genes which activate PI3K-AKT signaling. We hypothesize that testing alpelisib in combination with repurposed approved drugs, targeting alternative mechanisms of PI3K-AKT pathway activation, may be used to treat a cohort of TNBC patients. TNBC patient genetic data from the MSK (N=168), METABRIC (N=209), and TCGA (N=171) datasets were analyzed using cBioPortal to identify frequently occurring mutations. Differential expression analyses based on mutational status were performed on CCLE cell line (N=705) microarray and TCGA BC patient (N=981) RNA-sequencing data using R. Dysregulated genes were functionally analyzed using DAVID Ontology and those associated with poorer survival in TNBC were identified using KM-Plotter. STRING database was used to investigate protein interactions. The antiproliferative effects of targeted drugs tested alone and in combination in a panel of 6 TNBC cell lines were determined using acid phosphatase assays. Reverse-phase protein array analysis was carried out on protein lysates from cells following 24 hour treatment with drugs used alone and in combination.TP53 is the most frequently mutated gene in TNBC and mutations were associated with significant genetic dysregulation. A comprehensive analysis of upregulated genes in TP53-MUT TNBC led to the identification of 5 potential therapeutic targets - MET, S100A9, LCN2, CA9, TACSTD2, and KLK5 - based on their association with poorer survival in TNBC, protein interactions with TP53 and PIK3CA, and availability of targeted drugs. MET was selected for in vitro analysis based on its strong correlation with PI3K-AKT signaling. The presence of a TP53 mutation was associated with decreased antiproliferative effects in TNBC cell lines treated with alpelisib. Treatment with alpelisib in combination with a MET inhibitor, crizotinib, demonstrated greater antiproliferative effects in TP53-MUT/PIK3CA-MUT cell lines when compared to TP53 or PIK3CA MUT only models.TP53 mutations are associated with genetic dysregulation in TNBC and may confer resistance to alpelisib. Targeting upregulated proteins in a cohort of TP53-MUT/PIK3CA-MUT patients may improve sensitivity to alpelisib via dual inhibition of PI3K-AKT pathway activation. Protein analysis will uncover the mechanisms of synergy observed between alpelisib and crizotinib, and the therapeutic potential of the combination will be experimentally investigated in vivo. Citation Format: Grace Colley, Alexander Eustace, Dalal AlSultan. Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2269.

Introduction: Computational approaches have aided in estimating cellular composition of the tumour microenvironment. The evaluation of immune composition in tumours before treatment may predict pathologic complete response (pCR). The aim of the study was to perform a meta-analysis of HER2-positive breast cancer subjects who received neoadjuvant trastuzumab to detect associations between immune cells measured by CIBERSORT and ESTIMATE and pCR. Methods: PubMed was used to identify transcriptomic data of HER2-positive breast cancer patients who received neoadjuvant trastuzumab. Baseline data from eight neoadjuvant studies (N=338) was downloaded from GEO. Data from each study was background corrected and quantile normalised using ‘limma’ or ‘oligo’ packages in R. Immune profiles per sample was generated using computational softwares CIBERSORT and ESTIMATE, and were then linked to pCR status. Correlations between immune contexture and pCR for each study were interpreted using statistical testing. Meta-analysis by a logistic regression model was conducted on studies which passed assumptions to identify CIBERSORT immune subsets robust to pCR. Results: CIBERSORT results showed that three studies had reduced T follicular helper cells (Tfh) (Brodsky p=0.38, CHER-LOB p=0.17, TransNOAH p=0.25) and two studies had reduced plasma cells (CHER-LOB p=0.15, Brodsky p=0.38) in the pCR group, but was not significant after multiple correction. ESTIMATE analysis showed that data from two studies had elevated immune infiltration in pCR (Brodsky p=0.19, CHER-LOB p=0.10) but was not significant. A meta-analysis of pooled data from four studies (TRIO-US B07, 03-311, TransNOAH, CHER-LOB) showed that low Tfh (p=0.053, OR=0.04, CI [0.0012-0.99]) and high memory B-cells (p=0.008, OR=2126.9, CI [8.12-7.65 × 10+5]) prior to trastuzumab treatment may be associated with a better chance of achieving pCR. Conclusion: Results from our meta-analysis proposed that memory B- and T follicular helper subsets may predict a role in achieving pCR. Incorporating studies with larger sample cohorts such as the CALGB-40601 (N=265) study can achieve statistical power of this analysis. Citation Format: Dalal AlSultan, Alex J. Eustace, Stephen F. Madden, John Crown. In-silico approaches that detect immune contexture to trastuzumab response in neo-adjuvant studies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-10.

Flow cytometry MFI and % positive cells data for fluorescently labelled trastuzumab and pertuzumab in SKBR3, HCC1954, T47D and MCF-7 treated with TKIs

Background: Anti-HER2 therapies such as trastuzumab used for the treatment of patients with HER2+ metastatic breast cancer (MBC) have led to significant improvements to disease progression. We previously identified cases from the “Thousand Patient HER2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin, of HER2+ MBC long-term durable complete responders to trastuzumab, and reported that Copy Number Aberration (CNA) burden may represent a novel prognostic predictor to trastuzumab response from the exome analysis of in HER2+ MBC “exceptional responders’’ (ExRs). However, whole-genome sequencing (WGS) allows a better understanding of how CNA affects the MBC genome and to-date, the complete genome of this “exceptional” cohort has never been described. Methods: We performed WGS analysis to characterise the CNA profiles of 9 ExRs from our HER2+ MBC cohort treated with trastuzumab. Samples were obtained from patients who never progressed/relapsed for more than 5 years (OS > 60 months). DNA was sequenced from tumours (primary or metastases) and matching control (blood or normal tissue) at a mean depth of 60X and 30X, respectively (18 samples). Somatic single nucleotide variants (SNV) were detected using GATK4 Mutect2 and CNA were identified using Control-FREEC. Results: Eighty-five HER2+ MBC were identified with OS > 60 months, of which 28 were ExRs with bone, lung, liver and lymph metastasis who responded exceptionally to trastuzumab, with a mean OS of 108 months (range 61-236 months). This cohort includes patients who were diagnosed between 31 and 80 years old (median=51). WGS analysis revealed CNA in chr6p21 with amplification of CCND3 and in chr17q12 with amplification of RAD51D. SNV were identified in genes involved in the DNA damage repair (DDR) pathway such as ATM, BRCA2, RAD50 and FANCA. On-going analysis will allow the CNA profiles of all ExRs to be presented and their CNA burden calculated in order to investigate the relationship between whole genome CNA burden and HER2+ MBC patient survival. Conclusion: To our knowledge, this is the first study to sequence the whole genome of HER2+ MBC, never relapse exceptional responders. The identification of the genomic aberrations of these metastatic patients increases our understanding of the mechanisms involved in MBC progression. CNA burden may represent a novel prognostic predictor to trastuzumab response and new outcomes for patients, particularly as MBC is generally termed incurable. Citation Format: Charlotte Andrieu, Laura P. Ivers, Jose Javier Berenguer Pina, Darko Skrobo, Jo Ballot, Alex J. Eustace, Cecily Quinn, Giuseppe Gullo, Naomi Walsh, John P. Crown. Whole genome sequencing of long-term, never relapse exceptional responders HER2+ advanced metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-24.

Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin has been used to identify HER2+ BC patients with durable complete response (never relapsed) to trastuzumab-based therapy. ~10% of met HER2+ BC patients achieve a durable complete response to trastuzumab, meaning the majority of patients progress on treatment. Higher stromal tumour immune infiltrate has been associated with longer OS in met HER2+ BC. There is limited tumor immune profile and PD-1 expression data available for patients with met HER2+ BC with short OS. Using the SVUH database, we have identified a preliminary cohort of 21 met HER2+ BC patients that received trastuzumab and had an OS < 30 months. This study examines the levels of pan T cell marker CD3, cytotoxic T cell marker CD8, Natural Killer (NK) cell marker CD56 and immune checkpoint PD-1 by immunohistochemistry (IHC) in this preliminary cohort. Methods: Formalin-fixed, paraffin-embedded (FFPE) biopsy specimens (n=21 primary, n=7 matched metastatic biopsies) and associated clinico-pathological data were curated. Tumor biopsies were processed for IHC staining of CD8 (Agilent IR62361-2), CD3 (Agilent IR50361-2), CD56 (Agilent IR62861-2) and PD-1 (Roche 07099029001). PD-1 staining was available for 20/21 samples. Staining was performed using the DAKO Link 48 Autostainer as per the manufacturer’s instructions using positive (tonsil tissue) and negative controls (isotype controls). Slides were processed using the Aperio AT2 Digital Slide Scanner (Leica Biosystems), reviewed using Aperio ImageScope 12.4 software (Leica Biosystems) and analyzed in QuPath (University of Edinburgh). Images were annotated to outline tumor areas and an algorithm was trained to identify cells and classify them as either tumor or stromal. Data was expressed as number of positively stained cells/mm2 breast tumor or stromal tissue. Survival studies utilized the Kaplan Meier method. The paired Student’s T test was utilized for primary vs metastatic site comparisons. Results: Designating samples with > 1 stained cell/mm2 breast tumor as positive (pos) and zero stained cells as negative (neg), 19/21 (90.5%) primary samples were pos for CD3, 15/21 (71.4%) for CD56, 14/21 (66.6%) for CD8, and 10/20 (50%) for PD-1. Within the stromal compartment, 20/21 (95.2%) primary samples were pos for CD8, 18/21 (85.7%) for CD56, 16/21 (76.2%) for CD3 and 8/20 (40%) for PD-1. PD-1 expression in the primary tumor (median OS PD-1pos 7.85 mo vs PD-1neg 5.39 mo, hazard ratio (HR) 0.642 (95% CI 0.256-1.613), p=0.346) or the stroma (median OS PD-1pos 8.84 mo vs PD-1neg 5.39 mo, HR 0.495 (95% CI 0.197-1.244), p=0.135) was not significantly associated with OS. When comparing matched primary and metastatic samples (n=7), increased stromal levels of CD3 (4/7), CD8 (4/7), CD56 (5/7) and PD-1 (4/7) were observed. Increased levels of CD3 and CD8 were observed for 2/7 samples, and increased levels of CD56 and PD-1 for 4/7 samples. With the exception of tumor CD8 levels which decreased, mean values for tumor and stromal CD3, CD56, PD-1 and stromal CD8 levels were higher in metastatic sites but all differences were not found to be significant (p>0.05). Conclusions: Our results suggest that met HER2+ BC patients with < 30 months OS have significant T cell and NK cell presence in the tumor and stromal compartments in both primary and metastatic sites. Further expansion of this limited dataset is planned to gain greater insight in to the immune cell profiles and PD-1 status of met HER2+ BC patients with short OS. Citation Format: Denis M. Collins, Janet McCormack, Laura P. Ivers, Jose Javier Berenguer Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex J. Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with < 30 months overall survival (OS). [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-11.