Recent studies of the tumor microenvironment (TME) have elucidated the heterogeneity of stromal cells, with increasing evidence suggesting that stromal subsets play an important role in regulating antitumor immunity in breast cancer. However, the functional diversity of these cells within the tumor-immune microenvironment and how they interact with immune cells in a spatial and clinical context remain poorly understood. We performed multiplex immunofluorescence on tumor microarrays from two cohorts consisting of 591 patients with breast cancer to assess the abundance and spatial co-localization of stromal and immune subsets and their correlation with clinicopathologic features and patient outcomes. We found that stromal cells were spatially distinct. A perivascular-like subset that was disseminated throughout the stroma rather than restricted to vessel-adjacent regions was enriched in an immune-cold environment and associated with T-cell exclusion. An inflammatory-like cancer-associated fibroblast subset was associated with segregation of T cells from cancer cells. Our findings highlight the differential impact of stromal subsets on immune infiltration and activation within the breast cancer TME with potential implications for patient outcomes. SIGNIFICANCE:This study characterized spatially defined interactions between stromal and immune subsets in large clinical cohorts of early breast cancer. Findings from this study will fill gaps in current knowledge in how diverse stromal cell subsets, particularly novel subsets, interact with immune cells in a clinically relevant context.

The Rho-associated coiled-coil containing protein kinases 1/2 (ROCK1/2) are key signalling proteins involved in the regulation of the actin cytoskeleton and control a variety of cellular processes. This includes cell proliferation, stemness, cell migration and invasion as well as actomyosin contraction and extracellular matrix (ECM) remodelling. Due to a lack of ROCK2 specific inhibitors, previous pharmacological studies of ROCK function have relied on pan-ROCK1/2 inhibition. Here, we use the ROCK2 specific inhibitor, GV101, in the context of breast cancer (BC) models to uncouple the effects of ROCK2 pharmacological inhibition on both epithelial and stromal fibroblast cell populations. We demonstrate that ROCK2 inhibition with GV101 reduces fibroblast-mediated remodelling of pre-existing and de novo synthesised ECM resulting in a reduction in biomechanical stiffness, while also limiting epithelial cell growth in 2D and 3D settings. We also demonstrate that ROCK2 inhibition exposes epithelial cell vulnerability to fluid flow-induced shear stress. Furthermore, using 3D co-cultures we reveal that GV101 reduces single-cell migratory capacity, while the disruption of stromal ECM architecture is sufficient to impede collective cell migration and invasion. Finally, we assessed ROCK2 expression in human BC patient tumours revealing that ROCK2 expression is up-regulated during BC progression and that high ROCK2 expression correlates with poor patient outcome in the triple-negative subtype of BC. Together, these results demonstrate that ROCK2 specific inhibition disrupts key functions in both epithelial cell and stromal fibroblast compartments warranting further assessment of ROCK2 in highly proliferative, fibrotic or metastatic diseases, such as BC. Highlights ### Competing Interest Statement P.T. and D.H. receive reagents from Equilibre Biopharmaceuticals and Graviton Biosciences (GV101 provided free of charge). P.T. receives reagents from Kadmon Inc., InxMed (consultant) Redx Pharma, and Amplia Therapeutics. Under a licensing agreement between Amplia Therapeutics and Garvan Institute of Medical Research, K.J.M., P.T. (consultant) and D.H. are entitled to milestone payments.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with poor prognosis and limited treatment options. One of the mechanisms contributing to this is the ability of TNBC tumors to evade anti-tumour response, limiting the success of immune-checkpoint therapy. In recent studies of the tumor microenvironment, stromal cells have emerged as potential important mediators of lymphocyte function in TNBC. Multi-omics studies from our group and others have elucidated the heterogeneity of stromal cells and their interactions with immune cells in TNBC. However, the clinical relevance and functional characteristics of these relationships remain poorly explored. Our previous work revealed functionally distinct subpopulations of stromal cells in breast cancer: endothelial cells, myofibroblast-like cancer-associated fibroblasts (myCAFs), inflammatory-CAFs (iCAFs), and perivascular-like cells which resemble pericyte and smooth muscle cells in immature and differentiated states (imPVLs; dPVLs), respectively. Here we directly explored the clinical relevance of these stromal subpopulations and their association with immune evasion in a large independent TNBC cohort with long-term survival data. Using markers derived from our single-cell multi-omics studies, we performed multiplex immunofluorescence using the OPAL9 platform on tumor microarrays from 222 TNBC patients to mark myCAFs, iCAFs, dPVLs, endothelial cells, CD8 and/or PD1 positive T-cells. Digital imaging analysis (QuPath) revealed a significant negative correlation between the abundance of stromal cells and CD8 T cells. This cytotoxic T-cell exclusion is primarily driven by smooth muscle dPVLs. Parallel to this T-cell exclusion, our functional studies demonstrate that stromal cells suppress T-cell proliferation. Using multiple ex vivo co-culture models of primary TNBC CAFs and peripheral blood mononuclear cells (PBMCs) from healthy and matched donors, we show that CAFs suppress the proliferative capability of CD4 and CD8 T-cells. ScRNA-seq of these co-culture models demonstrate that CAF educated T cells are driven into a LAG3+ exhausted state enriched for canonical pathways of immunosuppressive cytokine signaling. Our findings suggest that manipulation of these stromal subpopulations could elicit a more effective immune response in a subset of patients through inhibiting T-cell dysfunction and exclusion. Citation Format: Julia Chen, Sunny Wu, Travis Ruan, Iveta Slapetova, Ewan Millar, Elgene Lim, Alex Swarbrick. Stromal mediators of lymphocyte exclusion and dysfunction in triple negative 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 PD9-01.

WinPro: A window of opportunity study of endocrine therapy with and without prometrium in postmenopausal women with early-stage hormone receptor-positive breast cancer Authors Teesha Downton1,2,3, Davendra Segara3, Andrew Ong4, Janne Bingham5, Emma-Kate Carson4, Julia Chen1,2,3, Kate Middleton3, Geoffrey Lindeman6, Andrew Parker3, Elgene Lim1,2,3. Affiliations 1Garvan Institute of Medical Research, Darlinghurst NSW, Australia; 2School of Clinical Medicine, St Vincent’s Healthcare Clinical Campus, Faculty of Medicine and Health, University of New South Wales Sydney, Australia; 3St Vincent’s Hospital Sydney, Darlinghurst NSW, Australia; 4Campbelltown Hospital, Campbelltown NSW, Australia; 5Royal Adelaide Hospital, Adelaide SA, Australia; 6Walter & Eliza Hall Institute of Medical Research, Parkville VIC, Australia Disclosures T. Downton: None. D. Segara: None. A. Ong: None. J. Bingham: None. E. Carson: None. J. Chen: None. K. Middleton: None. G. Lindeman: None. A. Parker: None. E. Lim: Advisory Board for Pfizer, Astra Zeneca, Lilly, Roche, Novartis, Gilead Australia. Research Funding from Pfizer, Novartis, Bayer. Abstract Background: Preclinical studies have observed that progesterone has inhibitory effects on the estrogen-stimulated growth of estrogen receptor (ER)-positive, progesterone receptor (PR)-positive breast cancer. Mohammed H et al. (Nature 2015) identified that activated PR associates with the ER and modulates the interactions of the ER with chromatin, with a shift towards the transcription of genes associated with apoptosis and differentiation, and away from genes associated with proliferation. We hypothesize that the addition of prometrium, a microionized progesterone, may enhance the anti-proliferative effects of standard endocrine therapy in women with ER-positive PR-positive breast cancer. Trial Design: WinPro (NCT03906669) is an ongoing multicenter, phase II, randomized, open-label, window of opportunity study comparing the effect on standard endocrine therapy with or without prometrium on breast cancer cell proliferation. The study population is postmenopausal women with early-stage operable breast cancer where the tumor is ≥5mm on imaging, ER ≥10%, PR≥10%, and HER2-negative. Patients currently on hormone replacement therapy or the oral contraceptive pill, or who have a history of endometrial cancer or venous thromboembolism are not eligible. Patients are randomized 1:1:1 to letrozole 2.5mg daily, letrozole 2.5mg + prometrium 300mg daily, or tamoxifen 20mg + prometrium 300mg daily. Allocated treatment is taken for 14 days prior to surgery. Primary surgery and adjuvant treatment is as per standard of care. Objectives: The primary objective is to compare the geometric mean suppression of centrally assessed Ki67 between the diagnostic biopsy sample (pre-treatment) and the surgical sample (post-treatment). The secondary objective is to evaluate safety and tolerability. Tertiary objectives include defining genes predictive of a reduction in Ki67, and evaluating the changes in ER, PR, AR, FoxA1, Cyclin D1, and apoptotic markers in breast tumors post-intervention. Accrual: This study opened in February 2018 and as of 13 July 2022, 164 patients have been enrolled. Target accrual is 200 patients. Contact information: This study is led at St Vincent’s Hospital Sydney, Australia, and funded by the Cancer Council of NSW and the NHMRC Translational Breast Cancer Project grant. Contact: Elgene Lim MBBS FRACP PhD at e.lim@garvan.org.au. Citation Format: Teesha Downton, Davendra Segara, Andrew Ong, Janne Bingham, Emma-Kate Carson, Julia Chen, Kate Middleton, Geoffrey Lindeman, Andrew Parker, Elgene Lim. WinPro: A window of opportunity study of endocrine therapy with and without prometrium in postmenopausal women with early-stage hormone receptor-positive 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 OT2-01-10.

Taxane-based chemotherapy regimens are in widespread use as standard of care treatment for patients with early breast cancer, though rarely its use can be complicated by taxane-induced pneumonitis (TIP). While breast cancer is the most diagnosed cancer in women worldwide, TIP remains under-described in this setting. Key questions relate to its incidence, diagnosis and management, potential predictive biomarkers, and the balance between this life-threatening toxicity and curatively intended treatment. At a single Australian institution, 6 cases of TIP are identified among 132 patients treated with a paclitaxel-containing regimen for early breast cancer (4.55%, 95% confidence interval 1.69-9.63%). This review first outlines the presentation, management, and outcomes for these cases, then answers these questions and proposes an approach to suspected TIP in patients with breast cancer.

With the adoption of inhibitors of cyclin dependent kinases 4 and 6 (CDK4/6i) in combination with endocrine therapy as standard of care for the treatment of advanced and metastatic estrogen receptor positive (ER+) breast cancer, the search is now on for novel therapeutic options to manage the disease after the inevitable development of resistance to CDK4/6i. In this review we will consider the integral role that the p53/MDM2 axis plays in the interactions between CDK4/6, ER alpha, and inhibitors of these molecules, the current preclinical evidence for the efficacy of MDM2 inhibitors in ER+ breast cancer, and discuss the possibility of targeting the p53/MDM2 via inhibition of MDM2 in the CDK4/6i resistance setting.

Advances in human epidermal growth factor receptor 2 (HER2)-directed therapies have revolutionised the care of patients with HER2-positive breast cancer. While adjuvant trastuzumab in combination with chemotherapy has dramatically improved the prognosis for patients with early-stage disease, up to a quarter of patients will develop recurrent disease. The standard-of-care treatment paradigm has evolved with the introduction of newer HER2-directed therapies and increasing use of neoadjuvant systemic therapy, the latter providing us with important functional data to HER2-directed therapies and impacting subsequent adjuvant therapy decisions. However, these new strategies come at a cost of increased toxicity and economic burden, and only a subset of patients benefit from such approaches. Thus, ongoing work is required to identify predictive biomarkers of response, to de-escalate treatment in patients who may do just as well with less therapy, and new therapeutic approaches for patients who do not respond to currently used therapies. In this review, we will examine the current therapeutic landscape, summarise the latest evidence, and list the current treatment algorithms for early stage HER2-positive breast cancer.

Window of opportunity therapies, which involve short-term administration of systemic therapy between cancer diagnosis and surgery, have raised significant interest in recent years as a mean of assessing the sensitivity of a patient's cancer to therapy prior to surgery. There is now compelling evidence that in patients with early stage hormone-receptor positive breast cancer, a 2-week preoperative treatment with standard hormone therapies in a preoperative window period provides important prognostic information, which in turn helps to aid decision-making regarding treatment options. Changes in short-term biomarker endpoints such as cell proliferation measured by Ki-67 can act as surrogate markers of long-term outcomes. Paired tissues obtained pre- and post-investigational treatment, without having to subject the patient to additional biopsies, can then be used to conduct translational research to investigate predictive biomarkers and pharmacodynamics. In this review, we will examine the utility and challenges of window of opportunities therapies in breast cancer in the current literature, and the current Australian and international trial landscape in this clinical space.

TPS5095 Background: mCRPC progresses via adaptive mechanisms that allow ongoing androgen receptor (AR) signalling despite castrate levels of androgens. Bipolar androgen therapy (BAT), cycling between supraphysiologic and subphysiologic serum testosterone levels, aims to exploit these adaptations to induce tumor regression. Extensive clinical data demonstrate the safety and efficacy of BAT in men with asymptomatic mCRPC. However, de novo resistance is still common and predictive biomarkers to refine patient selection are lacking. Pre-clinical data suggest that the induction of double-stranded DNA (dsDNA) breaks by BAT may be crucial to its mechanism of action. DNA repair defects, such as HRD, are particularly relevant in CRPC patients. We hypothesize that CRPC patients with DNA repair deficits such as HRD, may be particularly responsive to BAT. Methods: This is a phase II prospective single arm interventional trial (NCT03522064). Up to 30 patients will be recruited based on a Simon two-stage design with a power of 90% to detect an increase in response rate from 20% to 40%. Key inclusion criteria include i) asymptomatic or minimally symptomatic mCRPC, ii) rising PSA despite a castrate serum testosterone and iii) HRD on germline, tumor and/or circulating tumor DNA (ctDNA) analysis. Key exclusion criteria include i) ADT < 1 year, ii) disease extent/sites that would cause significant risk if tumor flare occurs (e.g.: brain) and iii) significant cardiac disease. Previous PARP inhibitor therapy will be permitted in a subset. Participants will receive IM testosterone enanthate 500mg q4w in combination with ongoing LHRH antagonist/agonist or orchidectomy. The primary endpoint is PSA response rate defined as PSA reduction ≥50% from baseline. Secondary endpoints include time to PSA progression, quality of life, radiologic response and safety and tolerability. Exploratory endpoints include changes in ctDNA and tumoral DNA alterations from baseline to progression. Accrual is ongoing. Clinical trial information: NCT03522064.

This study determined the influence of prematurity on the manifestation and treatment of neonatal abstinence syndrome (NAS). This was a medical record review of Australian infants exposed to opiates in 2004 and 2007. Finnegan scores were obtained for 215 of 361 (59%) preterm infants under 37-week gestation and 694 of 1178 (59%) term infants. The mean and standard deviation (SD) gestational ages were 34 (3) and 38 (3) weeks for preterm and term infants, respectively. Maternal daily methadone doses were similar for the preterm and term infants with a mean (SD) of 79 mg (39) versus 72 mg (38) (p = 0.06). Maximum Finnegan scores were significantly lower in preterm infants (10 versus 11, p = 0.01), scores were positively correlated with gestation and fewer preterm infants were medicated for NAS (40% versus 48% p = 0.05). Maximum median daily and interquartile range morphine doses were lower for preterm than term infants (0.5 mg/kg/day (0.3–0.6) versus 0.5 mg/kg/day (0.4–0.7), p = 0.02). Preterm infants were just as likely to be monitored for withdrawal as term infants, but their Finnegan scores were lower and fewer preterm infants were treated for NAS. Whether this indicates decreased NAS severity or physiological immaturity is uncertain. Other means of evaluating NAS in preterm infants are warranted, especially long-term outcomes.

AimThis study determined the influence of prematurity on the manifestation and treatment of neonatal abstinence syndrome (NAS).MethodsThis was a medical record review of Australian infants exposed to opiates in 2004 and 2007. Finnegan scores were obtained for 215 of 361 (59%) preterm infants under 37-week gestation and 694 of 1178 (59%) term infants.ResultsThe mean and standard deviation (SD) gestational ages were 34 (3) and 38 (3) weeks for preterm and term infants, respectively. Maternal daily methadone doses were similar for the preterm and term infants with a mean (SD) of 79mg (39) versus 72mg (38) (p=0.06). Maximum Finnegan scores were significantly lower in preterm infants (10 versus 11, p=0.01), scores were positively correlated with gestation and fewer preterm infants were medicated for NAS (40% versus 48% p=0.05). Maximum median daily and interquartile range morphine doses were lower for preterm than term infants (0.5mg/kg/day (0.3-0.6) versus 0.5mg/kg/day (0.4-0.7), p=0.02).ConclusionPreterm infants were just as likely to be monitored for withdrawal as term infants, but their Finnegan scores were lower and fewer preterm infants were treated for NAS. Whether this indicates decreased NAS severity or physiological immaturity is uncertain. Other means of evaluating NAS in preterm infants are warranted, especially long-term outcomes.

Aim To determine the short-term outcomes of Australian buprenorphine-exposed mother/infant dyads.Methods Retrospective record review of drug-exposed mothers and infants in Australia. Groups were based on drug exposure: buprenorphine (55, 3.8%), non-buprenorphine opiates (O, 686, 48.6%) and non-opiates (NO, 671, 47.5%).Results More than 30% of buprenorphine mothers continued to use heroin (21, 38%) and benzodiazepines (16, 29%). They were more likely to have child at risk concerns (29, 52.7%, P = 0.019) and have previous children placed in out-of-home care (9, 16.3%, P = 049). Buprenorphine babies were less likely to be preterm (16% vs. 25% (O), P = 0.001 and 23% (NO), P = 0.004) and had higher birthweights (median: 3165g vs. 2842.5g (O), P < 0.001 and 2900g (NO), P = 0.004). Buprenorphine and non-buprenorphine opioid babies had similar maximum Finnegan scores (median 10 vs. 11(O), P = 0.144). The number of babies needing abstinence treatment (45% vs. 51% (O), P = 0.411) and length of hospital stay (median days 9 vs. 11(O), P = 0.067) were similar, but buprenorphine infants required lower maximum morphine doses (mg/kg/day) (median 0.4mg vs. 0.5mg (O), P = 0.009).Conclusions Short-term medical outcomes of infants of buprenorphine-using mothers are similar to those of non-buprenorphine opiate-using mothers, but interpretation of these results is confounded by the high rates of polydrug exposure in the buprenorphine group. This and other social concerns noted in buprenorphine mothers and infants warrant further study.