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Supplementary Methods

Supplementary Tables S1-5

Supplementary Table S8

Supplementary Figures

Supplementary Table S1-S12

SUPPLEMENTARY FIGURE (1-7) AND TABLE (1-12) LEGENDS

E2F activation metagene & Fluorescence activated cell sorting

Purpose:To investigate the presence of ESR1 mutations in primary estrogen-receptor–positive (ER+) breast cancer treated with extended (>4 weeks) neoadjuvant (presurgical) aromatase inhibitor (NAI) therapy and to identify patients who may gain less benefit from aromatase inhibition (AI) alone based upon on-treatment changes in gene expression.Experimental Design:We evaluated ER, progesterone receptor, and Ki67 by immunostaining, ESR1 mutations by droplet-digital PCR and expression of over 800 key breast cancer genes in paired pre- and post-NAI tumor samples from 87 ER+ breast cancer patients.Results:Cell proliferation and estrogen-regulated genes (ERG) remained suppressed in most tumors indicative of persistent response to NAI. Enrichment of ESR1 mutations was found in five tumors and predominantly in patients receiving therapy for >6 months. ESR1-mutant tumors showed increased expression of ESR1 transcript and limited suppression of ERGs and proliferation-associated genes in response to NAI. ESR1 wild-type tumors with high residual proliferation (Ki67r ≥10%; 15/87 tumors) showed lower ESR1/ER expression pre- and post-therapy and lower ERGs. Tumors with ESR1 mutations or Ki67r ≥10% showed less inhibition of estrogen response, cell cycle, and E2F-target genes.Conclusions:Ligand-independent ER signaling, as a result of ESR1 mutation or reduced ER dependence, identified after extended NAI therapy, can guide early selection of patients who would benefit from combination therapy.

Supplementary Figures S1-S7

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored. Colour contrast is added to unstained histological samples by using surface plasmon polaritons whose properties depend on the sample’s dielectric constant.

The Victorian Tumour Summits are an initiative of the Victorian Integrated Cancer Services to engage clinicians and consumers in identifying unwarranted variations in cancer care across the state. From the analysis presented at the Victorian Breast Tumour Summit in 2021, this study provides a state-wide overview of epidemiology and surgical care of breast cancer in Victoria to outline any variations in care across the state, and limitations in data reporting, which impacts the understanding of breast cancer burden and service planning.A retrospective analysis of Victorian breast cancer patients diagnosed between 2016 and 2018 was performed using a linked data set provided by the Department of Health. The linked data sources include Victorian Cancer Registry, Victorian Admitted Episodes Dataset and Victorian Radiotherapy Minimum Data Set, from which patient demographic details, tumor characteristics and treatment records were extracted. Pearson's chi-squared test was used to determine the statistical significance of relationships between various categorical parameters. Variables including demographics, types of surgery (breast-conserving vs. mastectomy), rates of neoadjuvant chemotherapy, and time to surgery were examined.One thousand nine hundred thirty-seven patients with ductal carcinoma in situ and 13,375 patients with invasive breast cancer (IBC) were included. Of 11,351 patients with stages I-III IBC (85%, N = 13,375) 66% underwent breast-conserving surgery (BCS), and 31% underwent mastectomy. The ratio of mastectomy to BCS increased with increasing disease stage. Neoadjuvant chemotherapy was utilized in 11% of early IBC patients who were surgically treated. Eighty-three percent of patients undergoing upfront breast surgery were treated within 5 weeks of diagnosis, with a significant difference in the median time to surgery between public and private sectors. Breast reconstruction was performed in 37% of mastectomy patients, of whom 83% underwent immediate breast reconstruction, and 17% underwent delayed breast reconstruction.Victorian breast cancer data show a high quality of surgical care coordination. Significant gaps in our data warrant future improvements in the Victorian breast cancer notification system and access to pharmaceutical data for an enhanced understanding of the breast cancer treatment pathways and care delivery.

Aromatase inhibitor (AI) therapy provides oncological benefits in postmenopausal women with oestrogen receptor-positive breast cancer. However, AI treatment has been associated with increased cardiovascular risk. In nonbreast cancer populations, experimentally induced low oestrogen states and natural transition to menopause have been associated with increases in visceral adipose tissue (VAT), a known surrogate marker for cardiometabolic risk. Given that AI treatment blocks oestradiol production, we hypothesized that AI treatment would increase VAT.We conducted a prospective 12-month cohort study of 52 postmenopausal women newly initiating AI treatment (median age: 64.5 years) and 52 women with breast pathology not requiring endocrine therapy (median age: 63.5 years). VAT area and other body composition parameters were measured at baseline, 6 months and 12 months using dual X-ray absorptiometry. Other risk markers of cardiometabolic health were also assessed.In women initiating AI treatment, there was no statistically significant difference in VAT area after 12 months when compared to controls, with a mean adjusted difference of -5.00 cmThese data provide reassurance that over the initial 12 months of AI therapy, AI treatment is not associated with metabolically adverse changes in body composition, hepatic steatosis or vascular reactivity. The impact of extended AI therapy on cardiometabolic health requires further study.

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

Introduction Neoadjuvant radiotherapy (NART) as part of a multi-modality approach for locally advanced breast cancer (LABC) requires further investigation. Importantly, this approach may allow for a single-staged surgical procedure, with mastectomy and immediate autologous reconstruction. Multiple other potential benefits of NART include improved pathological downstaging of breast disease, reduced overall treatment time, elimination of time period with breast tissue deficit and improved patient satisfaction. Methods This is a retrospective multi-institutional review of patients with LABC and high-risk breast disease undergoing NART. Eligible patients sequentially underwent neoadjuvant chemotherapy (NACT) with or without HER2-targeted therapy, NART, followed by mastectomy with immediate autologous breast reconstruction (BR) 4- to 6 weeks post-completion of radiotherapy. Patient and tumour characteristics were analysed using descriptive statistics. Surgical complications were assessed using the Clavien-Dindo Classification (Ann Surg 2004; 240: 205). Results From 3/2013 to 9/2019, 153 patients were treated with NART. The median age was 47 years (IQR 42-52), with median body mass index of 27. Eighteen patients experienced Grade 3 acute surgical complications. This included 13 Grade 3B breast-site events and 9 Grade 3B donor-site events, where further surgical intervention was required for management of wound infection, wound dehiscence, flap or mastectomy skin necrosis, haematoma and internal mammary venous anastomotic thrombosis. No autologous flap loss was observed. Conclusion Neoadjuvant radiotherapy facilitates a single-stage surgical procedure with mastectomy and immediate autologous BR, eliminating the delay to reconstructive surgery and thus shortening a woman's breast cancer journey. The findings of this review support the use of NART, with comparable rates of surgical complications to standard sequencing.

Introduction Breast cancer survivors are at greater risk for cardiovascular-related mortality compared to women without breast cancer. Accordingly, attention to reducing the risk of cardiovascular disease must be a priority in the long-term management of these patients. Objective To assess the efficacy of a smartphone-based model of care for exercise promotion, cardiovascular risk reduction and psychological wellbeing in women undergoing treatment for breast cancer. Methods Female patients with breast cancer across four tertiary Australian hospitals were screened for inclusion in this randomized controlled trial. Patients were randomized to standard care (SC), with or without an adjunctive smartphone application ‘BreastMate’ (SP) at time of diagnosis. The primary endpoint was change in exercise capacity, measured by the change in six-minute walk test (6MWT) distance at 12-months when compared to baseline, between groups. Secondary endpoints included changes in cardiovascular risk factors, psychological wellbeing, and major adverse cardiovascular events (MACE). Results Of the 103 patients recruited, complete follow-up data was available in 80 (77.7%) patients (age 60±12 years, SP=41, SC=39). At 12-months follow-up, the SP group had a statistically and clinically significant improvement in 6MWT distance (Median Δ46 (IQR 28 to 63) metres vs. Δ8 (-10 to 35) metres; p< 0.001). Compared to SC, patients in the SP group had a greater reduction in systolic blood pressure (Δ-5.1 (-10 to 0) vs 0 (-10 to 7) mmHg; p=0.03) and waist circumference (Δ-1 (-4 to 0) vs 0 (-1.5 to 1) centimetres; p=0.003) as well as a greater improvement in vitality as measured by the sf-36 questionnaire (48.1 ± 18.4 to 63.3±19.2 vs 48.3 ± 24.2 to 59.6±18.8; p=0.04). There was no significant difference in cholesterol levels, diabetes control, smoking cessation or incidence of MACE between groups (p-value for all=NS). Conclusion In patients with breast cancer, a smartphone-based cardiovascular risk reduction program, as an adjunct to standard care, improved exercise capacity, systolic blood pressure, waist circumference and patient-reported vitality at 12-months. This innovative model of care could be implemented to improve the cardiovascular risk profile of breast cancer survivors. Australia and New Zealand Clinical Trials Registry (ANZCTR12620000007932). Citation Format: Alexandra C. Murphy, Omar Farouque, Anoop N. Koshy, Belinda Yeo, Ron Dick, Voltaire Nadurata, Laura Roccisano, Christopher Reid, Matias Yudi. SMARTphone-Based Cardiovascular Risk Reduction in Breast Cancer Patients [SMART-BREAST]: A Randomized Controlled Trial [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 P3-03-29.

Background Tucatinib (TUC) is a highly selective oral tyrosine kinase inhibitor of HER2 with minimal inhibition of EGFR. It was recently approved by the FDA for patients (pts) with HER2+ metastatic breast cancer (MBC), including pts with brain metastases (BM) whose cancers have progressed on at least 1 prior anti-HER2 regimen in the metastatic setting. In the HER2CLIMB (NCT02614794) pivotal trial, pts with HER2+ MBC previously treated with trastuzumab (T), pertuzumab, and trastuzumab emtansine (T-DM1) were randomized to receive TUC or placebo in combination with T and capecitabine (C). The addition of TUC resulted in clinically meaningful and statistically significant improvements in overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) in HER2+ MBC pts. Primary methods and outcomes have been reported previously (Murthy, NEJM 2019). Here we present an exploratory analysis describing the outcomes in the HER2CLIMB trial based on hormone receptor (HR) status. Methods Pts were randomized 2:1 to receive TUC or placebo in combination with T and C. All pts had a baseline brain MRI and randomization was stratified by presence of BM, ECOG status, and geographic region. All pts with HER2+ MBC who were positive for either or both estrogen receptor and progesterone receptor (> 1%) were assigned to the HR “positive” subgroup. Pts not meeting the above criteria were assigned to the HR “negative” subgroup. For the exploratory HR+/HR- efficacy analysis presented here, PFS per RECIST 1.1 by blinded independent central review was evaluated in the first 480 pts enrolled. OS, PFS in pts with baseline BM, and confirmed ORR in pts with measurable disease were evaluated in the total study population. P values presented for PFS are nominal. Results Overall, 612 pts were enrolled to HER2CLIMB; 370 pts (60%) had HR+ and 242 (40%) had HR- tumors. Baseline demographics and disease characteristics in HR+/HR- subgroups were generally balanced between treatment arms. In the HR+ group, there was a 42% reduction in the risk of progression or death in the TUC arm (hazard ratio: 0.58; 95% CI: 0.42, 0.80; P=0.0008); median (95% CI) PFS was 7.6 mo (7.4, 9.5) in the TUC arm vs 5.6 mo (4.3, 7.4) in the control arm. In the HR- group, there was a 46% reduction in the risk of progression or death in the TUC arm (hazard ratio: 0.54; 95% CI: 0.34, 0.86; P=0.008); median (95% CI) PFS was 8.1 mo (7.0, 11.6) in the TUC arm vs 4.2 mo (3.1, 8.6) in the control arm. In the total population, median OS was 21.7 mo vs 18.2 mo in HR+ in the TUC arm vs control arm, respectively; median OS in HR- was 31.1 mo in the TUC arm vs 14.1 mo in the control arm. In pts with BM in the HR+ group (n=166 [45%]), there was a 52% reduction in the risk of progression or death (hazard ratio: 0.48; 95% CI: 0.31, 0.75; P=0.0008); median (95% CI) PFS was 7.5 mo (5.6, 9.5) in the TUC arm vs 5.1 mo (4.1, 5.7) in the control arm, and median OS was 18.1 mo vs 12.8 mo, respectively. In pts with BM in the HR- group (n=125 [52%]), there was a 50% reduction in the risk of progression or death (hazard ratio: 0.50; 95% CI: 0.27, 0.95; P=0.03); median (95% CI) PFS was 7.8 mo (6.1, 11.6) in the TUC arm vs 5.4 mo (2.9, 8.6) in the control arm, and median OS was 18.5 mo vs 11.5 mo, respectively. In the total population, ORR was numerically higher in the TUC arm vs the control arm regardless of HR status (HR+: 37.4% [95% CI: 30.8, 44.5] vs 27.1% [95% CI: 19.0, 36.6], respectively and HR-: 45.3% [95% CI: 36.7, 54.0] vs 15.6% [95% CI: 7.8, 26.9], respectively). Conclusions Among pts with HER2+ MBC previously treated with T, pertuzumab, and T-DM1, the addition of TUC to T and C showed clinically meaningful improvements of PFS, OS, and ORR independent of HR status. Furthermore, pts with HR+ and HR- MBC with BM derived similar benefit from the addition of TUC to T and C. Citation Format: Erika Hamilton, Mattea Reinisch, Sherene Loi, Alicia Okines, Paula R. Pohlmann, Eva Harder Brix, Hugues Bourgeois, Belinda Yeo, Cheryl Aylesworth, Melody Cobleigh, Anthony Goncalves, Rebecca Moroose, Katherine H.R. Tkaczuk, Michaela Tsai, Christine Simmons, Michael Andersson, Hatem Soliman, Michelina Cairo, Lisa A. Carey, David Cameron, Jorge Ramos, Wentao Feng, Mafalda Oliveira. Tucatinib vs placebo in combination with trastuzumab and capecitabine for patients with locally advanced unresectable or HER2-positive metastatic breast cancer (HER2CLIMB): Outcomes by hormone receptor status [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD3-08.

BackgroundThe development of anti-human epidermal growth factor receptor 2 (HER2) therapies has significantly improved disease outcomes in patients with HER2-positive advanced breast cancer (ABC). However, elderly patients are persistently under-represented in clinical trials, with only 2.4% of patients aged ≥75 years in the pivotal CLEOPATRA study. Despite a lack of research addressing treatment outcomes in elderly patients, advanced age at diagnosis is associated with a greater likelihood of receiving no initial systemic therapy for de novo metastatic breast cancer. Studies have also shown that older women diagnosed with metastatic breast cancer have a poorer prognosis and shorter life expectancy. We examined treatment patterns and outcomes in an elderly (defined as ≥70) ‘real world’ Australian population. MethodsData was extracted from the Treatment of Advanced Breast Cancer in the HER2-positive Australian Patient (TABITHA) multi-site clinical registry, and patients stratified according to age ( ResultsWe identified 319 patients, including 67 patients (21%) aged ≥70 years. Older patients were more likely to have an Eastern Cooperative Oncology Group performance status of ≥2 (16% vs 3%; p ConclusionElderly patients with HER2-positive ABC demonstrated shorter chemotherapy durations, poorer overall survival, and increased rates of adverse events despite having similar disease characteristics and treatment patterns. Prospective studies are required to improve outcomes in the elderly HER2 positive population. Citation Format: Nicole Evans, Angelyn Anton, Rachel Wong, Sheau Wen Lok, Richard De Boer, Laeeq Malik, Sally Greenberg, Belinda Yeo, Louise Nott, Gary Richardson, Ian M Collins, Javier Torres, Frances Barnett, Peter Gibbs, Bianca Devitt. Real world outcomes in elderly women with HER2 positive advanced breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-35.

Inflammatory breast cancer (IBC) describes a highly aggressive form of breast cancer of diverse molecular subtypes and clonal heterogeneity across individual tumors. Accordingly, IBC is recognized by its clinical signs of inflammation, associated with expression of interleukin (IL)-6 and other inflammatory cytokines. Here, we investigate whether sub-clonal differences between expression of components of the IL-6 signaling cascade reveal a novel role for IL-6 to mediate a proliferative response in trans using two prototypical IBC cell lines. We find that SUM149 and SUM 190 cells faithfully replicate differential expression observed in a subset of human IBC specimens between IL-6, the activated form of the key downstream transcription factor STAT3, and of the HER2 receptor. Surprisingly, the high level of IL-6 produced by SUM149 cells activates STAT3 and stimulates proliferation in SUM190 cells, but not in SUM149 cells with low IL-6R expression. Importantly, SUM149 conditioned medium or co-culture with SUM149 cells induced growth of SUM190 cells, and this effect was abrogated by the IL-6R neutralizing antibody Tocilizumab. The results suggest a novel function for inter-clonal IL-6 signaling in IBC, whereby IL-6 promotes in trans proliferation of IL-6R and HER2-expressing responsive sub-clones and, therefore, may provide a vulnerability that can be exploited therapeutically by repurposing of a clinically approved antibody.

BACKGROUND: A history of cancer is incorporated into the surgical risk assessment of patients undergoing surgical aortic valve replacement through the Society for Thoracic Surgeons score. However, the prognostic significance of cancer in patients treated with transcatheter aortic valve replacement (TAVR) is unclear. As the cancer survivorship population increases, it is imperative to establish the efficacy and safety of TAVR in patients with severe symptomatic aortic stenosis (AS) and a history of malignancy. OBJECTIVES: The primary goal of this study was to assess the periprocedural outcomes and long-term mortality in patients with a history of cancer undergoing TAVR. METHODS: A systematic review of PubMed, MEDLINE, and EMBASE was conducted to identify studies reporting outcomes in patients with a history of malignancy undergoing TAVR. A meta-analysis was performed using a random-effects model with a primary outcome of all-cause mortality and cardiac mortality at the longest follow-up. On secondary analyses, procedural safety was assessed. RESULTS: A total of 13 observational studies with 10,916 patients were identified in the systematic review. Seven studies including 6,323 patients were included in the quantitative analysis. Short-term mortality (relative risk [RR] 0.61, 95%CI 0.36-1.01; p = .06) and long-term all-cause mortality (RR 1.24, 95%CI 0.95-1.63; p = .11) were not significantly different when comparing patients with and without a history of cancer. No significant difference in the rate of periprocedural complications including stroke, bleeding, acute kidney injury, and pacemaker implantation was noted. CONCLUSION: In patients with severe AS undergoing TAVR, a history of cancer was not associated with adverse short or long-term survival. Based on these findings, TAVR should be considered in all patients with severe symptomatic AS, irrespective of their history of malignancy.

Background: HER2 targeted therapies, such as trastuzumab, pertuzumab and trastuzumab-emtansine (TDM-1), have improved survival of patients (pts) with HER2+ metastatic breast cancer (MBC). In the pivotal phase III study of pertuzumab/trastuzumab/docetaxel in MBC (NCT00567190), only 10% of pts received prior trastuzumab, whereas in modern practice the majority of recurrent HER2+ MBC pts have received neo(adjuvant) trastuzumab [NAT]. Survival outcomes in the real world setting have not been well described. Methods: Pts were part of a prospective national registry collecting clinicopathologic, treatment and outcome data for HER2+ MBC pts diagnosed between 10/2006 and 1/2019. Survival was estimated by the Kaplan-Meier method and compared among groups by log-rank test. A multivariable analysis was used to assess the impact of prior trastuzumab-use on survival. Analysis was stratified by estrogen receptor (ER) status to exclude the impact of concurrent endocrine therapy in the ER positive (ER+) subgroup. The TABITHA registry is sponsored by BioGrid Australia with financial support from Roche Products Pty Limited. Results: Of 254 HER2+ MBC pts in the registry, 225 received HER2 directed therapy in the first-line advanced disease setting and were included for analysis. Median age was 58 years, 100 (44%) had de novo metastatic disease, 131 (58%) had ER+ disease and 24 (11%) had brain metastases. Of the 125 (56%) pts with recurrent disease, 89 (71%) pts received NAT. First-line HER2 therapy was pertuzumab plus trastuzumab in 176 (78%), trastuzumab in 38 (17%), TDM-1 in 9 (4%), and lapatinib or pertuzumab alone in single pts ( The response rate to anti-HER2 therapy among all MBC pts evaluable for response was 42% in pts who received NAT, and 53% in pts who did not (p=0.20). Median progression free survival (PFS) was 16.4 months and 22.6 months, in pts who were trastuzumab-treated and trastuzumab-naïve, respectively (HR 1.44 [95%CI 0.99 - 2.10], p=0.05). The median overall survival (OS) from first-line HER2-directed therapy for MBC was 41.2 months in pts who had prior NAT, and 52.0 months in trastuzumab-naïve pts (HR 1.73 [95%CI 1.04 - 2.86], p=0.03). On multivariable analysis, prior trastuzumab-exposure was no longer significantly associated with survival. Factors significantly associated with PFS were ER status (HR 0.63, p=0.02) and age (HR 1.02, p=0.04), while only age was significantly associated with OS (see table). Among the subset of 93 ER negative (ER-) pts, prior NAT was significantly associated with inferior PFS (HR 1.97 [95%CI 1.11 - 3.52], p=0.02) and OS (HR 2.13 [95% CI 1.00 - 4.57], p=0.046). Multivariable analysis found only age and presence of brain metastases to be significantly associated with OS in the ER- subgroup. NAT exposure did not impact on PFS (HR 1.15, [95% CI 0.70 - 1.90], p=0.58) or OS (HR 1.37, [95% CI 0.68 - 2.73], p=0.37) in ER+ pts. Conclusions: In pts with HER2+ MBC, receipt of NAT was associated with inferior survival when HER2 targeted therapy was used in the metastatic setting on univariate analysis, but this finding was not seen in a multivariable analysis. Furthermore, the impact of NAT was only observed in the subset of pts with ER- tumors, raising the possibility that the disease-course in ER+ subgroup is not entirely HER2 driven. Survival rates of these HER2+ MBC pts treated in the community are comparable to that reported in clinical trials. Cox proportional hazard models for OS in MBC on HER2-targeted therapyUnivariableMultivariableHR95% CIPHR95% CIPAdjuvant trastuzumab Y vs N1.731.04 – 2.850.031.370.64 – 2.940.42De novo metastatic disease Y vs N0.610.37 – 1.020.060.810.38 – 1.730.59Estrogen receptor positive vs negative0.880.53 – 1.440.600.690.41 – 1.180.18Brain metastases Y vs N1.800.88 – 3.670.111.960.90 – 4.260.09Age (years)1.021.00 – 1.040.051.031.01 – 1.060.003 Citation Format: Yada Kanjanapan, Dan Tran, Sheau Wen Lok, Peter Gibbs, Richard De Boer, Belinda Yeo, Sally Greenberg, Frances Barnett, Louise Knott, Gary Richardson, Rachel Wong, Michelle Nottage, Ian Collins, Javier Torres, Janine Lombard, Julie Johns, Michael Harold, Laeeq Malik. Impact of prior (neo)adjuvant trastuzumab exposure on the efficacy of HER2 targeted therapy for metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-13.

The development of brain metastases occurs commonly in HER2-positive metastatic breast cancer and is associated with a poorer prognosis. The advent of HER2-targeted therapy has improved overall survival, but the benefit in patients with brain metastases is unclear, as these patients are often excluded from clinical trials. This study aimed to explore real-world outcomes in patients with brain metastases in HER2-positive MBC.Data was extracted from the TABITHA registry, which consists of patient data collected prospectively from 16 Australian sites from 24th February 2015 to 31st October 2021. Data analysed included characteristics of brain metastases, treatment received and survival outcomes.A total of 135 (37%) of 361 patients with HER2-positive MBC were diagnosed with brain metastases during their clinical course, including 45 (12%) with brain metastases at time of MBC diagnosis. 61 (45%) had ≥4 brain lesions. The most common local therapy given was whole brain radiation therapy (WBRT) (36%) followed by multi-modality treatment with both surgery and radiation therapy (27%). The majority of patients received first-line HER2-targeted treatment with trastuzumab and pertuzumab followed by second-line trastuzumab emtansine (T-DM1) but third-line therapy was heterogenous. The median overall survival in patients who developed brain metastases was significantly shorter than those who did not develop brain metastases (58.9 vs. 96.1 months, P = .02).Real-world patients diagnosed with brain metastases in HER2-positive MBC have a relatively poor prognosis, despite advances in HER2-targeted treatment. As the range of HER2-targeted treatment expands, it is important to pursue clinical trials that focus on patients with brain metastases.

Radiation therapy (RT) is an established treatment for breast cancer and is associated with a reduction in disease recurrence and death. Thoracic RT is associated with incidental cardiac exposure, which can lead to a spectrum of radiation-induced heart disease (RIHD) ([1][1]). The mechanisms

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

Optical barcoding reveals enhanced TNFα activation and polyclonality in lung, but not liver, metastases from breast cancer., Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor–α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.

Venetoclax, a potent and selective BCL2 inhibitor, synergizes with endocrine therapy in preclinical models of ER-positive breast cancer. Using a phase Ib 3 + 3 dose-escalation and expansion study design, 33 patients with ER and BCL2-positive metastatic disease (mean prior regimens, 2; range, 0–8) were treated with daily tamoxifen (20 mg) and venetoclax (200–800 mg). Apart from uncomplicated “on-target” lymphopenia, no dose-limiting toxicities or high-grade adverse events were observed in the escalation phase (15 patients), and 800 mg was selected as the recommended phase II dose (RP2D). In the expansion phase (18 patients), few high-grade treatment-related adverse events were observed. For 24 patients treated at the RP2D, the confirmed radiologic response rate was 54% and the clinical benefit rate was 75%. Treatment responses were preempted by metabolic responses (FDG-PET) at 4 weeks and correlated with serial changes in circulating tumor DNA. Radiologic responses (40%) and clinical benefit (70%) were observed in 10 patients with plasma-detected ESR1 mutations. Significance: In the first clinical study to evaluate venetoclax in a solid tumor, we demonstrate that combining venetoclax with endocrine therapy has a tolerable safety profile and elicits notable activity in ER and BCL2-positive metastatic breast cancer. These findings support further investigation of combination therapy for patients with BCL2-positive tumors. See related commentary by Drago et al., p. 323. This article is highlighted in the In This Issue feature, p. 305

Background: Aromatase inhibitors (AI), which block the conversion of androgen to estrogen, are the mainstay therapy for estrogen receptor–positive (ER+) breast cancer (BC). However, many patients relapse. Purpose: To identify molecular alterations associated with long-term neoadjuvant AI therapy of ER+ BC. Methods: We retrospectively identified 137 patients diagnosed with ER+ BC and treated with neoadjuvant AI for at least 1 month at the Royal Marsden Hospital (2003–2016). Paired pre- (diagnosis) and post-AI (surgery) biopsies with >40% invasive cell areas were available from 87 ER+ BC patients. In all samples, we evaluated ER, PR and Ki67 immunostaining, ESR1 hot-spot mutations by droplet digital PCR and expression of 801 genes associated with BC and response to endocrine therapy by NanoString. Results: Mean time on neoadjuvant treatment was 26 weeks (range 5.6–92.3). Cell proliferation remained suppressed in most tumours indicating little evidence for acquired resistance: 56/87 (64%) showed residual Ki67 (Ki67r) 10%. This was paralleled by reduced expression of proliferation genes, ER (immunostaining and gene expression) and estrogen-regulated genes (ERG) at surgery compared with the diagnostic samples (all p6 months, giving a prevalence in this cohort of 5/34 (15%). Tumours with ESR1 mutations showed less suppression of ERGs (p=0.002) and proliferation (p=0.039) and increased ESR1 (p=0.016) expression at surgery compared with tumours without mutation. Pathway analysis confirmed lack of inhibition of estrogen response (FDR>5%) and less inhibition of cell cycle [enrichment score: -0.49 vs -0.78) and E2F targets (-0.47 vs -0.8) in tumours with mutation. Tumours without ESR1 mutation but with Ki67r>10% also showed reduced ERGs response (p=0.006) compared to tumours with Ki67r Conclusion: Overall most tumours showed no evidence for the emergence of resistant disease after neoadjuvant AI therapy even after many months of treatment. However, we detected an enrichment of ESR1 mutations (15% of cases) after long-term treatment as a putative driver of ERG expression and proliferation and thus reduced AI response. Therefore, mutant ER appears to be associated with ligand-independent ERG activity supporting the clinical validity of dual blockade with a selective ER down-regulator combined with a CDK4/6 inhibitor targeting the RB/E2F axis in this scenario. Citation Format: Leal MF, Haynes BP, Schuster E, Yeo B, Afentakis M, Dodson A, Buus R, Cheang MC, Martin L-A, Dowsett M. Acquired ESR1 mutation and persistent expression of estrogen regulated genes in ER+ breast cancers on long-term neoadjuvant treatment with aromatase inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-15-01.

Malignant pleural effusions (MPE) are a common complication of advanced cancers, particularly those adjacent to the pleura such as lung and breast cancer and are a frequent complication in metastatic disease. The pathophysiology of MPE formation in advanced breast cancer remains poorly understood, and their composition and biology are understudied. To characterise the phenotypic diversity of malignant pleural effusion, we performed single-cell RNA sequencing on 10 MPEs from 7 metastatic breast cancer patients with diverse molecular subtypes: two triple negative (TNBC) patients, three luminal B patients including one with a rare inflammatory subtype, and two luminal A patients. For all patients, we sequenced cells from the entire MPE, without performing any enrichment or selection, in order to ascertain the cellular composition and molecular phenotypes in an unbiased manner. We identified pronounced differences in the relative proportions of malignant, mesothelial and immune cell populations: both TNBC and two Luminal B patients had extensive malignant cell populations, while the other three patients had few or no detectible malignant cells but extensive immune populations. We also observed heterogeneity in the expression of subtype-specific gene signatures and in copy number aberration patterns that captured variability across malignant cell populations both within and between patients. We observed that most malignant cells retained the molecular subtype diagnosed in the primary tumour, however sub-populations of malignant cells could be identified that mapped to different molecular subtypes, indicative of complexity in the molecular phenotypes of cancer cells disseminated to or metastasising in the pleural cavity. We distinguished mesothelial cell populations from malignant cells using key markers, expression of the PAM50 signatures, and copy number aberration patterns. We found that pleural mesothelial cells expressed a cancer associated fibroblast-like transcriptomic program that may support cancer growth within the pleural cavity through the secretion of growth factors that target malignant cells. Our dataset presents the first unbiased and unselected assessment of breast cancer associated MPEs at single cell resolution, providing the community with a vital resource for the study of MPEs. Our work highlights the molecular and cellular diversity captured in MPEs and advances the use of these clinically relevant biopsies both in monitoring disease progression and in the development of targeted therapeutics for patients with advanced breast cancer. Citation Format: Holly J. Whitfield, Jean Berthelet, Stefano Mangiola, Caroline Bell, Robin L. Anderson, Bhupinder Pal, Anthony T. Papenfuss, Belinda Yeo, Delphine Merino, Melissa J. Davis. Defining the cellular composition and associated molecular phenotypes of malignant and non-malignant cells in breast cancer pleural effusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6232.

International practice guidelines recommend administration of bone-modifying agents (BMA) in metastatic breast cancer (MBC) patients with bone metastases to reduce skeletal-related events (SRE). Optimal delivery of BMA in routine clinical practice, including agent selection and prescribing intervals, remains unclear.To describe real-world practice of Australian breast oncologists.Prospective data from February 2015 to July 2020 on BMA delivery to MBC patients with bone metastases was analysed from Treatment of Advanced Breast Cancer in the Human Epidermal Growth Factor Receptor 2 (HER2)-Positive Australian Patient (TABITHA), a multi-site Australian HER2+ MBC registry.Of 333 HER2+ MBC patients, 171 (51%) had bone metastases at diagnosis, with a mean age of 58.1 years (range, 32-87). One hundred and thirty (76%) patients received a BMA, with 90 (69%) receiving denosumab and 40 (31%) receiving a bisphosphonate. Patients who received a BMA were more likely to have received concurrent first-line systemic anti-HER2 therapy (95% vs 83%; P = 0.04), to present with bone-only metastases at diagnosis (24% vs 7%; P = 0.02) and less likely to have visceral metastases (51% vs 71%; P = 0.03). Ten of 40 (25%) bisphosphonate patients and 45 of 90 (50%) denosumab patients received their BMA at the recommended 4-weekly interval. Prescribing intervals varied over time. Adverse events reported were consistent with clinical trial data.Three-quarters of Australian HER2+ MBC patients with bone metastases receive a BMA, often at different schedules than guidelines recommend. Further studies, including all MBC subtypes, are warranted to better understand clinicians' prescribing rationale and potential consequences of current prescribing practice on SRE incidence.

Patients presenting with hormone receptor-positive (HR+ ), human epidermal growth factor receptor 2-negative (HER2- ) metastatic breast cancer (MBC) are usually treated with endocrine therapy (ET), except if there is a concern about endocrine resistance or a need to achieve rapid disease control due to visceral crisis. The combination of CDK4/6 inhibitor + ET has now replaced single-agent ET as the standard first-line treatment; and it can also be considered a standard option in the second-line setting. This review briefly summarizes recently reported efficacy findings from the key phase III clinical trials of CDK4/6 inhibitor + ET in patients with HR+ /HER2- MBC, including evidence that adding a CDK4/6 inhibitor to ET improves overall survival and does so without reducing patients' quality of life. There is still much to learn regarding the use of CDK4/6 inhibitors and how they may be optimally integrated into clinical practice. In particular, there is a need for specific biomarkers that help predict the likelihood of response or resistance to CDK4/6 inhibitor therapy; and for data to guide treatment decisions when a patient's disease progresses on a CDK4/6 inhibitor.

Venous thromboembolism (VTE) is a common cause of morbidity and mortality in cancer patients. Until recently, guidelines recommended the use of low-molecular weight heparin (LMWH) as standard of care for VTE in patients with cancer. Despite the proven efficacy of direct oral anticoagulants (DOACs) for treatment of VTE, there is equipoise supporting their use in cancer patients.A systematic review of PubMed, Medline and EMBASE identified four randomised controlled trials (RCTs) in patients with cancer and VTE comparing a factor Xa inhibitor (FXaI) to LMWH. A meta-analysis was performed with a primary outcome of VTE recurrence and key secondary outcomes of major bleeding, clinically relevant non-major bleeding (CRNMB) and gastrointestinal (GI) bleeding.Four RCTs with 2,907 patients were included. 1,451 patients were randomised to FXaI and 1,456 to LMWH. VTE recurrence was lower in the FXaI group (RR 0.62, 95%CI 0.44-0.87; p=0.01; IIn patients with cancer and VTE, the rate of VTE recurrence was significantly lower with FXaI than with LMWH without an increased risk of major bleeding. Our data supports the use of FXaIs as the standard of care for the treatment of VTE in this population.

AIM The majority of women diagnosed with early breast cancer have hormone-receptor positive (HR+)/HER2-negative disease. Adjuvant endocrine therapy provides substantial risk reduction benefits in virtually all patients. The role of adjuvant chemotherapy in certain subsets of patients is equivocal. This paper sought to evaluate the role of the IHC4+C score to aid this clinical decision in addition to providing an overview of the current molecular and non- molecular tools available in the adjuvant setting. METHODS This prospective study included 53 post-operative HR+/HER2- negative early breast cancer patients selected from the multidiscipliniary team meeting between August 2017 and January 2020. IHC4+C testing was requested by clinicians for patients in whom the availability of the score may have impacted adjuvant decision-making. Adjuvant treatment decisions were recorded at three time points (prior and post IHC4+C scoring as well as the patient's final decision). The primary goal was the proportion of patients who were spared chemotherapy following the availability of IHC4+C scores to impact on clinicians' recommendations for adjuvant systemic therapy. RESULTS A total of 34 patients (64%) were initially recommended to undergo chemotherapy or to consider chemotherapy. With the availability of the IHC4+C score, only 17 patients (32%) underwent chemotherapy, demonstrating a substantial reduction in the frequency of chemotherapy prescribing. CONCLUSION This study demonstrates that when utilized appropriately in a multidisciplinary setting, the IHC4+C algorithm is an alternative, reproducible and affordable tool with a proven capacity to stratify risk and to spare a large proportion of patients from undergoing chemotherapy.

BACKGROUND: Anti-HER2 therapy-related cardiotoxicity is well described in the context of clinical trials, particularly in the setting of early stage disease, but there is more limited data in advanced breast cancer and in the real world setting. MATERIAL AND METHODS: A prospectively-maintained registry database with 312 consecutive patients diagnosed with HER2 positive advanced breast cancer in Australia was analysed. RESULTS: 287 patients (92%) received anti-HER2 therapy, 17 (6%) experienced anti-HER2 therapy-related cardiotoxicity. Patients who experienced cardiotoxicity were more likely to have ≥2 risk factors for cardiotoxicity (OR 3.9 95% CI 1.4-11.3 p = 0.01). A prior diagnosis of cardiovascular disease was significantly associated with cardiotoxicity (OR 7.1 95% CI 1.3-39.5). Cardiotoxicity resolved on imaging in 65% of patients; there was no association between severity and resolution. 11 patients (65%) received cardiologist input. Of the patients who developed cardiotoxicity, 12 patients (71%) received further anti-HER2 therapy in the first- or second-line setting without recurrent cardiotoxicity. DISCUSSION AND CONCLUSION: Therapy-related cardiotoxicity is an uncommon complication of anti-HER2 therapy in the real world setting. Cardiac toxicity resolved in the majority of affected patients, and further anti-HER2 therapy was administered without recurrence of cardiac issues. Our data suggests anti-HER2 therapy can be safely given in routine care, even in patients with risk factors for toxicity.

Granulocyte-colony stimulating factor (G-CSF) is one of several cytokines that can expand and mobilize haematopoietic precursor cells from bone marrow. In particular, G-CSF mobilizes neutrophils when the host is challenged by infection or tissue damage. Severe neutropenia, or febrile neutropenia is a life-threatening event that can be mitigated by administration of G-CSF. Consequently, G-CSF has been used to support patients undergoing chemotherapy who would otherwise require dose reduction due to neutropenia. Over the past 10-15 years it has become increasingly apparent, in preclinical tumour growth and metastasis models, that G-CSF can support tumour progression by mobilization of tumour-associated neutrophils which consequently promote tumour dissemination and metastasis. With the increasing use of G-CSF in the clinic, it is pertinent to ask if there is any evidence of a similar promotion of tumour progression in patients. Here, we have reviewed the preclinical and clinical data on the potential contribution of G-CSF to tumour progression. We conclude that, whilst the evidence for a promotion of metastasis is strong in preclinical models and that limited data indicate that high serum G-CSF levels in patients are associated with poorer prognosis, no studies published so far have revealed evidence of increased tumour progression associated with supportive G-CSF use during chemotherapy in patients. Analysis of G-CSF receptor positive cohorts within supportive trials, as well as studies of the role of G-CSF blockade in appropriate tumours in the absence of chemotherapy could yield clinically translatable findings.

Inflammatory breast cancer is a rare, yet highly aggressive form of breast cancer, which accounts for less than 5% of all locally advanced presentations. The clinical presentation of inflammatory breast cancer often differs significantly from that of noninflammatory breast cancer; however, immunohistochemistry reveals few, if any, distinguishing features. The more aggressive triple-negative and HER2-positive breast cancer subtypes are overrepresented in inflammatory breast cancer compared with noninflammatory breast cancer, with a poorer prognosis in response to conventional therapies. Despite its name, there remains some controversy regarding the role of inflammation in inflammatory breast cancer. This review summarises the current molecular evidence suggesting that inflammatory signaling pathways are upregulated in this disease, including NF-κB activation and excessive IL-6 production among others, which may provide an avenue for novel therapeutics. The role of the tumor microenvironment, through tumor-associated macrophages, infiltrating lymphocytes, and cancer stem cells is also discussed, suggesting that these tumor extrinsic factors may help account for the differences in behavior between inflammatory breast cancer and noninflammatory breast cancer. While there are various novel treatment strategies already underway in clinical trials, the need for further development of preclinical models of this rare but aggressive disease is paramount.

Treatment strategies for hormone receptor-positive (HR+ ), human epidermal growth factor receptor 2-negative (HER2- ) metastatic breast cancer in young women (

Background Pertuzumab combined with trastuzumab and docetaxel is the standard first-line therapy for HER2-positive metastatic breast cancer, based on results from the phase III CLEOPATRA trial. PERUSE was designed to assess the safety and efficacy of investigator-selected taxane with pertuzumab and trastuzumab in this setting. Patients and methods In the ongoing multicentre single-arm phase IIIb PERUSE study, patients with inoperable HER2-positive advanced breast cancer (locally recurrent/metastatic) (LR/MBC) and no prior systemic therapy for LR/MBC (except endocrine therapy) received docetaxel, paclitaxel or nab-paclitaxel with trastuzumab [8mg/kg loading dose, then 6mg/kg every 3weeks (q3w)] and pertuzumab (840mg loading dose, then 420mg q3w) until disease progression or unacceptable toxicity. The primary end point was safety. Secondary end points included overall response rate (ORR) and progression-free survival (PFS). Results Overall, 1436 patients received at least one treatment dose (initially docetaxel in 775 patients, paclitaxel in 589, nab-paclitaxel in 65; 7 discontinued before starting taxane). Median age was 54years; 29% had received prior trastuzumab. Median treatment duration was 16months for pertuzumab and trastuzumab and 4months for taxane. Compared with docetaxel-containing therapy, paclitaxel-containing therapy was associated with more neuropathy (all-grade peripheral neuropathy 31% versus 16%) but less febrile neutropenia (1% versus 11%) and mucositis (14% versus 25%). At this preliminary analysis (52 months' median follow-up), median PFS was 20.6 [95% confidence interval (CI) 18.9–22.7] months overall (19.6, 23.0 and 18.1months with docetaxel, paclitaxel and nab-paclitaxel, respectively). ORR was 80% (95% CI 78%–82%) overall (docetaxel 79%, paclitaxel 83%, nab-paclitaxel 77%). Conclusions Preliminary findings from PERUSE suggest that the safety and efficacy of first-line pertuzumab, trastuzumab and taxane for HER2-positive LR/MBC are consistent with results from CLEOPATRA. Paclitaxel appears to be a valid alternative taxane backbone to docetaxel, offering similar PFS and ORR with a predictable safety profile. ClinicalTrials.gov NCT01572038.

To investigate the presence ofWe evaluated ER, progesterone receptor, and Ki67 by immunostaining,Cell proliferation and estrogen-regulated genes (ERG) remained suppressed in most tumors indicative of persistent response to NAI. Enrichment ofLigand-independent ER signaling, as a result of