Unsupervised hierarchical clustering showing the difference on gene expression modules scores from baseline to surgery in the POETIC-treated subset (gene expression changes: surgery-baseline). The module scores shown in this heatmap are those selected at baseline by two unpaired SAM analysis between Ki67 H-H versus H–L, categories in the POETIC subset and annotated by the main categories. ER, estrogen receptor; expression, gene expression; H-H, Ki67 Highbaseline- Ki67 Highsurgery; H-L, Ki67 Highbaseline- Ki67 Lowsurgery; Her2-E, Her2 enriched; LumB, Luminal B; LumA, Luminal A; 2 wk; 2-week time point.

Differential single-gene expression changes between baseline and surgery of the 17 genes included in the FOS and JUN module scores in the POETIC cohort. Differential expression in the treated samples from POETIC subset (A) and in the controls (B). In red, there are the significant genes by P values by paired t tests and log2FC. FDR, false discovery rate; log2 FC, log2 fold change; NS, nonsignificant; P, significant by P value paired t tests; P&log2FC, significant by P value and log2 fold change.

Boxplots showing changes in gene expression from baseline to surgery of the two immune-related signatures (“durvalumab” and “immune-tolerance”) among H-H and H-L Ki67 response categories in the POETIC-treated subset (A) and in the NeoAI study (B). Boxplots showing gene signature expression of the two immune-related signatures at surgery stratified by H-H and H-L tumors in the POETIC-treated subset (C) and in the NeoAI study (D). H-H, Ki67 Highbaseline- Ki67 Highsurgery; H-L, Ki67 Highbaseline- Ki67 Lowsurgery; Her2-E, Her2 enriched; LumB, Luminal B; LumA, Luminal A; 2 wk, 2-week time point.

Differences in intrinsic subtype classification from baseline to surgery in the POETIC subset and the NeoAI study. Changes of intrinsic subtype classifications in all the POETIC-treated samples (A); in POETIC Luminal B–treated samples (B); in POETIC control samples (C); in POETIC Luminal B control samples (D); in all the NeoAI study samples (E), and in NeoAI Luminal B samples (F). Her2-E, Her2 enriched; LumB, Luminal B; LumA, Luminal A; 2 wk, 2-week time point.

PURPOSE: Aromatase inhibitor (AI) treatment is the standard of care for postmenopausal women with primary estrogen receptor-positive breast cancer. The impact of duration of neoadjuvant endocrine therapy (NET) on molecular characteristics is still unknown. We evaluated and compared changes of gene expression profiles under short-term (2-week) versus longer-term neoadjuvant AIs. EXPERIMENTAL DESIGN: Global gene expression profiles from the PeriOperative Endocrine Therapy for Individualised Care (POETIC) trial (137 received 2 weeks of AIs and 47 received no treatment) and targeted gene expression from 80 patients with breast cancer treated with NET for more than 1 month (NeoAI) were assessed. Intrinsic subtyping, module scores covering different cancer pathways and immune-related genes were calculated for pretreated and posttreated tumors. RESULTS: The differences in intrinsic subtypes after NET were comparable between the two cohorts, with most Luminal B (90.0% in the POETIC trial and 76.3% in NeoAI) and 50.0% of HER2 enriched at baseline reclassified as Luminal A or normal-like after NET. Downregulation of proliferative-related pathways was observed after 2 weeks of AIs. However, more changes in genes from cancer-signaling pathways such as MAPK and PI3K/AKT/mTOR and immune response/immune-checkpoint components that were associated with AI-resistant tumors and differential outcome were observed in the NeoAI study. CONCLUSIONS: Tumor transcriptional profiles undergo bigger changes in response to longer NET. Changes in HER2-enriched and Luminal B subtypes are similar between the two cohorts, thus AI-sensitive intrinsic subtype tumors associated with good survival might be identified after 2 weeks of AI. The changes of immune-checkpoint component expression in early AI resistance and its impact on survival outcome warrants careful investigation in clinical trials.

Single-gene expression changes of genes in common between baseline and surgery in the different cohorts. A, Scatterplot of differentially expressed genes between baseline and surgery measured by log2 FC among the entire short-term and long-term AI cohorts. B, Scatterplot of differentially expressed genes between baseline and surgery measured by log2 FC among Luminal B–treated tumors only in the short-term and long-term AI cohorts C, Boxplot showing the intersections of common genes differentially expressed between baseline and surgery among different combinations of subgroups of sample patients within the POETIC and NeoAI cohorts: all treated patients in POETIC, only treated with Luminal A in POETIC, only treated with Luminal B in POETIC, all patients in NeoAI, only Luminal A in NeoAI, only Luminal B patients in NeoAI, only controls in POETIC. FDR, false discovery rate; log2 FC, log2 fold change.

BACKGROUND:Circulating tumour DNA (ctDNA)-based detection of molecular residual disease (MRD) presents a strategy to identify patients at high risk of relapse. In this article, we profile early breast cancer patients with an ultrasensitive, whole genome sequencing (WGS)-based, tumour-informed ctDNA platform. MATERIALS AND METHODS:We analysed 617 plasma samples (median 8, range 2-14) from 78 patients (23 triple-negative breast cancer, 35 human epidermal growth factor receptor 2-positive, 18 hormone receptor-positive, and 2 unknown). Samples were collected at diagnosis before therapy, cycle 2 of neoadjuvant chemotherapy, post-surgery after neoad'juvant therapy if administered, every 3 months during the first year, and every 6 months thereafter. Plasma DNA was analysed using the NeXT Personal MRD platform, a tumour-informed WGS approach to produce personalized ctDNA sequencing panels tracking a median of 1451 variants per patient. MRD detection was correlated with clinical outcomes. RESULTS:ctDNA was detected at levels ranging from 2.19 parts per million (PPM) to 204 900 PPM (median 405 PPM), with 39% of all ctDNA detections in the ultra-low range <100 PPM. Of patients with samples at diagnosis, 98% (49/50) had ctDNA detected before treatment. At a median follow-up of 76 months (range 5-118 months), detection of ctDNA was associated with high risk of future relapse (P < 0.0001; log-rank test) and shortened overall survival (P < 0.0001) with a median lead time from ctDNA detection to clinical relapse of 15 months (range 0.9-61.5 months). MRD was identified in 100% (11/11) of patients who relapsed, with a median level of ctDNA at first MRD detection of 13.1 PPM. No ctDNA-undetected patients relapsed throughout follow-up (64/64). Comparison with exome-powered MRD detection assays showed improved sensitivity and lead time. CONCLUSIONS:A whole genome-powered MRD assay detected breast cancer relapse with a long lead time over clinical relapse, and was strongly associated with relapse-free survival. Rates of ctDNA detection at diagnosis were higher than those reported with exome-based tumour-informed assays.

Module scores expression changes in the POETIC cohort. A, Barplots showing the significant module scores expression changes between baseline and after 2 weeks of AI in the POETIC dataset for all samples, for Luminal B samples only (B) and for controls (C). The x-axis shows the log2FC and the y-axis shows the significant module scores that changed. Bars are colored by the degree of significance of the P value by paired t test. FDR; false discovery rate; log2 FC, log2 fold change.

1010 Background: Circulating tumor DNA (ctDNA) based detection of Molecular Residual Disease (MRD) in breast cancer patients presents a strategy to identify patients at high risk of relapse, although current assays have limited sensitivity for low level ctDNA detection. In this study we profile early breast cancer patients with an ultra-sensitive, whole genome sequencing-based, tumor-informed ctDNA platform, and correlate the findings with clinical outcomes. Methods: We analysed 598 plasma samples (median 8 samples/patient, range 2-14) from 76 patients with early breast cancer (23 TNBC, 33 HER2+, 16 HR+ and 4 unknown) enrolled in the ChemoNEAR study. Samples were collected at diagnosis before therapy, at Cycle 2 of neoadjuvant chemotherapy (NAC), post-surgery after neoadjuvant therapy if administered, and every 3 months during follow-up for the first year, and subsequently every 6 months for up to five years. Plasma cfDNA was analysed using the NeXT Personal ctDNA based MRD platform, a tumour-informed approach leveraging whole-genome sequencing of tumor and normal samples to produce personalised ctDNA sequencing panels, with each bespoke panel consisting of up to ~1,800 selected variants that enable ultra-sensitive MRD detection. MRD detection was correlated with clinical outcomes and histopathological data. Results: We detected a broad range of ctDNA levels ranging from 204,900 PPM to 2.2 PPM (median 296 PPM). 40% of all ctDNA detections were in the ultra-low range of <100 PPM. 97.8% (45/46) of patients had ctDNA detected at baseline prior to treatment, including 100% TNBC, 100% HER2+, and 83% of HR+ patients. At a median follow-up of 76 months (range 5-113) from study entry, detection of ctDNA associated with high risk of future relapse (HR undefined, p<0.0001; log-rank test) and shortened overall survival (p<0.0001; log-rank test) with median lead-time from ctDNA detection to clinical relapse of 12.5 months (range 1-60). MRD was identified in 100% (10/10) of patients who relapsed. Of the relapsed patients, the median level of ctDNA at first MRD detection was 18.5 PPM. No ctDNA undetected patients relapsed throughout follow up (58/58). Three patients had ctDNA detected in follow-up but had not relapsed at the time of data cut off. For patients that were ctDNA undetected at post-surgical landmark, 94% (31/33) patients did not relapse. Conclusions: NeXT Personal detected breast cancer relapse with a long lead-time over clinical relapse, and strongly associated with relapse free survival. The assay demonstrated high rates of ctDNA detection at diagnosis. The strong negative predictive value at landmark suggests potential use in de-escalation studies. Several prospective, interventional trials are being conducted to assess whether treatment based on MRD detection improves outcome.

Abstract Introduction Untreated obstructive sleep apnoea (OSA) is associated with 3-fold increased risk of a motor vehicle collision. To date research in this area has focused on driving simulators, crash records and emergency room reports each of which has limitations. Predictors of poor on road driving performance with OSA are not well developed. Methods We are recruiting to a study (ODESA), to assess on-road driving performance, collected through a smartphone app developed by our industry partner (Sentiance). Participants are recruited from referrals for investigation of possible OSA. Here, we present preliminary data from the first participants to assess if our measures of driving distinguish varying levels of performance between drivers, and if any measure is correlated with levels of oxygen desaturation (ODI), and Epworth Sleepiness Scale (ESS). Driving scores of Hard Events, Hard Acceleration and Hard Turning were compared across participants using one-way ANOVA’s, and tests of correlation with ODI and ESS were computed using Spearman's Rho. Results 10 participants (51 +/- 12 years [mean +/- SD], 4 female), have recorded 683 journeys (68 +/- 36 [mean +/- SD]). Significant effects of driving scores between participants, were found for Hard Events (F[9,533]=16.3, p< 0.00001), and Hard Acceleration (F[9,592]=5.64, p< 0.00001), but not Hard Turning (F[9,590]=1.48, p= 0.15). There was no reliable relationship between either ODI (10.2 +/- 15.6 [mean +/- SD]) and Hard Events (rs=.18, p=.63), Hard Acceleration (rs=.25, p=.49) and Hard Turning (rs=-.14, p=.7), or EES (10.9 +/- 4.8 [mean +/- SD]) and Hard Events (rs= .08, p=.83), Hard Acceleration (rs=.31, p=.39) and Hard Turning (rs= -.02, p=.96). Conclusion This preliminary analysis shows that despite finding variation in driving performance measures of Hard Events and Acceleration between participants, neither OSA severity or subjective sleepiness (EES), was correlated with the current data configuration. The ODESA study plans to test if neurophysiological measures (EEG), and temporal experience traces (TET), are more informative measures of driving behaviour in patients with suspected OSA. Support (if any)

Detection of molecular residual disease (MRD) allows for the identification of breast cancer patients at high-risk of recurrence, with the potential that early initiation of treatment at early stages of relapse could improve patient outcomes. The Invitae Personalized Cancer Monitoring™ assay (PCM) is a newly developed next-generation sequencing approach that utilizes up to 50 patient-specific, tumor-informed DNA variants, to detect circulating tumor DNA (ctDNA). The ability of the PCM assay to detect MRD before clinical relapse was evaluated. The cohort included 61 female patients with high-risk breast cancer who underwent neoadjuvant chemotherapy. Plasma samples were collected before and during neoadjuvant therapy, after surgery and during monitoring. PCM was used to detect ctDNA at each time point. The sensitivity to detect ctDNA in plasma from patients who relapsed during the monitoring phase was 76.9

Background: In early breast cancer (EBC) patients, we aimed to determine whether circulating tumor DNA (ctDNA) analysis following primary surgery, before systemic therapy, identified molecular residual disease and was associated with risk of relapse and relapse-free survival (RFS). Methods: Plasma was collected, retrospectively, before surgery, 1-14 weeks post-operatively, and before adjuvant therapy, and in a subset of patients after adjuvant therapy. A personalized, tumor-informed, multiplex PCR next generation sequencing assay (SignateraTM) was used for ctDNA detection and quantification. The primary objective was to compare RFS and distant recurrence-free survival (DRFS) in patients with detected versus non-detected ctDNA. Results: A total of 48 patients with EBC (median age 50.5 years) [34 hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-), 5 HER2+, 9 triple-negative breast cancer) were included. ctDNA was detected in 64.5% (20/31) of patients before surgery, and 35.4% (17/48) after surgery. ctDNA detection before surgery was associated with tumor grade (P = 0.019), ctDNA detection after surgery was associated with receptor subtype (P = 0.01). Patients with ctDNA detected after surgery had worse DRFS [hazard ratio = 5.5, 95% confidence interval (CI) 1.1-28.5, P = 0.04]. RFS in patients with ctDNA detected after surgery was worse than in those with lack of ctDNA detection, although not statistically significant (hazard ratio = 3.7, 95% CI 0.9-15.7, P = 0.073). Patients with ctDNA detected preoperatively or post-operatively had a trend towards worse RFS (hazard ratio = 7.8, 95% CI 0.9-63.7, P = 0.05) and DRFS (hazard ratio = 6.8, 95% CI 0.8-57, P = 0.07) compared with those with ctDNA undetected at both timepoints. ctDNA detection anticipated clinical relapse with a median lead time of 16 months. Conclusions: In patients with treatment-naive EBC, ctDNA is detectable after surgery. The absence of ctDNA at a single post-surgical timepoint is associated with improved DRFS, supporting the development of future trials studying deescalation of systemic therapy.

Modern neoadjuvant systemic therapy (NST) can result in high pathologic complete response rates (pCR) in triple negative (TN) and human epidermal growth factor receptor 2 positive (HER2+) breast cancer. The role of surgery is, therefore, being reconsidered in this rapidly evolving field. This report presents oncological outcomes of seven patients with TN or HER2+ breast cancer, with exceptional response to NST, and a post-NST image-guided vacuum assisted biopsy showing no residual disease (ypT0), who opted not to have breast surgery. The median age was 49 (IQR 36-61) years and the median tumour size at diagnosis was 50 (IQR 16-65) mm. All patients received breast radiotherapy and continued adjuvant systemic therapies as appropriate. At a median follow-up of 67 (IQR 61-77) months, all patients were alive and free of disease. This small case series supports the need for further research in 'exceptional responders' to provide safe, individualized patient-centred care.

560 Background: The impeded anti-proliferative response of Estrogen Receptor positive (ER+) HER2+ breast cancer (BC) to endocrine therapy (ET) has been considered negated by treatment with anti-HER2 therapies but residual disease after anti-HER2 therapy remains at risk of recurrence. Studying the molecular changes (MolC) of ER+HER2+ BC in response to ET will provide clinical insights to treatment options. Methods: POETIC was a phase III trial of post-menopausal patients with ER+ BC randomized 2:1 to 2-weeks of peri-operative aromatase inhibitors (POAI) vs control, followed by standard-of-care. Paired pre-treatment (B) and on-treatment (2wk) samples from 313 ER+HER2+ BC (213 POAI/100 controls) were gene expression profiled (BC 360 codeset; NanoString). Association of MolC with Early biological response to AI was assessed by residual Ki67 2wk (low ≤10%; high >10%) by T-test, multiple testing corrected by Benjamini & Hochberg (FDR); with time to recurrence (TTR) was estimated using multivariable Cox regression models adjusted for post-surgery clinicopathological variables and age as adjuvant treatment surrogate. Results: In POAI tumours, immunity-related signatures and mammary stemness were significantly upregulated in responders while proliferation, DNA-damage repair (DDR), TP53mutational status and ER-signaling were downregulated (FDR<0.05). Controls had exclusive downregulation of PDL1 and upregulation of hypoxia (FDR < 0.05). We previously identified 5 new molecular subgroups based on baseline gene expression (1- Immune high, ESR1 low; 2- ECM, ESR1 low, highest ERBB2; 3- DDR deficiency, 4- Endocrine signalling high and 5- Endocrine and PI3K/MAPK/RAS signalling high) which were associated with different response to AI and differential outcome. Endocrine-signalling, PI3K/MAPK/RAS signalling, tumour-immunity and chemokines were upregulated at a higher magnitude in AI sensitive subgroups (3 and 4) while the molecular subgroup with early resistance to AI and poorer outcome at baseline (2) did not show significant changes. Intrinsic subtype (IS) shifting was significantly more prevalent in treated (37%, 79/213) than in controls (14%, 14/100) (p <0.001). In POAI most Luminal B (LumB) shifted to Luminal A (LumA; 79%, 59/75), driven by a reduction in proliferation. LumA 2wk was associated with better outcome compared to LumB 2wk (HR 0.2; CI95% 0.06-0.72, p=0.01). IS 2wk provides additional information predicting TTR, than IS B (AIC value = 217.2 vs 221.6). Conclusions: We provide a comprehensive picture on how ER+HER2+ BC gene expression profile changes in response to ET. Most LumB BC shift to LumA, being LumA 2wk IS significantly associated with better outcome. There is a clinical utility of peri-operative ET for ER+HER2+ BC, guiding who would have good prognosis for adjuvant ET and who may need additional therapy. Clinical trial information: NCT02338310 .

Clinicians and people with narcolepsy report varied access to higher-cost narcolepsy treatments in England associated with variations in national and local commissioning. There are no publicly available data quantifying use of these drugs to support policy decisions. We therefore aimed to describe national, regional and local prescribing trends for higher-cost narcolepsy drugs using new national databases. We used the English prescribing dataset and secondary care medicines data to quantify volumes of high-cost narcolepsy drugs issued between 01 January 2019 and 31 December 2022. Volumes were converted to World Health Organisation defined daily doses, to estimate the monthly number of defined daily doses of sodium oxybate, pitolisant and solriamfetol issued by each integrated care board and region. We compared national, integrated care board, and regional level issuance of each drug over time. Analysis of almost 6000 primary care prescriptions and 2000 cumulative months of secondary care pharmacy stock data, issued across 41/42 integrated care boards in England, revealed a 49.1% increase in issuance of high-cost narcolepsy drugs between 2019 and 2022. In 2022, sodium oxybate accounted for 52.66% of issuance, pitolisant 43.09% and solriamfetol 4.25%, with 22.31% of defined daily doses issued in primary care. Three integrated care boards (NHS Southeast London, NHS Cumbria and North-East, NHS Cheshire and Merseyside) predominate, issuing 56.33% of all defined daily doses. Variations between integrated care boards and regions differ substantially by drug and route of issuance. Our findings describe substantial variation in the use of specialist narcolepsy drugs in England, and highlight the untapped potential of using large, public domain datasets to publicly review higher-cost drug prescribing.

Abstract Background: Mechanisms of resistance to endocrine therapy are not well understood within ER+HER2+ breast cancer (BC). Our prior work suggested that intrinsic HER2-Enriched (HER2E) molecular subtype predicts early resistance to aromatase inhibitors (AI) (Bergamino eBioMedicine 2022) and high on-treatment (on-Txt) Ki67 levels predict poor survival (Smith Lancet Oncol 2020). Improved early detection of persistent proliferating tumor cells with endocrine resistance pathways could be targeted by pre-emptive personalized therapy and reduction in recurrence. In this study, we proposed to further identify additional alterations/features from genomic and spatial data to provide unprecedented new insight into intrinsic and adaptive resistant pathways in tumor cells that may assist to identify molecular targets for treatment. Materials: POETIC was a phase III trial of post-menopausal patients with ER/PR+ invasive BC (n = 4480) randomized 2:1 to 2 weeks of peri-operative AI (POAI) vs control, followed by standard-of-care treatment. Ki67 was assessed by IHC and intra-tumor heterogeneity was evaluated (5-15 regions) for all the POETIC POAI samples (N = 2487). ER+HER2+ samples were classified as good responders (GR) or poor responders (PR) based on a reduction in Ki67 between pre-treatment (pre-Txt) and 2-week on-Txt samples. Tumor-infiltrating lymphocytes were assessed; multiplex Immunofluorescence (mIF) was performed to measure immune cell densities in tumor and stroma compartments (CD3, CD20, CD68, FOXP3, and CD3 FOXP3 co-expression). Gene expression profiles by BC360™ (Nanostring) on all 210 pairs of POAI treated ER+/HER2+; whole exome sequencing (WES, 100X) were performed on pre-Txt tumor and blood samples from 13 GR, 17 PR, and 9 HER2E GR. We performed GeoMx Whole Transcriptome on 4 pairs (pre-Txt and on-Txt) of GR and GeoMx Proteins (77 including IO proteins) on 6 pairs of GRs and 6 pairs of PRs. Results: The most frequently mutated genes were TP53, PIK3CA, GATA3, and CHD4. Only TP53 was associated with PR (Fisher’s exact p=0.01). TP53 mutated cases had higher expression of TP53 mutant-like gene expression signature compared to wild-type cases (Wilcoxon test p=0.001), mIF FOXP3 (Wilcoxon test p = 0.0005), and CD68 (Wilcoxon test p = 0.019) density score. However, within the HER2-E subset, we found that TP53 mutations were associated with GR (Fisher’s exact p=0.02). We found spatial heterogeneity of Ki67 IHC levels across POAI samples. Examining IHC, while there was higher heterogeneity of Ki67 in the ER+HER2- samples (n = 2264) with 3% of pre-Txt and 9% on-Txt, 6% of ER+HER2+ samples (13/223, 6 LumA, 5 LumB, and 2 HER2E) showed heterogeneity of Ki67 exclusively on-Txt. Even in GR tumors with Ki67 < 10% on-Txt, we identified hotspots with retained proliferating Ki67+ cells after 2 weeks of POAI. The lobular tumors were GR and had characteristic CDH1 mutations. Importantly, cases with persistent areas of Ki67+ cells, regardless of Her2 status, were associated with late relapse. To further explore intratumoral heterogeneity, we performed spatial whole transcriptomics profiling on 95 regions from 4 pairs of GR samples (Ki67 > 10% at baseline and Ki67 < 10% on-Txt) and found low intratumoral heterogeneity in the pre-Txt samples that increased at 2 weeks on-Txt. In a larger set of samples including both GR and PR with the GeoMx protein method, we found increased intratumoral heterogeneity in the PR vs GR. Conclusion: While TP53 mutation was generally a predictor of poor response; in HER2-E it paradoxically was associated with a good early response to aromatase inhibitor which warrants further investigation. Ki67 levels in ER+HER2+ showed higher intratumoral heterogeneity in a subset of patients on treatment suggesting the potential of persistent, proliferating cells leading to later recurrence. Our spatial RNA and protein data further observe the intratumoral heterogeneity that identifies pathways for use as potential spatial biomarkers. Citation Format: Maggie Chon U Cheang, Xixuan Zhu, Orsolya Sipos, Anastasia Alataki, Mikayla Feldbauer, Elena López-Knowles, Holly Tovey, Lucy Kilburn, Milana Bergamino Sirvén, Dhrusti Patel, Hui Xiao, Perry Maxwell, Anthony Skene, Chris Holcombe, Manuel Salto-Tellez, Nicholas Turner, Andrew Dodson, Ian Smith, John Robertson, Judith Bliss, Gene Schuster, Roberto Salgado, Mitch Dowsett, Katherine A Hoadley. Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC Trial [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 PS09-08.

Supplementary Figure 1. Correlations between expression levels of ER, PgR, HER2 or EGFR and Ki67 in baseline tumor samples