Citations

Abstract Background: Androgen receptor (AR) is widely expressed in ER+ breast cancers. Accumulated evidence showed that AR is a tumor suppressor in ERα+ breast cancers. Enzalutamide (ENZ), an AR antagonist, blocks AR signalling, and was reported to inhibit estrogen-driven tumor growth as effectively as tamoxifen. Hence, this study was performed to further examine the mechanism of action of ENZ in ERα+ breast cancers. Methods: The AR gene was knocked out using the CRISPR-Cas9 system. Four crRNAs targeting AR and trans-activating crRNA were co-transfected to Cas9-positive MCF7 and T47D cells, two ERα+ breast cancer cell lines with relatively low AR levels based on RNA seq. ESR1 knock down was performed using a lentivirus system. MCF7 and T47D cells were infected with the ESR1-shRNA lentivirus and selected by puromycin for 2 weeks. Single clones of knockdown or knockout cells were tested using western blot. MCF7 and T47D cells were collected for RNA-seq and ChIP-seq after treated with DMSO or 10µM ENZ for 6 hours in complete medium. All sequences were aligned to the Human Reference Genome (hg38, UCSC). Differential gene expression was performed using limma package and upstream analysis was done in Ingenuity Pathway Analysis software. ChIP peaks were called using MACS2 and overlapping was analyzed using ChIPpeakAnno package in R. Tritium labeled ENZ or estradiol were incubated with ERα overnight. The bound radioactivity was measured using a Beckmann LS 6500 liquidscintillation counter. Results: We found the cell growth inhibition effect of ENZ was little impaired by AR gene knock out in MCF7 and T47D cells as was expected. As ENZ has been shown to inhibit estrogen-driven tumor growth, we performed studies to understand the mechanism of ENZ.We performed RNA-seq and identified 193 differential expressed genes (76 up and 117 down) between DMSO and ENZ-treated MCF7 cells (FC>2 or <0.5, FDR<0.05). Upstream analysis showed that ESR1 might be the upstream regulator of those differentially expressed genes and ESR1 signaling was predicted to be inhibited after ENZ treatment (P=1.69×10-10). To examine the alteration of ERα genomic binding, we performed global ERα ChIP-seq and found 39.58% (8,681/21,933) of ERα-bound sites were abolished by ENZ and the binding intensity was significantly decreased upon ENZ treatment in 12,868 shared peaks. Moreover, radiolabled binding assays showed that ENZ displaced estrogen binding to ERα and directly bound to ERα with a KD= 399.60nM. We further observed a reduction of proliferation inhibition of ENZ in ESR1-KD cells compared with control cells. This suggests that ENZ inhibits ER+ cell growth by displacing E2 from ERα binding sites and subsequently diminishes ERα genomic binding and blocks ERα signaling in ER+ cancers. Conclusion: We found that enzalutamide inhibits cell growth not through AR but by antagonizing ERα activity in MCF7 and T47D ER+ cells. These results reveal ER is the main factor affecting enzalutamide efficacy in these two ERα+ breast cancer cell lines. Further clinical trials are needed to investigate AR antagonists in ERα+ breast cancers. Citation Format: Lixuan Wei, Jia Yu, Huanyao Gao, Huan Zhang, Thanh Nguyen, Yayun Gu, Richard M. Weinshilboum, James N. Ingle, Liewei Wang. Estrogen receptor is a target of enzalutamide in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-03.

Abstract Background: Hormone receptor-positive (HR+) breast cancer (BC) accounts for ~60-70% of all BC cases. CDK4/6i combined with endocrine therapy (ET) is the standard of care for first- and second-line therapy of patients with HR+ metastatic breast cancer (MBC) based on substantial improvements in progression free survival (PFS) and overall survival compared to ET alone. Unfortunately, most patients (pts) develop resistance to CDK4/6i. The mechanisms underlying CDK4/6i resistance have not been fully elucidated. Robust in vivo CDK4/6i resistant animal models from pts with de novo and acquired CDK 4/6i resistance are lacking. Patient-derived xenografts (PDXs) are important preclinical tools in oncology to advance our understanding of cancer biology and to evaluate drug response phenotypes. Unfortunately, the establishment of sustainable PDXs from pts with HR+ BC is much more difficult than from HR-negative BC. In order to obtain clinically relevant models that can be utilized to better understand mechanisms of primary and acquired resistance to ET and CDK4/6i, we report the development of PDXs from a prospective study in which percutaneous biopsies were obtained from women receiving palbociclib for the treatment of HR+ advanced or MBC (PROMISE; NCT03281902). Methods: PROMISE (Prospective Study to Evaluate the Role of Tumor Sequencing in Women Receiving Palbociclib for Advanced Hormone Receptor (HR)-Positive Breast Cancer) is a prospective trial which enrolled pts with HR+, HER2-negative MBC who planned to start treatment with palbociclib and letrozole (1st line [FL]) or palbociclib and fulvestrant (2nd line [SL]). For pts enrolled at Mayo Clinic Rochester (MCR), serial tumor biopsies were obtained prior to the start of treatment and at disease progression to establish PDXs. Because baseline take rate was low, we amended the protocol to attempt PDX generation after the 2nd cycle (C2) of therapy. Tissues were received within an hour of excision and were orthotopically implanted in the mammary fat pad of 6~8-week-old nonobese diabetic (NOD)/SCID/IL-2g-receptor null (NSG) mice. Tumor growth was defined as tumor volume ≥ 50 mm3. Tumor take rate was defined as percent of patients with development of at least one stably transplantable (passed at least for two generations) xenograft that was pathologically confirmed as BC. Results: Of the 50 pts enrolled at MCR, 37 were FL pts (2 ineligible) and 13 were SL pts (1 ineligible). Fresh tumor samples obtained pre-treatment from 43 eligible pts (32 FL), after C2 from 17 eligible pts (13 FL), and at disease progression from 10 eligible pts (5 FL) were implanted. Tumor growth was seen in at least one mouse from a pre-treatment specimen in 1 (3.1%) FL pt (confirmed BC) and 4 (36.4%) SL pts (2 confirmed BC); from end of C2 specimens in 2 (15.4%) FL pts and 2 (50%) SL pts (1 confirmed BC); and at progression in 1 (20%) FL pt and 2 (40%) SL pts (1 confirmed BC). The take rate from pre-treatment samples among FL pts was 1/32 (3.1%; 95% CI: 0.08-16.2%). Take rates in other groups are pending histological review. Discussion: In pts receiving palbociclib for HR+ MBC, the take rate using a pre-treatment percutaneous biopsy was low (3.1%) in the FL setting. However, higher growth rates were seen in the SL setting. The observation of higher growth rates at the end of C2 and at progression may relate to the activation of G1/S that occurs with withdrawal of palbociclib, and should be further studied. PDX remains a valuable tool for the evaluation of tumor biology and the development of new therapeutics, especially for those models established from pts with palbociclib and ET resistance. Proteogenomic and sequencing studies are ongoing to fully characterize these PDXs. Citation Format: Jia Yu, Vera J. Suman, Jun He, Jason P. Sinnwell, Ann M. Moyer, Bo Qin, Yayun Gu, Huan Zhang, Jodi M. Carter, Krishna R. Kalari, Brendan P. McMenomy, Minetta C. Liu, Tufia C. Haddad, Kathryn J. Ruddy, Fergus J. Couch, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard Weinshilboum, Ciara O'Sullivan, Matthew P. Goetz, Liewei Wang. Patient-derived xenografts (PDXs) generated from hormone receptor-positive breast cancer (BC) before and after cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) treatment: Initial findings from the PROMISE study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-07.

Abstract Background: Data supporting high tumor mutational burden (TMB-H) as a lone biomarker for an immune responsive tumor microenvironment (TME) or of response to immune checkpoint inhibition (ICI) in metastatic breast cancer (MBC) is weak; yet the tumor agnostic approval in TMB-H advanced tumors makes ICI an option in the clinic. PDL-1 positivity (PDL-1+) is the only consistent biomarker for ICI benefit in metastatic triple-negative BC (mTNBC). We sought to further evaluate concurrent predictors of an immune responsive or non-responsive TME within TMB-H MBC. Methods: Tumor samples (N=5621) obtained from patients with MBC were analyzed by next-generation sequencing (NGS) of DNA (592-gene panel or whole exome sequencing) and RNA (whole transcriptome sequencing) at Caris Life Sciences (Phoenix, AZ). Friends of Cancer Research TMB Harmonization Project (Merino et al., 2020) recommendations, with the TMB-H threshold set to ≥ 10 muts/Mb, were used. PDL-1+ (≥1% immune cells stained) assessed by IHC (Ventana SP142). Deficient-mismatch repair (dMMR) and high microsatellite instability (MSI-H) were determined by IHC and NGS, respectively. Gene expression profiling assessed 21 immune-related signatures and TMEs using MCP-counter (Betch et al., 2016) and quanTIseq (Finotello et al., 2019). Immune cell types were classified as immune-responsive (CD8+ and CD4+ T cells, B cells, myeloid dendritic cells, M1 macrophages, NK cells, and neutrophils) or non-responsive (Tregs, M2 macrophages, and monocytes). P-values adjusted by the Benjamini-Hochberg procedure. Exploratory p-values reported for sub-analyses of TMB-H responsive vs nonresponsive TME and PDL1+ vs PDL1- subgroups. Results: TMB-H was identified in 461/5621 (8.2%) MBC samples. TMB-H tumors exhibit significant enrichment of dMMR/MSI-H (7 vs 0%, p<0.0001) and IHC-PDL1+ (36 vs 28%, p<0.05) compared to TMB-L. TMB-H was not associated with increased abundance or proportion of immune responsive cell types or immune response gene signatures (e.g. antigen presentation), yet positive trends were observed. To identify predictive biomarkers of immune responsive (hot) or non-responsive (cold) TME profiles within TMB-H MBC, we generated a composite TME score representing the overall proportion of hot vs cold immune cell types. APOBEC mutagenesis was common in TMB-H tumors (72%) and was more frequently associated with an immune cold TME (77 vs 66% in hot TME subgroup, p=0.068). Within TMB-H tumors, cold TMEs were enriched with mutations (muts) in PIK3CA (61 vs 47%), CHEK2 (5 vs 0%), CBFB (4 vs 0%), amplifications (amp) of CCND1 (13 vs 4%), FGF19 (16 vs 5%), FGF4 (11 vs 4%), and MMLT6 (6 vs 0%), and expression of IHC-AR (75 vs 58%) compared to hot TMEs (all p<0.05). Hot TMEs had increased frequency of MSI-H (12 vs 5%) and KMT2D muts (12 vs 3%) compared to cold TMEs (all p<0.05). Amongst TMB-H and TMB-L tumors, PDL1+ was significantly associated with increased proportion of immune responsive cell types (ie CD8+) compared to PDL1- tumors (0.14 vs -0.08 TME score, p<0.0001). Comparison of TMB-H tumors by PDL1 status revealed enrichment of TP53 (67 vs 53%) and B2M (8 vs 0%) muts in PDL1+ tumors, while IHC-ER positivity (68 vs 54%) and CDH1 muts (32 vs 16%) were increased in PDL1- tumors (all p<0.05). Conclusions: High TMB alone does not strongly correlate with immune infiltrate or immune response gene signatures in MBC. Within TMB-H MBC, concurrent mutations in MSI-H and KMT2D are associated with an immune responsive TME while mutations in PIK3CA, CHEK2, CBFB, amplifications of CCND1, FGF19, FGF4, and MMLT6, and expression of IHC-AR are associated with an immune non-responsive TME. Co-occurring biomarkers within TMB-H breast cancer warrant evaluation in prospective cohorts for response or resistance to ICI to help develop composite biomarkers in breast cancer. Citation Format: Sarah Sammons, Andrew Elliott, Jeremy Force, Saranya Chumsri, Carey Anders, Antoinette R Tan, Daniel Magee, Jia Zeng, W. Michael Korn, Mustafa Kahsraw, Evanthia T Roussos Torres. Concurrent predictors of an immune responsive tumor microenvironment within tumor mutational burden-high breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-08.

153 Background: The prognostic significance of DDR alterations in mCRPC remains unclear, with conflicting data from prior reports. Whether DDR alterations are predictive of outcomes with therapeutic agents other than PARP inhibitors in mCRPC is also poorly understood. With increasing use of molecular profiling in mCRPC, understanding the full prognostic and predictive utility of plasma DDR alterations is paramount. Methods: A next-generation sequencing Predicine liquid biopsy assay was used to profile cfDNA and germline DNA in 407 mCRPC patients (pts) from two independent international cohorts (n=162 Australia, n=245 US). DDR genes profiled were BRCA2, ATM, BRCA1, MLH1 and MSH2. Kaplan-Meier survival estimates and multivariable Cox regression analyses were used to assess associations between DDR alterations and clinical outcomes including PSA response rate (PSA RR), progression-free survival and overall survival (OS). Results: Median age was 74 (IQR 67-79), with median follow up 74 months and median OS 23 months. 65/407 (16%) harboured pathogenic DDR alterations, including 21 patients with ³1 alteration. Frequency of genomic aberrations are shown in the table. BRCA2 alterations were further classified as heterozygous loss (66%), homozygous loss (14%), monoallelic mutation (11%) and biallelic mutation/loss of heterozygosity (9%). Aberrations in any DDR gene, ATM, MLH1 + MSH2 or BRCA2 were associated with shorter OS on univariable analysis, but only any DDR or BRCA2 aberration remained significant upon adjusting for clinical prognosticators and ctDNA fraction (Table). Pre-treatment BRCA2 aberration was associated with significantly shorter OS and lower PSA RR compared to BRCA2 wt for pts receiving an AR pathway inhibitor (ARPI) (18 vs 32 months, p=0.006; 36 vs 60%, p=0.04 respectively) but not for taxane chemotherapy (17 vs 20 months, p=0.3; 45 vs 66, p=0.1 respectively). Conclusions: Detection of an aberration in any DDR gene or BRCA2 was an independent poor prognostic factor across 2 large independent cohorts of mCRPC patients. Intriguingly, patients with a BRCA2 alteration appeared to have worse outcomes with ARPI but not chemotherapy, suggesting predictive utility of DDR profiling. Our data collectively speak to the potential role of DDR alterations, in particular BRCA2, as prognostic and/ or predictive biomarkers in mCRPC.[Table: see text]

150 Background: With the evolving use of targeted therapies exploiting genomic vulnerabilities in mCRPC, screening patients for sensitizing alterations is of increasing relevance. However, the influence of age on the detection of relevant genomic alterations is incompletely understood. In this study, we compared the cell-free DNA (cfDNA) profiles of younger (age <70) versus older (age ≥70) men with mCRPC and assessed the relative prognostic impact of common genomic alterations. Methods: A next-generation sequencing-based Predicine cfDNA assay was used to profile 276 mCRPC patients from two independent international cohorts. The frequency of genomic alterations across age categories was compared using the chi-squared test, while circulating tumour DNA fraction (ctDNA%) was compared using the Mann–Whitney U test. Cox proportional hazards analysis with interaction testing was used to assess the association between age, genomic alteration and overall survival. Results: The median age of the combined cohort was 72 years (IQR 66-78), with 170 (62%) patients ≥70 years old. Despite differences in baseline characteristics (poorer performance status in older patients, higher Gleason score and de novo metastatic disease in younger patients), there was no significant difference in ctDNA% (median 22% vs 30%, p=0.6). We observed similar frequencies of genomic alterations across the 10 most commonly aberrant genes, including AR (<70 vs ≥70: 46% vs 49%, p=0.6), TP53 (40% vs 36%, p=0.6), BRCA2 (29% vs 21%, p=0.13) and PTEN (39% vs 35%, p=0.5). This similarity persisted when analysing mutations and copy number alterations in isolation. While driver alterations in both age categories were strongly associated with unfavourable outcomes (Table), an exploratory analysis revealed that copy number gain in PIK3CA may have a worse prognostic impact in younger men ( p for interaction = 0.03), with a lesser effect observed in AR gain ( p = 0.1) and MYC gain ( p = 0.1). Conclusions: The genomic profiles of pertinent alterations between younger and older men are comparable. Certain genomic alterations may carry a worse prognosis in younger men. Patients should be considered for precision medicine assessment and directed therapies, regardless of age. [Table: see text]

Background Standard therapy for COVID-19 is continuously evolving. Autopsy studies showed high prevalence of platelet-fibrin-rich microthrombi in several organs. The aim of the study was therefore to evaluate the safety and efficacy of antiplatelet therapy (APT) in hospitalised patients with COVID-19 and its impact on survival. Methods 7824 consecutive patients with COVID-19 were enrolled in a multicentre international prospective registry (Health Outcome Predictive Evaluation-COVID-19 Registry). Clinical data and in-hospital complications were recorded. Data on APT, including aspirin and other antiplatelet drugs, were obtained for each patient. Results During hospitalisation, 730 (9%) patients received single APT (93%, n=680) or dual APT (7%, n=50). Patients treated with APT were older (74±12 years vs 63±17 years, p<0.01), more frequently male (68% vs 57%, p<0.01) and had higher prevalence of diabetes (39% vs 16%, p<0.01). Patients treated with APT showed no differences in terms of in-hospital mortality (18% vs 19%, p=0.64), need for invasive ventilation (8.7% vs 8.5%, p=0.88), embolic events (2.9% vs 2.5% p=0.34) and bleeding (2.1% vs 2.4%, p=0.43), but had shorter duration of mechanical ventilation (8±5 days vs 11±7 days, p=0.01); however, when comparing patients with APT versus no APT and no anticoagulation therapy, APT was associated with lower mortality rates (log-rank p<0.01, relative risk 0.79, 95% CI 0.70 to 0.94). On multivariable analysis, in-hospital APT was associated with lower mortality risk (relative risk 0.39, 95% CI 0.32 to 0.48, p<0.01). Conclusions APT during hospitalisation for COVID-19 could be associated with lower mortality risk and shorter duration of mechanical ventilation, without increased risk of bleeding. Trial registration number NCT04334291.

A rare APOE variant APOE3 -Jac reduces self-aggregation and promotes healthy brain aging.

AbstractObjectiveThis study was undertaken to determine whether the vertical parasagittal approach or the lateral peri‐insular/peri‐Sylvian approach to hemispheric surgery is the superior technique in achieving long‐term seizure freedom.MethodsWe conducted a post hoc subgroup analysis of the HOPS (Hemispheric Surgery Outcome Prediction Scale) study, an international, multicenter, retrospective cohort study that identified predictors of seizure freedom through logistic regression modeling. Only patients undergoing vertical parasagittal, lateral peri‐insular/peri‐Sylvian, or lateral trans‐Sylvian hemispherotomy were included in this post hoc analysis. Differences in seizure freedom rates were assessed using a time‐to‐event method and calculated using the Kaplan–Meier survival method.ResultsData for 672 participants across 23 centers were collected on the specific hemispherotomy approach. Of these, 72 (10.7%) underwent vertical parasagittal hemispherotomy and 600 (89.3%) underwent lateral peri‐insular/peri‐Sylvian or trans‐Sylvian hemispherotomy. Seizure freedom was obtained in 62.4% (95% confidence interval [CI] = 53.5%–70.2%) of the entire cohort at 10‐year follow‐up. Seizure freedom was 88.8% (95% CI = 78.9%–94.3%) at 1‐year follow‐up and persisted at 85.5% (95% CI = 74.7%–92.0%) across 5‐ and 10‐year follow‐up in the vertical subgroup. In contrast, seizure freedom decreased from 89.2% (95% CI = 86.3%–91.5%) at 1‐year to 72.1% (95% CI = 66.9%–76.7%) at 5‐year to 57.2% (95% CI = 46.6%–66.4%) at 10‐year follow‐up for the lateral subgroup. Log‐rank test found that vertical hemispherotomy was associated with durable seizure‐free progression compared to the lateral approach (p = .01). Patients undergoing the lateral hemispherotomy technique had a shorter time‐to‐seizure recurrence (hazard ratio = 2.56, 95% CI = 1.08–6.04, p = .03) and increased seizure recurrence odds (odds ratio = 3.67, 95% CI = 1.05–12.86, p = .04) compared to those undergoing the vertical hemispherotomy technique.SignificanceThis pilot study demonstrated more durable seizure freedom of the vertical technique compared to lateral hemispherotomy techniques. Further studies, such as prospective expertise‐based observational studies or a randomized clinical trial, are required to determine whether a vertical approach to hemispheric surgery provides superior long‐term seizure outcomes.

Significance The sorting nexin proteins are critical for endosomal trafficking and signaling. SNX17 and SNX27 are two SNX proteins displaying high similarity in domain structure, including a common FERM domain; however, they mediate distinct endocytic recycling pathways. We now solve this mystery by demonstrating that the FERM domains of SNX17 and SNX27 have different functions. Whereas the FERM domain of SNX17 recognizes the NPxY/NxxY motif, the same domain in SNX27 binds to a novel “DLF” motif within the N termini of SNX1/2. The interaction between SNX27 and SNX1/2 not only helps efficient retrieval of multiple cargoes but also promotes endosomal recruitment of SNX27. We further demonstrate that the SNX27–SNX1/2 interaction is crucial to neuronal growth and brain development in zebrafish.