Citations

Abstract Objectives To develop and validate a standards-based phenotyping tool to author electronic health record (EHR)-based phenotype definitions and demonstrate execution of the definitions against heterogeneous clinical research data platforms. Materials and Methods We developed an open-source, standards-compliant phenotyping tool known as the PhEMA Workbench that enables a phenotype representation using the Fast Healthcare Interoperability Resources (FHIR) and Clinical Quality Language (CQL) standards. We then demonstrated how this tool can be used to conduct EHR-based phenotyping, including phenotype authoring, execution, and validation. We validated the performance of the tool by executing a thrombotic event phenotype definition at 3 sites, Mayo Clinic (MC), Northwestern Medicine (NM), and Weill Cornell Medicine (WCM), and used manual review to determine precision and recall. Results An initial version of the PhEMA Workbench has been released, which supports phenotype authoring, execution, and publishing to a shared phenotype definition repository. The resulting thrombotic event phenotype definition consisted of 11 CQL statements, and 24 value sets containing a total of 834 codes. Technical validation showed satisfactory performance (both NM and MC had 100% precision and recall and WCM had a precision of 95% and a recall of 84%). Conclusions We demonstrate that the PhEMA Workbench can facilitate EHR-driven phenotype definition, execution, and phenotype sharing in heterogeneous clinical research data environments. A phenotype definition that integrates with existing standards-compliant systems, and the use of a formal representation facilitates automation and can decrease potential for human error.

Abstract Glial fibrillary acidic protein (GFAP) is a promising candidate blood-based biomarker for Alzheimer’s disease (AD) diagnosis and prognostication. The timing of its disease-associated changes, its clinical correlates, and biofluid-type dependency will influence its clinical utility. We evaluated plasma, serum, and CSF GFAP in families with autosomal dominant AD (ADAD), leveraging the predictable age at symptom onset to determine changes by stage of disease. Plasma GFAP elevations appear a decade before expected symptom onset, after β-amyloid accumulation and prior to neurodegeneration and cognitive decline. Plasma GFAP distinguished β-amyloid-positive from β-amyloid-negative ADAD participants and showed a stronger relationship with β-amyloid load in asymptomatic than symptomatic ADAD. Higher plasma GFAP was associated with the degree and rate of neurodegeneration and cognitive impairment. Serum GFAP showed similar relationships, but these were less pronounced for CSF GFAP. Our findings support a role for plasma GFAP as a clinical biomarker for β-amyloid-associated cognitive deterioration in AD.

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults with CD19-targeting CAR T-cell therapy showing an excellent initial curative response. However, 10-20% of patients who receive anti-C19 CAR T-cell treatment experience relapse due to CD19 antigen loss. B-cell activating factor receptor (BAFF-R) that is expressed on the surface of normal and neoplastic B cells mediates the survival of mature peripheral B cells along with the B-cell receptor. Since B-CLL is characterized by apoptotic disruption and subsequent accumulation of mature B cells, BAFF-R is a logical, novel target for CAR T-cell therapy directed against B-CLL, especially if CD19 antigen loss occurs. In this study, we first manufactured BAFF-R CAR T-cells by infecting healthy donor Tnaïve/memory (Tn/mem) cells with BAFF-R CAR lentivirus before evaluating the antitumor activity of BAFF-R CAR T-cells on wild type MEC-1 (a B-CLL cell line), modified MEC-1, and primary cell lines. Antigen-specific cytotoxicity and activation of the BAFF-R CAR T-cells or control CD19 CAR T-cells were determined upon incubation with B-CLL cell lines and patient-derived primary B-CLL by measuring degranulation and IFN-γ release, respectively. BAFF-R CAR T-cells demonstrated substantial cytotoxicity against MEC-1, CD19-deficient MEC-1, and primary B-CLL cell lines; CD19 CAR T-cells showed no effect on CD19-deficient MEC-1 cells but did show effects with primary B-CLL and MEC-1 cells. Similarly, the release of IFN-γ was greater when BAFF-R CAR T-cells were co-cultured with CD19-deficient MEC-1 or primary B-CLL compared to CD19 CAR T-cells co-cultured with CD19-deficient MEC-1. In summary, BAFF-R CAR T-cells exert significant antitumor properties not only on patient-derived primary B-CLL cells but also on a CD19-deficient B-CLL cell line. We, therefore, assert BAFF-R-targeting CAR T-cells to be a novel therapeutic avenue for patients newly diagnosed with B-CLL and patients experiencing relapsed B-CLL after anti-CD19 therapy. Citation Format: Yan Luo, Kaihin Lui, Tommy T. To, Andrew liu, Farah Yassine, Mohamed A. Kharfan Dabaja, Martha E. Gadd, Hong Qin. Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia [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 2855.

2058 Background: Immunotherapies targeting the programmed cell death-1 (PD-1) pathway in recurrent glioblastoma (rGBM) have failed. We hypothesize that combining therapies targeting multiple immunosuppressive pathways with cytotoxic and antiangiogenic therapies will improve survival. Here, we evaluate the safety and efficacy of an anti-PD-1 monoclonal antibody (retifanlimab), hypofractionated radiotherapy (HFRT), and bevacizumab, with or without an oral IDO1 inhibitor (epacadostat), in patients with rGBM in a nonrandomized, noncomparative sequential two-arm Phase II study. Methods: This is an open-label Phase II study of 2 sequential cohorts (Table). Cohort A first examines retifanlimab + bevacizumab + HFRT in patients with IDH1/2-WT rGBM. After a toxicity monitoring period, Cohort B, which adds epacadostat, starts enrolling. Key inclusion criteria includes dexamethasone ≤ 4 mg/day at registration and candidacy for reirradiation. Previous bevacizumab use for radiation necrosis is permitted. The primary endpoint is OS probability at 9 months (OS-9). Secondary endpoints include PFS, OS, and toxicity. Exploratory endpoints include immunological phenotyping of blood and tissue. An increase of OS-9 from 38% (bevacizumab alone) to 60% is considered clinically relevant. Results: From 6/2020 to 12/2021, we have completed accrual for cohort A and the interim analysis results are presented here: 25 patients with rGBM enrolled, with 23 evaluable. Median age is 64.3 years (42.1-81.8), 30.4% female, 30.4% MGMT promotor methylated, median KPS 90 (range 70-100), baseline dexamethasone 0 mg (range 0-4) with 52.2% of patients on dexamethasone during the first 3 cycles, median baseline ALC 1,000 cells/µl (range 300-3,700). Patients received a median of 6 cycles to date (range 2-20). Median follow-up is 11.97 months per the reverse Kaplan-Meier method. Interim analysis shows a median PFS of 9.9 months (95%CI: 5.5 to not reached (NR)) and median OS of 12.2 months (95%CI 7.3-NR). Notably, Cohort A met the primary endpoint with an OS-9 of 71.4% (95%CI: 46.7% -86.1%). No dose limiting toxicities have been yet observed. Two treatment-related grade 3 toxicities have occurred (myositis, hypertension). Cohort B enrollment is ongoing, and correlative studies pending. Conclusions: Interim analysis suggests retifanlimab combined with HFRT and bevacizumab in patients with rGBM is well-tolerated and had encouraging OS and PFS at the time of data cutoff. Cohort B, which adds epacadostat, is currently enrolling. Clinical trial information: NCT03532295. [Table: see text]

Background: Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. Methods: Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms. Results: We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes‚ whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. Conclusions: Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

Phosphatidylinositol polyphosphates (PIPs) are essential cellular regulators. Each PIP transiently appears at specific membranes to regulate the spatial and temporal recruitment of unique downstream effectors. Importantly, most PIPs serve as both signaling lipids, and as precursors for other PIPs. These precursor‐product relationships likely form cascades of pathways, which would provide a way for PIPs to coordinate events both spatially and temporally. Here we uncover a pathway on endosomes, which is likely governed by a cascade of three phosphoinositide lipids. The pathway is specific for the recycling of some receptors to the plasma membrane and is mediated by sorting‐nexin SNX17, and the Retriever, WASH and CCC complexes. We discover that PIKfyve and an upstream PI3‐kinase, VPS34 positively regulates this pathway. VPS34 produces PI3P, which provides the substrate for PIKfyve to generate PI3,5P 2 , which is then further converted to PI5P by myotubularin 3‐phosphatases. We show that PIKfyve regulates recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH and CCC complexes on endosomes. Importantly, PIKfyve inhibition causes a loss of Retriever and CCC from endosomes. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration. More broadly, these findings provide more general insights into mechanisms for how cells regulate the levels of some receptors at the cell surface.

Abstract Branched-chain amino acid transaminase 1 (BCAT1) is upregulated selectively in human isocitrate dehydrogenase (IDH) wildtype (WT) but not mutant glioblastoma multiforme (GBM) and promotes IDHWT GBM growth. Through a metabolic synthetic lethal screen, we report here that α-ketoglutarate (AKG) kills IDHWT GBM cells when BCAT1 protein is lost, which is reversed by reexpression of BCAT1 or supplementation with branched-chain α-ketoacids (BCKA), downstream metabolic products of BCAT1. In patient-derived IDHWT GBM tumors in vitro and in vivo, cotreatment of BCAT1 inhibitor gabapentin and AKG resulted in synthetic lethality. However, AKG failed to evoke a synthetic lethal effect with loss of BCAT2, BCKDHA, or GPT2 in IDHWT GBM cells. Mechanistically, loss of BCAT1 increased the NAD+/NADH ratio but impaired oxidative phosphorylation, mTORC1 activity, and nucleotide biosynthesis. These metabolic alterations were synergistically augmented by AKG treatment, thereby causing mitochondrial dysfunction and depletion of cellular building blocks, including ATP, nucleotides, and proteins. Partial restoration of ATP, nucleotides, proteins, and mTORC1 activity by BCKA supplementation prevented IDHWT GBM cell death conferred by the combination of BCAT1 loss and AKG. These findings define a targetable metabolic vulnerability in the most common subset of GBM that is currently incurable. Significance: Metabolic synthetic lethal screening in IDHWT glioblastoma defines a vulnerability to ΑΚG following BCAT1 loss, uncovering a therapeutic strategy to improve glioblastoma treatment. See related commentary by Meurs and Nagrath, p. 2354

Abstract Background Facial regions with a high risk for causing injection-related visual comprise are dual-supply vascular areas such as the nose, glabella, and forehead. These regions have in common that they receive arterial blood supply both by branches of the internal (ICA) and the external carotid artery (ECA). Objective The authors sought to investigate the anastomotic pathways between ICA and ECA branches in the upper face. Methods Postmortem computed tomographic angiographic scans of n = 38 Chinese non-embalmed hemifaces (25 males, 13 females; mean age, 37.79 [11.8] years; mean BMI, 21.90 [2.3] kg/m2) were conducted. Data analysis relied on the calculation of depth, distances, and pathways of forehead and temporal arteries to investigate the number of anastomotic connections, the connecting branches, and the layer of connection between ICA and ECA territories. Results Between ICA and ECA territories, only 1 connection in 57.9%, 2 connections in 31.6%, 3 connections in 5.3%, and 4 and 5 connections in 2.6% each were identified. A superficial connection was observed in 15.8% whereas in 84.2% the anastomotic connection was identified to be both superficial and deep. Conclusions Adverse events following facial minimally invasive soft-tissue filler injections for aesthetic purposes are not frequent but devastating if they occur. Anatomic knowledge as presented in this study can help to increase awareness of 3-dimensional vascular anastomotic pathways and identify safer injection zones and safer fascial planes. Evidence-based injection techniques should be followed, and safety aspects should be placed over the aesthetic outcome.

Abstract Background Patients (pts) with ER+ HER2+ breast cancer (BC) are less likely to achieve pathological complete response (pCR) after neoadjuvant chemotherapy with dual HER2 blockade than pts with ER- HER2+ BC. Endocrine therapy (ET) plus trastuzumab is effective in advanced ER+ HER2+ BC, but pCR rate is low in the neoadjuvant setting. Inhibition of CDK4/6 and HER2 results in synergistic reduction in cell proliferation in preclinical studies. We therefore combined ET with CDK4/6 inhibition and trastuzumab in ER+ HER2+ BC as a chemotherapy-sparing regimen. Methods We evaluated the efficacy of palbociclib, letrozole, trastuzumab (PLT) in the neoadjuvant setting for pts with stages II or III ER+ HER2+ BC. Primary endpoint was pCR after 16 weeks of therapy. We assumed null of 15% pCR and pCR ≥ 30% warrants further investigation. To achieve 80% power at 1-sided 0.05 significance, 48 pts were to be enrolled. Evaluable population included pts who completed Cycle (C) 1 unless discontinued due to treatment-emergent adverse events (TEAEs) prior to completing C1. All who received one dose on study were considered evaluable for toxicity. Biopsies were collected at baseline (BL), C1 day 15 (C1D15), and surgery for RNA sequencing and central Ki67 assessment, for PAM50 subtype distribution, complete cell cycle arrest (CCCA: Ki67 ≤2.7%) at C1D15 and surgery, and treatment induced signaling changes. Results Accrual stopped early due to futility. 26 pts accrued were evaluable for efficacy and toxicity. pCR (residual cancer burden- [RCB] 0) was 7.7% (95% CI 0.9 - 25.1%) and RCB 0/I was 38.5% (95% CI 20.2 - 59.4%). TEAEs (n= 337) were seen in all pts (71.5% grade [G] 1, 19.3% G2, 8.6% G3, 0.6% G4); the most common were leukopenia (7.7%), neutropenia (7.1%), anemia (5.9%). G3/4 TEAEs occurred in 19 pts (73.1%). Among the 19, incidence of G3/4 neutropenia was 50%, hypertension 26.9%, leucopenia 7.7%. TEAEs (hypertension, ventricular tachycardia, pulmonary edema) leading to treatment discontinuation were reported in 1 pt. Two pts had at least one SAE. No treatment-related deaths occurred. Pt reported outcomes using NCI PRO-CTCAE revealed no differences in appetite, nausea, respiratory symptoms, edema, palpitations, rashes and dry skin, or concentration from BL to end of C4. Pts had worsening hair loss from BL to end of C4. Ki67 analysis indicated CCCA in 78% at C1D15, compared to 18% at surgery after only P had been discontinued approximately 4 weeks prior to surgery. RNA sequencing was performed on available biopsies collected at BL (N=16), C1D15 (N=5), and surgery (N=2) from 16 pts. Among 16 BL samples, PAM50 subtyping identified 5 (31.3%) basal-like, 2 (12.5%) HER2-E, 6 (37.5%) Lum B, and 3 (18.8%) normal. Subtype switching to Lum A at C1D15 (N=3, 1 each with HER2-E, Lum B, and normal at BL) or normal (N=2, 1 basal and 1 HER2-E at BL) was observed. 161 genes were differentially expressed (FDR p<0.05); 145 downregulated and 16 upregulated comparing C1D15 to BL. MKI67, TK1, CCNB1, AURKB, PLK1 were among the downregulated genes, consistent with CCCA for majority of the samples at C1D15 by Ki67. Analysis of the Molecular Signatures Database Hallmark gene-sets comparing C1D15 and BL samples demonstrated downregulated biological processes involved in proliferation (E2F targets, G2M checkpoint, MYC targets, mitotic spindle), signaling (Estrogen response, mTORC1 signaling), and DNA damage (DNA repair) at C1D15, consistent with the mechanisms of action of the study drugs. E2F targets were higher in BL samples of RCB II/III, compared to RCB I (FDR p=0.042). Conclusions PALTAN did not meet its primary endpoint of pCR. Neoadjuvant PLT showed a pCR of 7.7% but was well tolerated. RNA sequencing and Ki67 data indicated potent anti-proliferative effects of study treatments, despite significant heterogeneity of intrinsic subtypes. Clinical trial information: NCT02907918. Citation Format: Foluso O Ademuyiwa, Donald Northfelt, Tracey O'Connor, Ellis Levine, Jingqin Luo, Yu Tao, Jeremy Hoog, Marie Laury, Tracy Summa, Trish Hammerschmidt, Zhanfang Guo, Ashley Frith, Katherine Weilbaecher, Mateusz Opyrchal, Rebecca Aft, Katherine Clifton, Rama Suresh, Nusayba Bagegni, Ian S Hagemann, Cynthia X Ma. Phase 2 study of neoadjuvant palbociclib, letrozole, and trastuzumab in patients with ER+ HER2+ breast cancer (PALTAN) [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 P2-13-01.

Abstract Background. DCIS consists of a molecularly heterogeneous group of premalignant lesions, with variable risk of invasive progression. Understanding biomarkers for invasive progression could help individualize treatment recommendations based upon tumor biology. As part of the NCI Human Tumor Atlas Network (HTAN), we conducted comprehensive genomic analyses on two large DCIS case-control cohorts. Methods. We performed smart3-seq and low-pass whole genome sequencing on two independent, retrospective, longitudinally sampled DCIS case-control cohorts. TBCRC 038 was a multicenter cohort diagnosed with DCIS between 1998 and 2016 at one of the Translational Breast Cancer Research sites; the RAHBT (Resource of Archival Human Breast Tissue) cohort included women identified through the St. Louis Breast Tissue Repository, and the Women’s Health Repository diagnosed between 1997 and 2001. We studied the spectrum of molecular changes present and sought genomic predictors of subsequent ipsilateral breast events (iBEs: DCIS recurrence or invasive progression) in both DCIS epithelium and stroma in formalin fixed paraffin embedded tissue. We generated de novo tumor and stroma-centric subtypes for DCIS that represents fundamental transcriptomic organization. Copy number analysis was performed using low-pass DNA sequencing. Non-negative matrix factorization (NMF) was applied to the RNA expression of all coding genes to identify clusters. A negative-binomial regression model was used to identify differentially expressed genes. Results. We analyzed 677 DCIS samples from 481 patients with 7.1 years median follow-up. In TBCRC samples, we identified three clusters via NMF in TBCRC referred to as ER low, quiescent, and ER high. The ER-low cluster had significantly higher levels of ERBB2 and lower levels of ESR1 compared to quiescent and ER-high clusters. Quiescent cluster lesions were less proliferative and less metabolically active than ER high and ER low subtypes. These findings were replicated in the RAHBT cohort. Focusing on the stromal component of DCIS from laser capture microdissection in RAHBT samples, we identified four distinct DCIS-associated stromal clusters. A “normal-like” stromal cluster with ECM organization and PI3K-AKT signaling; a “collagen-rich” stromal cluster; a “desmoplastic” stromal cluster with high fibroblast and total myeloid abundance, mostly associated with macrophages and myeloid dendritic cells (mDC); and an “immune-dense” stromal cluster. Further, we compared differentially expressed genes in patients with or without subsequent iBEs within 5 years of diagnosis. Hypothesizing that the resulting 812 DE genes (DESeq2) represent multiple routes to subsequent iBEs, we leveraged NMF to identify paths to progression. In both TBCRC and RAHBT cohorts, poor outcome groups exhibited increased ER, MYC signaling, and oxidative phosphorylation, supporting that these pathways are important for DCIS recurrence and progression. Conclusion. Comprehensive genomic profiling in two independent DCIS cohorts with longitudinal outcomes shows distinct DCIS stromal expression patterns and immune cell composition. RNA expression profiles reveal underlying tumor biology that is associated with later iBEs in both cohorts. These studies provide new insight into DCIS biology and will guide the design of diagnostic strategies to prevent invasive progression. Citation Format: Siri H Strand, Belén Rivero-Gutiérrez, Kathleen E Houlahan, Jose A Seoane, Lorraine M King, Tyler Risom, Lunden Simpson, Sujay Vennam, Aziz Khan, Timothy Hardman, Bryan E Harmon, Fergus J Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F McAuliffe, Julie Nangia, Joanna Lee, Jennifer Tseng, Anna Maria Storniolo, Alastair Thompson, Gaorav Gupta, Robyn Burns, Deborah J Veis, Katherine DeSchryver, Chunfang Zhu, Magdalena Matusiak, Jason Wang, Shirley X Zhu, Jen Tappenden, Daisy Yi Ding, Dadong Zhang, Jingqin Luo, Shu Jiang, Sushama Varma, Cody Straub, Sucheta Srivastava, Christina Curtis, Rob Tibshirani, Robert Michael Angelo, Allison Hall, Kouros Owzar, Kornelia Polyak, Carlo Maley, Jeffrey R Marks, Graham A Colditz, E Shelley Hwang, Robert B West. The Breast PreCancer Atlas DCIS genomic signatures define biology and correlate with clinical outcomes: An analysis of TBCRC 038 and RAHBT cohorts [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 GS4-07.