Citations

Background: The Trans-Omics for Precision Medicine (TOPMed) program is performing whole-genome sequencing across multiple studies, including longitudinal cohorts, case-control, and case-only sample sets. Detecting the effects of low frequency variants requires large sample sizes, which can only be achieved by combining data across diverse study designs, including matching of case-only sample sets with controls from other studies. Here we present a strategy for combined analysis of venous thromboembolism (VTE) case status in 3 longitudinal cohorts and 3 case-only sample sets, in the context of whole-genome association studies. Methods: For each cohort study, we sampled ‘pure’ controls (without replacement) from the risk set of each incident case, within strata defined by sex, ancestry and birth-cohort. Pure controls had no event throughout their period of observation. A case’s risk set was defined as controls with no prior VTE history and under observation through an age at least as old as the case. For case-only sample sets, controls for each case were sampled from a cohort study, using the same risk set definition. Because of limited overlap in birth years between the cohort studies and the case-only sample sets, this matching was done within strata defined only by sex and ancestry group. Mixed model logistic regression will be used to account for relatedness as a random effect. Although conditional logistic regression is not practical for whole-genome association studies, case-control matching is implicitly recognized by a fixed effect for age-at-event for each matched set (1 case and >=1 matched controls). Additional fixed effects will include sample set and sex. We will also adjust for variations in case-control ratio among the matched sets. Results: In two cohort studies, we matched 1,231 cases to 4,820 controls (overall ratio = 1:3.9); only 500 controls and 5 cases could not be matched. For the case-only sample sets we matched 2,141 cases to 2,780 controls from one cohort study (overall ratio = 1:1.3); zero controls and only 14 cases could not be matched. Conclusions: We were successfully able to match nearly all cases to controls, and more than 90% of controls were also matched. By matching controls to cases based on age at event, we can account for the different risk of VTE by age clusters. Although this strategy does not provide an asymptotically unbiased estimated of the hazard ratio, compared to classical risk set sampling, it uses a large portion of the available data, it provides odds ratio estimates yielding the correct sign of association, and it reduces the potential influence of resampling subjects with rare variants. This strategy also enables combined analysis of multiple studies with different designs.

Abstract Introduction: The bone marrow (BM) niche promotes multiple myeloma (MM) growth, survival and drug resistance. Therapies targeting both cancer cells and the microenvironment are highly effective. We were interested in identifying novel signaling pathways supporting MM pathogenesis through MM-BM crosstalk. The transmembrane receptor Roundabout 1 (ROBO1) plays a role in growth and dissemination of solid tumors, however its function in MM is unknown. Material and Methods: We analyzed ROBO1 expression in cell lines and primary samples via western blot, immunohistochemistry (IHC) and gene expression profiling. We used short hairpin RNA and CRISPR-Cas9 for ROBO1 knock down (KD) and knock out (KO), respectively. For protein structure-function and rescue studies, we stably expressed full-length (FL) or mutant ROBO1 devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived G674* truncation, with a C-terminus FLAG tag. We used a 3D hydrogel encapsulation system to study proliferation; FLAG immunoprecipitation (IP) followed by mass spectrometry to identify ROBO1 interacting partners; and immunofluorescence to detect ROBO1 localization. To study tumor growth in vivo, we performed PET-CT of mice inoculated subcutaneously or intramedullary with WT or ROBO1 KO MM cells and retrieved tumors for RNA sequencing. To study dissemination and homing, KO and FL addback MM cells were injected intravenously in SCID mice. Mice were monitored for development of tumors or hindlimb paralysis and femora/tumors harvested once mice reached endpoint. Results: ROBO1 is highly expressed in MM cell lines and primary cells but low/absent in normal plasma cells and other hematologic cancer cell lines. ROBO1 KD is specifically cytotoxic for MM cells and ROBO1 KO decreases proliferation, a phenotype fully rescued by FL ROBO1. Compared to WT, ROBO1 KO significantly decreases intramedullary (mean tumor volume (MTV): 1323 vs 457 mm3, p value= 0.02) and extramedullary (MTV: 2684 vs 823 mm3, p value= 0.001) tumor growth in vivo. We further discovered that ROBO1 KO decreases adhesion of MM to BM endothelial and BMSC, which is fully rescued by FL ROBO1. In a disseminated mouse model, ROBO1 KO cells generate bone plasmacytoma with reduced BM invasion, as compared to the extensive BM infiltration observed with ROBO1 FL cells. Consistently, in primary samples from patients, we detected ROBO1 expression only in 1 out of 10 solitary plasmacytoma (dim staining) as compared to 14 out of 14 MM bone marrow biopsy samples tested (11 strong, 3 dim, p value= 0.0001). Mechanistically, we show for the first time that ROBO1 C-terminus is cleaved in a ligand-independent fashion; translocates to the nucleus; and is necessary and sufficient to rescue ROBO1 KO proliferative defect. Viceversa, mutants lacking the cytoplasmic domain, including the G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies point to a previously unknown function of ROBO1 in RNA processing, setting the bases for future studies. Conclusions: We show for the first time that ROBO1 is necessary for MM growth and homing to the BM. Cleaved ROBO1 cytosolic domain translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO proliferative defect, possibly by participating in RNA processing. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Citation Format: Giada Bianchi, Peter G. Czarnecki, Matthew Ho, Aldo M. Roccaro, Antonio Sacco, Yawara Kawano, Annamaria Gulla, Anil Aktas Samur, Tianzeng Cheng, Kenneth Wen, Yu-Tzu Tai, Maria Moscvin, Xinchen Wu, Gulden Camci-Unal, Matteo Claudio Da VIa, Niccolo Bolli, Ruben D. Carrasco, Irene M. Ghobrial, Kenneth C. Anderson. ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB217.

Abstract Introduction: We previously showed that an imbalance between polyubiquitinated proteins awaiting proteasomal degradation and proteasome activity is a main determinant of proteasome inhibitor (PI) sensitivity in MM. While PIs have radically improved survival of MM patients, resistance is inevitable over time and understanding the molecular mechanisms underlying it is critically important to develop novel therapies to improve patient outcomes. The proteasome stress response (PSR) is an evolutionarily conserved homeostatic mechanism triggered by insufficient proteasome activity and aimed at restoring homeostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency is cleaved by the aspartic protease DDI2, translocates to the nucleus, and induces proteasome subunit gene transcription. It is unknown whether blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM. Materials and methods: We used human MM cell lines with distinct baseline sensitivity to PIs. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2 and NRF1. Cell viability or tumor growth of DDI2/NRF1 KO cells versus cells edited with a non-targeting gRNA were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate upon incubation with protein lysates from DDI2 WT, KO, or addback cells. Results: We detected full-length and cleaved NRF1 in human MM cell lines, but not other hematologic cancer cell lines, suggesting constitutively active PSR in MM. Query of the dependency map (depmap.org) shows that MM is the cell line type most highly dependent on DDI2. Consistently, DDI2 or NRF1 KO is cytotoxic alone or in combination with PI across a panel of MM cells with distinct PI sensitivity, including de novo, PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NOD SCID mice and results in prolongation of animal survival. While biallelic DDI2 or NRF1 KO monoclones could not be generated across 7 distinct MM cell lines, consistent with these genes being essential, we successfully established DDI2 KO monoclones in AMO1-VR, an isogenic AMO1 cell line variant adapted to grow in continuous bortezomib. Even in this cell line model of acquired PI resistance, DDI2 KO sensitized cells to PI carfilzomib. In this model, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome recovery after irreversible PI treatment and sensitizing to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. Conclusions: Our data show that MM cells are intrinsically dependent on an intact DDI2/NRF1 axis to survive in baseline conditions and upon proteasomal inhibition. We propose that DDI2 is an unexplored, promising molecular target for MM treatment by disrupting the PSR and exacerbating proteotoxicity. This study provides the preclinical rationale for the development of novel therapeutics targeting the DDI2/NRF1 pathway to overcome acquired and de novo PI resistance and improve patient outcomes in MM. Citation Format: Tianzeng Chen, Matthew Ho, Jenna Briere, Maria Moscvin, Peter G. Czarnecki, Kenneth C. Anderson, T. Keith Blackwell, Giada Bianchi. Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB110.

Abstract The purpose of this work was to assess technical performance of a prototype high-ratio (r29), 80 line cm −1 grid for imaging conditions which mimic those for adult cardiovascular angiography. The standard equipment r15, 80 line cm −1 grid was used as a reference. Plastic Water ® LR phantoms with thickness in the range 20–44 cm were used to simulate adult patient attenuation and scatter. Grids were tested using x-ray field of view 20 and 25 cm and x-ray source to detector distance (SID) 107 and 120 cm. The primary transmission fraction ( T P ) was measured using both narrow beam geometry and a lead beam stop (BS) technique. Scatter transmission ( T S ) was measured with the lead BS technique. The quantum signal to noise ratio improvement factor ( K SNR ) was used to describe relative grid performance. The experimental conditions required revised theory to assess grid performance. Theory to account for the detector glare and underestimation of scatter intensity by the lead BS method was developed. Also, novel K SNR theory was developed to allow direct comparison of two grids operated at different SID. Mean T P was modestly lower for the r29 versus r15 grid (0.69 versus 0.75). When tested under equivalent scatter condition, T S of the r29 grid was approximately ½ that of the r15 grid (0.18 versus 0.34). K SNR of the r29 grid at SID 120 cm compared to the r15 grid at SID 107 cm increased linearly with phantom thickness (range 1.0 to ∼1.16). Findings of this work indicate that the r29 grid used at SID 120 cm is expected to provide improved image quality (or reduced patient radiation dose) when compared to the r15 grid used at SID 107 cm for adult cardiovascular patients and that the potential benefit of the r29 grid increases with patient thickness >20 cm.

AbstractBackgroundAdult medulloblastoma (MB) is rare, and management guidelines are largely based on pediatric clinical trials and retrospective series. Limited data exist with respect to clinical characteristics, prognostic factors, and outcomes based on first-line treatments.MethodsTwo hundred adults with MB seen at a single institution from January 1978 to April 2017 were identified and followed for a median of 8.4 y (7.1, 10.3).ResultsPatient’s median age at diagnosis was 29 y (18, 63). One hundred eleven (55.5%) were standard-risk, 59 (29.5%) were high-risk, and 30 (15.0%) were indeterminate. Most received post-operative radiation (RT) (184 [92.0%]), and 105 (52.5%) received first-line chemotherapy. Median overall survival (OS) was 8.8 y (7.2, 12.2) and median progression-free survival (PFS) was 6.6 y (4.9, 11.2). High-risk patients had inferior OS (Hazard ratio [HR] = 2.5 [1.5, 4.2], P = .0006) and PFS (HR = 2.3 [1.3, 3.9], P = .002) compared to standard-risk patients. Age, sex, and metastatic disease were not associated with survival. After adjusting for risk status, those who received RT plus adjuvant chemotherapy had superior PFS compared to RT plus neoadjuvant chemotherapy [HR = 0.46 (0.22, 0.95), P = .0357]. Within a subgroup for whom detailed clinical data were available, those who received RT plus adjuvant chemotherapy had improved PFS compared to RT only [HR = 0.24 (0.074–0.76), P = .016]. The substitution of cisplatin for carboplatin and the elimination of vincristine did not negatively affect outcomes.ConclusionThis is the largest single-institution retrospective study of adult MB to our knowledge and identifies standard-risk status, first-line RT and adjuvant chemotherapy as factors associated with improved outcomes.

The classic method for determining alveolar‐capillary membrane conductance (D M ) and pulmonary capillary blood volume (V C ) involves measurement of lung diffusing capacity for carbon monoxide (D LCO ) at multiple inspired O 2 tensions (F I O 2 ). Recently, the technique has been simplified by simultaneously measuring D LNO (nitric oxide) to determine values at a single F I O 2 . Controversy exists over equations for calculation of θ CO (reaction rate of CO with Hgb) and the correct diffusion ratio for NO and CO (α) in the calculation of D MCO . The purpose of this study was to investigate how various published equations and ratios influence D M & V C using both the multiple and single F I O 2 methods. Eight healthy males were studied. D LCO & D LNO were measured using the rebreathe method (inspired vol. ~75% of inspiratory capacity, F I O 2 18, 35, & 55%). D M & V C were first calculated using five reported equations for θ CO . Once D M & V C were optimized based on reported predicted values, the D LNO method was compared using a range of α values. Using the multiple F I O 2 method, depending on the equation for θ CO , D M ranged from 47 to 103 ml/min/mmHg, and V C from 74 to 102 ml. For the calculation of D M & V C using the D LNO method, α ranged from 1.5 to 4.0 in order to optimize D M & V C values to the classic method. The determination of D M & V C is critically dependent on the equations used for θ CO which in turn affects the optimal ratio used to correct the D LNO method. NIH HL71478

Estrogen receptor beta (βER) is present in platelets but little is known about how βER contributes to platelet physiology. Platelet mitochondrial function and circulating platelet‐derived microparticles were analyzed in aged (24 months old) female wild type (WT) and estrogen receptor beta knockout (βERKO) mice. In intact, unstimulated platelets, ERβ did not co‐localized with mitochondria. Platelet lactate production, mitochondrial membrane potential and superoxide generation were similar in both groups, but activities of cytochrome bc 1 and cytochrome c oxidase were significantly less (P<0.05) in platelets of βERKO mice. Total numbers of platelet‐derived microparticles and those positive for procoagulant phosphatidylserine (PS) were two to three times higher (P<0.05) in βERKO than WT mice. Greater than 40% of procoagulant microparticles were derived from cells other than platelets in both groups of mice. Thrombin generating capacity of microvesicles was also greater in βERKO mice. Thus, loss of βER leads to significant changes in activities of enzymes of the mitochondrial electron transport system, and to increases in circulating procoagulant microparticles which may contribute to an increased thrombotic risk with aging in persons with βER gene polymorphisms. This study is supported by HL78638, AHA30503Z and Mayo Foundation.

Introduction: Outcomes from endovascular procedures are highly dependent on the experience and skill of the operating physician. The multi-disciplinary CREST-2 Interventional Management Committee (IMC) was charged with credentialing a cohort of skilled interventionists with adequate contemporary case volumes. Methods: Applicants were required to submit 25 consecutive cases completed within 5 years as primary operator out of a required total experience of ≥ 50 cases (≥ 20 for operators completing training). Interventionists not approved on initial review were asked to submit additional cases (with procedural angiograms), the number depending on quality and recent-quantity of the cases. Results: The IMC has had 102 meetings, and 283 interventionists have been evaluated: 104 (37%) interventionists were cardiologists, 64 (23%) vascular surgeons, 42 (15%) neurosurgeons, 32 (11%) neuroradiologists, 26 (9%) neurologists, 9 (3%) interventional radiologists, and 6 (2%) other. The mean total experience among the 226 interventionists with available information was 220±263 carotid stent cases (median 135; range 10-2500). A total of 7037 cases have been reviewed by the IMC, dating from August 2001 to April 2016, with 3366 symptomatic, 3541 asymptomatic and 130 undetermined. The range of cases reviewed per interventionist was 5 to 50. Of the 251 interventionists with sufficient periprocedural follow-up data, no stroke events were reported by 152 (60.5%), and at least one or more stroke events were reported by 99 (39.5%). The IMC has approved 115 interventionists, 29 at the first review and 86 subsequently, based upon submission and review of 631 additional contemporary cases (mean=7 cases per interventionist); 122 have approval pending submission of additional cases; 33 have been denied; 8 have been deferred; 4 have been approved for the CREST-2 Companion Registry only; and 1 is pending decision. Discussion: Rigorous evaluation and credentialing of carotid stenters in CREST-2 has been demanding, for the candidates and for the evaluators. Yet the cohort of interventionists so selected should be able to provide the high-quality stenting outcomes necessary for acceptance of the trial results.

Objective: Diabetes is a common among patients with atrial fibrillation (AF). However, little is known on the impact of diabetes on symptoms, health status, care and outcomes among AF patients. We assessed whether symptoms, health status, care and outcomes differ between AF patients with and without diabetes. Methods: This observational cohort study included 9,749 patients with AF from the Outcomes Registry for Better Informed Treatment of Atrial Fibrillation registry, a prospective, nationwide, multicenter outpatient registry of patients with incident and prevalent AF enrolled at 174 sites between June 2010 and August 2011. The outcomes were symptoms, health status, and AF treatment, as well as 2-year risks of death, readmission, thrombomebolic events, heart failure (HF) and AF progression. Results: Among AF patients; those with diabetes (29.5%) were younger and more likely to have hypertension, chronic kidney disease, HF, coronary heart disease, and stroke. Patients with diabetes had a lower Atrial Fibrillation Effects on Quality of Life score (80.6 [IQR: 62.5 - 92.6] vs. 82.4 [IQR: 67.6 -93.5]; P = 0.025), but a higher CHA 2 DS 2 -VASc score (5 [IQR: 4 - 6] vs. 4 [2 - 5]; P <0.0001) and were more likely to receive anticoagulation ( P <0.001). Diabetes was associated with higher mortality, with a greater overall (adjusted hazard ratio [aHR]: 1.63 [95% CI: 1.04-2.56] for age<70 vs. 1.25 [1.09-1.44] for age ≥70) and cardiovascular (2.20 [1.22-3.98] for age<70 vs. 1.24 [1.02-1.51] for age ≥70) mortality risks before age 70 years. Diabetes conferred a higher risk of non-cardiovascular (CV) death (1.29 [1.06-1.56]), sudden cardiac death (1.53 [1.04-2.26]), hospitalization (1.15 [1.09-1.22]), CV hospitalization (1.13 [1.05-1.22]) and non-CV and non-bleeding-related hospitalization (1.19 [1.10-1.30]); but no increase in risks of thromboembolic events, bleeding related hospitalization, new-onset HF, and AF progression. The use of oral anticoagulants (OAC) modifies the relation of diabetes and all-cause hospitalization, with a higher risk among those on OAC (1.21 [1.12-1.29]). Conclusions: Among patients with AF, diabetes was associated with worse AF symptoms and lower quality of life, as well an increased risk of death and hospitalizations, but not with the risk of thromboembolic or bleeding events.

Introduction: Patient understanding of available therapies for atrial fibrillation (AF) is foundational to shared medical decision making and long term medication adherence. Yet, there is a paucity of data regarding the extent to which patients newly diagnosed with AF in routine community practice understand their options. Hypotheses: 1) Understanding of warfarin, novel oral anticoagulants (NOAC), rhythm control therapy, cardioversion and radio frequency ablation changes little from baseline to 6 months and 2) treatment rates at 6 months are associated with patient understanding of therapies at baseline. Methods: We analyzed survey data from SATELLITE, a substudy of new-onset AF patients enrolled at 56 US sites participating in the ORBIT-AF registry. Patients were surveyed at the baseline and 6 month follow up clinic visit using Likert scales. Agreement between time points was assessed with the McNemar test, and the relationship between understanding and treatment was assessed only for the subset not on treatment at baseline. Results: Of 1000 patients enrolled in SATELLITE, 506 had 6-month survey data (data collection is continuing). Among these, the median age was 69.0 years (IQR 63.0 - 76.0) and 93.7% (474 of 506) were white. There was evidence of improvement in the self-reported understanding of warfarin and NOACs from baseline to 6 months, but not for rhythm control, ablation or cardioversion. The proportion reporting high understanding improved significantly for warfarin (p<.0001) and NOACs (p<.0001) from 47% (223 of 474) and 51% (245 of 481) at baseline to 60% (284 of 474) and 69% (332 of 481) at 6 months respectively (Figure 1). Patients with high understanding of the benefits of ablation (p=0.0005) and options for ablation (p=0.0093) at baseline were more likely to have this therapy at the 6 month follow up (N=590), but improved understanding was not associated with increased use of warfarin/NOACs (N=83) or rhythm control (N=444). Conclusions: Patients with new-onset AF had improved self-reported understanding of some treatment options over the first 6-months from diagnosis; however, factors other than patient understanding may influence AF treatments received at 6 months. Patient understanding of AF treatments remains suboptimal at 6 months, and our results suggest a need for ongoing patient education.