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Background: Although the most common causes of left ventricular aneurysm (LVA) is ischemic disease, other infectious, traumatic, genetic and iatrogenic etiologies exist. With the improvement of medical therapy for ischemic disease and earlier interventions such as PCI, the incidence of large LVA (>3cm) and surgical treatment for it is increasingly rare. Case study: We describe a case report and literature review of a giant LVA in a patient, who presented with unclear etiology. A 61-year-old male was referred to our tertiary center. He underwent aneurysmectomy and mitral valve replacement for a giant (10cm x 10cm) LVA with severe mitral regurgitation. Conclusion: Surgery for LVA is becoming less common. Early intervention can restore cardiac geometry with good short and long-term surgical outcomes, especially in patients with preserved EF. Ultimately, a giant ventricular aneurysm remains an indication for surgical intervention. Patients with markedly reduced EF may derive reduced benefits from aneurysmectomy.

Purpose Previous studies assessing impact of acute respiratory distress syndrome (ARDS) on mortality have shown conflicting results. We sought to assess the independent association of ARDS with in-hospital mortality among intensive care unit (ICU) patients with sepsis. Methods We studied two prospective sepsis cohorts drawn from the Early Assessment of Renal and Lung Injury (EARLI; n = 474) and Validating Acute Lung Injury markers for Diagnosis (VALID; n = 337) cohorts. ARDS was defined by Berlin criteria. We used logistic regression to compare in-hospital mortality in patients with and without ARDS, controlling for baseline severity of illness. We also estimated attributable mortality, adjusted for illness severity by stratification. Results ARDS occurred in 195 EARLI patients (41%) and 99 VALID patients (29%). ARDS was independently associated with risk of hospital death in multivariate analysis, even after controlling for severity of illness, as measured by APACHE II (odds ratio [OR] 1.65 (95% confidence interval [CI] 1.02, 2.67), p = 0.04 in EARLI; OR 2.12 (CI 1.16, 3.92), p = 0.02 in VALID). Patients with severe ARDS (P/F

Although the most common causes of left ventricular aneurysm (LVA) is ischemic disease, other infectious, traumatic, genetic and iatrogenic etiologies exist. With the improvement of medical therapy for ischemic disease and earlier interventions such as PCI, the incidence of large LVA (3cm) and surgical treatment for it is increasingly rare. Case study: We describe a case report and literature review of a giant LVA in a patient, who presented with unclear etiology. A 61-year-old male was referred to our tertiary center. He underwent aneurysmectomy and mitral valve replacement for a giant (10cm x 10cm) LVA with severe mitral regurgitation. Conclusion: Surgery for LVA is becoming less common. Early intervention can restore cardiac geometry with good short and long-term surgical outcomes, especially in patients with preserved EF. Ultimately, a giant ventricular aneurysm remains an indication for surgical intervention. Patients with markedly reduced EF may derive reduced benefits from aneurysmectomy.

Objective The aim of the present systematic review was to assess current trends in use of falciform ligament in abdominal surgery. Background The falciform ligament has been proposed in surgical literature as a suitable pedicle or flap with acceptable surgical outcomes; however, it is underutilized in abdominal surgery. Methods We performed a literature search and meta-analysis. All English studies describing use of a falciform ligament were eligible for inclusion. Results Of the 547 articles mentioning use of falciform ligament, 32 full text articles were included in this review. The majority were case reports (n = 14). The mremaining papers included retrospective analysis (n = 10), randomized control trials (n = 2), reviews (n = 3), and technique descriptions (n = 6). Conclusion The falciform ligament appears to be a safe and easily accessible natural tissue with a variety of surgical applications. It is most useful as a pedicle flap and could have benefit in foregut surgery including hepatic, pancreatic, biliary, gastric, and esophageal operations.

Supplemental Digital Content is available in the text., OBJECTIVES: Unbiased global metabolomic profiling has not been used to identify distinct subclasses in patients with early sepsis and sepsis-associated acute respiratory distress syndrome. In this study, we examined whether the plasma metabolome reflects systemic illness in early sepsis and in acute respiratory distress syndrome. DESIGN: Plasma metabolites were measured in subjects with early sepsis. SETTING: Patients were admitted from the emergency department to the ICU in a plasma sample collected within 24 hours of ICU admission. Metabolic profiling of 970 metabolites was performed by Metabolon (Durham, NC). Hierarchical clustering and partial least squares discriminant clustering were used to identify distinct clusters among patients with early sepsis and sepsis-associated acute respiratory distress syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among critically ill patients with early sepsis (n = 197), three metabolically distinct subgroups were identified, with metabolic subtype driven by plasma lipids. Group 1, with 45 subjects (23% of cohort), had increased 60-day mortality (odds ratio, 2; 95% CI, 0.99–4.0; p = 0.04 for group 1 vs all others). This group also had higher rates of vasopressor-dependent shock, acute kidney injury, and met Berlin acute respiratory distress syndrome criteria more often (all p < 0.05). Conversely, metabolic group 3, with 76 subjects (39% of cohort), had the lowest risk of 60-day mortality (odds ratio, 0.44; 95% CI, 0.22–0.86; p = 0.01) and lower rates of organ dysfunction as reflected in a lower Simplified Acute Physiology Score II (p < 0.001). In contrast, global metabolomic profiling did not separate patient with early sepsis with moderate-to-severe acute respiratory distress syndrome (n = 78) from those with sepsis without acute respiratory distress syndrome (n = 75). CONCLUSIONS: Plasma metabolomic profiling in patients with early sepsis identified three metabolically distinct groups that were characterized by different plasma lipid profiles, distinct clinical phenotypes, and 60-day mortality. Plasma metabolites did not distinguish patients with early sepsis who developed acute respiratory distress syndrome from those who did not.

BRAFV600E mutations occur in approximately 40% of all patients with papillary thyroid cancer (PTC) and are associated with a worse prognosis in population studies. Treatment with single-agent BRAF inhibitors can result in nondurable partial responses (PRs) in clinical trials, but resistance inevitably develops. The mechanisms of resistance are not completely understood, but in non-thyroid tumors harboringBRAFV600E mutations, resistance has been ascribed to concurrent or acquired mutations inMEK1/2, RAC1, KRAS,andNRAS.This case report describes a patient with radioactive iodine–refractory metastatic PTC treated in a clinical trial with combination BRAF and MEK inhibition who achieved a durable PR. At time of progression, biopsy revealed an acquiredKRASG12V–activating mutation. The patient subsequently went on to have a PR to cabozantinib therapy in the clinical trial. This is the first reported case of an acquiredKRAS-activating mutation that developed during treatment with BRAF and MEK inhibition in a patient withBRAF-mutated PTC. TheKRASmutation was also detected in peripheral blood samples taken as part of the trial, indicating that resistant mutations may be identified through noninvasive means. The identification of resistant mutations in patients at time of progression is necessary to identify possible therapeutic options including potential clinical trials.ClinicalTrials.govidentifier: NCT01723202

e19587 Background: The treatment landscape in patients with mantle cell lymphoma (MCL) is changing with the introduction of Bruton’s tyrosine kinase inhibitors (BTKis) and bendamustine in Australia. We sought to analyze the practice impact of the introduction of publicly funded novel agents in MCL. The objective of this study was to describe the evolving treatment patterns of Australian patients with MCL over the last 10 years using population-wide prescription records. Methods: Patients who initiated a treatment for MCL between 01/01/2011 and 07/31/2021 were extracted from the Services Australia 10% Pharmaceuticals Benefits Scheme (PBS) dataset, which includes the dispensing records for 10% of the Australian population. This dataset captures all publicly funded treatments in Australia. The index date was defined as the commencement of any drug for MCL. First-line (1L) therapy was defined as the first treatments prescribed for MCL. A patient was defined as relapsed/refractory (R/R) if they had commenced a drug which was in a different therapeutic category, or if they re-started the same regimen after a gap of more than 180 days. Descriptive analyses were conducted to examine the use of treatment regimens for the overall 10-year population by line of therapy. Analyses by calendar year were also performed to assess the changes in the treatment patterns. Results: Overall, 241 patients with MCL were identified over the study period. The majority of patients were male (68.4%), age > 60 years (84.9%), and most being aged 70-79 years (42.1% of total). Many patients were receiving comedications at baseline, including antihypertensives (44.1%), anticoagulants (14.5%), and/or antipsychotics or antidepressants (12.5%). In the overall population (2011-2021), the majority of patients had received 1L treatment with bendamustine-rituximab (BR, 53.9%), rituximab plus other regimens (27.6%), or rituximab monotherapy (11.2%). The most commonly used regimens in R/R patients at any subsequent episode of treatment included BTKi (66.3%), rituximab monotherapy (52.8%) or rituximab plus other regimens (31.5%). A trend in adoption of novel agents was observed throughout the years following their PBS listing. Analysis by calendar year showed that from 2011 to 2020 for 1L therapy, the use of BR increased from 0% to 50% and use of rituximab-containing regimen except BR decreased from 100% to 16.7%; use of BTKi increased in R/R patients from 0% to 74.7%. Conclusions: MCL treatment patterns have significantly changed in Australia since the introduction of BTKis and bendamustine-containing regimens. The use of rituximab-containing regimens except BR in 1L MCL has decreased and use of BTKis in R/R patients has increased.

e18730 Background: MZL remains understudied with limited real-world evidence on how current tx conform to clinical guidelines. This study aimed to assess real-world tx patterns, costs, and healthcare resource utilization (HRU) in US MZL pts. Methods: A retrospective, observational study was conducted using the IBM MarketScan® commercial and Medicare supplemental claims dataset (2017-2020). Newly diagnosed adult MZL pts (≥18 yr) continuously enrolled 6 month pre- and 3 month post-index date, defined as the first diagnosis date, were included. Tx regimens were identified by line of therapy and mutually exclusively categorized as rituximab monotherapy (R-mono), bendamustine + rituximab (BR), rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), ibrutinib (ibr), or other. Descriptive analyses were conducted to assess pt sociodemographic/clinical characteristics and tx utilization patterns (frequency, duration, discontinuation). Costs and HRU assessed included inpatient, outpatient, and pharmacy visits per-patient-per-month (PPPM). Multivariable logistic regression was conducted to examine predictors of costs and HRU. Results: In 2491 MZL pts (median age = 63; 52% male), 59% were commercially insured (median age = 57) and 41% in Medicare (median age = 76). Common comorbidities were hypertension (44%), diabetes (17%), atrial fibrillation (AF; 16%), and gastroesophageal reflux disease (15%). Mean time from diagnosis to tx initiation was 223 days. A total of 1,781 (72%) pts received first-line (1L), 518 (29%) pts received second-line (2L) and 239 (13%) pts received third-line (3L) therapies. R-mono was the most common regimen across both commercial and Medicare pts and all tx lines (Table). R-CHOP and BR were the second most used regimen in 1L with decreased use in 2L+. Ibr was used more in 2L+ setting but had the lowest 1L PPPM cost (median $2958.9) than other regimens. Overall MZL pts had PPPM 4.6 outpatient visits, 0.5 hospitalization, and mean length of stay of 2.6 days. Total PPPM healthcare cost was $19,895.8. Multivariable regression showed that baseline comorbidities (AF, renal disease, neutropenia) and tx discontinuation were significant predictors of higher costs and HRU. Conclusions: This real-world data suggested that US MZL real-world tx pattern across lines of therapy follows the regimen recommendations by the National Comprehensive Cancer Network clinical practice guidelines and that MZL pts incur high economic burden. Future studies are needed to evaluate long-term outcomes and the impact of heterogenous MZL subtypes.[Table: see text]

e19514 Background: Zanubrutinib, a next-generation Bruton’s tyrosine kinase inhibitor (BTKi), demonstrated superior overall response rate and a trend toward improved progression-free survival (PFS) as compared to ibrutinib in patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) in the phase 3 ALPINE trial (NCT03734016) based on interim analysis. However, its comparative efficacy with other treatments is not known. The goal of this study was to estimate the relative efficacy of zanubrutinib compared with standard treatments for R/R CLL through a network meta-analysis (NMA). Methods: The efficacy of zanubrutinib from ALPINE (study of zanubrutinib and ibrutinib in patients with R/R CLL was compared to ibrutinib, acalabrutinib, bendamustine and rituximab (BR), and venetoclax + rituximab (V+R) using data from 4 RCTs. Relevant RCTs were identified using a systematic literature review. A network was constructed that composed RCTs (ALPINE, ELEVATE-RR [NCT02477696], ASCEND [NCT02970318], and MURANO [NCT02005471]). Bayesian network meta-analysis (NMA) models were used to simultaneously synthesize hazard ratios (HRs) and 95% credible intervals (CrIs) for investigator-assessed PFS and overall survival (OS) to estimate the relative efficacy of zanubrutinib versus comparators of interest. An assumption of constant hazard ratio was applied in the NMA analysis. Analyses were performed using well-established codes published by the National Institute for Health and Care Excellence (NICE) Decision Support Unit and were implemented with OpenBUGS (version 3.2.3). Results: The NMA suggested a significant improvement in PFS for zanubrutinib over acalabrutinib (HR=0.52, 95% CrI [0.30, 0.90]), ibrutinib (0.47, [0.29, 0.76]), and BR (0.13, [0.06, 0.26]). Zanubrutinib had a numerically better PFS than V+R (0.69, [0.32, 1.46]) while not reaching statistical significance. For OS, NMA results showed a trend favoring zanubrutinib over acalabrutinib (0.75, [0.35, 1.59]), ibrutinib (0.62, [0.31, 1.22]), and BR (0.52, [0.21, 1.24]). The difference was not statistically significant with wide CrIs, suggesting a high uncertainty as inherent limitation of the analysis. Conclusions: This NMA reports the first summary of comparative PFS of available treatments for R/R CLL. Indirect treatment comparisons suggest PFS on zanubrutinib may be superior to other BTKis and immunochemotherapies in R/R CLL patients.[Table: see text]

e19526 Background: Zanubrutinib is an oral, highly selective, next-generation Bruton tyrosine kinase inhibitor. Efficacy of zanubrutinib was compared with bendamustine + rituximab (BR) in adult patients with treatment-naïve (TN) chronic lymphocytic leukemia (CLL) in the phase 3 SEQUOIA trial (NCT03336333). However, efficacy of zanubrutinib compared with other frontline CLL treatments remains unknown. The goal of this network meta-analysis of randomized controlled trials (RCTs) was to estimate the relative efficacy of zanubrutinib compared with standard frontline treatments for CLL. Methods: A total of 4 relevant RCTs were identified by a targeted literature review conducted throughout January 2022, CLL11 (NCT01010061), ALLIANCE (NCT01886872), MABLE (NCT01056510), and SEQUOIA. A feasibility assessment was performed to ensure that RCTs included within each population strata did not differ with respect to effect modifiers including presence of mutation, age, del11, and del17 status. A network was constructed for the composed 4 RCTs to compare the efficacy of zanubrutinib with bendamustine + rituximab, chlorambucil + obinutuzumab, chlorambucil + rituximab, and ibrutinib in patients with previously untreated CLL. Bayesian NMA models were conducted to simultaneously synthesize hazard ratio (HR) and 95% credible intervals (CI) for investigator-assessed progression-free survival (PFS). A constant hazard ratio was assumed in the NMA analysis. Statistical analyses were performed using codes suggested by the National Institute for Health and Care Excellence. Results: The efficacy results of the NMA indicated a statistically significant improvement in PFS for zanubrutinib over bendamustine + rituximab (HR=0.42; [95% CI=0.27, 0.65]), chlorambucil + obinutuzumab (0.45 [0.23, 0.86]), and chlorambucil + rituximab (0.22 [0.12, 0.41]). Zanubrutinib achieved comparable PFS to ibrutinib (1.07 [0.59, 1.94]). Conclusions: This is the first NMA to compare the efficacy of zanubrutinib with all commonly utilized first-line treatment for TN CLL. Results from this indirect treatment comparison suggested that the PFS for zanubrutinib may be statistically significantly better than immunochemotherapy. Findings from this study require validation with further large scale RCTs with longer follow up time.[Table: see text]

e18729 Background: While the incidence of AF in CLL has been increasing, the implications of AF in real-world CLL patients remain understudied. This study aimed to assess the impact of AF on clinical and economic outcomes in CLL patients. Methods: This retrospective observational study used the US IBM MarketScan® commercial claims/Medicare supplement database (2017-2020) to identify newly diagnosed CLL patients (≥18 years) and incidence of AF after index date, defined as the first CLL diagnosis date. Patients were followed for ≥3-months pre-index, and from index to last follow-up or death. In addition to patient characteristics, clinical outcomes (incidence of heart failure [HF], bleeding and stroke) and economic outcomes (costs and healthcare resource utilization [HRU]) were compared between CLL patients with AF (CLL+AF) and CLL patients without AF (CLL-AF). HRU was evaluated by inpatient, outpatient, ER, and pharmacy visits. Multivariable regression analyses were conducted to examine the association between AF and clinical outcomes. Results: Among a total of 16,800 newly diagnosed CLL patients included in the study, 20% developed AF. CLL+AF were significantly older than CLL-AF (median: 77 vs 62 years; P < .001). Compared to CLL-AF, CLL+AF also had significantly more comorbidities at baseline, as shown by higher Charlson comorbidity index (CCI) (median: 1.0 vs 3.0; P < .001) and more patients with previous AF history (0.3% vs 8.4%; P < .001). Further assessing clinical outcomes in CLL+AF vs CLL-AF, significantly higher incidence of HF (26.3% vs 3.0%; P < .001), bleeding (12.2% vs 4.8%; P < .001) and stroke (6.6% vs 1.2%; P < .001) were observed. For HRU, CLL+AF were reported to have significantly higher rates of ER visits (29.4% vs 12.9%; P < .001) and hospitalizations (42.2% vs 14.5%; P < .001) than CLL-AF. In CLL+AF, the average total AF-related costs were $13,520.21 within 30 days after AF diagnosis, and $22,304.82 within 60 days after AF diagnosis. Controlling for demographics and comorbidities, multivariable regressions reported statistically significant associations between AF and HF, as well as AF and stroke (Table). Conclusions: This real-world study reported significantly higher incidence of HF, bleeding and stroke incurred by CLL patients who developed AF compared with those who did not. The presence of HF, bleeding and stroke further increased HRU and costs. These findings highlight the importance of better disease management and treatment selection to prevent AF in CLL patients.[Table: see text]

e18727 Background: BTKi therapies, approved for relapsed or refractory (R/R) MCL, have not been comprehensively evaluated in RW populations. This study aimed to assess patient characteristics, treatment patterns and associated outcomes in RW BTKi-treated MCL pts. Methods: The retrospective multicenter chart review was conducted in the Cardinal Health Oncology Provider Extended Network. EMR data were extracted for eligible pts diagnosed with MCL who initiated any of the approved BTKi (ibrutinib [ibr], acalabrutinib [acal], zanubrutinib [zanu]) from 2018 to 2021; pts enrolled in trials were excluded. Index date was defined as the use of any of the BTKis. Pts were followed 12-mo pre-index for medical history, and from index to last follow-up or death. Descriptive analyses were conducted to assess demographic/clinical characteristics, MCL baseline features, BTKi treatment patterns, adverse events (AE), and response rates by BTKi. Multivariable logistic regression was performed to assess factors associated with response and AE. Results: The study cohort consisted of 300 MCL pts (59% male; 69% white); most (64%) pts were covered by Medicare, 34% had commercial insurance. BTKis were given mainly as monotherapy (93%) and in R/R setting (86%). Pts in zanu group were significantly older (n = 100, median age = 71, range = 50-91) than pts in ibr (n = 100, median age = 69, range = 39-87) and acal (n = 100, median age = 70, range = 51-86) groups. Significantly fewer pts in the zanu group had baseline Ann Arbor stage I-II (4%) than ibr (10%) or acal (13%), while more zanu pts had presence of B symptoms (67%) than ibr (44%) or acal (57%). Pts in the zanu group also had significantly less with ECOG of 3+ (4%) compared to ibr (8%) or acal (6%). At BTKi initiation, significantly more pts in zanu group (18%) had history of atrial fibrillation than ibr (1%) or acal (5%). Multivariable regression reported a significant association of age, gender, extranodal/splenic involvement, and timing of BTKi initiation with response and AE (Table). Conclusions: This study provides the first RW evidence on comparative effectiveness of ibr, acal, zanu in MCL pts. While pts treated with zanu were older and had more complex MCL baseline features at initiation, multivariable regression suggested a trend favoring zanu over ibr or acal for both response and AE. Frontline initiation of BTKi therapy was also associated with improved tolerability. Future RW studies are needed to discern long-term outcomes.[Table: see text]

Astroblastomas (ABs) are rare brain tumors of unknown origin. We performed an integrative genetic and epigenetic analysis of AB-like tumors. Here, we show that tumors traceable to neural stem/progenitor cells (radial glia) that emerge during early to later brain development occur in children and young adults, respectively. Tumors with MN1-BEND2 fusion appear to present exclusively in females and exhibit overexpression of genes expressed prior to 25 post-conception weeks (pcw), including genes enriched in early ventricular zone radial glia and ependymal tumors. Other, histologically classic ABs overexpress or harbor mutations of mitogen-activated protein kinase pathway genes, outer and truncated radial glia genes, and genes expressed after 25 pcw, including neuronal and astrocyte markers. Findings support that AB-like tumors arise in the context of epigenetic and genetic changes in neural progenitors. Selective gene fusion, variable imprinting and/or chromosome X-inactivation escape resulting in biallelic overexpression may contribute to female predominance of AB molecular subtypes.

Children with brain tumors often carry germline mutations that have long been known to contribute to tumorigenesis and treatment response. However, less is known about how germline mutations may impact developmental and behavioral outcomes for children with tumors of the central nervous system (CNS). As the molecular mechanisms governing cancerous and normal tissue expand, we hypothesize that particular germline variants may impact baseline neurocognitive function and/or treatment-induced toxicities. In this preliminary study, 10 children on the Head Start 4 trial diagnosed with medulloblastoma were assessed for baseline adaptive functioning using the Adaptive Behavior Assessment System Third Edition (ABAS-III) and germline whole-exome sequencing was performed. After filtering for high impact variants, Welch’s T-tests were used to identify mutations associated with lower ABAS-III General Adaptive Composite (GAC) scores, reflecting developmental and adaptive behavior delays compared with peers their age. We found 20 genes with alterations associated with lower scores with P-values less than 0.05, although none met FDR significance cutoffs. Genes found to be significant included LAMC1 (P=0.04) and KRTAP1-1 (P=0.045), which encode members of the laminin and keratin family and are involved in extracellular matrix adhesion. Mutations in PITX1, a known suppressor of RAS, were also associated with lower ABAS-III GAC scores (P=0.007). We suspect that follow-up studies with more patients will reveal alterations in cell-to-cell communication and signal transduction pathways, as these are common molecular perturbations in tumors that would likely impact regular CNS function. Future research with larger cohorts is essential to validate these findings and to improve our understanding of the functional impact of germline variants on both tumor and regular tissue biology, allowing for a more comprehensive view of the spectrum of neurodevelopmental impairment and novel strategies to circumvent these impairments. Funding: Thrasher Research Fund (JF, AC). R01CA108633, RC2CA148190, U10CA180850(NCI), and OSUCCC (AC).

OBJECTIVE: This study was conducted to analyse emergency ophthalmology referrals to a Canadian tertiary academic centre during the current coronavirus disease 2019 (COVID-19) pandemic in comparison to prepandemic referrals. DESIGN: This was a retrospective chart review looking at emergency referrals seen by the ophthalmology service between March 18 to April 17, 2020 (representing the COVID-19 period), and March 18 to April 17, 2019 (representing the pre-COVID-19 period). METHODS: Data gathered from referral records included patient demographics, timing and site of referral, and ophthalmic diagnosis. Referrals were categorized as urgent or nonurgent, with urgent indicating the need for ophthalmic assessment within 24 hours. RESULTS: The total number of referrals decreased by 54.2% in the COVID-19 period versus the pre-COVID-19 period. There was a similar bimodal age distribution in both periods, with fewer patients over 65 years of age presenting during the pandemic. Tertiary hospital referrals decreased by 62% in the pandemic period, while nontertiary emergency department referral trends varied and outpatient clinic referrals increased by 16%. Overall, there was a significant shift in the distribution of referral sites (p = 0.04). The proportion of urgent referrals increased by 14% during the pandemic; this was not statistically significant. There was no significant change in the timing of referrals or in the distribution of diagnostic segments. CONCLUSIONS: This study offers insight into the impact of the COVID-19 pandemic on ophthalmology referral patterns in a Canadian context. Moving forward, it helps to guide resource allocation and public education on the importance of seeking necessary eye care.

Introduction: Prognostic testing, including immunoglobulin heavy-chain variable region gene (IgHV) mutation status, cytogenetic abnormalities by fluorescence in situ hybridization (FISH), and immunophenotyping has been recommended in all newly diagnosed patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) prior to treatment initiation, and even in previously treated patients in some settings. Recent data have shown that disease with high-risk genetic features is better managed with novel agents than traditional chemoimmunotherapy. As such, the need for testing has become more relevant for disease management. However, there is limited recent data on real-world patterns of testing for risk factor assessment and in-turn, patterns of evidence-based treatment selection. This study aimed to examine: (1) the frequency and results of testing, (2) timing of testing by line of therapy, and (3) factors associated with the receipt of testing. Methods: De-identified data from the Flatiron Health EHR-derived database was used to identify patients who were ≥18 years old with newly-diagnosed CLL/SLL, who had ≥6-month continuous enrollment, and no prior treatment from January 2014 to May 2021. Testing evaluated included IgHV mutation status, FISH cytogenetic (11q deletion [del(11q)], 13q deletion [del(13q)], 17p deletion [del(17p)], Trisomy 12 [+12]) and other biomarkers (including CD38 and ZAP-70) by immunophenotyping. Descriptive analyses were conducted to examine the frequency and results in the overall population, and compared by patient characteristics and across sociodemographic groups. Multivariable logistic regression was conducted to examine factors associated with the likelihood of receiving testing. Statistical significance was determined as a p-value of Results: A total of 3,037 CLL patients were included (median age=73, 62.3% male, 74.6% white). The majority of patients (92%) received treatment in community practices, with 54.1% commercially-insured. We observed over half of CLL patients did not receive risk factor testing (Figure 1): IgHV mutation analyses (76.2%, n=2,315), FISH (61.5%, n=1,868) and immunophenotyping (72.1%, n=2,190). Of those who had testing, the majority (99%) had it done once prior to starting first-line therapy. Significant differences in the receipt of testing were observed between different age, gender, race/ethnicity, and regional subgroups (Table 1). Among patients who received testing, the presence of high-risk biomarkers was as follows: unmutated IgHV (56.1%), del(17p) present (14.4%), del(11q) present (16.9%), and CD38 present (30.8%). Compared to patients Conclusions: The NCCN guidelines recommend novel agents for patients with high-risk CLL/SLL. Thus, all patients are advised to complete risk-factor testing for both prognostication and selection of optimal, evidence-based therapy. Our real-world data highlights not only a significant gap in testing, but that this suboptimal testing is more common in vulnerable populations. We observed that despite identification of del(17), a quarter of CLL patients failed to receive novel agents in the frontline setting. Our analysis identified an unmet need for further education and refinement of clinical practice. This is necessary to achieve the best clinical outcome in CLL patients through robust risk-assessment testing and optimal therapeutic triaging. Figure 1 Figure 1. Disclosures Chanan-Khan: BieGene, Jansen, Ascentage: Consultancy; Ascentage, Starton, Cellectar, NonoDev, Alpha2 Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Alpha2 Pharmaceuticals: Patents & Royalties: Tabi; Ascentage: Research Funding; Cellectar: Current equity holder in publicly-traded company; Alpha2 Pharmaceuticals, NonoDev, Starton: Current holder of stock options in a privately-held company; BeiGene, Jansen, Ascentage: Honoraria. Yang: BeiGene, Ltd.: Current Employment. Liu: BeiGene, Ltd.: Current Employment. Zimmerman: BeiGene, Ltd.: Current Employment. Tang: BeiGene, Ltd.: Current Employment. Ailawadhi: Ascentage: Research Funding; Medimmune: Research Funding; Pharmacyclics: Consultancy, Research Funding; GSK: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Genentech: Consultancy; Xencor: Research Funding; BMS: Consultancy, Research Funding; Karyopharm: Consultancy; Cellectar: Research Funding; Sanofi: Consultancy; Janssen: Consultancy, Research Funding; AbbVie: Consultancy; Beigene: Consultancy; Takeda: Consultancy.

Objectives Chemotherapy remains a cornerstone treatment in non-small cell lung cancer either in combination with checkpoint inhibitors or as subsequent therapy. Identifying molecular predictors of response allows for optimal treatment selection. We performed genomic analysis on tumor samples of patients treated with carboplatin and nab-paclitaxel as part of a phase II trial to evaluate the prognostic and predictive value of mutations in DNA repair pathway in patients treated with this regimen. Materials and methods Next-generation sequencing libraries were produced using a capture-based targeted panel covering the coding exons of 278 genes on patients treated on clinical trial NCT00729612. Overall survival (OS) and progression-free survival (PFS) were assessed as part of the clinical outcomes and correlated with mutation analysis. Results Of 63 patients enrolled, 25 patients had sufficient and acceptable DNA isolated from archival tumor samples for targeted sequencing. The most commonly altered pathways included DNA repair (DR) including Fanconi anemia and homologous recombination, JAK-STAT signaling, IGF-1, mTOR, and MAPK-ERK. Four patients with mutations in homologous recombination mutations had a shorter PFS (hazard ratio [HR] = 4.54, 95% CI 1.2, 17.1, p = 0.026) and OS (HR = 6.3, 95% CI 1.8, 21.3, p = 0.003). Conclusion In this analysis of patients with predominantly squamous cell non-small cell lung cancer treated with carboplatin and nab-paclitaxel in a phase II trial, patients with mutations in homologous recombination pathways had shorter overall and progression-free survival. Validation on additional datasets of patients treated with platinum-based chemotherapy and immunotherapy combinations is warranted.

Children with brain tumors often carry germline mutations known to contribute to tumorigenesis and treatment response; however, little is known about how these mutations impact developmental and behavioral outcomes. As the molecular mechanisms governing cancerous and normal tissues expand, we hypothesize that specific germline variants may impact baseline neurocognitive function and/or treatment-induced toxicities. In this pilot study, ten children on the Head Start 4 (HS4) clinical trial diagnosed with medulloblastoma were assessed for baseline adaptive functioning using the Adaptive Behavior Assessment System Third Edition (ABAS-III) and germline whole-exome sequencing was performed. After filtering for high impact variants, Welch’s T-tests were used to identify mutations associated with lower ABAS-III General Adaptive Composite (GAC) scores, reflecting developmental and adaptive behavior delays compared with peers their age. We found twenty genes with alterations associated with lower scores with p-values less than 0.05. Genes found to be significant included LAMC1 (p=0.04) and KRTAP1-1 (p=0.045), which encode members of the laminin and keratin family respectively and are involved in extracellular matrix adhesion. Mutations in PITX1, a known suppressor of RAS, were also associated with lower ABAS-III GAC scores (p=0.007). We hypothesize that additional analyses of HS4 patients will reveal alterations in cell-to-cell communication and signal transduction pathways, common molecular perturbations in tumors that would likely impact central nervous system function. Validation studies are essential to improve our understanding of the functional impact of germline variants on both tumor and regular tissue biology, allowing for novel strategies to circumvent these delays.

Introduction: The mutational profile of non–small-cell lung cancer (NSCLC) has become an important tool in tailoring therapy to patients, with clear differences according to the population of origin. African Americans (AAs) have higher lung cancer incidence and mortality than Caucasians, yet discrepant results have been reported regarding the frequency of somatic driver mutations. We hypothesized that NSCLC has a distinct mutational profile in this group. Methods: We collected NSCLC samples resected from self-reported AAs in five sites from Tennessee, Michigan, and Ohio. Gene mutations were assessed by either SNaPshot or next generation sequencing, and ALK translocations were evaluated by fluorescence in situ hybridization. Results: Two hundred sixty patients were included, mostly males (62.3%) and smokers (86.6%). Eighty-one samples (31.2%) were squamous cell carcinomas. The most frequently mutated genes were KRAS (15.4%), epidermal growth factor receptor ( EGFR, 5.0%), PIK3CA (0.8%), BRAF, NRAS, ERBB2, and AKT1 (0.4% each). ALK translocations were detected in two nonsquamous tumors (1.7%), totaling 61 cases (23.5%) with driver oncogenic alterations. Among 179 nonsquamous samples, 54 (30.2%) presented a driver alteration. The frequency of driver alterations altogether was lower than that reported in Caucasians, whereas no difference was detected in either EGFR or KRAS mutations. Overall survival was longer among patients with EGFR mutations. Conclusions: We demonstrated that NSCLC from AAs has a different pattern of somatic driver mutations than from Caucasians. The majority of driver alterations in this group are yet to be described, which will require more comprehensive panels and assessment of noncanonical alterations.

Electrical characterization remains a key element in technology development and manufacturing of integrated circuits. Contact chain is a well known part of the diagnostic set of test structures used across many generations of silicon processes. Implementation of such test structures becomes challenging in new technologies with 3-D devices, like FinFET. Contacts to active regions of such devices are inherently dependent on the architecture of epitaxial raised source and drain and for proper characterization require the presence of transistor gates, which set the environment for contacts. This paper describes a new type of test structure, so-called gated contact chains, developed for contact process characterization in FinFET technologies. Instead of simple chain of contacts, each structure contains a series of active devices with common gate electrode used to turn on the chain of transistors to enable measurement of chain resistance. To discriminate between chain failures caused by an open contact or by other mechanisms (e.g., bad transistor with very high threshold voltage) a series of measurement under various test conditions was performed and analysed. In order to overcome a limitation of the contact chain size and enable data collection from larger sample of contacts, we proposed to implement the gated chains in addressable arrays, increasing their density and failure rate observability. Finally, the paper presents the examples of electrical failure modes detected by those chains in FinFET process.

The Epstein–Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. Although most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing life-threatening diseases, such as Hodgkin lymphoma and posttransplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV infection in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1 as a potential target antigen for vaccine development. Primary tumors from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) express BZLF1 protein. Pulsing human dendritic cells (DC) with recombinant BZLF1 followed by incubation with autologous mononuclear cells led to expansion of BZLF1-specific CD8+ T cells in vitro and primed BZLF1-specific T-cell responses in vivo. In addition, vaccination of hu-PBL-SCID mice with BZLF1-transduced DCs induced specific cellular immunity and significantly prolonged survival from fatal EBV-LPD. These findings identify BZLF1 as a candidate target protein in the immunosurveillance of EBV and provide a rationale for considering BZLF1 in vaccine strategies to enhance primary and recall immune responses and potentially prevent EBV-associated diseases. Cancer Immunol Res; 3(7); 787–94. ©2015 AACR.

© 2015 the American Physiological Society. The early sequence of events leading to the development of the acute respiratory distress syndrome (ARDS) in patients with sepsis remains inadequately understood. The purpose of this study was to identify changes in gene expression early in the course of illness, when mechanisms of injury may provide the most relevant treatment and prognostic targets. We collected whole blood RNA in critically ill patients admitted from the Emergency Department to the intensive care unit within 24 h of admission at a tertiary care center. Whole genome expression was compared in patients with sepsis and ARDS to patients with sepsis alone. We selected genes with >1 log2 fold change and false discovery rate

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruch’s membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity.

Metastasis of solid tumors is associated with poor prognosis and bleak survival rates. Tumor-infiltrating myeloid cells (TIMs) are known to promote metastasis, but the mechanisms underlying their collaboration with tumor cells remain unknown. Here, we report an oncogenic role for microRNA (miR) in driving M2 reprogramming in TIMs, characterized by the acquisition of pro-tumor and pro-angiogenic properties. The expression of miR-21, miR-29a, miR-142-3p and miR-223 increased in myeloid cells during tumor progression in mouse models of breast cancer and melanoma metastasis. Further, we show that these miRs are regulated by the CSF1-ETS2 pathway in macrophages. A loss-of-function approach utilizing selective depletion of the miR-processing enzyme Dicer in mature myeloid cells blocks angiogenesis and metastatic tumor growth. Ectopic expression of miR-21 and miR-29a promotes angiogenesis and tumor cell proliferation through the downregulation of anti-angiogenic genes such as Col4a2, Spry1 and Timp3, whereas knockdown of the miRs impedes these processes. miR-21 and miR-29a are expressed in Csf1r+ myeloid cells associated with human metastatic breast cancer, and levels of these miRs in CD115+ non-classical monocytes correlates with metastatic tumor burden in patients. Taken together, our results suggest that miR-21 and miR-29a are essential for the pro-tumor functions of myeloid cells and the CSF1-ETS2 pathway upstream of the miRs serves as an attractive therapeutic target for the inhibition of M2 remodeling of macrophages during malignancy. In addition, miR-21 and miR-29a in circulating myeloid cells may potentially serve as biomarkers to measure therapeutic efficacy of targeted therapies for CSF1 signaling.

Summary: Orchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cell-cycle-regulated transcription has improved significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G0, G1-S, and S-G2-M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the single-cell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development. : The study of E2Fs in vivo has been challenging. Cuitiño et al. reconstruct the spatiotemporal expression of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the mammalian cell cycle and propose that orchestrated accumulation of different E2F combinations control gene expression in proliferating (E2F3A-8-4) and differentiating (E2F3A-4) cells. Keywords: E2F, cell cycle, transcription, RNA-seq, E2F-tagged knockin mice, immunostaining, confocal microscopy, image analysis, deep learning, FUCCI mouse embryos

Entry into the cell cycle is mediated by cyclin-dependent kinase 4/6 (CDK4/6) activation, followed by CDK2 activation. We found that pharmacologic inhibition of the Flt3 internal tandem duplication (ITD), a mutated receptor tyrosine kinase commonly found in patients with acute myelogenous leukemia (AML), led to the down-regulation of cyclin D2 and D3 followed by retinoblastoma protein (pRb) dephosphorylation and G1 cell-cycle arrest. This implicated the D-cyclin-CDK4/6 complex as a downstream effector of Flt3 ITD signaling. Indeed, single-agent PD0332991, a selective CDK4/6 inhibitor, caused sustained cell-cycle arrest in Flt3 ITD AML cell lines and prolonged survival in an in vivo model of Flt3 ITD AML. PD0332991 caused an initial cell-cycle arrest in well-established Flt3 wild-type (wt) AML cell lines, but this was overcome by down-regulation of p27Kip and reactivation of CDK2. This acquired resistance was not observed in a Flt3 ITD and a Flt3 wt sample from a patient with primary AML. In summary, the mechanism of cell-cycle arrest after treatment of Flt3 ITD AML with a Flt3 inhibitor involves down-regulation of cyclin D2 and D3. As such, CDK4/6 can be a therapeutic target in Flt3 ITD AML but also in primary Flt3 wt AML. Finally, acquired resistance to CDK4/6 inhibition can arise through activation CDK2.

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized.

Contact Chain is a well known element of the diagnostic set of test structures used across many generations of silicon processes. Implementation of such test structures becomes challenging in new technologies with 3D devices, like FinFET. Contacts to active regions of such devices are inherently dependent on the architecture of epitaxial raised source and drain and for proper characterization require the presence of transistor gates, which set the environment for contacts. This paper describes examples of test structures for contact process development for FinFET technologies. Instead of simple chain of contacts, each structure contains a series of active devices with common gate electrode used to turn on the chain of transistors to enable measurement of chain resistance. To discriminate between chain failures caused by an open contact or by other mechanisms (e.g. bad transistor with very high threshold voltage) a series of measurement under various test conditions is performed and analysed to extract the contact failure rate. We also demonstrate the application of such structures in FinFET process characterization.

Chronic inflammation is a key pathogenic process in age-related macular degeneration (AMD). Amyloid-beta (Aβ) is a constituent of AMD drusen and promotes the activation of NLRP3 inflammasome which facilitates the production of cytokines. We investigated the role of transcription factor NF-κB in the activation of inflammasome in the RPE and the effect of vinpocetine, a dietary supplement with inhibitory effect on NF-κΒ. ARPE19/NF-κB-luciferase reporter cells treated with Aβ demonstrated enhanced NF-κB activation that was significantly suppressed by vinpocetine. Intraperitoneal injection of vinpocetine (15 mg/kg) inhibited NF-κB nuclear translocation and reduced the expression and activation of NLRP3, caspase-1, IL-1β, IL-18, and TNF-α in the RPE of adult rats that received intraocular Αβ, as measured by retinal immunohistochemistry and Western blot. Cytokine level in the vitreous was assayed using multiplex suspension arrays and revealed significantly lower concentration of MIP-3α, IL-6, IL-1α, IL-1β, IL-18, and TNF-α in vinpocetine treated animals. These results suggest that the NF-κB pathway is activated by Aβ in the RPE and signals the priming of NLRP3 inflammasome and the expression of pro-inflammatory cytokines including the inflammasome substrates IL-1β and IL-18. NF-κB inhibition may be an effective approach to stem the chronic inflammatory milieu that underlies the development of AMD. Vinpocetine is a potentially useful anti-inflammatory agent that is well-tolerated in long term use.

Purpose of Study: Research over the past decade has established a critical role for the tumor microenvironment in facilitating tumor growth and promoting invasiveness. Cells of the myeloid lineage, including macrophages, have been known to be key mediators of tumor progression and facilitate metastasis. Recent work from our lab highlights the Ets transcription factors, PU.1 and ETS2, as important players in tumor associated macrophages (TAMs) during breast cancer progression. Both transcription factors are downstream effectors of the Colony Stimulating Factor 1 (CSF1) signaling pathway, which is not only important for myeloid cell survival, but also has a well-established role within the tumor microenvironment. Here, we investigated the requirement of PU.1 in TAMs in a mouse mammary tumor model and have begun to address the role of PU.1 during metastasis. Research Method: We employed cre-loxP technology to conditionally delete PU.1 in the myeloid cell compartment. Syngeneic mice were orthotopically injected with mammary tumor cells directly into the fat pad. To model breast cancer brain metastasis in the mouse, we employed an intracranial injection model which recapitulates the biology of the human disease. We were able to isolate tumor associated macrophages from either site (mammary or brain tumor) for downstream applications. Novel Findings: Macrophage-specific deletion of PU.1 resulted in a significant reduction in mammary tumor growth as well as tumor angiogenesis. Our results suggest that ETS2 and PU.1, acting downstream of the CSF1 signaling pathway, cooperatively regulate the expression of pro-tumor genes along with ‘oncogenic’ microRNA. Conventional ChIP assays show that both transcription factors occupy enhancer regions adjacent to a set of selected genes and cooperatively regulate expression in tumor infiltrating macrophages. We focused on miR-21 and miR-29a for a more detailed investigation as both are expressed in CSF1R+ myeloid cells within the brain metastatic microenvironment. In our intracranial injection model, macrophages (F4/80 positive cells) are recruited to the growing tumor, and both miR-21 and miR-29a are upregulated in TAMs isolated by FACS from tumor-bearing mice as compared to controls. Conclusions and Implications: Given our present data, we believe that the Ets factors, PU.1 and Ets2, regulate a transcriptional program, including microRNAs, that is critical for CSF1 action in tumor infiltrating macrophages. Currently, clinical trials are underway using CSF1R inhibitors for several solid tumor types [clinicaltrials.gov]. The macrophage-specific microRNA, including miR-21 and miR-29a, may serve as indicators for to the efficacy of CSF1R inhibition. Citation Format: Katie Thies, David A. Taffany, Haritha Mathsyaraja, Sudarshana M. Sharma, Walter Hans Meisen, Tom Liu, Cynthia Timmers, Jose Otero, Balveen Kaur, Michael C. Ostrowski. The CSF1-PU.1 pathway in tumor associated macrophages promotes breast cancer growth and progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C30.

The Epstein-Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. While most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing lifethreatening pathology such as Hodgkin’s lymphoma and post-transplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1, a transcription factor that initiates latency to lytic cycle transition, as a potential target antigen for vaccine development. IHC assays on primary lymphoma samples from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) revealed significant expression of BZLF1 protein (>10% tumor cells). Other EBV-positive lymphomas that exhibited staining for BZLF1 included lymphomatoid granulomatosis (1/3) and diffuse large B-cell lymphoma (5/10); whereas EBV(+) Hodgkin’s and EBV(+) unclassifiable lymphomas failed to demonstrate any BZLF1 signal. To examine the immunomodulatory properties of BZLF1, PBMCs were isolated from 24 healthy, EBV-seropositive donors of various HLA types and cultured in vitro with adenovirus encoding recombinant BZLF1 (rAd5F35/BZLF1) or null control (rAd5F35/null) for 18 hrs. Quantification of the number of IFN-γ secreting cells by ELISPOT revealed increased immune activation in response to BZLF1 exposure in the majority (20/24) of donors. These results led us to hypothesize that enhanced recognition of BZLF1 by antigen-specific cellular immunity may provide a protective effect against EBV. To address this, we next investigated whether DCs loaded with recombinant BZLF1 protein could mediate the expansion of BZLF1-specific CD8(+) T-cells in vitro. DCs were generated in vitro from healthy PBMC monocytes. Coculture of rBZLF1-loaded DCs and autologous PBMCs led to increased frequencies of BZLF1-specific CD8(+) T-cells as determined by tetramer flow assays. To determine whether BZLF1 vaccination could prime BZLF1-specific T-cell immunity in vivo, SCID mice depleted of murine NK cells were engrafted with EBV-positive PBMCs (hu-PBL-SCID) and injected simultaneously with rBZLF1 (100 µg/mouse). Four weeks post-vaccination, ex vivo splenocytes were reexposed to BZLF1-presenting DCs in vitro and assayed for IFN-γ secretion by ELISPOT. In comparison to vehicle, vaccination with rBZLF1 alone significantly enhanced BZLF1-specific IFN-γ responses (p=0.0007, n=3). In order to generate broader, more sustained immune responses against BZLF1, we next explored BZLF1 virally-transduced DC vaccination as a potential approach to prevent EBV-LPD. Monocyte-derived DCs were infected with rAd5F35/BZLF1 or rAd5F35/null (MOI=10) and cocultured with autologous PBMCs for 7 days. Exposure to BZLF1-transduced DCs induced a robust expansion of BZLF1-specific CD8(+) cells that was comparatively higher than recombinant protein presentation. Finally, we examined whether vaccination of hu-PBL-SCID mice with rAd5F35/BZLF1-transduced DCs would positively enhance cellular immunity and improve survival against fatal EBV-LPD. Hu-PBL-SCID mice were injected IP with 5 x 106 BZLF1-transduced DCs (or control). Mice in the “booster group” received a second dose of either rAd5F35/BZLF1 (or control) DCs as a booster. Five weeks post-vaccination, ex-vivo splenocytes were cultured 1:1 with autologous lymphoblastoid cell lines (LCLs) and assayed for IFN-γ secretion by ELISPOT. Mice vaccinated with rAd5F35/BZLF1-transduced DCs showed significantly higher responsiveness to LCLs relative to vector control mice (p Disclosures No relevant conflicts of interest to declare.