Impact of C-reactive Protein on Anticoagulation Monitoring in Extracorporeal Membrane Oxygenation.

OBJECTIVE: To evaluate the impact of inflammation on anticoagulation monitoring for patients supported with extracorporeal membrane oxygenation (ECMO). DESIGN: Prospective single-center cohort study. SETTING: University-affiliated tertiary care academic medical center. PARTICIPANTS: Adult venovenous and venoarterial ECMO patients anticoagulated with heparin/ MEASUREMENTS AND MAIN RESULTS: C-Reactive protein (CRP) was used as a surrogate for overall inflammation. The relationship between CRP and the partial thromboplastin time (PTT, seconds) was evaluated using a CRP-insensitive PTT assay (PTT-CRP) in addition to measurement using a routine PTT assay. Data from 30 patients anticoagulated with heparin over 371 ECMO days was included. CRP levels (mg/dL) were significantly elevated (median, 17.2; interquartile range [IQR], 9.2-26.1) and 93% of patients had a CRP of ≥5. The median PTT (median 58.9; IQR, 46.9-73.3) was prolonged by 11.3 seconds compared with simultaneously measured PTT-CRP (median, 47.6; IQR, 40.1-55.5; p < 0.001). The difference between PTT and PTT-CRP generally increased with CRP elevation from 2.7 for a CRP of <5.0 to 13.0 for a CRP between 5 and 10, 17.7 for a CRP between 10 and 15, and 15.1 for a CRP of >15 (p < 0.001). In a subgroup of patients, heparin was transitioned to argatroban, and a similar effect was observed (median PTT, 62.1 seconds [IQR, 53.0-78.5 seconds] vs median PTT-CRP, 47.6 seconds [IQR, 41.3-57.7 seconds]; p < 0.001). CONCLUSIONS: Elevations in CRP are common during ECMO and can falsely prolong PTT measured by commonly used assays. The discrepancy due to CRP-interference is important clinically given narrow PTT targets and may contribute to hematological complications.

p53 immunohistochemistry as an ancillary tool for rapid assessment of residual disease in TP53-mutated acute myeloid leukemia and myelodysplastic syndromes.

OBJECTIVES: Measurable residual disease flow cytometry (MRD-FC) and molecular studies are the most sensitive methods for detecting residual malignant populations after therapy for TP53-mutated acute myeloid leukemia and myelodysplastic neoplasms (TP53+ AML/MDS). However, their sensitivity is limited in suboptimal aspirates or when the immunophenotype of the neoplastic blasts overlaps with erythroids or normal maturing myeloid cells. In this study, we set out to determine if p53 immunohistochemistry (IHC) correlates with MRD-FC and next-generation sequencing (NGS) in the posttherapy setting and to determine the utility of p53 IHC to detect residual disease in the setting of negative or equivocal MRD-FC. METHODS: We retrospectively identified 28 pre- and posttherapy bone marrow biopsy specimens from 9 patients with TP53+ AML/MDS and a p53 overexpressor phenotype by IHC (strong 3+ staining at initial diagnosis). Next-generation sequencing and/or MRD-FC results were collected for each specimen. RESULTS: Using a threshold of more than ten 2-3+ cells in any one 400× field, p53 IHC detected residual disease with a sensitivity of 94% and a specificity of 89%. The threshold used in this study showed a high degree of concordance among 6 blinded pathologists (Fleiss κ = 0.97). CONCLUSIONS: Our study suggests that p53 IHC can be used as a rapid tool (within 24 hours) to aid in the detection of residual disease that may complement MRD-FC or NGS in cases in which the flow cytometry immunophenotype is equivocal and/or the bone marrow aspirate is suboptimal.

The highs and lows of cyclic thrombocytopenia.

Cyclic thrombocytopenia (CTP) is characterized by periodic platelet oscillation with substantial amplitude. Most CTP cases have a thrombocytopenic background and are often misdiagnosed as immune thrombocytopenia with erratically effective treatment choices. CTP also occurs during hydroxyurea treatment in patients with myeloproliferative diseases. While the aetiology of CTP remains uncertain, here we evaluate historical, theoretical and clinical findings to provide a framework for understanding CTP pathophysiology. CTP retains the intrinsic oscillatory factors defined by the homeostatic regulation of platelet count, presenting as reciprocal platelet/thrombopoietin oscillations and stable oscillation periodicity. Moreover, CTP patients possess pathogenic factors destabilizing the platelet homeostatic system thereby creating opportunities for external perturbations to initiate and sustain the exaggerated platelet oscillations. Beyond humoral and cell-mediated autoimmunity, we propose recently uncovered germline and somatic genetic variants, such as those of MPL, STAT3 or DNMT3A, as pathogenic factors in thrombocytopenia-related CTP. Likewise, the JAK2 V617F or BCR::ABL1 translocation that drives underlying myeloproliferative diseases may also play a pathogenic role in hydroxyurea-induced CTP, where hydroxyurea treatment can serve as both a trigger and a pathogenic factor of platelet oscillation. Elucidating the pathogenic landscape of CTP provides an opportunity for targeted therapeutic approaches in the future.

Diagnostic impact of RNA-based next-generation sequencing fusion panel for solid tumors: A single-institution experience.

OBJECTIVES: Gene rearrangements frequently act as oncogenic driver mutations and determine the onset and progression of cancer. RNA-based next-generation sequencing (NGS) is being used with increasing frequency for solid tumors. The purpose of our study is to investigate the feasibility and utility of an RNA-based NGS fusion panel for solid tumors. METHODS: We conducted a retrospective, single-institution review of fusion panels requested between May 2022 and March 2023. Demographic, clinical, pathologic, and molecular findings of the patients were reviewed. The utility of the RNA-based NGS fusion panel for the pathologic diagnosis of solid tumors was assessed. RESULTS: Our study included 345 cases, and a fusion event was identified in 24.3% (78/321) of cases. Among the 110 cases submitted for diagnostic purposes, a fusion event was detected in 42.7% (47/110) of cases. The results led to refinement or clarification of the initial diagnosis in 31.9% (15/47) of cases and agreement or support for the initial diagnosis in 59.6% (28/47) of cases. Furthermore, our study indicated that the overall cellularity (tumor and normal tissue) of the tested specimen influences the success of the testing process. CONCLUSIONS: In summary, this study demonstrated the feasibility and utility of an RNA-based NGS fusion panel for a wide variety of solid tumors in the appropriate clinicopathologic context. These findings warrant further validation in larger studies involving multiple institutional patient cohorts.

Study of ß1-transferrin and ß2-transferrin using microprobe-capture in-emitter elution and high-resolution mass spectrometry.

Cerebrospinal fluid (CSF) leak can be diagnosed in clinical laboratories by detecting a diagnostic marker ß2-transferrin (ß2-Tf) in secretion samples. ß2-Tf and the typical transferrin (Tf) proteoform in serum, ß1-transferrin (ß1-Tf), are Tf glycoforms. An innovative affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with high-resolution mass spectrometry (HR-MS) to study the Tf glycoforms and the primary structures of ß1-Tf and ß2-Tf. To implement MPIE, an analyte is first captured on the surface of a microprobe, and subsequently eluted from the microprobe inside an electrospray emitter. The capture process is monitored in real-time via next-generation biolayer interferometry (BLI). When electrospray is established from the emitter to a mass spectrometer, the analyte is immediately ionized via electrospray ionization (ESI) for HR-MS analysis. Serum, CSF, and secretion samples were analyzed using MPIE-ESI-MS. Based on the MPIE-ESI-MS results, the primary structures of ß1-Tf and ß2-Tf were elucidated. As Tf glycoforms, ß1-Tf and ß2-Tf share the amino acid sequence but contain varying N-glycans: (1) ß1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611; and (2) ß2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611. The resolving power of the innovative MPIE-ESI-MS method was demonstrated in the study of ß2-Tf as well as ß1-Tf. Knowing the N-glycan structures on ß2-Tf allows for the design of more novel test methods for ß2-Tf in the future.

The role of genetics in refractory immune thrombocytopenia.

Patients with refractory immune thrombocytopenia (rITP) have increased morbidity and mortality. Currently, there is limited understanding of the cause of refractoriness and no markers to help direct novel treatment options. Understanding the reason(s) for refractoriness is crucial to determining novel treatment options. The pathogenesis underlying rITP has generally been thought to be an underlying genetic predisposition with an environmental trigger. Familial ITP remains rare, and there are few twin studies, suggesting that a simple genetic cause is unlikely. However, genetic mutations provide the background for several autoimmune diseases. In this review, we explore the evidence of either an inherited genetic cause of rITP or an acquired mutation, in particular one resulting in clonal expansion of cytotoxic T cells.

Genomic landscape of T-large granular lymphocyte leukemia and chronic lymphoproliferative disorder of NK cells: a single institution experience.

LGLL is a rare and chronic lymphoproliferative disorder including T-LGLL and CLPD-NK. Here, we investigated the genomic profiles of LGLL with a focus on STAT3 and STAT5B mutations in a cohort of 49 patients (41 T-LGLL, 8 CLPD-NK). Our study indicated that STAT3 was identified in 38.8% (19/49) of all patients, while STAT5B occurred in only 8.2% (4/49) of patients. We found that STAT3 mutations were associated with lower ANC in T-LGLL patients. The average number of pathogenic/likely pathogenic mutations in STAT3/STAT5B-mutated patients was significantly higher than that in WT patients (1.78 ± 1.17 vs 0.65 ± 1.36, p = 0.0032). Additionally, TET2-only mutated T-LGLL (n = 5) had a significant reduction in platelet values compared with the WT (n = 16) or STAT3-only mutated T-LGLL (n = 12) (p < 0.05). In conclusion, we compared the somatic mutational landscape between STAT3/STAT5B WT and mutated patients and correlate with their distinct clinical characteristics.

Enkurin: a novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.

Impaired protein homeostasis, though well established in age-related disorders, has been recently linked with the pathogenesis of myeloproliferative neoplasms (MPNs). However, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34+ cultures from patient bone marrow and healthy cord/peripheral blood specimens), we extend our prior data from platelet RNA sequencing in patients with MPN and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelet, parent megakaryocyte, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin (ENKUR), a calcium mediator protein originally implicated only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across specimens from patients with MPN and experimental models (including upon treatment with thapsigargin, an agent that causes protein misfolding in the ER by selective loss of calcium), with a concomitant upregulation of a cell cycle marker, CDC20. Silencing of ENKUR using short hairpin RNA in CD34+-derived megakaryocytes further confirms this association with CDC20 at both RNA and protein levels and indicates a likely role for the PI3K/Akt pathway. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis and ER and protein folding stress in MPN transformation.

Minimal/Measurable Residual Disease Monitoring in Patients with Lymphoid Neoplasms by High-Throughput Sequencing of the T-Cell Receptor.

High-throughput sequencing of the T-cell receptor beta (TRB) and gamma (TRG) loci is increasingly utilized due to its high sensitivity, specificity, and versatility in the diagnosis of various T-cell malignancies. Application of these technologies for tracking disease burden can be valuable in detecting recurrence, determining response to therapy, guiding future management of patients, and establishing endpoints for clinical trials. In this study, the performance of the commercially available LymphoTrack high-throughput sequencing assay was assessed for determining residual disease burden in patients with various T-cell malignancies. A custom bioinformatics pipeline and database was also developed to facilitate minimal/measurable residual disease analysis and clinical reporting. This assay demonstrated excellent test performance characteristics, achieving a sensitivity of 1 of 100,000 T-cell equivalents for the DNA inputs evaluated and high concordance with orthogonal testing methods. This assay was further utilized to correlate disease burden in several patients, demonstrating its potential utility for monitoring patients with T-cell malignancies.

Smoking status in acute myeloid leukemia is associated with worse overall survival and independent of prior nonhematopoietic malignancies, cytogenetic abnormalities, and WHO category.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy with several patient- and disease-associated variables known to impact prognosis. Tobacco smoking is an environmental factor associated with a greater incidence of AML, but there have been limited studies that evaluated smoking toward overall survival. We retrospectively searched for AML cases and collected clinical and diagnostic data for each case. We also used an independent next-generation sequencing (NGS) data set to assess for a distinct mutational signature associated with smoking. When stratified by smoking status, there was a greater number of males, patients aged ≥60 years, and patients with ≥2 comorbidities within the smoking category (P < .05). Survival analysis demonstrated decreased survival probability in the smokers, male smokers, smokers with 1 other comorbidity, and smokers without a prior history of nonhematopoietic malignancy (P < .05) as compared to nonsmokers. Smoking was associated with a decrease in survival within the World Health Organization categories of AML, not otherwise specified (AML NOS; P = .035) and AML with recurrent genetic abnormalities (AML RGA; P = .002). Multivariate analysis showed that patients who were smokers had a greater hazard ratio than nonsmokers after adjusting for the other covariates. Our findings demonstrated that smoking was independently associated with decreased overall survival after adjusting for other potentially confounding factors. In addition, our results suggest that a mutational signature can be recognized using NGS data in a subset of AML patients who smoke.

The role of CD8+ T-cell clones in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.

Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34 + cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin ( ENKUR ), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20 . Silencing of ENKUR by shRNA in CD34 + derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation. Key Points: Enkurin, a calcium adaptor protein, is identified as a novel marker of pathogenesis in MPNs.MPN megakaryocyte and platelet expression of enkurin at RNA and protein levels is inversely associated with a cell differentiation cycle gene, CDC20.Likely role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation.

Potential pitfalls in multiplex PCR-based next-generation sequencing: a case-based report.

Amplicon-based next-generation sequencing (NGS) assays employ highly sensitive, rapid, and cost-effective methods to detect clinically actionable mutations for the diagnosis, prognosis, and treatment of patients with cancer. However, recognition of certain limitations inherent to amplicon-based NGS assays is crucial for the correct interpretation and reporting of variants in the clinical setting. In this report, we illustrate three different potential pitfalls related to amplicon-based NGS assays based on our institutional experience and highlight how the risk of such events can be minimised.

Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3 and MPL mutations.

Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to 2 patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients, rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.

Neutral-Coating Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Top-Down Identification of Hemoglobin Variants.

BACKGROUND: Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathy. However, conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of structural characterization. We describe the use of neutral-coating capillary electrophoresis coupled with high-resolution mass spectrometry (CE-HR-MS) to achieve high-performance top-down identification of Hb variants. METHODS: An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 capillary electrophoresis (CE) unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis. Deconvolution of raw mass spectrometry data was carried out to merge multiple charge states and isotopic peaks of an analyte to obtain its monoisotopic mass. RESULTS: The neutral-coating CE could baseline separate individual Hb subunits dissociated from intact Hb forms, and the HR-MS could achieve both intact-protein analysis and top-down analysis of analytes. A number of patient samples that contain Hb subunit variants were analyzed, and the variants were successfully identified using the CE-HR-MS method. CONCLUSIONS: The CE-HR-MS method has been demonstrated as a useful tool for top-down identification of Hb variants. With the ability to characterize the primary structures of Hb subunits, the CE-HR-MS method has significant advantages to complement or partially replace the conventional methods for the identification of Hb variants.

Prevalence, mutational spectrum and clinical implications of clonal hematopoiesis of indeterminate potential in plasma cell dyscrasias.

Clonal hematopoiesis of indeterminate potential (CHIP) is common both in healthy individuals and patients with hematological cancers. Recent studies have showed worse prognosis for patients with multiple myeloma (MM) and non-Hodgkin lymphoma undergoing stem cell transplant, that have concomitant presence of CHIP. Data regarding the clinical and biological role of CHIP in plasma cell dyscrasias (PCDs) is rapidly increasing. However, the prevalence and prognostic implication of CHIP in patients with MM outside of the transplant setting, and in those with other more indolent PCDs remains elusive. Here we explored the prevalence and clinical implications of CHIP detected through next-generation sequencing in 209 patients with PCDs including MM, light chain (AL) amyloidosis (ALA), monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma (SMM). To avoid attributing the mutations to the plasma cell clone, CHIP was defined as the presence of DNMT3A, TET2, or ASXL1 mutations in the peripheral blood or bone marrow (DTA-CH). The prevalence of DTA-CH was 19% in patients with PCDs, with no difference between each PCD. TET2 (23%) and DNMT3A (22%), were the most frequently mutated genes. DTA-CH correlated with older age in MM (P = .001) and MGUS/SMM (P = 0.0007), as well as with coronary artery disease or congestive heart failure in MM (P = .03). DTA-CH did not predict worse OS or PFS in either MM or ALA, nor it predict higher risk of progression to MM in patients with MGUS/SMM. Our results overall further elucidate the prevalence and mutational spectrum of CHIP in PCDs, providing more information regarding the clinical relevance of CHIP in this patient population.

SARS-CoV-2 Brain Regional Detection, Histopathology, Gene Expression, and Immunomodulatory Changes in Decedents with COVID-19.

Brains of 42 COVID-19 decedents and 107 non-COVID-19 controls were studied. RT-PCR screening of 16 regions from 20 COVID-19 autopsies found SARS-CoV-2 E gene viral sequences in 7 regions (2.5% of 320 samples), concentrated in 4/20 subjects (20%). Additional screening of olfactory bulb (OB), amygdala (AMY) and entorhinal area for E, N1, N2, RNA-dependent RNA polymerase, and S gene sequences detected one or more of these in OB in 8/21 subjects (38%). It is uncertain whether these RNA sequences represent viable virus. Significant histopathology was limited to 2/42 cases (4.8%), one with a large acute cerebral infarct and one with hemorrhagic encephalitis. Case-control RNAseq in OB and AMY found more than 5000 and 700 differentially expressed genes, respectively, unrelated to RT-PCR results; these involved immune response, neuronal constituents, and olfactory/taste receptor genes. Olfactory marker protein-1 reduction indicated COVID-19-related loss of OB olfactory mucosa afferents. Iba-1-immunoreactive microglia had reduced area fractions in cerebellar cortex and AMY, and cytokine arrays showed generalized downregulation in AMY and upregulation in blood serum in COVID-19 cases. Although OB is a major brain portal for SARS-CoV-2, COVID-19 brain changes are more likely due to blood-borne immune mediators and trans-synaptic gene expression changes arising from OB deafferentation.

Multiplex Epstein-Barr virus BALF2 genotyping detects high-risk variants in plasma for population screening of nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) exhibits unusual geographic restriction despite ubiquitous lifelong infection. Screening programs can detect most NPC cases at an early stage, but existing EBV diagnostics are limited by false positives and low positive predictive value (PPV), leading to excess screening endoscopies, MRIs, and repeated testing. Recent EBV genome-wide association studies (GWAS) suggest that EBV BALF2 variants account for more than 80% of attributable NPC risk. We therefore hypothesized that high-risk BALF2 variants could be readily detected in plasma for once-lifetime screening triage. METHODS: We designed and validated a multiplex genotyping assay to detect EBV BALF2 polymorphisms in human plasma. Targeted next-generation sequencing was used to validate this assay, conduct association studies with clinical phenotype, and longitudinally genotype plasma to assess within-host haplotype stability. We examined the association between NPC and BALF2 haplotypes in a large non-endemic population and three prior EBV GWAS. Finally, we estimated NPC mortality reduction, resource utilization, and cost-effectiveness of BALF2 variant-informed screening using a previously-validated cohort model. RESULTS: Following analytical validation, the BALF2 genotyping assay had 99.3% concordance with sequencing in a cohort of 24 NPC cases and 155 non-NPC controls. BALF2 haplotype was highly associated with NPC in this non-endemic population (I613V: odds ratio [OR] 7.9; V317M: OR 178.8). No other candidate BALF2 polymorphisms were significantly associated with NPC or hematologic disorders. Longitudinal genotyping revealed 97.8% within-host haplotype concordance, indicative of lifelong latent infection. In a meta-analysis of 755 NPC cases and 981 non-NPC controls, BALF2 I613V and V317M were significantly associated with NPC in both endemic and non-endemic populations. Modeled variant-informed screening strategies achieved a 46% relative increase in PPV with 7% decrease in effective screening sensitivity, thereby averting nearly half of screening endoscopies/MRIs among endemic populations in east/southeast Asia. CONCLUSIONS: EBV BALF2 haplotypes are temporally stable within hosts and can be readily detected in plasma via an inexpensive multiplex genotyping assay that offers near-perfect sequencing concordance. In endemic and non-endemic populations, I613V and V317M were highly associated with NPC and could be leveraged to develop variant-informed screening programs that mitigate false positives with small reductions in screening sensitivity.