A novel XNA-based Luminex assay to detect UBA1 somatic mutations associated with VEXAS syndrome.

Objectives: Patients with VEXAS syndrome carry mutations of UBA1 gene coding for the E1 enzyme. The three most frequent mutations are p.M41T(122T > C), p.M41V (c.121A > G), and p.M41L (c.121A > C) in codon 41 of exon 3. Currently, sanger sequencing was mainly used to detect these mutations, which has low sensitivity and low throughput. There is a need of high sensitivity, simple and high throughput method to characterize patients with VEXAS syndrome. Methods: Based on our proprietary XNA technology, we have developed a QClamp® Plex platform to detect eight mutations in a single reaction using the Luminex xMap technology. The assay sensitivity, specificity and precision were subsequently evaluated. Furthermore, the reference interval and clinical sensitivity/specificity were estimated using clinical healthy/positive DNA samples and the sanger sequencing method was used for comparison. Results: With spiking synthetic mutant DNA in wildtype GM24385 cell line DNA, this assay can detect UBA1 mutations with a detection limit of variant allele frequency (VAF) at 0.1-5%. Our assay shows 100% concordance with Sanger sequencing results when used for analyzing 15 positive and 19 negative clinical samples. Conclusions: The QClamps® Plex UBA1 Mutation Detection Assay is a quicker, simpler, and more sensitive assay that can accurately detect the UBA1 mutations even at early stages with low mutation frequency.

How I diagnose and manage VEXAS syndrome.

OBJECTIVES: To discuss VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome, including the clinical and pathologic features, diagnostic challenges, and treatment options. METHODS: A case-based approach and pertinent literature review were used to highlight the features of VEXAS syndrome, describe how to make the diagnosis, and discuss available therapies. RESULTS: VEXAS syndrome is an adult-onset, progressive systemic inflammatory disorder with overlapping rheumatologic and hematologic manifestations, including an increased risk of myelodysplastic neoplasms and plasma cell neoplasms. The disorder is associated with a somatic mutation of the X-linked UBA1 gene involved in ubiquitylation, typically involving p.Met41; however, rare variations have been identified outside this region. Patients often present with complex histories and see physicians from multiple specialties before receiving the diagnosis, which is often delayed. Symptoms are related to inflammation as well as cytopenias, particularly macrocytic anemia. Characteristic cytoplasmic vacuoles are present in myeloid (granulocytic, monocytic) and erythroid precursors in the vast majority of cases. CONCLUSIONS: Either clinicians or pathologists may suspect a diagnosis of VEXAS syndrome depending on the clinical presentation and bone marrow findings. More studies are needed to determine the best therapeutic options, which are currently limited.

DeepHeme: A generalizable, bone marrow classifier with hematopathologist-level performance.

Morphology-based classification of cells in the bone marrow aspirate (BMA) is a key step in the diagnosis and management of hematologic malignancies. However, it is time-intensive and must be performed by expert hematopathologists and laboratory professionals. We curated a large, high-quality dataset of 41,595 hematopathologist consensus-annotated single-cell images extracted from BMA whole slide images (WSIs) containing 23 morphologic classes from the clinical archives of the University of California, San Francisco. We trained a convolutional neural network, DeepHeme, to classify images in this dataset, achieving a mean area under the curve (AUC) of 0.99. DeepHeme was then externally validated on WSIs from Memorial Sloan Kettering Cancer Center, with a similar AUC of 0.98, demonstrating robust generalization. When compared to individual hematopathologists from three different top academic medical centers, the algorithm outperformed all three. Finally, DeepHeme reliably identified cell states such as mitosis, paving the way for image-based quantification of mitotic index in a cell-specific manner, which may have important clinical applications.

Clinicopathologic characteristics, genetic features, and treatment options for acute lymphoblastic leukemia with JAK2 rearrangement-A 10-case study and literature review.

JAK2 rearrangement (JAK2-R) in acute lymphoblastic leukemia (ALL) is rare and often categorized as B-ALL with BCR::ABL1-like features based on the World Health Organization classification. We report 10 patients with JAK2-R ALL, 9 males and 1 female, with a median age 40.5 years. Eight patients presented with marked leukocytosis (median WBC, 63 × 10 9/L) and hypercellular (>95%) bone marrow with increased lymphoblasts (72%-95%). There was no evidence of bone marrow fibrosis or hypereosinophilia. Immunophenotypic analysis showed 9 B-cell and 1 T-cell neoplasms. Using fluorescence in situ hybridization (FISH) and RNA sequencing analysis, JAK2 partners were identified for 7 cases and included PCM1 (n = 4), ETV6 (n = 2) and BCR (n = 1). All patients received upfront polychemotherapy. Additionally, 2 patients received ruxolitinib, 2 received allogeneic stem cell transplant, and 1 received CAR-T therapy. The 1- and 3-year overall survival rates were 55.6% and 22.2%, respectively. A literature review identified 24 B-ALL and 4 T-ALL cases with JAK2-R reported, including 16 males, 6 females and 6 gender not stated. Many JAK2 partner-genes were reported with the most common being PAX5 (n = 7), ETV6 (n = 5), BCR (n = 4) and PCM1 (n = 2). Survival data on 13 reported cases showed 1- and 3-year overall survival rates of 41.7% and 41.7%, respectively. In summary, JAK2-R ALL occurs more often in adult males, are mostly of B-cell lineage, and associated with an aggressive clinical course. Absence of eosinophilia and bone marrow fibrosis and no evidence of preexisting/concurrent JAK2-R myeloid neoplasms distinguish JAK2-R ALL from other myeloid/lymphoid neoplasms with eosinophilia and JAK2-R.

Clinical Response to Upfront Targeted Tyrosine Kinase Inhibitors among Patients with Myeloid/Lymphoid Neoplasms with Eosinophilia and Tyrosine Kinase Gene Fusion.

INTRODUCTION: Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusion (MLN-TK) is an entity encompassed of a heterogeneous group of rare hematopoietic neoplasms that are driven by gene fusion involving PDGDRA/B, FGFR1, JAK2, FLT3 or ETV6::ABL1. Though patients presenting with chronic phase MLN-TK with PDGFRA fusion display a favorable outcome in response to upfront TK inhibitor (TKI) therapy, the outcomes of MLNs driven by other TK fusions are not well described. In this study, we aimed to critically analyze the treatment outcomes of patients with MLN-TK, focusing on the role of upfront TKIs in both chronic- and blast-phase diseases. METHODS: The retrospective study included patients with confirmed MLN-TK from 3 centers and assessed demographic and clinical variables, treatment, and outcomes. RESULTS: Forty-two patients with confirmed MLN-TK [PDGFRA (n = 22), PDGFRB (n = 4), FGFR1(n = 10), JAK2 (n = 2); and FLT3 (n = 3)] were included. Fifteen of 25 (60%) chronic-phased patients received upfront TKI therapy had a long-term remission. Nine of 16 (60%) blast-phase patients with upfront TKIs also achieved complete remission and remained alive at a median follow-up of 20 months. All 3 patients with blast phase disease who received upfront chemotherapy without positive response did not respond to subsequent TKI therapy, emphasizing the importance of initiating TKI therapy early. Upfront TKI therapy was associated with longer overall survival in univariate analyses (HR, 0.054 [95% CI, 0.007-0.42]) and multivariate analyses (HR, 0.03 [95% CI, 0.002-0.47]). CONCLUSION: The outcomes of upfront TKI therapy are excellent for MLN-TK in both chronic and blast phases, regardless of gene abnormalities.

Novel assays to investigate the mechanisms of latent infection with HIV-2.

Although there have been great advancements in the field of HIV treatment and prevention, there is no cure. There are two types of HIV: HIV-1 and HIV-2. In addition to genetic differences between the two types of HIV, HIV-2 infection causes a slower disease progression, and the rate of new HIV-2 infections has dramatically decreased since 2003. Like HIV-1, HIV-2 is capable of establishing latent infection in CD4+ T cells, thereby allowing the virus to evade viral cytopathic effects and detection by the immune system. The mechanisms underlying HIV latency are not fully understood, rendering this a significant barrier to development of a cure. Using RT-ddPCR, we previously demonstrated that latent infection with HIV-1 may be due to blocks to HIV transcriptional elongation, distal transcription/polyadenylation, and multiple splicing. In this study, we describe the development of seven highly-specific RT-ddPCR assays for HIV-2 that can be applied to the study of HIV-2 infections and latency. We designed and validated seven assays targeting different HIV-2 RNA regions along the genome that can be used to measure the degree of progression through different blocks to HIV-2 transcription and splicing. Given that HIV-2 is vastly understudied relative to HIV-1 and that it can be considered a model of a less virulent infection, application of these assays to studies of HIV-2 latency may inform new therapies for HIV-2, HIV-1, and other retroviruses.

Association of Vitamin D Status and COVID-19-Related Hospitalization and Mortality.

BACKGROUND: The relationship between vitamin D status and COVID-19-related clinical outcomes is controversial. Prior studies have been conducted in smaller, single-site, or homogeneous populations limiting adjustments for social determinants of health (race/ethnicity and poverty) common to both vitamin D deficiency and COVID-19 outcomes. OBJECTIVE: To evaluate the dose-response relationship between continuous 25(OH)D and risk for COVID-19-related hospitalization and mortality after adjusting for covariates associated with both vitamin D deficiency and COVID-19 outcomes. DESIGN: Retrospective cohort study. PATIENTS: Veteran patients receiving care in US Department of Veteran Affairs (VA) health care facilities with a positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test and a blood 25(OH)D test between February 20, 2020, and November 8, 2020, followed for up to 60 days. MAIN MEASURES: Exposure was blood 25(OH)D concentration ascertained closest to and within 15 to 90 days preceding an index positive SARS-CoV-2 test. Co-primary study outcomes were COVID-19-related inpatient hospitalization requiring airborne, droplet, contact, or other isolation and mortality ascertained within 60 days of an index positive SARS-CoV-2 test. KEY RESULTS: Of 4,599 veterans with a positive SARS-CoV-2 test, vitamin D deficiency (< 20 ng/mL) was identified in 665 (14.5%); 964 (21.0%) were hospitalized; and 340 (7.4%) died. After adjusting for all covariates, including race/ethnicity and poverty, there was a significant independent inverse dose-response relationship between increasing continuous 25(OH)D concentrations (from 15 to 60 ng/mL) and decreasing probability of COVID-19-related hospitalization (from 24.1 to 18.7%, p=0.009) and mortality (from 10.4 to 5.7%, p=0.001). In modeling 25(OH)D as a log-transformed continuous variable, the greatest risk for hospitalization and death was observed at lower 25(OH)D concentrations. CONCLUSIONS: Continuous blood 25(OH)D concentrations are independently associated with COVID-19-related hospitalization and mortality in an inverse dose-response relationship in this large racially and ethnically diverse cohort of VA patients. Randomized controlled trials are needed to evaluate the impact of vitamin D supplementation on COVID-19-related outcomes.

A new testing platform using fingerstick blood for quantitative antibody response evaluation after SARS-CoV-2 vaccination.

Testing and vaccination have been major components of the strategy for combating the ongoing COVID-19 pandemic. In this study, we have developed a quantitative anti-SARS-CoV-2 spike (S1) IgG antibody assay using a fingerstick dried blood sample. We evaluated the feasibility of using this high-throughput and quantitative anti-SARS-CoV-2 spike (S1) IgG antibody testing assay in vaccinated individuals. Fingerstick blood samples were collected and analyzed from 137 volunteers before and after receiving the Moderna or Pfizer mRNA vaccine. Anti-SARS-CoV-2 S1 IgG antibody could not be detected within the first 7 days after receiving the first vaccine dose, however, the assay reliably detected antibodies from day 14 onwards. In addition, no anti-SARS-CoV-2 nucleocapsid (N) protein IgG antibody was detected in any of the vaccinated or healthy participants, indicating that the anti-SARS-CoV-2 S1 IgG assay is specific for the mRNA vaccine-induced antibodies. The S1 IgG levels detected in fingerstick samples correlated with the levels found in venous blood plasma samples and with the efficacy of venous blood plasma samples in the plaque reduction neutralization test (PRNT). The assay displayed a limit of quantification (LOQ) of 0.59 µg/mL and was found to be linear in the range of 0.51-1000 µg/mL. Finally, its clinical performance displayed a Positive Percent Agreement (PPA) of 100% (95% CI: 0.89-1.00) and a Negative Percent Agreement (NPA) of 100% (95% CI: 0.93-1.00). In summary, the assay described here represents a sensitive, precise, accurate, and simple method for the quantitative detection and monitoring of post-vaccination anti-SARS-CoV-2 spike IgG responses.

Association between household exposure and cycle threshold in COVID-19 infected health care workers.

OBJECTIVE: Household SARS-COV-2 contact constitutes a high-risk exposure for health care workers (HCWs). Cycle threshold (Ct) of reverse transcriptase-polymerase chain reaction testing provides an estimate of COVID-19 viral load, which can inform clinical and workplace management. We assessed whether Ct values differed between HCWs with COVID-19 with and without household exposure. METHODS: We analyzed HCW COVID-19 cases whose Ct data could be compared. We defined low Ct at a cut-point approximating a viral load of 4.6 × 106 copies per ml. Logistic regression tested the association of household exposure and symptoms at diagnosis with a low Ct value. RESULTS: Of 77 HCWs with COVID-19, 20 were household exposures cases and 34 were symptomatic at testing (7 were both household-exposed and symptomatic at testing). Among household exposures, 9 of 20 (45%) manifested lower Ct values compared to 14 of 57 (25%) for all others. In a bivariate model, household exposure was not statistically associated with lower Ct (Odds Ratio [OR] 1.20; 95% Confidence Interval [CI] 0.97-1.51). In multivariable modelling both household exposure (OR] 1.3; 95% CI 1.03-1.6) and symptoms at diagnosis (OR 1.4; 95% CI 1.15-1.7) were associated with a low Ct value. DISCUSSION: Household exposure in HCWs with newly diagnosed COVID-19 was associated with lower Ct values, consistent with a higher viral load, supporting the hypothesis that contracting COVID-19 in that manner leads to a greater viral inoculum.

Diagnosis of anaplastic lymphoma kinase-negative anaplastic large cell lymphoma on pleural fluid cytology.

ALK-negative anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T-cell lymphoma. Compared to ALK-positive ALCL, patients with ALK-negative ALCL typically are older, present with nodal disease, and have lower survival rates. We report a unique presentation of ALK-negative ALCL in a pleural fluid. Cell block preparation enabled both confirmation of the diagnosis via immunohistochemical staining and gene rearrangement testing for prognostic information.