Differential Gene Expression in the Upper Respiratory Tract following Acute COVID-19 Infection in Ambulatory Patients That Develop Long COVID.

Background: Post-acute sequelae of COVID-19, or long COVID, is a condition characterized by persistent COVID-19 symptoms. As long COVID is defined by clinical criteria after an elapsed period, an opportunity for early intervention may aid in future prophylactic approaches; however, at present, the pathobiological mechanisms are multifactorial. By analyzing early virally infected upper respiratory tract tissue prior to eventual clinical diagnosis, it may be possible to identify biomarkers of altered immune response to facilitate future studies and interventions. Methods: This is a sub-group analysis of samples collected from those with confirmed COVID-19. RNA extraction from nasopharyngeal/mid-turbinate samples, sequencing, and bioinformatic analysis were performed to analyze long COVID and non-long COVID cohorts at day 14 post infection. Differences in mean viral load at various timepoints were analyzed as well as serological data. Results: We identified 26 upregulated genes in patients experiencing long COVID. Dysregulated pathways including complement and fibrinolysis pathways and IL-7 upregulation. Additionally, genes involved in neurotransmission were dysregulated, and the long COVID group had a significantly higher viral load and slower viral clearance. Conclusions: Uncovering early gene pathway abnormalities associated with eventual long COVID diagnosis may aid in early identification. We show that, post acute infection, in situ pathogenic deviations in viral response are associated with patients destined to meet consensus long COVID diagnosis that is entirely dependent on clinical factors. These results identify an important biological temporal window in the natural history of COVID-19 infection and long COVID pathogenesis amenable to testing from standard-of-care upper respiratory tract specimens.

Diagnosis of systemic mastocytosis with cryptic deletion of TET2 and DNMT3A resulting from unbalanced translocation.

Systemic mastocytosis (SM) is a rare haematological neoplasm associated with the gain of function mutation KIT D816V in 90% of adult patients. Classically, cytogenetic aberrations are not common except in cases of SM associated with another haematological neoplasm. We highlight here an unusual clinical presentation of SM and demonstrate the utility of advanced cytogenetic analysis (optical genome mapping, OGM) in detecting a novel cytogenetic abnormality resulting in an unusual mechanism of DNMT3A and TET2 loss of function.

Clinical Features and Long-Term Outcomes of a Pan-Canadian Cohort of Adolescents and Young Adults with Myeloproliferative Neoplasms: A Canadian MPN Group Study.

Myeloproliferative neoplasms (MPNs) are a group of chronic hematologic malignancies that lead to morbidity and early mortality due to thrombotic complications and progression to acute leukemia. Clinical and mutational risk factors have been demonstrated to predict outcomes in patients with MPNs and are used commonly to guide therapeutic decisions, including allogenic stem cell transplant, in myelofibrosis. Adolescents and young adults (AYA, age ≤45 years) comprise less than 10% of all MPN patients and have unique clinical and therapeutic considerations. The prevalence and clinical impact of somatic mutations implicated in myeloid disease has not been extensively examined in this population. We conducted a retrospective review of patients evaluated at eight Canadian centers for MPN patients diagnosed at ≤45 years of age. In total, 609 patients were included in the study, with median overall survival of 36.8 years. Diagnosis of prefibrotic or overt PMF is associated with the lowest OS and highest risk of AP/BP transformation. Thrombotic complications (24%), including splanchnic circulation thrombosis (9%), were frequent in the cohort. Mutations in addition to those in JAK2/MPL/CALR are uncommon in the initial disease phase in our AYA population (12%); but our data indicate they may be predictive of transformation to post-ET/PV myelofibrosis.

The Clinical Utility of FLT3 Mutation Testing in Acute Leukemia: A Canadian Consensus.

FMS-like tyrosine kinase 3 (FLT3) mutations are detected in approximately 20-30% of patients with acute myeloid leukemia (AML), with the presence of a FLT3 internal tandem duplication (FLT3-ITD) mutation being associated with an inferior outcome. Assessment of FLT3 mutational status is now essential to define optimal upfront treatment in both newly diagnosed and relapsed AML, to support post-induction allogeneic hematopoietic stem cell transplantation (alloSCT) decision-making, and to evaluate treatment response via measurable (minimal) residual disease (MRD) evaluation. In view of its importance in AML diagnosis and management, the Canadian Leukemia Study Group/Groupe canadien d'étude sur la leucémie (CLSG/GCEL) undertook the development of a consensus statement on the clinical utility of FLT3 mutation testing, as members reported considerable inter-center variability across Canada with respect to testing availability and timing of use, methodology, and interpretation. The CLSG/GCEL panel identified key clinical and hematopathological questions, including: (1) which patients should be tested for FLT3 mutations, and when?; (2) which is the preferred method for FLT3 mutation testing?; (3) what is the clinical relevance of FLT3-ITD size, insertion site, and number of distinct FLT3-ITDs?; (4) is there a role for FLT3 analysis in MRD assessment?; (5) what is the clinical relevance of the FLT3-ITD allelic burden?; and (6) how should results of FLT3 mutation testing be reported? The panel followed an evidence-based approach, taken together with Canadian clinical and laboratory experience and expertise, to create a consensus document to facilitate a more uniform approach to AML diagnosis and treatment across Canada.

Host Expression Profiling From Diagnostic Coronavirus Disease 2019 Swabs Associates Upper Respiratory Tract Immune Responses With Radiologic Lung Pathology and Clinical Severity.

Background: COVID-19 presents with a breadth of symptomatology including a spectrum of clinical severity requiring intensive care unit (ICU) admission. We investigated the mucosal host gene response at the time of gold standard COVID-19 diagnosis using clinical surplus RNA from upper respiratory tract swabs. Methods: Host response was evaluated by RNA-sequencing, and transcriptomic profiles of 44 unvaccinated patients including outpatients and in-patients with varying levels of oxygen supplementation were included. Additionally, chest X-rays were reviewed and scored for patients in each group. Results: Host transcriptomics revealed significant changes in the immune and inflammatory response. Patients destined for the ICU were distinguished by the significant upregulation of immune response pathways and inflammatory chemokines, including cxcl2 which has been linked to monocyte subsets associated with COVID-19 related lung damage. In order to temporally associate gene expression profiles in the upper respiratory tract at diagnosis of COVID-19 with lower respiratory tract sequalae, we correlated our findings with chest radiography scoring, showing nasopharygeal or mid-turbinate sampling can be a relevant surrogate for downstream COVID-19 pneumonia/ICU severity. Conclusions: This study demonstrates the potential and relevance for ongoing study of the mucosal site of infection of SARS-CoV-2 using a single sampling that remains standard of care in hospital settings. We highlight also the archival value of high quality clinical surplus specimens, especially with rapidly evolving COVID-19 variants and changing public health/vaccination measures.

Recent Thymic Emigrants Require RBPJ-Dependent Notch Signaling to Transition into Functionally Mature Naive T Cells.

Recent thymic emigrant (RTE) cells are nascent T cells that continue their post-thymic maturation in the periphery and dominate T cell immune responses in early life and in adults having undergone lymphodepletion regimens. However, the events that govern their maturation and their functionality as they transition to mature naive T cells have not been clearly defined. Using RBPJind mice, we were able to identify different stages of RTE maturation and interrogate their immune function using a T cell transfer model of colitis. As CD45RBlo RTE cells mature, they transition through a CD45RBint immature naive T (INT) cell population that is more immunocompetent but shows a bias toward IL-17 production at the expense of IFN-γ. Additionally, the levels of IFN-γ and IL-17 produced in INT cells are highly dependent on whether Notch signals are received during INT cell maturation or during their effector function. IL-17 production by INT cells showed a total requirement for Notch signaling. Loss of Notch signaling at any stage of INT cells resulted in an impaired colitogenic effect of INT cells. RNA sequencing of INT cells that had matured in the absence of Notch signals showed a reduced inflammatory profile compared with Notch-responsive INT cells. Overall, we have elucidated a previously unknown INT cell stage, revealed its intrinsic bias toward IL-17 production, and demonstrated a role for Notch signaling in INT cell peripheral maturation and effector function in the context of a T cell transfer model of colitis.

Determining the Longitudinal Serologic Response to COVID-19 Vaccination in the Chronic Kidney Disease Population: A Clinical Research Protocol.

Background: People living with chronic kidney disease (CKD) have been disproportionately affected by the coronavirus disease 2019 (COVID-19) pandemic, including higher rates of infection, hospitalization, and death. Data on responsiveness to COVID-19 vaccination strategies and immunogenicity are limited, yet required to inform vaccination strategies in this at-risk population. Objective: The objective of this study is to characterize the longitudinal serologic response to COVID-19 vaccination. Design: This is a prospective observational cohort study. Setting: Participating outpatient kidney programs within Ontario and British Columbia. Patients: Up to 2500 participants with CKD G3b-5D receiving COVID-19 vaccination, including participants receiving dialysis and kidney transplant recipients (CKD G1T-5T). Measurements: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies (anti-spike, anti-receptor binding domain, anti-nucleocapsid) will be detected by ELISA (enzyme-linked immunosorbent assay) from serum or dried blood spot testing. In a subset of participants, neutralizing antibodies against novel variants of concern will be evaluated. Peripheral blood mononuclear cells will be collected for exploratory immune profiling of SARS-CoV-2 specific cellular immunity. Methods: Participants will be recruited prior to or following any COVID-19 vaccine dose and have blood sampled for serological testing at multiple timepoints: 1, 3, 6, 9, and 12 months post vaccination. When possible, samples will be collected prior to a dose or booster. Participants will remain in the study for at least 1 year following their last COVID-19 vaccine dose. Strengths and limitations: The adaptive design of this study allows for planned modification based on emerging evidence or rapid changes in public health policy surrounding vaccination. Limitations include incomplete earlier timepoints for blood collection due to rapid vaccination of the population. Conclusions: This large multicenter serologic study of participants living with kidney disease will generate data on the kinetics of SARS-CoV-2 immune response to vaccination across the spectrum of CKD, providing insights into the amplitude and duration of immunity conferred by COVID-19 vaccination and allowing for characterization of factors associated with immune response. The results of this study may be used to inform immunization guidelines and public health recommendations for the 4 million Canadians living with CKD.

Contexte: Les personnes atteintes d'insuffisance rénale chronique (IRC) ont été touchées de façon disproportionnée par la pandémie de COVID-19 ayant notamment présenté des taux plus élevés d'infection, d'hospitalisation et de décès. Les données sur la réactivité aux stratégies de vaccination de la COVID-19 et à l'immunogénicité sont limitées, mais elles sont nécessaires pour développer des stratégies de vaccination dans cette population à risque. Objectif: Caractériser la réponse sérologique longitudinale à la vaccination contre la COVID-19. Conception: Étude de cohorte observationnelle prospective. Cadre: Les programmes ambulatoires de santé rénale participants en Ontario et en Colombie-Britannique. Sujets: Jusqu'à 2 500 personnes atteintes d'IRC G3B-5D recevant un vaccin contre la COVID-19, y compris des patients suivant des traitements de dialyse et des receveurs d'une greffe rénale (IRC G1T-5T). Mesures: Les anticorps IgG anti-SARS-CoV-2 (anti-spike, anti-domaine de liaison au récepteur, anti-nucléocapside) seront détectés par ELISA à partir du sérum ou de taches de sang séché. Un sous-groupe de sujets participera également à l'évaluation d'anticorps neutralisants dirigés contre les nouveaux variants préoccupants. Des cellules mononuclées de sang périphérique seront prélevées pour établir un profil immunitaire exploratoire de l'immunité cellulaire spécifique au SARS-CoV-2. Méthodologie: Les sujets seront recrutés avant ou après toute dose du vaccin contre la COVID-19 et se soumettront à des prélèvements sanguins pour les tests sérologiques à 1, 3, 6, 9 et 12 mois post-vaccination. Lorsque possible, des échantillons seront prélevés avant l'administration d'une dose ou d'un rappel. Les sujets demeureront dans l'étude pendant au moins un an après leur dernière dose de vaccin contre la COVID-19. Points forts et limites: La conception adaptative de l'étude permet d'apporter des modifications planifiées fondées sur de nouvelles données ou des changements rapides dans les politiques de santé publique entourant la vaccination. Les résultats sont limités par l'absence de certains prélèvements sanguins antérieurs (point temporels) en raison de la vaccination rapide de la population. Conclusion: Cette vaste étude sérologique multicentrique menée auprès de personnes atteintes de néphropathie fournira des données sur la cinétique de la réponse immunitaire à la vaccination contre le SARS-CoV-2 dans l'ensemble du spectre de l'IRC. Elle fournira des informations sur l'amplitude et la durée de l'immunité conférée par la vaccination contre la COVID-19 et permettra de caractériser les facteurs associés à la réponse immunitaire. Ces résultats serviront à orienter les recommandations de santé publique et les lignes directrices en matière d'immunisation pour les quatre millions de Canadiens et Canadiennes qui vivent avec l'IRC.

A Lower Frequency of Spliceosome Mutations Distinguishes Clonal Cytopenias of Undetermined Significance From Low-Risk Myelodysplastic Syndromes, Despite Inherent Similarities in Genomic, Laboratory, and Clinical Features.

Clonal cytopenias of undetermined significance (CCUS) are associated with an increased risk of developing a myelodysplastic syndrome (MDS); however, the mechanism and factors associated with evolution remain unclear. We propose that next-generation sequencing (NGS) of cytopenic cases with equivocal morphologic dysplasia will improve patient clinical care and that serial sequencing of such equivocal cases could identify the factors that predict evolution to MDS. We performed targeted NGS of samples from 193 individuals with confirmed or suspected MDS or MDS/myeloproliferative neoplasm, including sequential investigation for 28 individuals at the time of diagnosis and during follow-up. NGS facilitated the diagnosis of all suspicious cases as myeloid neoplasm (21%), CCUS (34%), or idiopathic cytopenias of undetermined significance (45%) when no variants were detected. We found that there was no significant difference in most measured clinical features or clonal phenotypes, such as cell counts, number of variants, variant allele frequencies, and overall survival, between CCUS and International Prognostic Scoring System-Revised-defined low-risk MDS. However, there was a significant difference in the types of variants between CCUS and low-risk MDS, with a significantly lower number of splicing factor mutations in CCUS cases (P < .001). Moreover, we observed an increased probability of evolution to MDS of individuals with CCUS compared with that in those with idiopathic cytopenias of undetermined significance over the first 5 years (P = .045). Our analyses revealed no conclusive pattern associating clonal expansion or the number of variants with the evolution of CCUS to MDS, perhaps further supporting the similarity of these diseases and the clinical importance of recognizing and formally defining CCUS as a category of precursor myeloid disease state in the next revision of the World Health Organization guidelines.

Janus kinases restrain chronic lymphocytic leukemia cells in patients on ibrutinib: Results of a phase II trial.

Preclinical observations that killing of chronic lymphocytic leukemia (CLL) cells was dexamethasone (DEX) were enhanced by concomitant inhibition of Bruton's tyrosine kinase and janus kinases (JAKs) motivated a phase II trial to determine if clinical responses to ibrutinib could be deepened by DEX and the JAK inhibitor ruxolitinib. Patients on ibrutinib at 420 mg daily for 2 months or with abnormal serum ß2M levels after 6 months or with persistent lymphadenopathy or splenomegaly after 12 months were randomized to receive DEX 40 mg on days 1-4 of a 4-week cycle for six cycles alone (three patients) or with ruxolitinib 15 mg BID on days 1-21 of each cycle (five patients). Ruxolitinib dosing was based on a previous phase I trial. Steroid withdrawal symptoms and significantly decreased serum IgG levels occurred in all patients regardless of their exposure to ruxolitinib. A fatal invasive fungal infection was seen in a patient taking DEX without ruxolitinib. Complete responses anticipated with addition of ruxolitinib were not seen. Gene expression studies suggested ruxolitinib had turned off interferon signaling in CLL cells and turned on genes associated with the activation of NFκB by TNF-α. Ruxolitinib increased blood levels of TNF-α by cycle 3 and decreased the inhibitory cytokine IL-10. These results suggest ruxolitinib releases activating signals for CLL cells that persist in patients on ibrutinib. This inhibitory JAK signaling may contribute to the therapeutic activity of ibrutinib. Thus JAK inhibitors provide no added value with ibrutinib for disease control and should be used with caution in CLL patients. Combining glucocorticoids with ibrutinib may increase the risk of serious infects.

Recognition of Long-COVID-19 Patients in a Canadian Tertiary Hospital Setting: A Retrospective Analysis of Their Clinical and Laboratory Characteristics.

A proportion of patients with COVID-19 have symptoms past the acute disease phase, which may affect quality of life. It is important for clinicians to be aware of this "long-COVID-19" syndrome to better diagnose, treat, and prevent it. We reviewed clinical and laboratory characteristics of a COVID-19 cohort in a Toronto, Ontario tertiary care center. Demographic, clinical, and laboratory data were collected, and patients were classified as "long-COVID-19" or "non-long-COVID-19" using consensus criteria. Of 397 patients who tested positive for COVID-19, 223 met inclusion criteria, and 62 (27%) had long-COVID-19. These patients had a similar age distribution compared to non-long-COVID-19 patients overall but were younger in the admitted long COVID-19 group. The long-COVID-19 group had more inpatients compared to the non-long-COVID-19 group (39% vs. 25%) and more frequent supplemental oxygen or mechanical ventilation use. However, long-COVID-19 patients did not differ by duration of mechanical ventilation, length of stay, comorbidities, or values of common laboratory tests ordered. The most frequent symptoms associated with long-COVID-19 were fatigue and weakness, as reported most commonly by the infectious disease, respirology and cardiology disciplines. In conclusion, by retrospective chart review, 27% of COVID-19 patients presenting to a tertiary care center in Toronto, Canada, were found to meet criteria for long-COVID-19. Past medical history and routine laboratory testing at presentation did not predict for long-COVID-19 development.

Association of frailty with clinical outcomes in myelofibrosis: a retrospective cohort study.

There is limited understanding of the impact of frailty on clinical outcomes in patients with myelofibrosis (MF). In this retrospective cohort study on 439 chronic phase MF patients [mean age: 68·7 ± 12 years; median follow-up: 3·4 years (IQR 0·4-8·6)] from 2004 till 2018, we used a 35-variable frailty index (FI) to categorise patient's frailty status as fit (FI < 0·2, reference), prefrail (FI 0·2-0·29) or frail (FI ≥ 0·3). The association of frailty with overall survival (OS) and cumulative JAK inhibitor (JAKi) therapy failure was measured using hazard ratio (HR, 95% CI). In multivariable analysis, prefrail (HR 1·7, 1·1-2·5) and frail patients (HR 2·9, 1·6-5·5), those with higher DIPSS score (HR 2·5, 1·6-3·9) and transfusion dependency (HR 1·9, 1·3-2·9) had shorter OS. In a subset analysis of patients on JAKi treatment (n = 222), frail patients (HR 2·5, 1·1-5·7), patients with higher DIPSS score (HR 1·7, 1·0-3·1) and transfusion dependence (HR 1·7, 1·1-2·7) had higher cumulative incidence of JAKi failure. Age, comorbidities, ECOG performance status, and MPN driver mutations did not impact outcomes. Thus, higher frailty scores are associated with worse OS and increased JAKi failure in MF, and is a superior indicator of fitness in comparison to age, comorbidities, and performance status.

Effect of Ibrutinib on the IFN Response of Chronic Lymphocytic Leukemia Cells.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has profound activity in chronic lymphocytic leukemia (CLL) but limited curative potential by itself. Residual signaling pathways that maintain survival of CLL cells might be targeted to improve ibrutinib's therapeutic activity, but the nature of these pathways is unclear. Ongoing activation of IFN receptors in patients on ibrutinib was suggested by the presence of type I and II IFN in blood together with the cycling behavior of IFN-stimulated gene (ISG) products when IFN signaling was blocked intermittently with the JAK inhibitor ruxolitinib. IFN signaling in CLL cells from human patients was not prevented by ibrutinib in vitro or in vivo, but ISG expression was significantly attenuated in vitro. ISGs such as CXCL10 that require concomitant activation of NF-κB were decreased when this pathway was inhibited by ibrutinib. Other ISGs, exemplified by LAG3, were decreased as a result of inhibited protein translation. Effects of IFN on survival remained intact as type I and II IFN-protected CLL cells from ibrutinib in vitro, which could be prevented by ruxolitinib and IFNR blocking Abs. These observations suggest that IFNs may help CLL cells persist and specific targeting of IFN signaling might deepen clinical responses of patients on ibrutinib.

Human Aging Alters the Spatial Organization between CD34+ Hematopoietic Cells and Adipocytes in Bone Marrow.

Age-related clonal hematopoiesis is a major risk factor for myeloid malignancy and myeloid skewing is a hallmark of aging. However, while it is known that non-cell-autonomous components of the microenvironment can also influence this risk, there have been few studies of how the spatial architecture of human bone marrow (BM) changes with aging. Here, we show that BM adiposity increases with age, which correlates with increased density of maturing myeloid cells and CD34+ hematopoietic stem/progenitor cells (HSPCs) and an increased proportion of HSPCs adjacent to adipocytes. However, NGFR+ bone marrow stromal cell (NGFR+ BMSC) density and distance to HSPCs and vessels remained stable. Interestingly, we found that, upon aging, maturing myeloid cell density increases in hematopoietic areas surrounding adipocytes. We propose that increased adjacency to adipocytes in the BM microenvironment may influence myeloid skewing of aging HSPCs, contributing to age-related risk of myeloid malignancies.

Confounding effect of therapeutic protamine and heparin levels on routine and special coagulation testing.

: The management of a patient with hemophilia undergoing cardiovascular surgery relies on accurate coagulation test results. Both unfractionated heparin (UFH) and protamine sulfate used during cardiac surgery can interfere with factor and inhibitor assays. Here we describe the effects of UFH and protamine sulfate on routine coagulation, factor activity, and inhibitor assays. Pooled normal plasma (PNP) with UFH, PNP with protamine sulfate, PNP with both protamine sulfate and UFH were tested for the activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), UFH anti-Xa, one-stage factor VIII (FVIII) activity, one-stage factor IX (FIX) activity, and Bethesda inhibitor assays for FVIII and FIX. UFH had a dose-dependent effect with TT, aPTT, and PT. On Bethesda inhibitor testing, FIX inhibition was detected at 1 U/ml UFH and 3 U/ml UFH for FVIII. Increasing protamine sulfate concentration in PNP prolonged the PT and aPTT in a dose-dependent manner, decreased FVIII and FIX activity and did not affect TT or UFH anti-Xa. At protamine sulfate doses of at least 200 µg/ml there was weak FVIII and FIX inhibition detected. At lower ratios of protamine sulfate to UFH (0.6 : 1-0.8 : 1), the aPTT decreased, suggesting reversal of UFH. However, at protamine sulfate to UFH ratios of 1.0 : 1 and higher, aPTT prolongation was observed. Inhibition of FVIII and FIX was detected at low ratios of protamine sulfate to UFH (below 0.4 : 1) and disappeared at higher ratios. UFH and protamine sulfate, alone or in combination, impact factor activity and inhibitor testing for both FVIII and FIX. Hence, factor activity and inhibitor assay results should be interpreted with caution when UFH or protamine sulfate are present.

Persistent janus kinase-signaling in chronic lymphocytic leukemia patients on ibrutinib: Results of a phase I trial.

Methods to deepen clinical responses to ibrutinib are needed to improve outcomes for patients with chronic lymphocytic leukemia (CLL). This study aimed to determine the safety and efficacy of combining a janus kinase (JAK)-inhibitor with ibrutinib because JAK-mediated cytokine-signals support CLL cells and may not be inhibited by ibrutinib. The JAK1/2 inhibitor ruxolitinib was prescribed to 12 CLL patients with abnormal serum beta-2 microglobulin levels after 6 months or persistent lymphadenopathy or splenomegaly after 12 months on ibrutinib using a 3 + 3 phase 1 trial design (NCT02912754). Ibrutinib was continued at 420 mg daily and ruxolitinib was added at 5, 10, 15, or 20 mg BID for 3 weeks out of five for seven cycles. The break was mandated to avoid anemia and thrombocytopenia observed with ruxolitinib as a single agent in CLL. The combination was well-tolerated without dose-limiting toxicities. Cyclic changes in platelets, lymphocytes, and associated chemokines and thrombopoietic factors were observed and partial response criteria were met in 2 of 12 patients. The results suggest that JAK-signaling helps CLL cells persist in the presence of ibrutinib and ruxolitinib with ibrutinib is well-tolerated and may be a useful regiment to use in combination therapies for CLL.