IκBε deficiency accelerates disease development in chronic lymphocytic leukemia.

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

B cell receptor signaling proteins as biomarkers for progression of CLL requiring first-line therapy.

The molecular landscape of chronic lymphocytic leukemia (CLL) has been extensively characterized, and various potent prognostic biomarkers were discovered. The genetic composition of the B-cell receptor (BCR) immunoglobulin (IG) was shown to be especially powerful for discerning indolent from aggressive disease at diagnosis. Classification based on the IG heavy chain variable gene (IGHV) somatic hypermutation status is routinely applied. Additionally, BCR IGH stereotypy has been implicated to improve risk stratification, through characterization of subsets with consistent clinical profiles. Despite these advances, it remains challenging to predict when CLL progresses to requiring first-line therapy, thus emphasizing the need for further refinement of prognostic indicators. Signaling pathways downstream of the BCR are essential in CLL pathogenesis, and dysregulated components within these pathways impact disease progression. Considering not only genomics but the entirety of factors shaping BCR signaling activity, this review offers insights in the disease for better prognostic assessment of CLL.

Association of Altered Plasma Lipidome with Disease Severity in COVID-19 Patients.

The severity of COVID-19 is linked to an imbalanced immune response. The dysregulated metabolism of small molecules and bioactive lipids has also been associated with disease severity. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyze over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). This is the third publication in a series, and it reports the results of comprehensive lipidome profiling using targeted LC-MS/MS. We identified 1076 lipid features across 25 subclasses, including glycerophospholipids, sterols, glycerolipids, and sphingolipids, among which 531 lipid features were dramatically changed in the plasma of intensive care unit (ICU) patients compared to patients in the ward. Patients in the ICU showed 1.3-57-fold increases in ceramides, (lyso-)glycerophospholipids, diglycerides, triglycerides, and plasmagen phosphoethanolamines, and 1.3-2-fold lower levels of a cyclic lysophosphatidic acid, sphingosine-1-phosphates, sphingomyelins, arachidonic acid-containing phospholipids, lactosylceramide, and cholesterol esters compared to patients in the ward. Specifically, phosphatidylinositols (PIs) showed strong fatty acid saturation-dependent behavior, with saturated fatty acid (SFA)- and monosaturated fatty acid (MUFA)-derived PI decreasing and polystaturated (PUFA)-derived PI increasing. We also found ~4000 significant Spearman correlations between lipids and multiple clinical markers of immune response with |R| ≥ 0.35 and FDR corrected Q < 0.05. Except for lysophosphatidic acid, lysophospholipids were positively associated with the CD4 fraction of T cells, and the cytokines IL-8 and IL-18. In contrast, sphingosine-1-phosphates were negatively correlated with innate immune markers such as CRP and IL-6. Further indications of metabolic changes in moderate COVID-19 disease were demonstrated in recovering ward patients compared to those at the start of hospitalization, where 99 lipid species were altered (6 increased by 30-62%; 93 decreased by 1.3-2.8-fold). Overall, these findings support and expand on early reports that dysregulated lipid metabolism is involved in COVID-19.

Immune dysregulation as a leading principle for lymphoma development in diverse immunological backgrounds.

Lymphoma is a heterogeneous group of malignancies arising from lymphocytes, which poses a significant challenge in terms of diagnosis and treatment due to its diverse subtypes and underlying mechanisms. This review aims to explore the shared and distinct features of various forms of lymphoma predisposing conditions, with a focus on genetic, immunological and molecular aspects. While diseases such as autoimmune disorders, inborn errors of immunity and iatrogenic immunodeficiencies are biologically and immunologically distinct, each of these diseases results in profound immune dysregulation and a predisposition to lymphoma development. Interestingly, the increased risk is often skewed towards a particular subtype of lymphoma. Patients with inborn errors of immunity in particular present with extreme forms of lymphoma predisposition, providing a unique opportunity to study the underlying mechanisms. External factors such as chronic infections and environmental exposures further modulate the risk of lymphoma development. Common features of conditions predisposing to lymphoma include: persistent inflammation, recurrent DNA damage or malfunctioning DNA repair, impaired tumor surveillance and viral clearance, and dysregulation of fundamental cellular processes such as activation, proliferation and apoptosis. Our growing understanding of the underlying mechanisms of lymphomagenesis provides opportunities for early detection, prevention and tailored treatment of lymphoma development.

High-throughput Proteomics Identifies THEMIS2 as Independent Biomarker of Treatment-free Survival in Untreated CLL.

It remains challenging in chronic lymphocytic leukemia (CLL) to distinguish between patients with favorable and unfavorable time-to-first treatment (TTFT). Additionally, the downstream protein correlates of well-known molecular features of CLL are not always clear. To address this, we selected 40 CLL patients with TTFT ≤24 months and compared their B cell intracellular protein expression with 40 age- and sex-matched CLL patients with TTFT >24 months using mass spectrometry. In total, 3268 proteins were quantified in the cohort. Immunoglobulin heavy-chain variable (IGHV) mutational status and trisomy 12 were most impactful on the CLL proteome. Comparing cases to controls, 5 proteins were significantly upregulated, whereas 3 proteins were significantly downregulated. Of these, only THEMIS2, a signaling protein acting downstream of the B cell receptor, was significantly associated with TTFT, independently of IGHV and TP53 mutational status (hazard ratio, 2.49 [95% confidence interval, 1.62-3.84]; P < 0.001). This association was validated on the mRNA and protein level by quantitative polymerase chain reaction and ELISA, respectively. Analysis of 2 independently generated RNA sequencing and mass spectrometry datasets confirmed the association between THEMIS2 expression and clinical outcome. In conclusion, we present a comprehensive characterization of the proteome of untreated CLL and identify THEMIS2 expression as a putative biomarker of TTFT.

Antibodies against angiotensin II receptor type 1 and endothelin A receptor are increased in COVID-19 patients.

Background: Increased titers of autoantibodies targeting the G-protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endotelin-1 type A receptor (ETAR) are associated with severe coronavirus disease 2019 (COVID-19) infection. The aim of this study was to determine whether 1) these antibodies are specifically related to COVID-19 disease pathogenesis or increased during any severe respiratory illness, 2) if they are formed during illness, and 3) if they correlate with inflammatory markers or long-term symptoms. Methods: Antibodies against AT1R, ETAR, and antinuclear antibodies (ANAs) were measured in n=40 prospectively enrolled COVID-19 patients and n=207 COVID-19 patients included in a biobank. Clinical and laboratory findings were prospectively and retrospectively assessed in both cohorts, and results were combined for analysis. The presence of auto-antibodies against AT1R or ETAR in peripheral blood was compared between hospitalized patients with COVID-19 and controls (n=39). Additionally, AT1R and ETAR titers were compared between patients with an unfavorable disease course, defined as intensive care admission and/or death during hospital admission (n=121), to those with a favorable disease course (n=126). A subset of intubated patients with severe COVID-19 were compared to intubated patients with acute respiratory distress syndrome (ARDS) due to any other cause. Results: Significantly increased AT1R and ETAR antibody titers were found in COVID-19 patients compared to controls, while titers were equal between favorable and unfavorable COVID-19 disease course groups. On ICU, intubated patients with COVID-19 had significantly increased AT1R and ETAR titers compared to patients with ARDS due to any other cause. The titers did not correlate with baseline inflammatory markers during admission or with diffusion capacity, cognitive impairment, or fatigue measured at 3 months follow-up. Conclusions: In patients hospitalized for COVID-19, antibodies against AT1R and ETAR are increased compared to controls and patients with ARDS due to other causes than COVID-19. The baseline antibody titers do not correlate with inflammatory markers or long-term symptoms in this study.

BRAF V600E is associated with higher incidence of second cancers in adults with Langerhans cell histiocytosis.

In this retrospective study, BRAF mutation status did not correlate with disease extent or (event-free) survival in 156 adults with Langerhans cell histiocytosis. BRAFV600E was associated with an increased incidence of second malignancies, often comprising hematological cancers, which may be clonally related.

EuroClonality-NGS Recommendations for Evaluation of B-Cell Clonality Analysis by Next-Generation Sequencing: A Structured Approach with the DEPART Algorithm.

Next-generation sequencing (NGS)-based clonality analysis allows in-depth assessment of the clonal composition of a sample with high sensitivity for detecting small clones. Within the EuroClonality-NGS Working Group, a protocol for NGS Ig clonality analysis was developed and validated previously. This NGS-based approach was designed to generate small amplicons, making it suitable for samples with suboptimal DNA quality, especially material derived from formalin-fixed, paraffin-embedded tissue. Using expert assessment of NGS Ig clonality results as a reference, a structured algorithmic approach to the assessment of NGS-amplicon-based B-cell clonality analysis was developed. A structured approach with the Detection of clonality through Evaluation of sample quality and assessment of Pattern, Abundance and RaTio (DEPART) algorithm was proposed, which consecutively evaluates sample quality, the pattern of the clonotypes present, the abundance of the most dominant clonotypes, and the ratio between the dominant clonotypes and the background to evaluate the different Ig gene targets. Specific issues with respect to evaluation of the various Ig targets as well as the integration of results of individual targets into a molecular clonality conclusion are discussed and illustrated with case examples. Finally, the importance of interpretation of NGS-based clonality results in clinical and histopathologic contexts is discussed. It is expected that these recommendations will have clinical utility to facilitate proper evaluation of clonality assessment.

Context-dependent T-cell Receptor Gene Repertoire Profiles in Proliferations of T Large Granular Lymphocytes.

T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.

Fixed-duration venetoclax plus obinutuzumab improves quality of life and geriatric impairments in FCR-unfit patients with CLL.

Chronic lymphocytic leukemia (CLL)-related symptoms and morbidity related to the advanced age at diagnosis impairs the well-being of older adult patients. Therefore, it is essential to tailor treatment according to geriatric characteristics and aim for an improvement in health-related quality of life (HRQoL) as a primary treatment goal. In the HOVON139/GiVe trial, 12 cycles of fixed-duration venetoclax plus obinutuzumab (Ven-O) were shown to be effective and tolerable in FCR (fludarabine, cyclophosphamide, rituximab)-unfit patients with CLL (n = 67). However, prolonged venetoclax exposure as consolidation treatment led to increased toxicity with limited effect on minimal residual disease. To assess the impact of geriatric assessment on treatment outcomes and the patients' HRQoL, patient-reported outcomes (PROs), including function, depression, cognition, nutrition, physical performance, muscle parameters, comorbidities, and the European Organization for Research and Treatment of Cancer C30 and CLL17 questionnaires were assessed. At baseline, geriatric impairments were present in >90% of patients and ≥2 impairments present in 60% of patients predicted grade ≥3 nonhematological toxicity. During treatment, the number of geriatric impairments diminished significantly and clinically relevant improvements in HRQoL subscales were reached for global health status, physical functioning, role functioning, emotional functioning, fatigue, dyspnea, physical condition or fatigue, and worries or fears related to health and functioning. These improvements were comparable for patients receiving venetoclax consolidation and patients in whom treatment could mostly be discontinued. Collectively, frontline fixed-duration Ven-O improves overall PROs in older, unfit patients with CLL with and without geriatric impairments. This study was registered at EudraCT as 2015-004985-27 and the Netherlands Trial Register as NTR6043.

ACVIM consensus statement guidelines on diagnosing and distinguishing low-grade neoplastic from inflammatory lymphocytic chronic enteropathies in cats.

BACKGROUND: Lymphoplasmacytic enteritis (LPE) and low-grade intestinal T cell lymphoma (LGITL) are common diseases in older cats, but their diagnosis and differentiation remain challenging. OBJECTIVES: To summarize the current literature on etiopathogenesis and diagnosis of LPE and LGITL in cats and provide guidance on the differentiation between LPE and LGITL in cats. To provide statements established using evidence-based approaches or where such evidence is lacking, statements based on consensus of experts in the field. ANIMALS: None. METHODS: A panel of 6 experts in the field (2 internists, 1 radiologist, 1 anatomic pathologist, 1 clonality expert, 1 oncologist) with the support of a human medical immunologist, was formed to assess and summarize evidence in the peer-reviewed literature and complement it with consensus recommendations. RESULTS: Despite increasing interest on the topic for clinicians and pathologists, few prospective studies were available, and interpretation of the pertinent literature often was challenging because of the heterogeneity of the cases. Most recommendations by the panel were supported by a moderate or low level of evidence. Several understudied areas were identified, including cellular markers using immunohistochemistry, genomics, and transcriptomic studies. CONCLUSIONS AND CLINICAL IMPORTANCE: To date, no single diagnostic criterion or known biomarker reliably differentiates inflammatory lesions from neoplastic lymphoproliferations in the intestinal tract of cats and a diagnosis currently is established by integrating all available clinical and diagnostic data. Histopathology remains the mainstay to better differentiate LPE from LGITL in cats with chronic enteropathy.

Targeted multiomics in childhood-onset SLE reveal distinct biological phenotypes associated with disease activity: results from an explorative study.

OBJECTIVE: To combine targeted transcriptomic and proteomic data in an unsupervised hierarchical clustering method to stratify patients with childhood-onset SLE (cSLE) into similar biological phenotypes, and study the immunological cellular landscape that characterises the clusters. METHODS: Targeted whole blood gene expression and serum cytokines were determined in patients with cSLE, preselected on disease activity state (at diagnosis, Low Lupus Disease Activity State (LLDAS), flare). Unsupervised hierarchical clustering, agnostic to disease characteristics, was used to identify clusters with distinct biological phenotypes. Disease activity was scored by clinical SELENA-SLEDAI (Safety of Estrogens in Systemic Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index). High-dimensional 40-colour flow cytometry was used to identify immune cell subsets. RESULTS: Three unique clusters were identified, each characterised by a set of differentially expressed genes and cytokines, and by disease activity state: cluster 1 contained primarily patients in LLDAS, cluster 2 contained mainly treatment-naïve patients at diagnosis and cluster 3 contained a mixed group of patients, namely in LLDAS, at diagnosis and disease flare. The biological phenotypes did not reflect previous organ system involvement and over time, patients could move from one cluster to another. Healthy controls clustered together in cluster 1. Specific immune cell subsets, including CD11c+ B cells, conventional dendritic cells, plasmablasts and early effector CD4+ T cells, differed between the clusters. CONCLUSION: Using a targeted multiomic approach, we clustered patients into distinct biological phenotypes that are related to disease activity state but not to organ system involvement. This supports a new concept where choice of treatment and tapering strategies are not solely based on clinical phenotype but includes measuring novel biological parameters.

Recent revelations and future directions using single-cell technologies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a clinically and biologically heterogeneous disease with varying outcomes. In the last decade, the application of next-generation sequencing technologies has allowed extensive mapping of disease-specific genomic, epigenomic, immunogenetic, and transcriptomic signatures linked to CLL pathogenesis. These technologies have improved our understanding of the impact of tumor heterogeneity and evolution on disease outcome, although they have mostly been performed on bulk preparations of nucleic acids. As a further development, new technologies have emerged in recent years that allow high-resolution mapping at the single-cell level. These include single-cell RNA sequencing for assessment of the transcriptome, both of leukemic and non-malignant cells in the tumor microenvironment; immunogenetic profiling of B and T cell receptor rearrangements; single-cell sequencing methods for investigation of methylation and chromatin accessibility across the genome; and targeted single-cell DNA sequencing for analysis of copy-number alterations and single nucleotide variants. In addition, concomitant profiling of cellular subpopulations, based on protein expression, can also be obtained by various antibody-based approaches. In this review, we discuss different single-cell sequencing technologies and how they have been applied so far to study CLL onset and progression, also in response to treatment. This latter aspect is particularly relevant considering that we are moving away from chemoimmunotherapy to targeted therapies, with a potentially distinct impact on clonal dynamics. We also discuss new possibilities, such as integrative multi-omics analysis, as well as inherent limitations of the different single-cell technologies, from sample preparation to data interpretation using available bioinformatic pipelines. Finally, we discuss future directions in this rapidly evolving field.

Extranodal marginal zone lymphoma clonotypes are detectable prior to eMZL diagnosis in tissue biopsies and peripheral blood of Sjögren's syndrome patients through immunogenetics.

Introduction: Activated B cells play a key role in the pathogenesis of primary Sjögren's syndrome (pSS) through the production of autoantibodies and the development of ectopic germinal centers in the salivary glands and other affected sites. Around 5-10% of pSS patients develop B-cell lymphoma, usually extranodal marginal zone lymphomas (eMZL) of the mucosa-associated lymphoid tissue (MALT). The aim of the current study is to investigate if the eMZL clonotype is detectable in prediagnostic blood and tissue biopsies of pSS patients. Methods/Results: We studied prediagnostic tissue biopsies of three pSS patients diagnosed with eMZL and four pSS controls through immunoglobulin (IG) gene repertoire sequencing. In all three cases, we observed the eMZL clonotype in prediagnostic tissue biopsies. Among controls, we observed transient elevation of clonotypes in two pSS patients. To evaluate if eMZL clonotypes may also be detected in the circulation, we sequenced a peripheral blood mononuclear cell (PBMC) sample drawn at eMZL diagnosis and two years prior to eMZL relapse in two pSS patients. The eMZL clonotype was detected in the peripheral blood prior to diagnosis in both cases. Next, we selected three pSS patients who developed eMZL lymphoma and five additional pSS patients who remained lymphoma-free. We sequenced the IG heavy chain (IGH) gene repertoire in PBMC samples taken a median of three years before eMZL diagnosis. In two out of three eMZL patients, the dominant clonotype in the prediagnostic PBMC samples matched the eMZL clonotype in the diagnostic biopsy. The eMZL clonotypes observed consisted of stereotypic IGHV gene combinations (IGHV1-69/IGHJ4 and IGHV4-59/IGHJ5) associated with rheumatoid factor activity, a previously reported feature of eMZL in pSS. Discussion: In conclusion, our results indicate that eMZL clonotypes in pSS patients are detectable prior to overt eMZL diagnosis in both tissue biopsies and peripheral blood through immunogenetic sequencing, paving the way for the development of improved methods of early detection of eMZL.