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.

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.

Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Detecting measurable residual disease beyond 10-4 by an IGHV leader-based NGS approach improves prognostic stratification in CLL.

The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between patients with CLL with durable remission and those at risk of early relapse. We herein present an academically developed immunoglobulin heavy-chain variable (IGHV) leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. We demonstrate, based on measurements in contrived MRD samples, that the linear range of detection and quantification of our assay reaches beyond MRD 10-5. If provided with sufficient DNA input, MRD can be detected down to MRD 10-6. There was high interassay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative polymerase chain reaction (PCR) (r = 0.92 [95% confidence interval {CI}, 0.86-0.96]) and droplet digital PCR (r = 0.93 [95% CI, 0.88-0.96]) on contrived MRD samples. In a cohort of 67 patients from the CLL11 trial, using MRD 10-5 as a cutoff, undetectable MRD was associated with superior progression-free survival (PFS) and time to next treatment. More important, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. PFS of patients in this MRD range was significantly shorter, compared with patients with MRD <10-5 (hazard ratio [HR], 4.0 [95% CI, 1.6-10.3]; P = .004), but significantly longer, compared with patients with MRD ≥10-4 (HR, 0.44 [95% CI, 0.23-0.87]; P = .018). These results support the clinical utility of the IGHV leader-based NGS assay.

High-risk subtypes of chronic lymphocytic leukemia are detectable as early as 16 years prior to diagnosis.

Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a CLL precursor state with a prevalence of up to 12% in aged individuals; however, the duration of MBL and the mechanisms of its evolution to CLL remain largely unknown. In this study, we sequenced the B-cell receptor (BcR) immunoglobulin heavy chain (IGH) gene repertoire of 124 patients with CLL and 118 matched controls in blood samples taken up to 22 years prior to diagnosis. Significant skewing in the BcR IGH gene repertoire was detected in the majority of patients, even before the occurrence of lymphocytosis and irrespective of the clonotypic IGH variable gene somatic hypermutation status. Furthermore, we identified dominant clonotypes belonging to major stereotyped subsets associated with poor prognosis up to 16 years before diagnosis in 14 patients with CLL. In 22 patients with longitudinal samples, the skewing of the BcR IGH gene repertoire increased significantly over time to diagnosis or remained stable at high levels. For 14 of 16 patients with available samples at diagnosis, the CLL clonotype was already present in the prediagnostic samples. Overall, our data indicate that the preclinical phase of CLL could be longer than previously thought, even in adverse-prognostic cases.

Treatment Approaches to Chronic Lymphocytic Leukemia With High-Risk Molecular Features.

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Over the past decades, several cytogenetic, immunogenetic and molecular features have emerged that identify patients suffering from CLL with high-risk molecular features. These biomarkers can clearly aid prognostication, but may also be capable of predicting the efficacy of various treatment strategies in subgroups of patients. In this narrative review, we discuss treatment approaches to CLL with high-risk molecular features. Specifically, we review and provide a comprehensive overview of clinical trials evaluating the efficacy of chemotherapy, chemoimmunotherapy and novel agent-based treatments in CLL patients with TP53 aberrations, deletion of the long arm of chromosome 11, complex karyotype, unmutated IGHV, B cell receptor stereotypy, and mutations in NOTCH1 or BIRC3. Furthermore, we discuss future pharmaceutical and immunotherapeutic perspectives for CLL with high-risk molecular features, focusing on agents currently under investigation in clinical trials.

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia.

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Reading the B-cell receptor immunome in chronic lymphocytic leukemia: revelations and applications.

B-Cell receptor (BCR) sequencing has been the force driving many recent advances in chronic lymphocytic leukemia (CLL) research. Here, we discuss the general principles, revelations, and applications of reading the BCR immunome in the context of CLL. First, IGHV mutational status, obtained by measuring the mutational imprint on the IGHV gene of the CLL clonotype, is the cornerstone of CLL risk stratification. Furthermore, the discovery of "BCR-stereotyped" groups of unrelated patients that share not only a highly similar BCR on their leukemic clone, but also certain clinical characteristics has provided insights key to understanding disease ontogeny. Additionally, whereas the BCR repertoire of most CLL patients is characterized by a single dominant rearrangement, next-generation sequencing (NGS) has revealed a rich subclonal landscape in a larger than previously expected proportion of CLL patients. We review the mechanisms underlying these "multiple dominant" cases, including V(D)J-recombination errors, failure of allelic exclusion, intraclonal diversification, and "true" bi- or oligoclonality, and their implications, in detail. Finally, BCR repertoire sequencing can be used for sensitive quantification of minimal residual disease to potentially unprecedented depth. To surmount pitfalls inherent to this approach and develop internationally harmonized protocols, the EuroClonality-NGS Working Group has been established.

Disseminated cryptococcal disease during treatment with idelalisib and corticosteroids for follicular lymphoma.

A patient on a regimen of idelalisib and corticosteroids for a relapse of follicular lymphoma presented to our emergency ward with a fever of unknown origin. Despite the initiation of broad-spectrum antibiotics and fluids, the patient's clinical condition deteriorated. Eventually, a diagnosis of disseminated cryptococcosis was established and immunophenotyping revealed complete absence of circulating B and CD4+-T lymphocytes, and a markedly diminished CD8+-T lymphocyte count. In this case, treatment with idelalisib and corticosteroids likely resulted in profound lymphopenia and the first reported instance of disseminated cryptococcosis under this regimen. After the withdrawal of idelalisib and steroids and initiation of antifungal therapy, lymphocyte counts partially recovered. After clinical improvement, the patient could be discharged from the hospital. This case highlights that the combination of idelalisib and corticosteroids can cause significant immunocompromise and opportunistic infections. Additionally, we illustrate the rate of lymphocyte reconstitution after withdrawal from idelalisib and corticosteroids.

Immunoglobulin gene analysis in chronic lymphocytic leukemia in the era of next generation sequencing.

Twenty years after landmark publications, there is a consensus that the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is an important cornerstone for accurate risk stratification and therapeutic decision-making in patients with chronic lymphocytic leukemia (CLL). The IGHV SHM status has traditionally been determined by conventional Sanger sequencing. However, NGS has heralded a new era in medical diagnostics and immunogenetic analysis is following this trend. There is indeed a growing demand for shifting practice and using NGS for IGHV gene SHM assessment, although it is debatable whether it is always justifiable, at least taking into account financial considerations for laboratories with limited resources. Nevertheless, as this analysis impacts on treatment decisions, standardization of both technical aspects, and data interpretation becomes essential. Also, the need for establishing new recommendations and providing dedicated education and training on NGS-based immunogenetics is greater than ever before. Here we address potential and challenges of NGS-based immunogenetics in CLL. We are convinced that this perspective helps the hematological community to better understand the pros and cons of this new technological development for CLL patient management.

[Risk of thrombosis in reactive thrombocytosis]. / Risico op trombose bij reactieve trombocytose.

Reactive thrombocytosis (RT; thrombocyte count: > 450 x 109/l) is a condition in which an increase in platelet production, stimulated by cytokines in the bone marrow, is secondary to some condition or circumstance. Although RT does often occur in children, the risk of thromboembolic complications is negligible in this group if they have no other risk factors for thrombosis. In the absence of additional risk factors for thrombosis it seems as though RT in adults does not predispose them to thromboembolic complications either. Patients with RT caused by a non-myeloproliferative malignancy do have an increased risk of thromboembolic complications; antithrombotic prophylaxis might be effective in this group, but there is no scientific evidence for this. We advise a watch-and-wait approach in children and adults with RT who have no other risk factors for thromboembolism, even in patients with extreme thrombocytosis (thrombocyte count: > 1000 x 109/l). In patients who do have an increased risk of thromboembolic complications we advise tailoring prescription or non-prescription of antithrombotic prophylaxis to the individual patient.

Disease understanding in patients with intermittent claudication: A qualitative study.

The aim of our qualitative study was to investigate the understanding of patients with intermittent claudication (IC) regarding the etiology and atherosclerotic nature of their disease. Patients were recruited from participants of the SUPER study, a randomized trial comparing angioplasty and supervised exercise therapy for alleviation of IC owing to an iliac artery obstruction. Patients were submitted to explorative, semistructured, in-depth interviews that were fully transcribed, coded, and categorized. We interviewed 19 patients. The majority of respondents (79%) recognized smoking as a major risk factor contributing to the etiology of IC. However, nearly one-half (47%) underestimated the effects of unhealthy dietary and exercise patterns. In contrast, a substantial number of respondents (42%) overestimated the contribution of genetics to the etiology of their disease. Most respondents (79%) were unaware of the fact that IC implies systemic atherosclerosis.This study shows that the patients' interpretation of the etiology and nature of IC was mostly incorrect. Therefore, we suggest that health care providers enhance counseling about etiologic factors and the systemic nature of IC to optimize outcomes of lifestyle adjustments.