Analysis of 3760 hematologic malignancies reveals rare transcriptomic aberrations of driver genes.

BACKGROUND: Rare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging. METHODS: To address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes. RESULTS: We found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities. CONCLUSIONS: Altogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers.

TNF-α promoter hypomethylation is frequent in oncopediatric patients who recovered from mucositis.

The aim of this study was to investigate the DNA methylation profile in genes encoding catalase (CAT) and superoxide dismutase (SOD3) enzymes, which are involved in oxidative stress mechanisms, and in genes encoding pro-inflammatory cytokines interleukin-6 (IL6) and tumor necrosis factor-alpha (TNF-α) in the oral mucosa of oncopediatric patients treated with methotrexate (MTX®). This was a cross-sectional observational study and the population comprised healthy dental patients (n = 21) and those with hematological malignancies (n = 64) aged between 5 and 19 years. Oral conditions were evaluated using the Oral Assessment Guide and participants were divided into 4 groups: 1- healthy individuals; 2- oncopediatric patients without mucositis; 3- oncopediatric patients with mucositis; 4- oncopediatric patients who had recovered from mucositis. Methylation of DNA from oral mucosal cells was evaluated using the Methylation-Specific PCR technique (MSP). For CAT, the partially methylated profile was the most frequent and for SOD3 and IL6, the hypermethylated profile was the most frequent, with no differences between groups. For TNF-α, the hypomethylated profile was more frequent in the group of patients who had recovered from mucositis. It was concluded that the methylation profiles of CAT, SOD3, and IL6 are common profiles for oral cells of children and adolescents and have no association with oral mucositis or exposure to chemotherapy with MTX®. Hypomethylation of TNF-α is associated with oral mucosal recovery in oncopediatric patients who developed oral mucositis during chemotherapy.

Germline Predisposition in Hematologic Malignancies: Testing, Management, and Implications.

Although numerous barriers for clinical germline cancer predisposition testing exist, the increasing recognition of deleterious germline DNA variants contributing to myeloid malignancy risk is yielding steady improvements in referrals for testing and testing availability. Many germline predisposition alleles are common in populations, and the increasing number of recognized disorders makes inherited myeloid malignancy risk an entity worthy of consideration for all patients regardless of age at diagnosis. Germline testing is facilitated by obtaining DNA from cultured skin fibroblasts or hair bulbs, and cascade testing is easily performed via buccal swab, saliva, or blood. Increasingly as diagnostic criteria and clinical management guidelines include germline myeloid malignancy predisposition, insurance companies recognize the value of testing and provide coverage. Once an individual is recognized to have a deleterious germline variant that confers risk for myeloid malignancies, a personalized cancer surveillance plan can be developed that incorporates screening for other cancer risk outside of the hematopoietic system and/or other organ pathology. The future may also include monitoring the development of clonal hematopoiesis, which is common for many of these cancer risk disorders and/or inclusion of strategies to delay or prevent progression to overt myeloid malignancy. As research continues to identify new myeloid predisposition disorders, we may soon recommend testing for these conditions for all patients diagnosed with a myeloid predisposition condition.

Biological relevance of alternative splicing in hematologic malignancies.

Alternative splicing (AS) is a strictly regulated process that generates multiple mRNA variants from a single gene, thus contributing to proteome diversity. Transcriptome-wide sequencing studies revealed networks of functionally coordinated splicing events, which produce isoforms with distinct or even opposing functions. To date, several mechanisms of AS are deregulated in leukemic cells, mainly due to mutations in splicing and/or epigenetic regulators and altered expression of splicing factors (SFs). In this review, we discuss aberrant splicing events induced by mutations affecting SFs (SF3B1, U2AF1, SRSR2, and ZRSR2), spliceosome components (PRPF8, LUC7L2, DDX41, and HNRNPH1), and epigenetic modulators (IDH1 and IDH2). Finally, we provide an extensive overview of the biological relevance of aberrant isoforms of genes involved in the regulation of apoptosis (e. g. BCL-X, MCL-1, FAS, and c-FLIP), activation of key cellular signaling pathways (CASP8, MAP3K7, and NOTCH2), and cell metabolism (PKM).

Long non-coding RNA MALAT1 in hematological malignancies and its clinical applications.

ABSTRACT: Metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) is a well-established oncogenic long non-coding RNA, the higher expression of which is strongly correlated with cancer events such as tumorigenesis, progression, metastasis, drug resistance, and treatment outcome in solid cancers. Recently, a series of studies has highlighted its potential role in hematological malignancies in terms of these events. Similar to solid cancers, MALAT1 can regulate various target genes via sponging and epigenetic mechanisms, but the miRNAs sponged by MALAT1 differ from those identified in solid cancers. In this review, we systematically describe the role and underlying mechanisms of MALAT1 in multiple types of hematological malignancies, including regulation of cell proliferation, metastasis, stress response, and glycolysis. Clinically, MALAT1 expression is related to poor treatment outcome and drug resistance, therefore exhibiting potential prognostic value in multiple myeloma, lymphoma, and leukemia. Finally, we discuss the evaluation of MALAT1 as a novel therapeutic target against cancer in preclinical studies.

GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

Genome-wide DNA methylation-analysis of blastic plasmacytoid dendritic cell neoplasm identifies distinct molecular features.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) constitutes a rare and aggressive malignancy originating from plasmacytoid dendritic cells (pDCs) with a primarily cutaneous tropism followed by dissemination to the bone marrow and other organs. We conducted a genome-wide analysis of the tumor methylome in an extended cohort of 45 BPDCN patients supplemented by WES and RNA-seq as well as ATAC-seq on selected cases. We determined the BPDCN DNA methylation profile and observed a dramatic loss of DNA methylation during malignant transformation from early and mature DCs towards BPDCN. DNA methylation profiles further differentiate between BPDCN, AML, CMML, and T-ALL exhibiting the most striking global demethylation, mitotic stress, and merely localized DNA hypermethylation in BPDCN resulting in pronounced inactivation of tumor suppressor genes by comparison. DNA methylation-based analysis of the tumor microenvironment by MethylCIBERSORT yielded two, prognostically relevant clusters (IC1 and IC2) with specific cellular composition and mutational spectra. Further, the transcriptional subgroups of BPDCN (C1 and C2) differ by DNA methylation signatures in interleukin/inflammatory signaling genes but also by higher transcription factor activity of JAK-STAT and NFkB signaling in C2 in contrast to an EZH2 dependence in C1-BPDCN. Our integrative characterization of BPDCN offers novel molecular insights and potential diagnostic applications.

Non-invasive prenatal test identifies circulating cell-free DNA chromosomal abnormalities derived from clonal hematopoiesis in aggressive hematological malignancies.

Liquid biopsy is a minimally invasive diagnostic tool for identification of tumor-related mutations in circulating cell-free DNA (cfDNA). The aim of this study was to investigate feasibility, sensitivity, and specificity of non-invasive prenatal test (NIPT) for identification of chromosomal abnormalities in cfDNA from a total of 77 consecutive patients with non-Hodgkin B-cell lymphomas, Hodgkin lymphoma (HL), or plasma cell dyscrasia. In this case series, half of patients had at least one alteration, more frequently in chromosome 6 (23.1%), chromosome 9 (20.5%), and chromosomes 3 and 18 (16.7%), with losses of chromosome 6 and gains of chromosome 7 negatively impacting on overall survival (OS), with a 5-year OS of 26.9% and a median OS of 14.6 months, respectively (P = 0.0009 and P = 0.0004). Moreover, B-cell lymphomas had the highest NIPT positivity, especially those with aggressive lymphomas, while patients with plasma cell dyscrasia with extramedullary disease had a higher NIPT positivity compared to conventional cytogenetics analysis and a worse outcome. Therefore, we proposed a NIPT-based liquid biopsy a complementary minimally invasive tool for chromosomal abnormality detection in hematological malignancies. However, prospective studies on larger cohorts are needed to validate clinical utility of NIPT-based liquid biopsy in routinely clinical practice.

Genetic polymorphisms and clinical parameters associated with renal toxicity in Thai hematologic malignancy patients receiving high dose methotrexate.

High-dose methotrexate (HD-MTX) is a widely used chemotherapy regimen for hematologic malignancies such as lymphomas and acute lymphoblastic leukemia, but its use can lead to adverse effects, including acute kidney injury (AKI), impaired liver function, and mucositis, causing extended hospital stays and delayed subsequent chemotherapy. Our study aimed to investigate the predictive factors for renal toxicities associated with HD-MTX in Thai patients undergoing treatment for hematologic malignancies. We enrolled 80 patients who underwent MTX-containing regimens, analyzing 132 chemotherapy cycles. The most common disease was primary central nervous system lymphoma (33%). Genetic polymorphisms were examined using the MassARRAY® system, identifying 42 polymorphisms in 25 genes. Serum creatinine and MTX levels were measured 24 and 48 h after MTX administration. For the primary outcome, we found that the allele A of MTRR rs1801394 was significantly related to renal toxicity (odds ratio 2.084 (1.001-4.301), p-value 0.047). Patients who exceeded the MTX threshold levels at 24 h after the dose had a significantly higher risk of renal toxicity (OR (95%CI) = 6.818 (2.350-19.782), p < 0.001). Multivariate logistic regression analysis with a generalized estimated equation revealed hypertension and age as independent predictors of increased MTX levels at 24 h after the given dose.

SLC27A2 is a potential immune biomarker for hematological tumors and significantly regulates the cell cycle progression of diffuse large B-cell lymphoma.

BACKGROUND: Research on the fatty acid metabolism related gene SLC27A2 is currently mainly focused on solid tumors, and its mechanism of action in hematological tumors has not been reported. METHOD: This study aims to explore the pathological and immune mechanisms of the fatty acid metabolism related gene SLC27A2 in hematological tumors and verify its functional role in hematological tumors through cell experiments to improve treatment decisions and clinical outcomes of hematological tumors. RESULT: This study identified the fatty acid metabolism related gene SLC27A2 as a common differentially expressed gene between DLBCL and AML. Immune microenvironment analysis showed that SLC27A2 was significantly positively correlated with T cell CD4 + , T cell CD8 + , endothelial cells, macrophages, and NK cells in DLBCL. In AML, there is a significant negative correlation between SLC27A2 and B cells, T cell CD8 + , and macrophages. SLC27A2 participates in the immune process of hematological tumors through T cell CD8 + and macrophages. The GESA results indicate that high expression of SLC27A2 is mainly involved in the fatty acid pathway, immune pathway, and cell cycle pathway of DLBCL. The low expression of SLC27A2 is mainly involved in the immune pathway of AML. Therefore, SLC27A2 is mainly involved in the pathological mechanisms of hematological tumors through immune pathways, and cell experiments have also confirmed that SLC27A2 is involved in the regulation of DLBCL cells. CONCLUSION: In summary, our research results comprehensively report for the first time the mechanism of action of SLC27A2 in the immune microenvironment of DLBCL and AML, and for the first time verify the cycle and apoptotic effects of the fatty acid related gene SLC27A2 in DLBCL cells through cell experiments. Research can help improve the treatment of AML and DLBCL patients.

Putting the STING back into BH3-mimetic drugs for TP53-mutant blood cancers.

TP53-mutant blood cancers remain a clinical challenge. BH3-mimetic drugs inhibit BCL-2 pro-survival proteins, inducing cancer cell apoptosis. Despite acting downstream of p53, functional p53 is required for maximal cancer cell killing by BH3-mimetics through an unknown mechanism. Here, we report p53 is activated following BH3-mimetic induced mitochondrial outer membrane permeabilization, leading to BH3-only protein induction and thereby potentiating the pro-apoptotic signal. TP53-deficient lymphomas lack this feedforward loop, providing opportunities for survival and disease relapse after BH3-mimetic treatment. The therapeutic barrier imposed by defects in TP53 can be overcome by direct activation of the cGAS/STING pathway, which promotes apoptosis of blood cancer cells through p53-independent BH3-only protein upregulation. Combining clinically relevant STING agonists with BH3-mimetic drugs efficiently kills TRP53/TP53-mutant mouse B lymphoma, human NK/T lymphoma, and acute myeloid leukemia cells. This represents a promising therapy regime that can be fast-tracked to tackle TP53-mutant blood cancers in the clinic.

Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens.

ABSTRACT: Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.

DNA damage response defects in hematologic malignancies: mechanistic insights and therapeutic strategies.

ABSTRACT: The DNA damage response (DDR) encompasses the detection and repair of DNA lesions and is fundamental to the maintenance of genome integrity. Germ line DDR alterations underlie hereditary chromosome instability syndromes by promoting the acquisition of pathogenic structural variants in hematopoietic cells, resulting in increased predisposition to hematologic malignancies. Also frequent in hematologic malignancies are somatic mutations of DDR genes, typically arising from replication stress triggered by oncogene activation or deregulated tumor proliferation that provides a selective pressure for DDR loss. These defects impair homology-directed DNA repair or replication stress response, leading to an excessive reliance on error-prone DNA repair mechanisms that results in genomic instability and tumor progression. In hematologic malignancies, loss-of-function DDR alterations confer clonal growth advantage and adverse prognostic impact but may also provide therapeutic opportunities. Selective targeting of functional dependencies arising from these defects could achieve synthetic lethality, a therapeutic concept exemplified by inhibition of poly-(adenosine 5'-diphosphate ribose) polymerase or the ataxia telangiectasia and Rad 3 related-CHK1-WEE1 axis in malignancies harboring the BRCAness phenotype or genetic defects that increase replication stress. Furthermore, the role of DDR defects as a source of tumor immunogenicity, as well as their impact on the cross talk between DDR, inflammation, and tumor immunity are increasingly recognized, thus providing rationale for combining DDR modulation with immune modulation. The nature of the DDR-immune interface and the cellular vulnerabilities conferred by DDR defects may nonetheless be disease-specific and remain incompletely understood in many hematologic malignancies. Their comprehensive elucidation will be critical for optimizing therapeutic strategies to target DDR defects in these diseases.

Evaluation of the pathogenic potential of germline DDX41 variants in hematopoietic neoplasms using the ACMG/AMP guidelines.

Clinical use of gene panel testing for hematopoietic neoplasms in areas, such as diagnosis, prognosis prediction, and exploration of treatment options, has increased in recent years. The keys to interpreting gene variants detected in gene panel testing are to distinguish between germline and somatic variants and accurately determine whether the detected variants are pathogenic. If a variant is suspected to be a pathogenic germline variant, it is essential to confirm its consistency with the disease phenotype and gather a thorough family history. Donor eligibility must also be considered, especially if the patient's variant is also detected in the expected donor for hematopoietic stem cell transplantation. However, determining the pathogenicity of gene variants is often complicated, given the current limited availability of databases covering germline variants of hematopoietic neoplasms. This means that hematologists will frequently need to interpret gene variants themselves. Here, we outline how to assess the pathogenicity of germline variants according to criteria from the American College of Medical Genetics and Genomics/Association for Molecular Pathology standards and guidelines for the interpretation of variants using DDX41, a gene recently shown to be closely associated with myeloid neoplasms with a germline predisposition, as an example.

Fifth Edition of the World Health Organization Classification of Tumors of the Hematopoietic and Lymphoid Tissues: Acute Lymphoblastic Leukemias, Mixed-Phenotype Acute Leukemias, Myeloid/Lymphoid Neoplasms With Eosinophilia, Dendritic/Histiocytic Neoplasms, and Genetic Tumor Syndromes.

This manuscript represents a review of lymphoblastic leukemia/lymphoma (acute lymphoblastic leukemia/lymphoblastic lymphoma), acute leukemias of ambiguous lineage, mixed-phenotype acute leukemias, myeloid/lymphoid neoplasms with eosinophilia and defining gene rearrangements, histiocytic and dendritic neoplasms, and genetic tumor syndromes of the 5th edition of the World Health Organization Classification of Tumors of the Hematopoietic and Lymphoid Tissues. The diagnostic, clinicopathologic, cytogenetic, and molecular genetic features are discussed. The differences in comparison to the 4th revised edition of the World Health Organization classification of hematolymphoid neoplasms are highlighted.

A novel method for simultaneous detection of hematological tumors and infectious pathogens by metagenomic next generation sequencing of plasma.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) is valuable for pathogen identification; however, distinguishing between infectious diseases and conditions with potentially similar clinical manifestations, including malignant tumors, is challenging. Therefore, we developed a method for simultaneous detection of infectious pathogens and cancer in blood samples. METHODS: Plasma samples (n = 244) were collected from 150 and 94 patients with infections and hematological malignancies, respectively, and analyzed by mNGS for pathogen detection, alongside human tumor chromosomal copy number variation (CNV) analysis (≥5Mbp or 10Mbp CNV region). Further, an evaluation set, comprising 87 plasma samples, was analyzed by mNGS and human CNV analysis, to validate the feasibility of the method. RESULTS: Among 94 patients with hematological malignancy, sensitivity values of CNV detection for tumor diagnosis were 69.15 % and 32.98 % for CNV region 5Mbp and 10Mbp, respectively, with corresponding specificities of 92.62 % and 100 % in the infection group. Area under the ROC curve (AUC) values for 5Mbp and 10Mbp region were 0.825 and 0.665, respectively, which was a significant difference of 0.160 (95 % CI: 0.110-0.210; p < 0.001), highlighting the superiority of 5Mbp output region data. Six patients with high-risk CNV results were identified in the validation study: three with history of tumor treatment, two eventually newly-diagnosed with hematological malignancies, and one with indeterminate final diagnosis. CONCLUSIONS: Concurrent CNV analysis alongside mNGS for infection diagnosis is promising for detecting malignant tumors. We recommend adopting a CNV region of 10Mbp over 5Mbp for our model, because of the lower false-positive rate (FPR).

The role of GATA family transcriptional factors in haematological malignancies: A review.

GATA transcriptional factors are zinc finger DNA binding proteins that regulate transcription during development and cell differentiation. The 3 important GATA transcription factors GATA1, GATA2 and GATA3 play essential role in the development and maintenance of hematopoietic systems. GATA1 is required for the erythroid and Megakaryocytic commitment during hematopoiesis. GATA2 is crucial for the proliferation and survival of early hematopoietic cells, and is also involved in lineage specific transcriptional regulation as the dynamic partner of GATA1. GATA3 plays an essential role in T lymphoid cell development and immune regulation. As a result, mutations in gene encoding the GATA transcription factor or alteration in the protein expression level or their function have been linked to a variety of human haematological malignancies. This review presents a summary of recent understanding of how the disrupted biological function of GATA may contribute to hematologic diseases.

The landscape of cytogenetic and molecular genetic methods in diagnostics for hematologic neoplasia.

Improvements made during the last decades in the management of patients with hematologic neoplasia have resulted in increase of overall survival. These advancements have become possible through progress in our understanding of genetic basis of different hematologic malignancies and their role in the current risk-adapted treatment protocols. In this review, we provide an overview of current cytogenetic and molecular genetic methods, commonly used in the genetic characterization of hematologic malignancies, describe the current developments in the cytogenetic and molecular diagnostics, and give an outlook into their future development. Furthermore, we give a brief overview of the most important public databases and guidelines for sequence variant interpretation.

Hairy cell leukemia 2024: Update on diagnosis, risk-stratification, and treatment-Annual updates in hematological malignancies.

DISEASE OVERVIEW: Hairy cell leukemia (HCL) and HCL-like disorders, including HCL variant (HCL-V) and splenic diffuse red pulp lymphoma (SDRPL), are a very heterogenous group of mature lymphoid B-cell disorders characterized by the identification of hairy cells, a specific genetic profile, a different clinical course and the need for appropriate treatment. DIAGNOSIS: Diagnosis of HCL is based on morphological evidence of hairy cells, an HCL immunologic score of 3 or 4 based on the CD11c, CD103, CD123, and CD25 expression, the trephine biopsy which makes it possible to specify the degree of tumoral bone marrow infiltration and the presence of BRAFV600E somatic mutation. RISK STRATIFICATION: Progression of patients with HCL is based on a large splenomegaly, leukocytosis, a high number of hairy cells in the peripheral blood, and the immunoglobulin heavy chain variable region gene mutational status. VH4-34 positive HCL cases are associated with a poor prognosis, as well as HCL with TP53 mutations and HCL-V. TREATMENT: Patients should be treated only if HCL is symptomatic. Chemotherapy with risk-adapted therapy purine analogs (PNAs) are indicated in first-line HCL patients. The use of chemo-immunotherapy combining cladribine (CDA) and rituximab (R) represents an increasingly used therapeutic approach. Management of relapsed/refractory disease is based on the use of BRAF inhibitors (BRAFi) plus R, MEK inhibitors (MEKi), recombinant immunoconjugates targeting CD22, Bruton tyrosine kinase inhibitors (BTKi), and Bcl-2 inhibitors (Bcl-2i). However, the optimal sequence of the different treatments remains to be determined.