Disruption of the ZFP574-THAP12 complex suppresses B cell malignancies in mice.

Despite the availability of life-extending treatments for B cell leukemias and lymphomas, many of these cancers remain incurable. Thus, the development of new molecular targets and therapeutics is needed to expand treatment options. To identify new molecular targets, we used a forward genetic screen in mice to identify genes required for development or survival of lymphocytes. Here, we describe Zfp574, an essential gene encoding a zinc finger protein necessary for normal and malignant lymphocyte survival. We show that ZFP574 interacts with zinc finger protein THAP12 and promotes the G1-to-S-phase transition during cell cycle progression. Mutation of ZFP574 impairs nuclear localization of the ZFP574-THAP12 complex. ZFP574 or THAP12 deficiency results in cell cycle arrest and impaired lymphoproliferation. Germline mutation, acute gene deletion, or targeted degradation of ZFP574 suppressed Myc-driven B cell leukemia in mice, but normal B cells were largely spared, permitting long-term survival, whereas complete lethality was observed in control animals. Our findings support the identification of drugs targeting ZFP574-THAP12 as a unique strategy to treat B cell malignancies.

Motive and opportunity: MYC rearrangements in high-grade B-cell lymphoma with MYC and BCL2 rearrangements (an LLMPP study).

ABSTRACT: Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements ("double hit"; HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of and mechanisms driving IG vs non-IG MYC rearrangements have not been elucidated. Here, we used custom targeted capture and/or whole-genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, although BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because 1 IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B-cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.

Detection of disease-specific signatures in B cell repertoires of lymphomas using machine learning.

The classification of B cell lymphomas-mainly based on light microscopy evaluation by a pathologist-requires many years of training. Since the B cell receptor (BCR) of the lymphoma clonotype and the microenvironmental immune architecture are important features discriminating different lymphoma subsets, we asked whether BCR repertoire next-generation sequencing (NGS) of lymphoma-infiltrated tissues in conjunction with machine learning algorithms could have diagnostic utility in the subclassification of these cancers. We trained a random forest and a linear classifier via logistic regression based on patterns of clonal distribution, VDJ gene usage and physico-chemical properties of the top-n most frequently represented clonotypes in the BCR repertoires of 620 paradigmatic lymphoma samples-nodular lymphocyte predominant B cell lymphoma (NLPBL), diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL)-alongside with 291 control samples. With regard to DLBCL and CLL, the models demonstrated optimal performance when utilizing only the most prevalent clonotype for classification, while in NLPBL-that has a dominant background of non-malignant bystander cells-a broader array of clonotypes enhanced model accuracy. Surprisingly, the straightforward logistic regression model performed best in this seemingly complex classification problem, suggesting linear separability in our chosen dimensions. It achieved a weighted F1-score of 0.84 on a test cohort including 125 samples from all three lymphoma entities and 58 samples from healthy individuals. Together, we provide proof-of-concept that at least the 3 studied lymphoma entities can be differentiated from each other using BCR repertoire NGS on lymphoma-infiltrated tissues by a trained machine learning model.

Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement.

High-grade B-cell lymphoma (HGBCL), the subtype of non-Hodgkin lymphoma, to be relapsed or refractory in patients after initial therapy or salvage chemotherapy. Dual dysregulation of MYC and BCL2 is one of the important pathogenic mechanisms. Thus, combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine. It has been shown to be a potential therapeutic target for multiple diseases. In this study, the DHODH inhibitor brequinar exhibited growth inhibition, cell cycle blockade, and apoptosis promotion in HGBCL cell lines with MYC and BCL2 rearrangements. The combination of brequinar and BCL2 inhibitors venetoclax had a synergistic inhibitory effect on the survival of DHL cells through different pathways. Venetoclax could upregulate MCL-1 and MYC expression, which has been reported as a resistance mechanism of BCL2 inhibitors. Brequinar downregulated MCL-1 and MYC, which could potentially overcome drug resistance to venetoclax in HGBCL cells. Furthermore, brequinar could downregulate a broad range of genes, including ribosome biosynthesis genes, which might contribute to its anti-tumor effects. In vivo studies demonstrated synergetic tumor growth inhibition in xenograft models with brequinar and venetoclax combination treatment. These results provide preliminary evidence for the rational combination of DHODH and BCL2 blockade in HGBCL with abnormal MYC and BCL2.

Challenging our understanding of B-cell lymphomagenesis and risk: Paediatric high-grade B-cell lymphoma, not otherwise specified with a DDX3X::MLLT10 fusion and an IGH deletion.

We report a unique case of high-grade B-cell lymphoma, not otherwise specified in a 5-year-old child. Whole-genome sequencing revealed a DDX3X::MLLT10 fusion, usually seen in T-cell acute lymphoblastic leukaemia (ALL). This suggests the novel idea that MLLT10 fusions are capable of driving B-cell malignancies. An IGH deletion usually only seen in adults was also found. These unique genetic findings provide novel insights into B-cell lymphomagenesis. The child remains in remission 7 year post chemotherapy, which demonstrates that novel complex molecular findings do not always denote high-risk disease.

Assessment of Bone Marrow Involvement in B-Cell non-Hodgkin Lymphoma Using Immunoglobulin Gene Rearrangement Analysis with Next-Generation Sequencing.

BACKGROUND: Assessment of bone marrow involvement (BMI) in non-Hodgkin lymphoma (NHL) is crucial for determining patient prognosis and treatment strategy. We assessed the prognostic value of next-generation sequencing (NGS)-based immunoglobulin (Ig) gene clonality analysis as an ancillary test for BMI evaluation in NHL. METHODS: A retrospective cohort of 124 patients newly diagnosed with B-cell NHL between 2019 and 2022 was included. NGS-based Ig clonality analysis was conducted using LymphoTrak IGH FR1 Assay and IGK Assay (Invivoscribe Technologies, San Diego, CA, USA) on BM aspirate samples, and the results were compared with those of histopathological BMI (hBMI). RESULTS: Among the 124 patients, hBMI was detected in 16.9% (n = 21). The overall agreement of BMI between Ig clonality analyses and histopathological analysis for IGH, IGK, and either IGH or IGK was 86.3%, 92.7%, and 90.3%. The highest positive percent agreement was observed with clonal rearrangements of either IGH or IGK gene (90.5%), while the highest negative percent agreement was observed with clonal rearrangement of IGK gene (96.1%). For the prediction of hBMI, positive prediction value ranged between 59.1% and 80.0% and the negative prediction value ranged between 91.3% and 97.9%. CONCLUSION: NGS-based clonality analysis is an analytic platform with a substantial overall agreement with histopathological analysis. Assessment of both IGH and IGK genes for the clonal rearrangement analysis could be considered for the optimal diagnostic performance of BMI detection in B-cell NHL.

Pathogenic Variants Associated with Epigenetic Control and the NOTCH Pathway Are Frequent in Classic Hodgkin Lymphoma.

Classic Hodgkin lymphoma (cHL) constitutes a B-cell neoplasm derived from germinal center lymphocytes. Despite high cure rates (80-90%) obtained with the current multiagent protocols, a significant proportion of cHL patients experience recurrences, characterized by a lower sensitivity to second-line treatments. The genomic background of chemorefractory cHL is still poorly understood, limiting personalized treatment strategies based on molecular features. In this study, using a targeted next-generation sequencing (NGS) panel specifically designed for cHL research, we compared chemosensitive and chemorefractory diagnostic tissue samples of cHL patients. Furthermore, we longitudinally examined paired diagnosis-relapsesamples of chemorefractory cHL in order to define patterns of dynamic evolution and clonal selection. Pathogenic variants in NOTCH1 and NOTCH2 genes frequently arise in cHL. Mutations in genes associated with epigenetic regulation (CREBBP and EP300) are particularly frequent in relapsed/refractory cHL. The appearance of novel clones characterized by mutations previously not identified at diagnosis is a common feature in cHL cases showing chemoresistance to frontline treatments. Our results expand current molecular and pathogenic knowledge of cHL and support the performance of molecular studies in cHL prior to the initiation of first-line therapies.

The Evolving Role of Bruton's Tyrosine Kinase Inhibitors in B Cell Lymphomas.

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase crucial for B cell development and function, acts downstream of the B cell receptor (BCR) in the BCR pathway. Other kinases involved downstream of the BCR besides BTK such as Syk, Lyn, PI3K, and Mitogen-activated protein (MAP) kinases also play roles in relaying signals from the BCR to provide pro-survival, activation, and proliferation cues. BTK signaling is implicated in various B-cell lymphomas such as mantle cell lymphoma, Waldenström Macroglobulinemia, follicular lymphoma, and diffuse large B cell lymphoma, leading to the development of transformative treatments like ibrutinib, the first-in-class covalent BTK inhibitor, and pirtobrutinib, the first-in-class noncovalent BTK inhibitor. However, kinase-deficient mutations C481F, C481Y, C481R, and L528W in the BTK gene confer resistance to both covalent and non-covalent BTK inhibitors, facilitating B cell survival and lymphomagenesis despite kinase inactivation. Further studies have revealed BTK's non-catalytic scaffolding function, mediating the assembly and activation of proteins including Toll-like receptor 9 (TLR9), vascular cell adhesion protein 1 (VCAM-1), hematopoietic cell kinase (HCK), and integrin-linked kinase (ILK). This non-enzymatic role promotes cell survival and proliferation independently of kinase activity. Understanding BTK's dual roles unveils opportunities for therapeutics targeting its scaffolding function, promising advancements in disrupting lymphomagenesis and refining B cell lymphoma treatments.

B-cell lymphoma research in Malaysia - A narrative review.

Lymphomas are a diverse group of malignant proliferations that arise as discrete tissue masses. The most widely accepted taxonomy for lymphoma is the World Health Organization classification of tumours of haematopoietic and lymphoid tissues, the 5th edition of which was released in June 2022. Most (85% to 90%) lymphoid neoplasms are of B cell origin. Mature B-cell neoplasms are a heterogeneous group of malignancies with similar disease courses and treatment paradigms. This review focuses on the various mature B-cell lymphomas in Malaysia, including Hodgkin lymphoma. A literature search was performed in various bibliographic databases. A total of 64 papers were included in this review. We found 15 papers on Hodgkin lymphoma, 14 on follicular lymphoma, 12 on Burkitt lymphoma, 5 on mucosa-associated lymphoid tissue (MALT) lymphoma, 4 on plasmablastic lymphoma, 3 on mantle cell lymphoma, 1 each on primary mediastinal large B-cell lymphoma, B-lymphoblastic lymphoma, and 3 on other unspecified B-cell lymphomas. The site, age, distribution, prognostic markers, and the various subclassification of B cell lymphomas were studied from these papers. Prognostic genetic markers in B-cell lymphomas include C-MYC, BCL2 and BCL6 as they are the most prevalent mutations in this condition. Anecdotal outcomes range from rapid fatality to unexplained spontaneous remission. This review adds to the existing literature on lymphoma in Malaysia by compiling the evidence that may lead to further research on the diagnosis and treatment of lymphoma in Malaysia and worldwide.

[Clinicopathological characteristics of angioimmunoblastic T-cell lymphoma with B-cell proliferation or neoplasms].

Objective: To investigate the clinical, pathological and immunophenotypic features, and differential diagnosis of angioimmunoblastic T-cell lymphoma (AITL) with B-cell proliferation or neoplasms. Methods: Eight qualified cases were collected from the Department of Pathology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China from January 2019 to July 2023. One case was diagnosed with AITL and diffuse large B-cell lymphoma (DLBCL) and the other seven cases were diagnosed with AITL and B-cell proliferation. Clinical characteristics and pathological morphology were summarized. Immunohistochemical analysis, fluorescence in situ hybridization and gene rearrangement detection were performed. Results: The patients' average age was 58 years. Five of them were male. Biopsies of the enlarged cervical lymph nodes showed structural destruction and exhibited various histologic patterns. Some cases revealed Burkitt-like morphology, a moderate tumor volume and slightly irregular nuclei. Some cases showed prominent nucleoli. High endothelial venules and expanded follicular dendritic cells were detected. Tumor cells derived from T-follicular helper (TFH) cells were positive for two or more TFH biomarkers. Nodular or diffuse patchy proliferation of B cells was noted around the tumor tissue, which was initially considered as B-cell lymphoma. All of the 8 cases showed monoclonal rearrangements of the T-cell receptor genes while 5 of them also showed clonal rearrangements of the Ig genes. Seven of the 8 cases were subject to the detection of C-MYC gene breakage and were all negative. EBV-positive cells were seen in 6 cases. Neoplastic B cells were positive for C-MYC (>40%), while proliferative B cells were negative for C-MYC (<40%). Conclusions: The histological morphology of AITL with B-cell proliferation or lymphoma may be different from AITL. An integrated analysis, incorporating clinical, morphologic, immunophenotypic, and molecular assessment, helps reach an accurate diagnosis. This group of cases demonstrated the clinical and pathological characteristics of AITL accompanied by B-cell proliferation and B-cell lymphoma. The findings suggest that C-MYC maybe a feasible indicator for distinguishing B-cell proliferation from B-cell lymphoma, and provide a simple and feasible immunohistochemical marker for the diagnosis and research of composite lymphoma.

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

Use of genome-wide DNA methylation analysis to identify prognostic CpG site markers associated with longer survival time in dogs with multicentric high-grade B-cell lymphoma.

BACKGROUND: DNA methylation analysis might identify prognostic CpG sites in CHOP-treated dogs with multicentric high-grade B-cell lymphoma (MHGL) with heterogenous prognosis. OBJECTIVE: To identify prognostic CpG sites of MHGL through genome-wide DNA methylation analysis with pyrosequencing validation. ANIMALS: Test group: 24 dogs. Validation group: 100 dogs. All client-owned dogs were diagnosed with MHGL and treated with CHOP chemotherapy. METHODS: Cohort study. DNA was extracted from lymph node samples obtained via FNA. Genome-wide DNA methylation analysis using Digital Restriction Enzyme Analysis of Methylation (DREAM) was performed on the test group to identify differentially methylated CpG sites (DMCs). Bisulfite pyrosequencing was used to measure methylation status of candidate DMCs in the validation group. Median survival times (MST) were analyzed using Kaplan-Meier (log-rank) product limit method. RESULTS: DREAM analyzed 101 576 CpG sites. Hierarchical clustering of 16 262 CpG sites in test group identified group with better prognosis (MST = 55-477 days vs 10-301 days, P = .007). Volcano plot identified 1371 differentially methylated CpG sites (DMCs). DMC near the genes of FAM213A (DMC-F) and PHLPP1 (DMC-P) were selected as candidates. Bisulfite-pyrosequencing performed on validation group showed group with methylation level of DMC-F < 40% had favorable prognosis (MST = 11-1072 days vs 8-1792 days, P = .01), whereas group with the methylation level combination of DMC-F < 40% plus DMC-P < 10% had excellent prognosis (MST = 18-1072 days vs 8-1792 days, P = .009). CONCLUSION AND CLINICAL IMPORTANCE: Methylation status of prognostic CpG sites delineate canine MGHL cases with longer MST, providing owners with information on expectations of potential improved treatment outcomes.

AID in non-Hodgkin B-cell lymphomas: The consequences of on- and off-target activity.

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

Follicular lymphoma B cells exhibit heterogeneous transcriptional states with associated somatic alterations and tumor microenvironments.

Follicular lymphoma (FL) is an indolent non-Hodgkin lymphoma of germinal center origin, which presents with significant biologic and clinical heterogeneity. Using RNA-seq on B cells sorted from 87 FL biopsies, combined with machine-learning approaches, we identify 3 transcriptional states that divide the biological ontology of FL B cells into inflamed, proliferative, and chromatin-modifying states, with relationship to prior GC B cell phenotypes. When integrated with whole-exome sequencing and immune profiling, we find that each state was associated with a combination of mutations in chromatin modifiers, copy-number alterations to TNFAIP3, and T follicular helper cells (Tfh) cell interactions, or primarily by a microenvironment rich in activated T cells. Altogether, these data define FL B cell transcriptional states across a large cohort of patients, contribute to our understanding of FL heterogeneity at the tumor cell level, and provide a foundation for guiding therapeutic intervention.

Lysine demethylase LSD1 is associated with stemness in EBV-positive B cell lymphoma.

EBV-infected lymphoma has a poor prognosis and various treatment strategies are being explored. Reports suggesting that B cell lymphoma can be induced by epigenetic regulation have piqued interest in studying mechanisms targeting epigenetic regulation. Here, we set out to identify an epigenetic regulator drug that acts synergistically with doxorubicin in EBV-positive lymphoma. We expressed the major EBV protein, LMP1, in B-cell lymphoma cell lines and used them to screen 100 epigenetic modifiers in combination with doxorubicin. The screening results identified TCP, which is an inhibitor of LSD1. Further analyses revealed that LMP1 increased the activity of LSD1 to enhance stemness ability under doxorubicin treatment, as evidenced by colony-forming and ALDEFLUOR activity assays. Quantseq 3' mRNA sequencing analysis of potential targets regulated by LSD1 in modulating stemness revealed that the LMP1-induced upregulation of CHAC2 was decreased when LSD1 was inhibited by TCP or downregulated by siRNA. We further observed that SOX2 expression was altered in response to CHAC2 expression, suggesting that stemness is regulated. Collectively, these findings suggest that LSD1 inhibitors could serve as promising therapeutic candidates for EBV-positive lymphoma, potentially reducing stemness activity when combined with conventional drugs to offer an effective treatment approach.

Phosphatidylserine synthesis controls oncogenic B cell receptor signaling in B cell lymphoma.

Cancer cells harness lipid metabolism to promote their own survival. We screened 47 cancer cell lines for survival dependency on phosphatidylserine (PS) synthesis using a PS synthase 1 (PTDSS1) inhibitor and found that B cell lymphoma is highly dependent on PS. Inhibition of PTDSS1 in B cell lymphoma cells caused a reduction of PS and phosphatidylethanolamine levels and an increase of phosphoinositide levels. The resulting imbalance of the membrane phospholipidome lowered the activation threshold for B cell receptor (BCR), a B cell-specific survival mechanism. BCR hyperactivation led to aberrant elevation of downstream Ca2+ signaling and subsequent apoptotic cell death. In a mouse xenograft model, PTDSS1 inhibition efficiently suppressed tumor growth and prolonged survival. Our findings suggest that PS synthesis may be a critical vulnerability of malignant B cell lymphomas that can be targeted pharmacologically.

Patient-specific computational models predict prognosis in B cell lymphoma by quantifying pro-proliferative and anti-apoptotic signatures from genetic sequencing data.

Genetic heterogeneity and co-occurring driver mutations impact clinical outcomes in blood cancers, but predicting the emergent effect of co-occurring mutations that impact multiple complex and interacting signalling networks is challenging. Here, we used mathematical models to predict the impact of co-occurring mutations on cellular signalling and cell fates in diffuse large B cell lymphoma and multiple myeloma. Simulations predicted adverse impact on clinical prognosis when combinations of mutations induced both anti-apoptotic (AA) and pro-proliferative (PP) signalling. We integrated patient-specific mutational profiles into personalised lymphoma models, and identified patients characterised by simultaneous upregulation of anti-apoptotic and pro-proliferative (AAPP) signalling in all genomic and cell-of-origin classifications (8-25% of patients). In a discovery cohort and two validation cohorts, patients with upregulation of neither, one (AA or PP), or both (AAPP) signalling states had good, intermediate and poor prognosis respectively. Combining AAPP signalling with genetic or clinical prognostic predictors reliably stratified patients into striking prognostic categories. AAPP patients in poor prognosis genetic clusters had 7.8 months median overall survival, while patients lacking both features had 90% overall survival at 120 months in a validation cohort. Personalised computational models enable identification of novel risk-stratified patient subgroups, providing a valuable tool for future risk-adapted clinical trials.

Cytogenetic and pathologic characterization of MYC-rearranged B-cell lymphomas in pediatric and young adult patients.

MYC-rearranged B-cell lymphoma (BCL) in the pediatric/young adult (YA) age group differs substantially in disease composition from adult cohorts. However, data regarding the partner genes, concurrent rearrangements, and ultimate diagnoses in these patients is scarce compared to that in adult cohorts. We aimed to characterize the spectrum of MYC-rearranged (MYC-R) mature, aggressive BCL in the pediatric/YA population. A retrospective study of morphologic, immunophenotypic, and fluorescence in situ hybridization (FISH) results of patients age ≤ 30 years with suspected Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBCL), and a MYC-R by FISH between 2013-2022 was performed. Two-hundred fifty-eight cases (129 (50%) pediatric (< 18 years) and 129 (50%) YA (18-30 years)) were included. Most MYC-R BCL in pediatric (89%) and YA (66%) cases were BL. While double-hit (DH) cytogenetics (MYC with BCL2 and/or BCL6-R, HGBCL-DH) was rare in the pediatric population (2/129, 2%), HGBCL-DH increased with age and was identified in 17/129 (13%) of YA cases. Most HGBCL-DH had MYC and BCL6-R, while BCL2-R were rare in both groups (3/258, 1%). MYC-R without an IG partner was more common in the YA group (14/116 (12%) vs 2/128 (2%), p = 0.001). The pediatric to YA transition is characterized by decreasing frequency in BL and increasing genetic heterogeneity of MYC-R BCL, with emergence of DH-BCL with MYC and BCL6-R. FISH to evaluate for BCL2 and BCL6 rearrangements is likely not warranted in the pediatric population but should continue to be applied in YA BCL.