An immunohistochemical atlas of necroptotic pathway expression.

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo.

Germinal center cytokine driven epigenetic control of Epstein-Barr virus latency gene expression.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.

Targeting latent viral infection in EBV-associated lymphomas.

Epstein-Barr virus (EBV) contributes to the development of a significant subset of human lymphomas. As a herpes virus, EBV can transition between a lytic state which is required to establish infection and a latent state where a limited number of viral antigens are expressed which allows infected cells to escape immune surveillance. Three broad latency programs have been described which are defined by the expression of viral proteins RNA, with latency I being the most restrictive expressing only EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) and latency III expressing the full panel of latent viral genes including the latent membrane proteins 1 and 2 (LMP1/2), and EBNA 2, 3, and leader protein (LP) which induce a robust T-cell response. The therapeutic use of EBV-specific T-cells has advanced the treatment of EBV-associated lymphoma, however this approach is only effective against EBV-associated lymphomas that express the latency II or III program. Latency I tumors such as Burkitt lymphoma (BL) and a subset of diffuse large B-cell lymphomas (DLBCL) evade the host immune response to EBV and are resistant to EBV-specific T-cell therapies. Thus, strategies for inducing a switch from the latency I to the latency II or III program in EBV+ tumors are being investigated as mechanisms to sensitize tumors to T-cell mediated killing. Here, we review what is known about the establishment and regulation of latency in EBV infected B-cells, the role of EBV-specific T-cells in lymphoma, and strategies to convert latency I tumors to latency II/III.

Pediatric primary lymphoma of bone in epiphysis case report.

Primary lymphoma of the bone (PLB) is a rare entity, with a majority of pediatric cases presenting in the metaphysis of long bones. There have been only seven reported cases to date of pediatric lymphoma of the bone arising from the epiphysis, of which only two have been described in the proximal tibia. We report a pediatric case of PLB in the tibial epiphysis which presented initially with knee pain. Imaging was performed with X-ray, MRI, CT, and PET-CT with bone biopsies revealing diffuse large B-cell lymphoma. This patient also showed a second, synchronous lesion in the left iliac bone, which was also biopsy proven to diffuse large B-cell lymphoma. Lymphoma in the epiphysis for children is rare and often confused with infectious etiologies or other types of tumors. Misdiagnosis may result in inappropriate treatment and possible progression of the disease, thus making early identification important to initiate therapy.

Mature B-cell lymphomas in adolescents and young adults.

Pediatric non-Hodgkin lymphoma includes over 30 histologies (many with subtypes), with approximately 800 cases per year in the US, compared to >60,000 cases of adult NHL annually. Improvements in survival in pediatric and adolescent mature B cell NHL over the past 5 decades align with the overall success of the cooperative trial model with dramatic improvements in outcomes through dose escalation of chemotherapy and, more recently, targeted therapy with rituximab. Pediatric dose-intense strategies carry risks of long-term consequences, but treatment failure is nearly universally fatal. By comparison, adult mature B cell lymphoma is typically less aggressive and treated with less intense chemotherapy. Optimizing therapy for adolescents and young adults remains a major challenge that requires creative solutions, including engineering study groups to combine biologically comparable adult and pediatric populations and developing effective salvage strategies that will ultimately be required for investigations of front-line dose reduction. In this review, we discuss challenges and opportunities for improving outcomes for adolescents and young adults with high-grade mature B cell lymphomas, diffuse large B cell lymphoma, and primary mediastinal B cell lymphoma.

Children's Oncology Group's 2023 blueprint for research: Non-Hodgkin lymphoma.

Pediatric non-Hodgkin lymphoma (NHL) includes over 30 histologies (many with subtypes), with approximately 800 cases per year in the United States. Improvements in survival in NHL over the past 5 decades align with the overall success of the cooperative trial model with dramatic improvements in outcomes. As an example, survival for advanced Burkitt lymphoma is now >95%. Major remaining challenges include survival for relapsed and refractory disease and long-term morbidity in NHL survivors. Langerhans cell histiocytosis (LCH) was added to the NHL Committee portfolio in recognition of LCH as a neoplastic disorder and the tremendous unmet need for improved outcomes. The goal of the Children' Oncology Group NHL Committee is to identify optimal cures for every child and young adult with NHL (and LCH). Further advances will require creative solutions, including engineering study groups to combine rare populations, biology-based eligibility, alternative endpoints, facilitating international collaborations, and coordinated correlative biology.

Children's Oncology Group's 2023 blueprint for research: Hodgkin lymphoma.

The goal of therapy in pediatric Hodgkin lymphoma (HL) is to maximize overall survival while minimizing the morbidity of curative therapy. Key findings from recent Children's Oncology Group (COG) trials include: (i) superior event-free survival with the addition of brentuximab vedotin (Bv) in frontline regimens for high-risk disease, (ii) successful reduction in myeloablative regimens with demonstrated safety and efficacy of Bv and checkpoint inhibitor therapy in relapsed disease, and (ii) the potential to select a population that can be salvaged after relapse without receiving a stem cell transplant. The COG HL committee will lead a National cancer Institute National Clinical Trials Network phase 3 trial to evaluate the combination of Bv/nivolumab in early-stage disease. Ongoing advances in technology and blood biomarkers are increasing the ability to deliver biologically driven, personalized treatment for HL.

Does congenital cytomegalovirus infection contribute to the development of acute lymphoblastic leukemia in children?

Cytomegalovirus (CMV) is a ubiquitous herpesvirus that has a profound impact on the host immune system. Congenital cytomegalovirus (cCMV) infection modulates neonatal immune cell compartments, yet the full impact of in utero exposure on developing fetal immune cells remains poorly characterized. A series of recent studies have identified a potential link between cCMV infection and the development of acute lymphoblastic leukemia (ALL) in childhood. Here, we review the emerging evidence linking CMV and ALL risk, discuss what is known about the causes of childhood ALL, and propose how CMV infection in early life may confer increased ALL risk.

Diagnosis and management of Hodgkin lymphoma in children, adolescents, and young adults.

Advances in the management of Hodgkin lymphoma in children, adolescents and young adult have resulted in survival outcomes exceeding 90%. The risk of late toxicity, however, remains a significant concern for survivors of HL and the focus of modern trials have been to advance cure rates while reducing long term toxicity. This has been accomplished through response-adapted treatment approaches and the incorporation of novel agents, many of which target the unique interaction between the Hodgkin and Reed Sternberg cells and the tumor microenvironment. In addition, an improved understanding of prognostic markers, risk stratification, and the biology of this entity in children and AYAs may allow us to further tailor therapy. This review focuses on the current management of HL in the upfront and relapsed settings, recent advances in novel agents that target HL and the tumor microenvironment, and promising prognostic markers that may help guide the future management of HL.

Primary Mediastinal B-Cell Lymphoma in Children and Young Adults.

Primary mediastinal B-cell lymphoma (PMBCL) is a rare but aggressive mature B-cell lymphoma that arises from thymic B cells and most commonly affects adolescents and young adults. PMBCL is now recognized by the WHO as a distinct entity from diffuse large B-cell lymphoma (DLBCL), not otherwise specified, with a unique clinical presentation and distinct morphologic features and molecular alterations. Similar to classic Hodgkin lymphoma, PMBCL tumors are characterized by alterations in the nuclear factor-κB and JAK/STAT pathways. These tumors also exhibit an immune evasion phenotype marked by upregulation of PD-L1 and loss of B2M. Historic data indicates that outcomes for pediatric patients with PMBCL are inferior compared with pediatric patients with DLBCL treated on the same protocols, and there is no current standard approach to initial treatment. Common regimens used for children with PMBCL include multiagent chemotherapy regimens designed for Burkitt lymphoma, such as Lymphomes Malins B (LMB)-based or Berlin-Frankfurt-Münster (BFM)-based chemotherapy ± rituximab. Based on initial data in adults showing excellent outcomes with the use of DA-EPOCH-R regimens, these regimens have also been adopted in pediatrics, although with mixed results. Novel agents are currently being studied in PMBCL with the goal of improving outcomes and reducing reliance on radiation and/or high-dose chemotherapy. Immune checkpoint blockade with PD-1 inhibition is of particular interest given the upregulation of PD-L1 in PMBCL and the known efficacy of these agents in the relapsed setting. Future efforts in PMBCL will also seek to determine the role of FDG-PET in evaluating response to therapy and the role of biomarkers in risk stratification.

Brentuximab vedotin after autologous transplantation in pediatric patients with relapsed/refractory Hodgkin lymphoma.

Outcomes for children and adolescents with relapsed and refractory Hodgkin lymphoma (HL) are poor, with ∼50% of patients experiencing a subsequent relapse. The anti-CD30 antibody-drug conjugate brentuximab vedotin improved progression-free survival (PFS) when used as consolidation after autologous stem cell transplantation (ASCT) in adults with high-risk relapsed/refractory HL. Data on brentuximab vedotin as consolidative therapy after ASCT in pediatric patients with HL are extremely limited, with data of only 11 patients reported in the literature. We performed a retrospective analysis of 67 pediatric patients who received brentuximab vedotin as consolidation therapy after ASCT for the treatment of relapsed/refractory HL to describe the experience of this regimen in the pediatric population. This is the largest cohort reported to date. We found that brentuximab vedotin was well tolerated with a safety profile similar to that of adult patients. With a median follow-up of 37 months, the 3-year PFS was 85%. These data suggest a potential role for the use of brentuximab vedotin as consolidation therapy after ASCT for children with relapsed/refractory HL.

Molecular Evolution of Classic Hodgkin Lymphoma Revealed Through Whole-Genome Sequencing of Hodgkin and Reed Sternberg Cells.

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells in classic Hodgkin lymphoma (cHL) limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Using this approach, we now report the whole-genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events leading to cHL. We identified alterations in driver genes not previously described in cHL, APOBEC mutational activity, and the presence of complex structural variants including chromothripsis. We found that high ploidy in cHL is often acquired through multiple, independent chromosomal gains events including whole-genome duplication. Evolutionary timing analyses revealed that structural variants enriched for RAG motifs, driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID-driven mutagenesis usually preceded large chromosomal gains. This study provides a temporal reconstruction of cHL pathogenesis. SIGNIFICANCE: Previous studies in cHL were limited to coding sequences and therefore not able to comprehensively decipher the tumor complexity. Here, leveraging cHL whole-genome characterization, we identify driver events and reconstruct the tumor evolution, finding that structural variants, driver mutations, and AID mutagenesis precede chromosomal gains. This article is highlighted in the In This Issue feature, p. 171.

Methionine metabolism controls the B cell EBV epigenome and viral latency.

Epstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B cell compartment, and reactivate. Within memory B cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas, to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic, and CRISPR approaches to identify that an abundant methionine supply and interconnecting methionine and folate cycles maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes and de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens in vivo. These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B cell life cycle and suggest therapeutic approaches.

Therapeutic options for CTLA-4 insufficiency.

BACKGROUND: Heterozygous germline mutations in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) impair the immunomodulatory function of regulatory T cells. Affected individuals are prone to life-threatening autoimmune and lymphoproliferative complications. A number of therapeutic options are currently being used with variable effectiveness. OBJECTIVE: Our aim was to characterize the responsiveness of patients with CTLA-4 insufficiency to specific therapies and provide recommendations for the diagnostic workup and therapy at an organ-specific level. METHODS: Clinical features, laboratory findings, and response to treatment were reviewed retrospectively in an international cohort of 173 carriers of CTLA4 mutation. Patients were followed between 2014 and 2020 for a total of 2624 months from diagnosis. Clinical manifestations were grouped on the basis of organ-specific involvement. Medication use and response were recorded and evaluated. RESULTS: Among the 173 CTLA4 mutation carriers, 123 (71%) had been treated for immune complications. Abatacept, rituximab, sirolimus, and corticosteroids ameliorated disease severity, especially in cases of cytopenias and lymphocytic organ infiltration of the gut, lungs, and central nervous system. Immunoglobulin replacement was effective in prevention of infection. Only 4 of 16 patients (25%) with cytopenia who underwent splenectomy had a sustained clinical response. Cure was achieved with stem cell transplantation in 13 of 18 patients (72%). As a result of the aforementioned methods, organ-specific treatment pathways were developed. CONCLUSION: Systemic immunosuppressants and abatacept may provide partial control but require ongoing administration. Allogeneic hematopoietic stem cell transplantation offers a possible cure for patients with CTLA-4 insufficiency.