MYC Controls the Epstein-Barr Virus Lytic Switch.

Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.

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.

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.

Subsequent malignant neoplasms among children with Hodgkin lymphoma: a report from the Children's Oncology Group.

Survivors of Hodgkin lymphoma (HL) have an increased risk of subsequent malignant neoplasms (SMNs). Response-adapted treatment may decrease this risk by reducing exposure to therapy associated with SMN risk. The Children's Oncology Group study AHOD0031 evaluated response-adapted therapy for children and adolescents with intermediate-risk HL. We report the SMNs among 1711 patients enrolled in AHOD0031. Patients were treated with 4 cycles of doxorubicin, bleomycin, vincristine, etoposide, prednisone, and cyclophosphamide with or without involved-field radiation therapy (RT). Patients with a slow early response to initial chemotherapy were randomized to 2 additional cycles of dexamethasone, etoposide, cisplatin and cytarabine or no additional chemotherapy, and all received RT. At a median follow-up of 7.3 years, an analysis of SMNs was performed. The 10-year cumulative incidence of SMN was 1.3% (95% confidence interval [CI], 0.6-2.0). SMNs included 3 patients with acute myeloid leukemia (AML), 11 with solid tumors, and 3 with non-Hodgkin lymphoma. Sixteen of 17 patients with an SMN had received combined modality therapy. The standardized incidence ratio for SMN was 9.5 (95% CI, 4.5-15.2) with an excess absolute risk of 1.2 per 1000 person-years. The cumulative incidence of SMNs was higher among patients who received RT (P = .037). In multivariate analysis, RT, B symptoms, and race were associated with SMN risk. Given the latency from exposure, we have likely captured all cases of secondary leukemia and myelodysplastic syndrome (MDS). Longer follow-up is needed to determine the risk of solid tumors. Avoidance of RT without sacrificing disease control should remain a goal for future therapeutic approaches. This trial was registered at www.clinicaltrials.gov as #NCT00025259.

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.

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.

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.

Epigenetic reprogramming sensitizes immunologically silent EBV+ lymphomas to virus-directed immunotherapy.

Despite advances in T-cell immunotherapy against Epstein-Barr virus (EBV)-infected lymphomas that express the full EBV latency III program, a critical barrier has been that most EBV+ lymphomas express the latency I program, in which the single Epstein-Barr nuclear antigen (EBNA1) is produced. EBNA1 is poorly immunogenic, enabling tumors to evade immune responses. Using a high-throughput screen, we identified decitabine as a potent inducer of immunogenic EBV antigens, including LMP1, EBNA2, and EBNA3C. Induction occurs at low doses and persists after removal of decitabine. Decitabine treatment of latency I EBV+ Burkitt lymphoma (BL) sensitized cells to lysis by EBV-specific cytotoxic T cells (EBV-CTLs). In latency I BL xenografts, decitabine followed by EBV-CTLs results in T-cell homing to tumors and inhibition of tumor growth. Collectively, these results identify key epigenetic factors required for latency restriction and highlight a novel therapeutic approach to sensitize EBV+ lymphomas to immunotherapy.

How I treat primary mediastinal B-cell lymphoma.

The World Health Organization now recognizes primary mediastinal B-cell lymphoma (PMBCL) as a unique clinical and biologic entity. PMBCL is distinct from other B-cell non-Hodgkin lymphoma subtypes and has features that overlap with classical Hodgkin lymphoma, including a peak incidence in the adolescent and young adult population, mediastinal presentation of disease, and molecular alterations in JAK2 and programmed death ligands. Because PMBCL is rare, there are few prospective clinical trials to guide therapy, resulting in no single standard of care. Given the long life expectancy of survivors of PMBCL, treatment approaches must balance maximizing cure while minimizing long-term toxicity. In this article, I review my approach to the treatment of PMBCL, incorporating data from adult and pediatric studies, as well as recent advances in our understanding of the molecular basis of PMBCL.

CXCL12 expression is an adverse predictor for disease recurrence in patients with metastatic non-seminomatous testicular germ cell tumors.

BACKGROUND: To validate the utility of the chemokine ligand 12 (CXCL12) as prognostic marker in patients with localized and metastatic germ cell tumors (GCT). METHODS: CXCL12 expression was analyzed on a tissue microarray consisting of 750 tissue cores of different histological tumor components, Germ cell neoplasia in situ (GCNIS) and adjacent normal tissue of 263 testicular cancer patients using a semi-quantitative score. The association between CXCL12 expression and recurrence-free survival (RFS) as well as overall survival (OS) was assessed using Kaplan-Meier curves with log-rank tests. RESULTS: CXCL12 expression was absent in all seminomas but was found in 52 of 99 (52.5%) non-seminomas. Follow-up was available for 260 patients of which 36 (13.8%) recurred. In patients with stage 1 non-seminoma GCT, CXCL12 expression was not associated with higher risk of disease recurrence (p = 0.270). In contrast, post chemotherapy RFS of patients with metastatic non-seminoma and positive CXCL12 expression was significantly shorter compared to CXCL12 negative patients (p = 0.003). OS differences were not statistically different between patients with CXCL12 positive or negative tumors for either localized or metastatic disease. CONCLUSIONS: CXCL12 is almost exclusively expressed in non-seminoma. Pure seminoma, GCNIS and adjacent normal testicular tissue are CXCL12 negative. Our analysis suggests that patients with metastatic disease and a CXCL12-positive non-seminoma are at higher risk for disease recurrence after first-line chemotherapy and might thus be candidates for more intensive treatment and/or closer follow-up.

Systemic inflammatory markers have independent prognostic value in patients with metastatic testicular germ cell tumours undergoing first-line chemotherapy.

BACKGROUND: The prognostic utility of systemic inflammatory markers has so far not been investigated in patients with metastatic testicular germ cell tumours (GCTs). METHODS: International Germ Cell Cancer Cooperative Group (IGCCCG) risk groups and blood-based systemic inflammatory markers (haemoglobin, leukocytes, platelets (P), neutrophils (N), lymphocytes (L), C-reactive protein (CRP) and albumin) of 146 patients undergoing first-line chemotherapy for GCT were retrieved. In addition, N to L ratio (NLR), P to L ratio and the systemic immune-inflammation index (SII=N × P/L) were calculated. The prognostic ability of these markers for overall survival (OS) were assessed using regression analyses and Kaplan-Meier curves with log-rank tests. RESULTS: In univariate Cox regression, low haemoglobin and albumin as well as high leukocytes, N, NLR, SII and CRP were associated with a shorter OS. In multivariable Cox regression analyses, high leukocyte (hazard ratio (HR) 1.274 (95% confidence interval (CI) 1.057-1.535); P=0.011) and N count (1.470 (1.092-1.980); P=0.011), higher NLR (84.5 (2.2-3193.4); P=0.017) and SII (12.15 (1.17-126.26); P=0.037) remained independent prognostic predictors for OS besides the IGCCCG risk groups. CONCLUSIONS: Systemic inflammatory markers might have prognostic utility for patients with metastatic GCT. The planned IGCCCG update could be an opportunity to test these markers in a larger data set.

Outcomes of adults and children with primary mediastinal B-cell lymphoma treated with dose-adjusted EPOCH-R.

Treatment with dose-adjusted EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone) chemotherapy and rituximab (DA-EPOCH-R) has become the standard of care for primary mediastinal B-cell lymphoma (PMBCL) at many institutions despite limited data in the multi-centre setting. We report a large, multi-centre retrospective analysis of children and adults with PMBCL treated with DA-EPOCH-R to characterize outcomes and evaluate prognostic factors. We assessed 156 patients with PMBCL treated with DA-EPOCH-R across 24 academic centres, including 38 children and 118 adults. All patients received at least one cycle of DA-EPOCH-R. Radiation therapy was administered in 14·9% of patients. With median follow-up of 22·6 months, the estimated 3-year event-free survival (EFS) was 85·9% [95% confidence interval (CI) 80·3-91·5] and overall survival was 95·4% (95% CI 91·8-99·0). Outcomes were not statistically different between paediatric and adult patients. Thrombotic complications were reported in 28·2% of patients and were more common in paediatric patients (45·9% vs. 22·9%, P = 0·011). Seventy-five per cent of patients had a negative fluorodeoxyglucose positron emission tomography (FDG-PET) scan at the completion of DA-EPOCH-R, defined as Deauville score 1-3. Negative FDG-PET at end-of-therapy was associated with improved EFS (95·4% vs. 54·9%, P < 0·001). Our data support the use of DA-EPOCH-R for the treatment of PMBCL in children and adults. Patients with a positive end-of-therapy FDG-PET scan have an inferior outcome.

Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells.

Classical Hodgkin lymphoma (cHL) is characterized by sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells amid reactive host background, complicating the acquisition of neoplastic DNA without extensive background contamination. We overcame this limitation by using flow-sorted HRS and intratumor T cells and optimized low-input exome sequencing of 10 patient samples to reveal alterations in genes involved in antigen presentation, chromosome integrity, transcriptional regulation, and ubiquitination. ß-2-microglobulin (B2M) is the most commonly altered gene in HRS cells, with 7 of 10 cases having inactivating mutations that lead to loss of major histocompatibility complex class I (MHC-I) expression. Enforced wild-type B2M expression in a cHL cell line restored MHC-I expression. In an extended cohort of 145 patients, the absence of B2M protein in the HRS cells was associated with lower stage of disease, younger age at diagnosis, and better overall and progression-free survival. B2M-deficient cases encompassed most of the nodular sclerosis subtype cases and only a minority of mixed cellularity cases, suggesting that B2M deficiency determines the tumor microenvironment and may define a major subset of cHL that has more uniform clinical and morphologic features. In addition, we report previously unknown genetic alterations that may render selected patients sensitive to specific targeted therapies.

Verification and feedback for medical students: an observational study during general practice rotations.

PURPOSE OF THE STUDY: A general practice and family medicine rotation is mandatory as part of undergraduate medical education. However, little is known about the student-teacher interaction in this specific setting of ambulatory teaching. In this study we analysed how frequently preceptors verified students' history taking and clinical examination skills and how often they gave feedback. The type of feedback given was also categorised. METHODS: From April to December 2012, 410 individual patient consultations were observed in 12 teaching practices associated with the Philipps University Marburg, Germany. Material was collected using structured field-note forms and videotaping. Descriptive data analysis was performed. Informed, written consent was provided by all participants. RESULTS: We analysed 410 consultations which lasted 14.8 min on average. In 130 (31.7%) consultations students took the patient's medical history; 124 (95.4%) of these were verified by the general practitioner (GP). Physical examination was performed by students in 202 (49.3%) of consultations; 169 (81.9%) of these were verified by the GP. Feedback occurred in 132 (32.2%) of the 410 patient consultations. Feedback was mostly non-specific and positive (68.9%), and occurred during consultation with the patient present. Specific, negative feedback also occurred relatively frequently (29.5%). Specific, positive and non-specific, negative responses were rarely given. CONCLUSIONS: GPs should give feedback more frequently and when doing so, focus on specific feedback. GPs should be further instructed in different feedback techniques.

Recent molecular and therapeutic advances in B-cell non-Hodgkin lymphoma in children.

Paediatric B-cell non-Hodgkin lymphoma (B-NHL) compromises a heterogeneous group of histological entities of which Burkitt lymphoma is the most common. In resource-rich countries, the expected cure rate is in excess of 85% with application of risk-adapted short intensive chemotherapy. In recent years, large paediatric cooperative group trials have sought to improve upon outcomes by decreasing the intensity of cytotoxic treatment as well as introducing targeted therapies, such as rituximab. These efforts have resulted in excellent outcomes, however there remains a group of high-risk patients for whom novel treatment approaches are needed. In this review, we will summarize the recent paediatric clinical trials in B-NHL as well as compare treatment approaches across the major cooperative groups. We will also highlight our current understanding of the molecular biology of paediatric B-NHL with a focus on how this may help guide future rational targeted therapy.

Teaching points-do they occur and what do they contain? An observation study concerning the general practice rotation.

BACKGROUND: A general practice rotation is mandatory in most undergraduate medical education programs. However, little is known about the student-teacher interaction which takes place in this setting. In this study we analyzed occurrence and content of teaching points. METHODS: From April to December 2012, 410 individual patient consultations were observed in twelve teaching practices associated with the Philipps University Marburg, Germany. Material was collected using structured field-note forms and videotaping. Data analysis was descriptive in form. A teaching point is defined here as a general rule or specific, case-related information divulged by the teaching practitioner. RESULTS: According to the analysis of 410 consultations, teaching points were made in 66.3% of consultations. During these consultations, 74.3% general- and 46.3% case related teaching points occurred; multiple categorizations were possible. Of seven possible topics, therapy was most common, followed, in frequency of occurrence, by patient history, diagnostic procedure, physical examination, disease pathology, differential diagnosis, risk factors and case presentation. CONCLUSIONS: The majority of consultations conducted within student presence contained teaching points, most frequently concerning therapy. General teaching points were more common than specific teaching points. Whilst it is encouraging that most consultations included teaching points, faculty development aimed at raising awareness for teaching and learning techniques is important.