Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Spontaneous tumor lysis syndrome (STLS) during biopsy for burkitt lymphoma: a case report.

BACKGROUND: Tumor lysis syndrome (TLS) is a hematologic oncological emergency characterized by metabolic and electrolyte imbalances. On breakdown of tumor cells, enormous amounts of potassium, phosphate, and nucleic acids are released into systemic circulation. TLS mainly occurs during chemotherapy. However, there are rare incidences of spontaneous tumor lysis syndrome (STLS) prior to commencement of therapy. CASE PRESENTATION: In the case being reported, the child had just undergone a biopsy. As the incision was being closed, there was a sudden onset of high fever, arrhythmia, severe hyperkalemia, hypocalcemia, and acidosis. Following timely symptomatic treatment and continuous renal replacement therapy(CRRT), the child's laboratory results improved, and organ function was restored to normal. The final pathological diagnosis confirmed Burkitt lymphoma. The boy is currently on maintenance chemotherapy. CONCLUSIONS: TLS is a potentially life-threatening complication in hematologic oncology. Several important conclusions can be drawn from this case, reminding clinicians to: (1) be fully aware of the risk factors of TLS and evaluate the level of risk; (2) pay attention to the possibility of STLS during operation, if surgical procedures are necessary and operate with minimal trauma and in the shortest time possibly; (3) take preoperative prophylaxis actively for high-risk TLS patients, including aggressive fluid management and rational use of diuretics and uric-acid-lowering drugs. In addition, this case confirms the effectiveness of CRRT for severe STLS.

The Treatment of Burkitt Lymphoma With the Berlin-Frankfurt-Münster Protocol With Rituximab and Consolidative Autologous Transplantation.

INTRODUCTION: Intensive treatment approaches are required for adult patients with Burkitt lymphoma (BL), although an univocal standard of care still does not exist. The use of frontline autologous stem cells transplantation (ASCT) is debated. PATIENTS AND METHODS: Between 2004 and 2020, 50 patients with BL were treated with the Berlin-Frankfurt-Münster (BFM). Treatment plan consisted of 3 blocks, A (ifosfamide, vincristine, methotrexate, etoposide, and cytarabine), B (vincristine, cyclophosphamide, methotrexate, and doxorubicin), and C (vindesine, methotrexate, etoposide, and cytarabine), each repeated twice, every 28 days. Rituximab was given at day 1 each block. Intrathecal prophylaxis was given once per each block. ASCT was scheduled at the end of the 6 blocks after conditioning. RESULTS: Median age at onset was 38 years (range 16-72); stages III-IV disease was observed in 82% of cases; bulky disease occurred in 44% of the patients, with B-symptoms in 38%. Stem cell harvest was performed in 72% of patients, who all received a subsequent ASCT. The full 6 blocks treatment was completed in 70% of the patients. The overall response rate was 74%, with a complete response rate of 60%. Ten-year overall survival and progression-free survival were 83.7% and 76.0%, respectively, without reaching the median. Ten-year disease-free survival was 80.3%. Grades 3-4 neutropenia, thrombocytopenia, anemia, and mucositis were seen in 96%, 60%, 32%, and 24% of patients. Infections occurred in 60% of patients. CONCLUSION: Intensive treatment according to BFM protocol, with rituximab and ASCT, appears feasible, safe, and highly effective in adult patients with BL, as confirmed by long-term survival rates reflecting response maintenance.

Bone Marrow Aplasia after CAR-T-Cell Therapy for Relapsed/Refractory Burkitt's Lymphoma.

Chimeric antigen receptor T-cells (CAR-T) are now a standard approach for treating relapsed/refractory B-cell lymphomas. Immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) is a newly described entity that can manifest following CAR-T. Bone marrow (BM) aplasia is an uncommon manifestation of IEC-HS reported after CAR-T-cell therapy and is defined as the reduction or absence of hematopoietic progenitor cells resulting in severe pancytopenia. We describe the case of a 44-year-old female with relapsed/refractory Burkitt lymphoma (BL) who received treatment with lisocabtagene maraleucel with her post-CAR-T course complicated by cytokine release syndrome (CRS) and IEC-HS ultimately leading to persistent BM aplasia. She underwent a rescue allogeneic stem cell transplant but ultimately succumbed to progressive disease. IEC-HS is an increasingly recognized complication that occurs after CAR-T treatments that can result in aplasia, a dangerous complication with serious sequelae including infection, transfusion dependence, and high risk for hemorrhage. The underlying mechanism is poorly understood, and further studies are needed to understand how to treat it better.

Case Report: Unedited allogeneic chimeric antigen receptor T cell bridging to conditioning-free hematopoietic stem cell transplantation for a child with refractory Burkitt lymphoma.

Purpose: Burkitt lymphoma (BL) is the most common tumor of non-Hodgkin's lymphoma (NHL) in children, accounting for about 40% of cases. Although different combined short-course chemotherapies have achieved a good effect, refractory/relapsed BL has a poor prognosis with cure rates less than 30%. Chimeric antigen receptor T cell (CAR-T) therapy has developed rapidly in recent years and achieved excellent results in acute lymphoblastic leukemia (ALL). However, in some cases, there is a failure to produce autologous CAR-T cells because of T-cell dysfunction. In such cases, allogeneic CAR-T therapy has to be considered. Methods: A 17-year-old boy with stage II BL did not respond to extensive chemotherapy and sequential autologous CAR-T therapy. Lentiviral vectors containing anti-CD20-BB-ζ (20CAR) and anti-CD22-BB-ζ (22CAR) transgenes were used to modify the T cells from an HLA-identical matched unrelated donor. Flow cytometry was used to assess the cytokine analyses and CAR-T cell persistence in peripheral blood, enumerated by qPCR as copies per ug DNA. Informed consent for autologous/allogeneic CAR-T therapy was obtained from the patient and his legal guardian. Results: Unedited HLA-matched allogeneic CD20 and CD22 CAR-T cells were infused after lymphodepletion chemotherapy with cyclophosphamide and fludarabine. The patient experienced Grade IV cytokine release syndrome (CRS) and went into complete remission (CR) after anti-inflammatory treatment including tocilizumab. Because of persistent pancytopenia and full donor chimerism, the same donor's conditioning-free peripheral blood stem cells were successfully transplanted 55 days post CAR-T. Neutrophils were engrafted at day +11 and platelets were rebuilt at day +47 without obvious acute graft-versus-host disease (GVHD), but there was mild chronic GVHD in the skin and eyes. Currently, active anti-rejection therapy is still underway. Conclusion: Unedited HLA-matched allogeneic CAR-T cell therapy could be an innovative, effective, and safe treatment for children with refractory/relapse BL without obvious acute GVHD. Conditioning-free allogeneic hematopoietic stem cell transplantation (HSCT) from the same donor is feasible for a patient with full donor T-cell chimerism after allogeneic CAR-T. It cannot be ignored that close GVHD monitoring is needed post HSCT.

Childhood Burkitt Lymphoma in Nigeria.

Burkitt lymphoma (BL) is a curable, aggressive pediatric cancer, yet in low- and middle-income countries like Nigeria, children with BL face poor survival outcomes. Identifying barriers to and facilitators of cancer care for.

Clinical Outcomes and Health Care Utilization in Patients With Burkitt Lymphoma.

PURPOSE: Burkitt lymphoma is an aggressive B-cell lymphoma requiring intensive therapy, which places patients at risk for severe toxicity. However, few studies have described these patients' clinical outcomes and health care utilization, particularly among older adults. METHODS: We conducted a retrospective analysis of adults 40 years and older with Burkitt lymphoma at Massachusetts General Hospital and Dana-Farber Cancer Institute from February 1999 to December 2020 (N = 97). We abstracted patient characteristics, clinical outcomes, and health care utilization (unplanned hospitalizations, intensive care unit [ICU] admissions) during therapy from the electronic health record. Using univariate logistic regression, we examined factors associated with rates of unplanned hospitalization and ICU admission during therapy. RESULTS: Among evaluable patients (median age, 69 years; 23.7% female; 19.3% with bone marrow involvement), 45.8% (38 of 83) experienced unplanned hospitalization and 23.2% (19 of 82) experienced ICU admission during therapy. Among those 70 years and older, rates of unplanned hospitalization and ICU admission were 36.8% (14 of 38) and 29.0% (11 of 38), respectively. Bone marrow involvement (odds ratio [OR], 3.00; P = .069) was associated with a nonsignificantly greater likelihood of unplanned hospitalization. Older age (OR, 1.06; P = .039), Charlson comorbidity index >0 (OR, 3.14; P = .038), and hypoalbuminemia (OR, 3.22; P = .035) were associated with greater likelihood of ICU admission. Overall, 8.7% (8 of 92) of patients died during treatment, all of whom were 70 years and older. CONCLUSION: Adults with Burkitt lymphoma experience substantial rates of unplanned hospitalizations and ICU admissions, with older adults at especially high risk for ICU admission and death during treatment. Our findings underscore the need to develop supportive care interventions for patients with Burkitt lymphoma to help improve clinical outcomes and health care utilization.

Epstein‒Barr virus-associated cellular immunotherapy.

Epstein‒Barr virus (EBV) is a human herpes virus that is saliva-transmissible and universally asymptomatic. It has been confirmed that more than 90% of the population is latently infected with EBV for life. EBV can cause a variety of related cancers, such as nasopharyngeal carcinoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Currently, many clinical studies have demonstrated that EBV-specific cytotoxic T lymphocytes and other cell therapies can be safely and effectively transfused to prevent and treat some diseases caused by EBV. This review will mainly focus on discussing EBV-specific cytotoxic T lymphocytes and will touch on therapeutic EBV vaccines and chimeric antigen receptor T-cell therapy briefly.

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.

Introduction: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. Methods: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Results: Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. Discussion: The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

Case report: Sandwich therapy of CAR-T combined with ASCT: Sequential CAR-T cell therapy with ASCT after remission with CAR-T therapy caused long-term survival in a patient with relapsed/refractory Burkitt's lymphoma with TP53 mutations.

Burkitt's lymphoma (BL) with TP53 mutation often has poor outcome after standard chemoimmunotherapy. Adoptive chimeric antigen receptor (CAR)-T cell therapy may be a new paradigm for treating refractory/relapsed (r/r) BL, but its therapeutic effects remain inconclusive. Here, we report a patient with r/r BL who failed to achieve complete remission (CR) and progressed rapidly after multiple protocol chemotherapy. The patient achieved CR with CAR19 and CAR22 T-cell cocktail therapy and obtained long-term disease-free survival after autologous hematopoietic stem cells (ASCT) and subsequential CAR19 and CAR22 T-cell cocktail therapy. The clinical evolution and genetic features of this case may provide some guidance for CAR-T therapy in overcoming relapses associated with TP53 gene mutations.

Molecular characterization and clinical outcome of B-cell precursor acute lymphoblastic leukemia with IG-MYC rearrangement.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Burkitt lymphoma after solid-organ transplant: Treatment and outcomes in the paediatric PTLD collaborative.

Burkitt lymphoma arising in paediatric post-solid-organ transplantation-Burkitt lymphoma (PSOT-BL) is a clinically aggressive malignancy and a rare form of post-transplant lymphoproliferative disorder (PTLD). We evaluated 35 patients diagnosed with PSOT-BL at 14 paediatric medical centres in the United States. Median age at organ transplantation was 2.0 years (range: 0.1-14) and age at PSOT-BL diagnosis was 8.0 years (range: 1-17). All but one patient had late onset of PSOT-BL (≥2 years post-transplant), with a median interval from transplant to PSOT-BL diagnosis of 4.0 years (range: 0.4-12). Heart (n = 18 [51.4%]) and liver (n = 13 [37.1%]) were the most frequently transplanted organs. No patients had loss of graft or treatment-related mortality. A variety of treatment regimens were used, led by intensive Burkitt lymphoma-specific French-American-British/Lymphomes Malins B (FAB/LMB), n = 13 (37.1%), and a low-intensity regimen consisting of cyclophosphamide, prednisone and rituximab (CPR) n = 12 (34.3%). Median follow-up was 6.7 years (range: 0.5-17). Three-year event-free and overall survival were 66.2% and 88.0%, respectively. Outcomes of PSOT-BL patients receiving BL-specific intensive regimens are comparable to reported BL outcomes in immunocompetent children. Multi-institutional collaboration is feasible and provides the basis of prospective data collection to determine the optimal treatment regimen for PSOT-BL.

Successful pseudo-autologous stem cell transplantation for donor-derived Burkitt lymphoma occurring 9 years after allogeneic transplantation.

Donor-derived hematological malignancies have been recognized as rare but serious late complications in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Most cases in the literature were diagnosed as myelodysplastic syndrome or acute leukemia, with very few malignant lymphoma reported. We herein present another case of donor-derived Burkitt lymphoma that occurred 9 years after allo-HSCT under continued administration of immunosuppressants for chronic graft-versus-host disease (GVHD). The patient achieved a partial response after rituximab-combined intensive chemotherapy. To reduce the risk of relapse and to avoid organ toxicities due to repeated chemotherapies, we performed upfront high-dose chemotherapy followed by stem cell rescue using donor-derived CD34+ cells, called pseudo-autologous HSCT (pASCT), and adjusted immunosuppressants appropriately. The patient remained disease-free for 23 months after pASCT without exacerbation of cGVHD. Although the observation period has been relatively short and longer follow-up is needed, pASCT may be a feasible option for donor-derived lymphoma even in patients with active cGVHD.

Inflammatory Non-CpG Antisense Oligonucleotides Are Signaling Through TLR9 in Human Burkitt Lymphoma B Bjab Cells.

Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability. This compelled us to develop a more convenient high-throughput assay. In this study, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPBMC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs through comparison to known benchmark PS-ASO controls that were previously shown to be safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO TLR9-dependent innate immune activation.

[Epidemiological and clinical profile childhood cancers with pleural and pulmonary lesions in the pediatric oncology unit of Treichville University Hospital (Abidjan, Côte d'Ivoire)]. / Profil épidémiologique et clinique des cancers pédiatriques avec atteintes pleurales et pulmonaires au service de pédiatrie du CHU de Treichville (Abidjan, Côte d'Ivoire).

INTRODUCTION: Pediatric cancers are a major public health problem in sub-Saharan Africa. However, they are seldom studied, especially as regards in their extensive forms. METHODOLOGY: An eight-year retrospective and descriptive study was carried out so as to specify the epidemiological and clinical characteristics of cancers with pleural and pulmonary involvement in children of 0 to 14years of age in the pediatric oncology unit at the University Hospital of Treichville, Côte d'Ivoire (Ivory Coast). RESULTS: The frequency of pleural and pulmonary involvement in pediatric cancers was 13.8%. Children's average age was 7.2years, with sex ratio at 2.11. Solid tumors were predominant, with a predominance of Burkitt's lymphoma (39.3%) and nephroblastoma (35.7%). The most affected age groups were 10 to 15years (Burkitt's lymphoma) and 0 to 5years (nephroblastoma). Time to diagnosis ranged from 31 and 60days in 40.4% of cases, and time to treatment was at most 30 days, for the overwhelming majority (97.1%) of the children. Chemotherapy was initiated in 67.9% of patients. Hospital mortality was 73.2%. CONCLUSION: Through this study, the authors established the profile of childhood cancers with pleural and pulmonary involvement. Comparative studies of mortality in pediatric cancers with and without pleural and pulmonary involvement could further underline the importance of early management before dissemination.

EFFICACY OF AUTOLOGOUS STEM CELL TRANSPLANTATION IN ADULT BURKITT/BURKITT-LIKE LYMPHOMA: A SYSTEMATIC REVIEW.

BACKGROUND: Burkitt and Burkitt like lymphoma (BL/BLL) are highly proliferative germinal or post-germinal B cell tumors. Few studies have evaluated the impact of autologous stem cell transplantation (ASCT) on disease outcomes. AIM: We performed a systematic review to analyze the efficacy of ASCT as frontline consolidation and for treatment of relapsed/refractory cases in adult BL/BLL. MATERIALS AND METHODS: Eligible studies with clear outcome measures on the efficacy of ASCT in adult patients with BL/BLL were identified through systematic search. The overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and progression/relapse were used to assess the efficacy. RESULTS: For patients who underwent ASCT in first CR, 5-year PFS and OS ranged between 70-78% and 70-83% respectively. For relapsed/refractory disease, 5-year PFS and OS were 27% and 31%, respectively. Patients undergoing ASCT for chemoresistant disease fared poorly with 3-year OS of 7% vs 37% for chemosensitive disease (p ≤ 0.00001). The overall response rate to ASCT for patients transplanted in first CR ranged between 71% and 93% and was 37% for patients who were transplanted in disease status other than first CR. Disease progression/relapse was observed in 16-29% of the patients transplanted in first CR, and 55% to 60% in relapsed disease. CONCLUSION: We found insufficient evidence to support ASCT over chemotherapy alone in the first remission for adult BL/BLL. Evidence supports guidelines recommending ASCT for chemosensitive disease but suggests there is no benefit to ASCT for chemoresistant disease.

Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells.

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins  that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.