Childhood non-Hodgkin Lymphoma Research Articles

Non-Hodgkin Lymphoma in Children.

The non-Hodgkin lymphomas (NHLs) of childhood include high-grade mature B cell lymphoma [Burkitt lymphoma (BL), diffuse large B cell lymphoma (DLBCL), and primary mediastinal large B cell lymphoma (PMLBCL)], anaplastic large cell lymphoma (ALCL), and lymphoblastic lymphoma (LL). The prognosis for children with NHL is generally excellent, although there are some higher risk groups. In this regard, PMLBCL is generally associated with a poorer outcome than BL or DLBCL of comparable stage. The long-term event-free survival for children with ALCL is approximately 70%. Novel biological agents, including those that target CD-30 or ALK, may hold promise for improving treatment results. Children with LL are treated with regimens derived from those used to treat acute lymphoblastic leukemia (ALL). Recent biological study of LL may provide insights into revising treatment stratification. The challenge in pediatric NHL, a group that already has a relatively good prognosis, is to improve treatment outcome without increasing concerning late effects.

Non-Hodgkin lymphoma in children with an associated inherited condition: A retrospective analysis of the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP).

Inherited conditions affecting genetic aberration, viral oncogenesis, reduced immune surveillance, and long-lasting antigen stimulation may build the way to lymphomagenesis in humans. We extracted from the database of 4 consecutive trials for pediatric non-Hodgkin lymphoma (NHL) all cases with an associated genetic disease. Among 1,430 patients, 34 (2.4%) had an associated inherited condition and a mature B-lineage (n = 28), anaplastic large cell lymphoma (n = 4), or T-lineage (n = 2) NHL. Their median age at the diagnosis was 9.3 years (range, 2.6-17.8 years). In 14 cases (41%) the underlying condition was considered to be a potential cause for undue toxicity if the expected therapy was applied. Thus, treatment modification had been planned in advance. The overall survival was 89% (standard error [SE] 1%), 73% (SE 10%), and 73% (SE 23%) at 3 years for registered patients with no inherited condition associated, with genetic abnormalities and with underlying condition causing an immune deficiency, respectively (P = 0.003). In our cohort, patients with NHL with an underlying constitutional condition represent the 2.4% of the cases. In the subset of patients with primary immune deficiency, which may have contributed to lymphomagenesis, allogeneic hematopoietic stem cell transplantation may be required. In the remaining patients, the association with lymphoma remains apparently unexplained and could be not causative. Detailed reporting of such cases may contribute to disclose even rare and fully unexpected association, which may have implications for research in the field of lymphomagenesis.

Late outcomes among adult survivors of childhood non-Hodgkin lymphoma (NHL): A report from the St. Jude Lifetime Cohort Study.

10064 Background: Adult survivors of childhood NHL are at risk for chronic conditions likely underestimated by patient reported outcomes. Prevalence and severity based on direct clinical assessment are lacking. Methods: Clinical, laboratory, and performance-based evaluations were obtained on 200 adult survivors of pediatric NHL at St. Jude Children’s Research Hospital. Chronic conditions were graded per CTCAE criteria. Impaired physical function was defined as performance below the 10th percentile of normative data. Multivariable Poisson regression models were used to investigate associations [relative risk (RR), 95% confidence intervals (CI)] between patient characteristics, therapies, and clinical outcomes. Results: Survivors (66% male, 87% white) were a median age of 10 years (range 1-19) at diagnosis and 34 years (20-58) at evaluation. Forty-six (23%) received radiation to the brain, 69(35%) high dose methotrexate, and 161(81%) steroids. Most (93%) had ≥ 1 chronic condition, 77% ≥ 2 chronic conditions...

International Pediatric Non-Hodgkin Lymphoma Response Criteria.

Response criteria are well established for adult patients with non-Hodgkin lymphoma (NHL). A revised set of response criteria in adults with NHL was recently published. However, NHL in children and adolescents involves different histologies, primary sites of disease, patterns of metastatic spread, approaches to therapy, and responses to treatment compared with adult NHL. However, there are no standardized response criteria specific to pediatric NHL. Therefore, we developed international standardized methods for assessing response to therapy in children and adolescents with NHL. An international multidisciplinary group of pediatric oncologists, pathologists, biologists, and radiologists convened during and after the Third and Fourth International Childhood, Adolescent and Young Adult NHL Symposia to review existing response and outcome data, develop methods for response evaluation that reflect incorporation of more sensitive technologies currently in use, and incorporate primary and metastatic sites of disease for the evaluation of therapeutic response in children and adolescents with NHL. Using the current adult NHL response criteria as a starting point, international pediatric NHL response criteria were developed incorporating both contemporary diagnostic imaging and pathology techniques, including novel molecular and flow cytometric technologies used for the determination of minimal residual disease. Use of the international pediatric NHL response criteria in children and adolescents receiving therapy for NHL incorporates data obtained from new and more sensitive technologies that are now being widely used for disease evaluation, providing a standardized means for reporting treatment response.

Revised International Pediatric Non-Hodgkin Lymphoma Staging System.

Treatment and prognosis of pediatric non-Hodgkin lymphoma (NHL) have improved dramatically in the last 30 years. However, the St Jude NHL staging classification for pediatric NHL was developed more than 35 years ago. The most recent Lugano lymphoma staging classification focused on adult lymphoma. Furthermore, major limitations of the current pediatric NHL staging classification include lack of consideration of new distinct pediatric NHL histologic entities; absence of recognition of frequent skin, bone, kidney, ovarian, and other organ involvement; and lack of newer precise methods to detect bone marrow and CNS involvement, minimal disease quantification, and highly sensitive imaging technologies. An international multidisciplinary expert panel convened in Frankfurt, Germany, in 2009 at the Third International Childhood, Adolescent and Young Adult NHL Symposium to develop a revised international pediatric NHL staging system (IPNHLSS), addressing limitations of the current pediatric NHL staging system and creating a revised classification. Evidence-based disease distribution and behavior were reviewed from multiple pediatric cooperative group NHL studies. A revised IPNHLSS was developed incorporating new histologic entities, extranodal dissemination, improved diagnostic methods, and advanced imaging technology. This revised IPNHLSS will facilitate more precise staging for children and adolescents with NHL and facilitate comparisons of efficacy across different treatment strategies, various institutions, multicenter trials, and cooperative groups by allowing for reproducible pediatric-based staging at diagnosis and relapse.

Pediatric mature B-cell non Hodgkin lymphoma treatment with LMB-96 protocol. The Children Cancer Hospital Egypt experience

Purpose: Burkitt lymphoma (BL) is a highly aggressive mature B-cell non-Hodgkin lymphoma (NHL) and is the fastest growing human tumor. The outcome of childhood NHL has improved steadily over the past decades through the use of intensive sequential multi-agent chemotherapy regimens. Methods: A retrospective study having all patients 18 years old or younger diagnosed with mature B cell NHL and treated at Children Cancer Hospital Egypt (CCHE). All children were treated according to the modified (LMB 96) protocol during the period between July 2007 and December 2012. Patients were followed up till June 2013. Results: Three hundred and seventy-seven patients were diagnosed with mature B cell NHL and received the LMB96 treatment protocol. The majorities were males (76.4%) with a median age of 5.3 years, and ranged from 0.1-18.0 years. The median follow-up period was 28.2 months (range 0.9-72 months). Burkitt lymphoma was the most predominant pathologic subtype (79.6%, n = 300), and abdominal mass as a primary site was the most common presentation (71.3%). Twenty seven patients (7.2%) were treated as group A, 268 (71.0%) as group B, and 82 (21.8%) patients as high risk group C. Seventy-one (18.8%) patients suffered adverse events. Major adverse events were early deaths in 17 patients (4.5%), death during induction chemotherapy seen in 18 patients (4.7%), and during maintenance therapy in 7 patients (1.8%), tumor progression in 19 patients (5.0%), and relapse in 10 patients (3.7%). Sixty-three patients (16.7%) died during the study period. The main causes of death were tumor lysis syndrome (TLS) in 25.3%, and severe sepsis during chemotherapy in 41.3% of the patients. The 3 years OS and EFS were 83.3% and 80.4% respectively for the whole groups of patients. OS and EFS were 100% for group A, and 87.5%±3.9% and 85.9±4.3% for group B. For group C BM + /CNS - patients, OS was 55.62%±15.8%, and EFS of 53.8%±15.6%. For BM + /CNS + patients, OS and EFS were 63.2%±21.76% and 57.9%±22.1% respectively. BM - /CNS + patients had OS 72.4%±18.8% and EFS 67.6%±19.7% at 36 months. Conclusion: TLS and chemotherapy related toxicity remains a major challenge affecting the outcome of pediatric mature B cell NHL. We identified bone marrow involvement as a risk factor affecting treatment outcome. Aggressive supportive care measures are mandatory to avoid unacceptable high toxicity related mortality.

Non-Hodgkin Lymphoma in Children with Primary Immunodeficiencies: Clinical Manifestations, Diagnosis, and Management, Belarusian Experience

Introduction. Non-Hodgkin lymphoma (NHL) is the most frequent malignancy associated with primary immune deficiency disease (PID). We aimed to present the clinical characteristics and outcomes of Belarusian children with PID who developed NHL. Procedure. We reviewed 16 patients with PID and NHL. Eight patients had combined PID: 5—Nijmegen breakage syndrome, 1—Bloom syndrome, 1—Wiskott-Aldrich syndrome, and 1—Х-linked lymphoproliferative syndrome. Results. In 75% cases PID was diagnosed simultaneously or after the NHL was confirmed. PID-associated NHL accounted for 5.7% of all NHL and was characterized by younger median age (6.3 versus 10.0 years, P<0.05) and by prevalence of large-cell types (68.8% versus 24.5%, P<0.001). Children with combined PID had median age of 1.3 years; 5 of them developed EBV-associated diffuse large B-cell lymphoma with lung involvement. Five of 6 patients with chromosomal breakage syndrome developed T-NHL. Six patients died of infections; two died after tumor progression; one child had early relapse; two died of second NHL and one of secondary hemophagocytic syndrome. Overall, 4 children are alive and disease-free after a follow-up from 1.4 to 5.7 years. Conclusions. PID needs to be diagnosed early. Individualized chemotherapy, comprehensive supportive treatment, and hematopoietic stem cell transplantation may improve survival of children with PID and NHL.

Клиническое значение и методы определения минимальной диссеминированной и минимальной остаточной болезни при неходжкинских лимфомах у детей: обзор литературы

In spite of good treatment results of childhood Non-Hodgkin lymphomas (NHL) and lower toxicity of current chemotherapy regimens relapse rate remains the same and prognosis after disease failure is mostly unfavorable. Minimal disseminated and minimal residual disease analysis may be useful as the strong predictor of lymphoma recurrence. Due to the rarity and heterogenity of NHL large studies need to be performed to determine the priority target, the best method for its detection, positivity cut-off and timepoints for minimal disease assessment in every NHL type.

Maternal and birth anthropometric characteristics in relation to the risk of childhood lymphomas: a Swedish nationwide cohort study.

This Swedish nationwide cohort study aims to examine the role of maternal characteristics (maternal age, education, smoking, BMI, diabetes, and preeclampsia) and multiple intrauterine growth measures on the risk of childhood lymphomas. A total of 3 444 136 singleton live births registered in the Swedish Medical Birth Register were analyzed, among whom there were 515 incident non-Hodgkin lymphoma (NHL) cases and 169 Hodgkin lymphoma (HL) cases aged 0-14 years at diagnosis (1973-2007) identified through linkage with the Swedish Cancer Register. Proportional hazards models were used to estimate the hazard ratio (HR) and 95% confidence intervals (95% CI) of NHL and HL. Male sex (HR=2.00, 95% CI: 1.66-2.41), older maternal age (HR=1.03, 95% CI: 1.00-1.06, per 1-year increase), and large for gestational age compared with appropriate for gestational age (AGA) birth weight (HR=1.83, 95% CI: 1.20-2.79) were correlated with the risk of NHL; of note, in subanalysis by sex, the latter association was confined to girls (HR=3.37, 95% CI: 1.90-5.97, Pinteraction by sex=0.008). The risk of childhood HL overall was more evident among boys (HR=2.03, 95% CI: 1.46-2.81), whereas indices of accelerated fetal growth were not convincingly associated with the risk of HL. Apart from the established association with sex, the findings point to accelerated intrauterine growth as a risk factor for childhood NHL that may differ by sex. Given the rarity of this condition at birth, however, further studies with more elaborate indices are needed to conclude on its association with rare diseases such as HL.

Comparative Study of Obinutuzumab (GA101) Vs. Rituximab Against CD20+ rituximab-Sensitive and -Resistant Burkitt (BL) and Acute Lymphoblastic Leukemia (B-ALL): Potential Targeted Therapy in Patients with High Risk BL and Pre-B-ALL

Background: Aggressive non-Hodgkin lymphoma (NHL) represents >90% of all NHL that occur in children and adolescents. Among all NHLs, Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5 yrs, EFS) following short but intense multi-agent chemotherapy (Cairo et al. Blood, 2007). Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-ALL) have a dismal prognosis, often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013). Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe and well-tolerated and is associated with >90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ B-NHL/ALL to an increase risk of relapse and/or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012,). Obinutuzumab, a novel glycoengineered type II CD20 antibody, has been shown to enhance cell death and ADCC vs. RTX (Herter et al, Clinc Canc Res, 2013), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil.Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant and sensitive BL and pre-B-ALL tumor targets in-vitro and in-vivo in xenografted NSG mice.Methods: Raji (CD20+) and Loucy (T-ALL, CD20-), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD and analysis by flow-cytometry. Loucy cells (CD20-) were used as the negative control. ADCC were performed with K562-IL-15-41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer).The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy for in vivo evaluation by BLI. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice, bred in-house under pathogen free conditions, were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg, obinutuzumab (30 mg/kg), and RTX (30 mg/kg).Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH, U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were then injected every 7 days with the respective therapy for 8 weeks. Mice were monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system.Results: Obinutuzumab compared to RTX (100 mg/ml, 24hrs), significantly enhanced cell death in Raji 45.1±3.3% vs 32.7±6.8%, (p=0.005), Raji4RH 15.8±2.2% vs 2.1±1.5% (p=0.001) and U698-M 40.5±2.9 % vs 26.36±2.6% (p=0.001) n=6. Obinutuzumab vs RTX also elicited a significant increase ADCC with K562-IL15-41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% (p=0.001), Raji-4RH 40.0±1.6% vs 0.5±1.1%, (p=0.001), and U-698-M 70.0±6 % vs. 45.56± 0.1% (p=0.001) n=3.Further, we found that, in vivo, obinutuzumab was significantly more effective than RTX when administered at the same doses in BL (RTX resistant/sensitive) and pre-B-ALL xenografts. Overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in BL; Raji (p=0.05), Raji4RH (p=0.024) and U698-M (p=0.03) (Figure1: A, B and C).Conclusion: Obinutuzumab significantly enhances cell death and NK mediates ADCC in sensitive and RTX resistant CD20+ B-NHL and B-ALL compared to RTX. These preliminary studies also demonstrate that RTX sensitive/resistant BL and pre-B-ALL xenografted mice display significantly increased survival when given 30 mg/kg of obinutuzumab and decreased tumor burden in BL and Pre-B-ALL xenografts compared to an equal dose of RTX. Obinutuzumab may be a novel agent to investigate as adjuvant therapy in patients with relapsed refractory CD20+ B-NHL and/or B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New Insights into Potential Driver Mutations in Pediatric Burkitt Lymphoma

IntroductionBurkitt Lymphoma (BL) is the most common subtype of Non-Hodgkin Lymphoma (NHL) in childhood and adolescence. On the molecular level translocations involving the MYC oncogene are known as the hallmark of BL. However, as this event is not sufficient for BL pathogenesis, the search for additional driver mutations is ongoing. Recently published next-generation sequencing studies introduced various recurrently mutated genes in BL and reported the finding of mutations in Inhibitor of DNA-binding 3 (ID3) (Richter et al., Nat Genet 2012; Schmitz et al., Nature 2012; Love et al., Nat Genet 2012). Within the same pathway also the functional partner of ID3, TCF3, and the downstream target CCND3 were shown to harbor recurrent mutations. Functional analyses of the altered proteins hint at increased cellular growth and proliferation, among others by activating PI3K and CDK4/6.MethodsTo characterize the frequency and clinical relevance we investigated these candidate genes in a well-defined cohort of 63 pediatric BL that were uniformly diagnosed and treated according to NHL-BFM protocols. Also we screened the second most common pediatric B cell malignancies, precursor B cell leukemias (pB-ALL) and Diffuse Large B-cell Lymphoma (DLBCL), for mutations within this pathway. Initial pretreatment tumor samples from 63 BL (including 14 Burkitt leukemias), 15 DLBCL, 6 cases with features intermediate between BL and DLBCL (B-NHL nfc), 96 pB-ALL and an extended group of another 10 relapsed BL were available for analysis. Tumor cell content for DNA extraction was estimated to be at least 60%. Sanger sequencing was performed for the full coding region of ID3 in all entities. Additionally, TCF3 exon 16 and CCND3 exon 5 were investigated in all BL, DLBCL and B-NHL nfc cases. Clinical data were available from the NHL-BFM and ALL-BFM study center. This study was approved by the Ethical Advisory Board of the University of Giessen (A89/11 Amendment 2013).ResultsMutation frequencies for the 60/63 MYC translocation positive BL were 48 (80%) for ID3, TCF3 11 (13%) and CCND3 22 (37%) respectively. Most of the cases presented with more than one ID3 mutation and all affected cases had at least one mutation affecting the functional binding helices of ID3. At least one of the three candidate genes was altered by a potential protein-changing mutation in 53/60 cases (88%). In the group of 15 DLBCL three presented with ID3 mutations. Remarkably, in two of these cases a translocation involving MYC was reported in the study database. There were no ID3 mutations in 96 pB-ALL cases, but two cases presented with non-protein changing variants.With respect to clinical characteristics and patient outcome there were no significant differences with respect to ID3, TCF3 and CCND3 mutation status. To further analyze whether ID3, TCF3 and CCND3 mutations at initial diagnosis have prognostic impact on outcome, ten additional cases of initial tumor samples from patients who suffered relapse were analyzed. However, the ID3, TCF3 and CCND3 mutation status at diagnosis did not differ significantly among the 19 relapsed and the 52 non-relapsed BL patients.DiscussionThe aim of this study was to determine the relevance of ID3, TCF3 and CCND3 gene mutations in a well-defined cohort of pediatric BL. Most interestingly we found ID3 mutations in BL with a significantly higher frequency than previously published in the index studies including patients of all age groups by Richter et al., Schmitz et al. and Love et al., with frequencies ranging between 34 and 68%.The finding that ID3 mutations were virtually exclusive for MYC translocation positive tumors offers the prospect of the ID3 mutation status as a criterion of interest for analysis of intermediate cases between BL and DLBCL.As expected, no ID3 mutations were found in pB-ALL cases, supporting the assumption of exclusive occurrence within the pathogenesis of mature B cell lymphoma.While there was no evidence for a certain role with respect to clinical manifestations and patient outcome, the high frequency of 88% BL cases with at least one mutation in ID3, TCF3 or CCND3 leads to the proposal that this pathway plays an emerging role especially in pediatric BL and it might represent the second hit in BL pathogenesis. This is of special clinical interest as already available kinase inhibitors might successfully address the downstream targets PI3K and CDK4/6. DisclosuresNo relevant conflicts of interest to declare.

Statistical analysis of relation between plasma methotrexate concentration and toxicity in high-dose methotrexate therapy of childhood nonHodgkin lymphoma.

Plasma monitoring of Methotrexate (MTX) levels is a standard approach to predict MTX-related toxicities in a high-dose (HD) MTX monotherapy for childhood acute lymphoblastic leukemia. However, it is uncertain whether plasma MTX levels can predict MTX-related toxicity in the HDMTX plus additional chemotherapy for childhood B-cell nonHodgkin lymphoma (B-NHL). To statistically analyze the relationship between MTX pharmacokinetic parameters and MTX-related toxicities, we collected data from patients with delayed MTX elimination (≥1 µM at 48 hr and/or ≥0.5 µM at 72 hr) in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) BNHL 03 study. Blood MTX levels were measured at 24, 48, and 72 hr after 3 or 5 g/m2 HD-MTX administration for 24 hr. Three hundred and four patients received 2-4 courses of the HDMTX plus additional chemotherapy, and delayed MTX elimination was observed in 165 courses of 127 patients. In those, nephrotoxicity was significantly correlated with plasma MTX levels for each patient (P = 0.03), and also for each course (P = 0.009), but no other toxicities were correlated. Another analysis according to HDMTX courses showed no significant correlation between the first high plasma MTX levels and subsequent MTX levels in later course. It also showed that incidence of liver and gastrointestinal toxicities was most frequent in the first HDMTX course, and then sharply decreased in later courses (P < 0.001). Our results suggest that plasma MTX level is not a reliable predictor for adverse events except for nephrotoxicity in multiple HDMTX therapy courses in childhood B-NHL. Pediatr Blood Cancer 2015;62:279-284. © 2014 Wiley Periodicals, Inc.

Abstract 3112: Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma

Abstract BACKGROUND: Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo et al., BJH, 2012). Children with BL who relapsed or progressed have chemotherapy resistant disease and can rarely be cured after salvage therapy (Cairo et al.,JCO, 2012). Bruton's tyrosine Kinase (BTK) is a major regulator of normal B-cell development and functions through activation of the BCR, MAPK, NF-κB and AKT signaling pathways (Qiu et al., Oncogene, 2000). Chronic active B-cell receptor signaling through activation of the BTK, can be specifically inhibited by a selective BTK inhibitor, ibrutinib (Davis et al., Nature, 2010). However, the mechanism(s) and function of BTK is still poorly understood in B-cells, especially in BL. Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in a variety of organisms and stem cells for new experimental and therapeutic tools (Sander et al, Nat. Biotech., 2011; Carlson et al., Mol Ther., 2012). OBJECTIVES: We hypothesize that 1) TALEN nuclease technology is suitable for the modification of endogenous BTK gene expression in BL cells, and 2) a TALENs-mediated BTK knockout model will provide a useful tool for understanding the mechanism and function of BTK and the development of therapeutic agents that target BTK in BL. METHODS: BTK TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011; Lee/Cairo et al., ASH, 2012) for BTK gene disruption. BTK TALENs validities in 293 and Raji cells were confirmed by surveyor assay and sequencing analysis. Western blotting was performed and statistical significance was determined by one-tailed Student t test using GraphPad Prism software. RESULTS: Two pairs of functional BTK TALENs (BTK-T1 and BTK-T2) targeting endogenous BTK gene were constructed and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293 cells. We confirmed that entire BTK coding sequences were deleted by PCR genotyping and sequencing analysis in stable BTK-knockout Raji cell clones (BTK+/-). BTK-T12-25 and BTK-T12-29 showed a significant decrease of BTK protein expression (50±1.1% reduction, p&amp;lt;0.0005; 27±0.7% reduction, p&amp;lt;0.0005, respectively) compared to empty vector-transfected Raji cells as mock control. There was a significant decrease in Erk phosphorylation in BTK knockout Raji cells compared to mock control (30±1.6%, p&amp;lt;0.001). CONCLUSIONS: These preliminary data suggest that BTK-TALENs are useful tools for the disruption of endogenous BTK gene expression. This TALEN mediated BTK knockout model will be used for studying BTK function in BL and the efficacy of BTK inhibitors as potential therapeutic agents in BL. Citation Format: Sanghoon Lee, Changhong Yin, Timmy O'Connell, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3112. doi:10.1158/1538-7445.AM2014-3112

Non Hodgkin Lymphoma and Hepatitis C Virus in Egyptian Children: One Centre Study

Non Hodgkin lymphoma (NHL) represents a major health problem in the world. In Egypt, where both, hepatitis C virus is highly endemic and NHL has high incidence, questions arise about the association of both diseases especially in children. The primary aim of this study was to describe the epidemiological situation of NHL in children associated with HCV in Mansoura, Egypt. Pediatric patients provisionally diagnosed as suffering from NHL were recruited from hematology unit at Mansoura University children hospital (MUCH) from January 2013 to March 2014. Hematologists collected demographic characteristics, clinical history, and laboratory and treatment data. Blood samples were collected from each participant in the study and subjected to complete virological profiles to detect hepatitis C specific IgG, hepatitis B Surface antigen (HBsAg) and anti hepatitis B core IgM (HBcIgM) and HIV specific immunoglobulin G. Sera samples were subjected to real time polymerase chain reaction to determine active infection. The present study was carried out on 92 children with NHL. They were mainly males (69.9%) with mean age 6.9± 3.7 years. The lymphoma was mainly Burkitt type (54.3%). The patients were mainly in stage 3 (80.5%). HCV IgG was positive in 41.3% of the patients and HCV viremia was detected in 43.5% of the patients, while none of the control subjects had any positive HCV markers. In comparative study between NHL associated with active HCV and those not associated with active HCV, there was statistically significant elevations of ALT (P=0.002), AST (P=0.03) and bilirubin (P=0.005). History of blood transfusions, though was common among NHL associated with HCV (75%) than those not associated with HCV (42.3%), this was statistically insignificant difference (P=0.13). HCV viremia was associated with 30% of patients with recent incidence of NHL. There was statistically significant association of HCV IgG with HCV viremia (90%-P=0.0001). The results presented here describe a strong association of chronic HCV infection with NHL. Much remains unknown about the natural history of HCV infection and its possible contribution to carcinogenesis; however, our data suggest that NHL may be among the malignant complications of chronic HCV infection in children. It is strongly recommended to screen all children, in endemic areas for HCV, affected with NHL for HCV IgG and when positive to evaluate them by PCR.

Updated investigations of cancer excesses in individuals born or resident in the vicinity of Sellafield and Dounreay.

Background:Earlier studies have shown raised risks of leukaemia and non-Hodgkin lymphoma in children, teenagers and young adults resident either at birth or diagnosis in Seascale. Some increases in cancer risk in these age groups have also been noted among those living around Dounreay. We aimed to update previous analyses relating to areas close to these nuclear installations by considering data from an additional 16 years of follow-up.Methods:Cross-sectional analyses compared cancer incidence rates for 1963–2006 among those aged 0–24 years at diagnosis living in geographically specified areas around either Sellafield or Dounreay with general population rates. Cancer incidence for the period 1971–2006 among the cohort of Cumbrian births between 1950 and 2006 was compared to national incidence for 1971–2006 using person-years analysis. Cancer among those born in the postcode sector closest to Dounreay was compared with that among those born in the three adjoining postcode sectors. Analyses considered both cancer overall and ICD-O-3 defined diagnostic subgroups including leukaemia, central nervous system tumours and other malignancies.Results:Apart from previously reported raised risks, no new significantly increased risks for cancer overall or any diagnostic subgroup were found among children or teenagers and young adults living around either nuclear installation. Individuals born close to the installations from 1950 to 2006 were not shown to be at any increased risk of cancer during the period 1971 to date.Conclusions:Analysis of recent data suggests that children, teenagers and young adults currently living close to Sellafield and Dounreay are not at an increased risk of developing cancer. Equally, there is no evidence of any increased cancer risk later in life among those resident in these areas at birth.

Epstein-Barr virus viral load and serology in childhood non-Hodgkin's lymphoma and chronic inflammatory conditions in Uganda: implications for disease risk and characteristics.

Epstein-Barr virus (EBV) has been linked to malignancies and chronic inflammatory conditions. In this study, EBV detection was compared in children with non-Hodgkin's lymphoma and children with chronic inflammatory conditions, using samples and data from a case-control study carried out at the Mulago National Referral Hospital between 2004 and 2008. EBV viral load was measured in saliva, whole blood and white blood cells by real-time PCR. Serological values for IgG-VCA, EBNA1, and EAd-IgG were compared in non-Hodgkin's lymphoma and chronic inflammatory conditions; and in Burkitt's lymphoma and other subtypes of non-Hodgkin's lymphoma. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated. Of the 127 children included (87 males and 40 females; median age 7 years, range 2-17), 96 had non-Hodgkin's lymphoma (46 Burkitt's lymphoma and 50 other non-Hodgkin's lymphoma), 31 had chronic inflammatory conditions, and only 10% were HIV-positive. The most common clinical presentations for all disease categories considered were fever, night sweats, and weight loss. EBV viral load in whole blood was elevated in Burkitt's lymphoma compared to other non-Hodgkin's lymphoma (OR 6.67, 95% CI 1.32, 33.69; P-value = 0.04), but EBV viral loads in saliva and white blood cells were not different in any of the disease categories considered. A significant difference in EAd-IgG was observed when non-Hodgkin's lymphoma was compared with chronic inflammatory conditions (OR 0.19, 95% CI 0.07, 0.51; P-value = 0.001). When compared to chronic inflammatory conditions, EBV viral load was elevated in Burkitt's lymphoma, and EA IgG was higher in non-Hodgkin's lymphoma. This study supports an association between virological and serological markers of EBV and childhood non-Hodgkin's lymphoma, irrespective of subtype, in Uganda.

Inferior outcomes of stage III T lymphoblastic lymphoma relative to stage IV lymphoma and T-acute lymphoblastic leukemia: long-term comparison of outcomes in the JACLS NHL T-98 and ALL T-97 protocols.

T cell lymphoblastic lymphoma (T-LBL) accounts for 30 % of all childhood non-Hodgkin's lymphomas (NHL) in Japan. Twenty-nine patients with T-LBL in stages III and IV were eligible for and enrolled in the JACLS NHL-T98 trial (1998-2002), and 72 patients with T-ALL were enrolled in the JACLS ALL-T97 trial (1997-2001). The 10-year overall survival (OS) (61.1 ± 11.5 %) and the 10-year event-free survival (EFS) (44.4 ± 11.7 %) of stage III LBL were lower than those of other diseases, and the OS and EFS were nearly the same when comparing stage IV LBL and ALL (OS: stage IV LBL, 80.0 ± 12.7 % vs. ALL, 80.2 ± 4.9 %; EFS: stage IV, LBL 70.0 ± 14.5 % vs. ALL, 70.7 ± 5.5 %). Outcomes were worse for stage III LBL than for stage IV LBL or T-ALL. Given that the treatment results of T-ALL and LBL stage IV did not differ when compared with previous reports, LBL stage III in Japanese children may differ from LBL stage III in children in other countries.

Burkitt Lymphoma and MYC

Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children and adolescents, but at least 30% of cases occur in patients older than 60 years, and the absolute number of BL cases in adults exceeds those in childhood. BL is described as a monomorphic proliferation of medium-sized transformed B cells with round nuclei, clumped chromatin, basophilic cytoplasm, and squared-off cell borders, cytoplasmic vacuoles, medium-sized paracentral nucleoli, and a starry sky pattern. Translocation involving MYC is characteristic but not specific for BL. No single parameter is the gold standard for diagnosis; morphology, cytogenetics, immunophenotype, and gene expression profiles all may contribute to the diagnosis. Although neither EBV nor MYC are sufficient to cause BL there is increasing information from techniques such as complete RNA sequencing that identify essential pathways that are activated in the pathogenesis of BL. These findings suggest novel opportunities for improved therapeutic intervention.

Non-Hodgkin lymphomas in childhood: how to move on?

Non-Hodgkin lymphomas of childhood represent a diverse group of neoplasms with different clinical, pathological, immunophenotypical and genetic features. A vast majority of childhood non-Hodgkin lymphomas could be classified into one of the three major histological subgroups: mature B-cell neoplasms, lymphoblastic lymphomas or anaplastic large cell lymphomas. Modern therapeutic strategies lead to cure in more than 80% of patients. Conversely, refractory diseases, as well as disease relapse convey a dismal prognosis. This fact requires much better stratification based on prognostic markers which would ideally recognize distinct groups of patients requiring different therapeutic regimens. Defining novel diagnostic and prognostic markers should improve diagnosis and prognosis as well as patient follow-up. It should also allow introduction of individually tailored treatment regimens in selected groups of patients with non-Hodgkin lymphomas, with the main goal of improving treatment results and decreasing short- and long-term complications.

Clinical characteristics and treatment outcomes of children with anaplastic large cell lymphoma: a single center experience.

BackgroundAnaplastic large cell lymphoma (ALCL) is uncommon in children, accounting for approximately 15% of all cases of childhood non-Hodgkin lymphoma. Despite many studies attempting new treatment strategies, treatment outcomes have not significantly improved, and the optimal treatment for pediatric ALCL has not been established.MethodsThe records of newly diagnosed ALCL patients at our institute between July 1998 and April 2013 were reviewed. We evaluated the general characteristics of the patients, chemotherapy regimens, overall survival (OS) rates, and event-free survival (EFS) rates.ResultsTwenty-eight ALCL patients were eligible. The median age at diagnosis was 10.8 years. Lymph node involvement was the most common presentation (79%). CCG-5941, a multi-agent T-cell lineage chemotherapy, was the predominant treatment regimen (57%). The five-year OS and EFS rates were 88% and 69%, respectively. Stage, the presence of B symptoms, lung involvement, and bone marrow involvement were significant prognostic factors for EFS (P=0.02, 0.01, 0.01, and 0.02, respectively). Eight patients relapsed, and three died during the study period. Four of the eight patients who relapsed were treated with high-dose chemotherapy and autologous stem cell transplantation (HDCT-ASCT). Two of the four who had undergone HDCT-ASCT developed secondary relapses and were subsequently treated with allogeneic SCT or brentuximab.ConclusionWe found that treatment outcomes with multi-agent chemotherapy in children with ALCL were similar to those of previous reports, and that relapsed patients could be salvaged with HDCT-ASCT or allogeneic SCT. A prospective, larger cohort study is warranted to define the optimal treatment for pediatric ALCL.