Genomic Pathways and Potential Therapeutic Targets in Pediatric Burkitt Lymphoma (PBL): A Children's Oncology Group Report

Pediatric Burkitt Lymphoma (PBL) is the most common histological subtype of NHL in children and adolescents (40%). The prognosis for PBL has doubled from 40–80% 3yr EFS over the past 30 years secondary to the introduction of short and intensive multiagent chemotherapy including fractionated cyclophosphamide, high dose methotrexate and moderate to high dose cytosine arabinoside (Cairo et al Leukemia, 2002, Cairo et al Blood, 2007 and Cairo et al JCO, 2011). However, 20% of children still relapse and the prognosis is dismal in this subgroup (<20% OS) and more importantly the morbidity is extremely high resulting in a high incidence of severe mucositis, systemic infections and prolonged hospitalizations (Cairo et al, Blood 2007). Novel targeted therapy such as rituximab has been recently investigated in PBL (Cairo et al ASCO, 2010). However, additional targeted approaches are needed to reduce both morbidity and prevent relapse. Dave/Staudt et al (NEJM, 2006) at the NCI first identified a group of C-MYC target genes in BL. Subsequently, Klapper et al (Blood, 2008) within the BFM developed a molecular Burkitt lymphoma (MBL) index in PBL.

We therefore investigated and compared the genomic expression patterns and more importantly the common cellular functional pathways in PBL specimens obtained from children and adolescents treated on COG-ANHLO1P1 and compared the results to PBL specimens from the Dave/Staudt NCI and Klapper BFM experiences.

Children and adolescents with newly diagnosed and de-novo advanced PBL were consented and treated on the COG-ANHLO1P1 chemotherapy (Rituximab and FAB chemotherapy) (Cairo et al, ASCO, 2010). Briefly, total RNA was isolated from patient tumor samples,cDNA was generated from 100ng of total RNA, and cDNA was biotin-labeled by in vitro transcription (Affymetrix) as we have previously described (Jiang/Cairo, J. Immun, 2004, Exp Hem, 2009). Fragmented biotin labeled cDNA was hybridized to GeneChip U133A2 and subsequently scanned by GeneChip® Scanner 3000 (Affymetrix). The data (log values) was imported into GeneSpring GX 10 (Silicon Genetics) and Partek Ingenuity Pathway Analysis IPA 6.5. Additionally COG samples were validated by building a prediction model with Support Vector Machines using the Klapper BFM PBL specimens as training database (NCBI GEO GSEI0172 and GSE4475) and subsequently compared to Dave/Staudt et al. NCI PBL specimen (GSE 4732) data and analyzed by a one-way ANOVA followed by the Tukey multiple comparison test. Furthermore, expression of selected genes was measured by qRT-PCR (Applied Biosystems, ABI7500) and compared to genomic profiling results.

Within the 3 groups of PBL specimens there were 1,565 genes identified. There were 375 genes that were significantly over expressed among the 3 cohorts of PBL specimens representing a cross sectional PBL genetic signature. Utilizing the Ingenuity Pathway analysis software (IPA 6.5) we identified that 75% of the overexpressed genes were either protein kinases, receptor / cytokine binding proteins or transcriptional proteins (Figure 1). We further examined several signaling pathways including, JAK-STAT, MAPK and toll-like receptor (TLR) pathways. There was a significant increase in the expression of several antiapoptotic genes in the JAK-STAT pathway including PTPN11 (↑26F), STAT1 (↑9F) and PIM1 (↑6F). Within the MAPK pathway there were several cell growth and proliferation genes significantly overexpressed including MAP2K (↑12F), RAF1 (↑9F) and MAPK9 (↑7F). Lastly within the TLR pathway there was a significant over expression of IRAK1 (↑23F) and NF- kβIA (↑15F). BL classifier genes were also overexpressed: C-MYC (↑27F) and DLEU1 (↑10F). Comparative qRT-PCR vs. genomic profiling expression demonstrated consistent results: PTPN11: ↑35F vs. 26F, PIM1: ↑4.7 vs. 5.4F, STAT1: ↑10.3 vs. 9.2F, MAP2K1 ↑8.7 vs. 11.8F, RAF1: ↑21 vs. 9F and DLEU1 ↑8.1 vs. 9.9F.

These comparative genomic data and analyses suggest a common genetic signature of children and adolescents with sporadic PBL in North America and Europe. Furthermore, there are a number of signal transduction pathways with overexpressed protein kinases that maybe amenable to small molecular tyrosine kinase inhibitors for future molecularly based targeted therapeutic approaches in children and adolescents with BL.

No relevant conflicts of interest to declare.