ALBERT

Description: Introduction: Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma with a translocation involving MYC and immunoglobulin(Ig) loci. It is most common in children, but also affects adults, and occurs in sporadic, endemic and HIV-associated forms. The Epstein-Barr virus (EBV)-associated endemic subtype is the most common pediatric cancer in equatorial Africa, but also occurs in other parts of the world, for example in the rain forest of Brazil. Intensive chemotherapy is effective, but the associated toxicity requires supportive care that is not readily available in resource-poor regions. Previously published molecular characterization of small numbers of tumors indicated that the mutation profiles of endemic and sporadic cases are similar, but not identical. One goal of the BLGSP is to conduct comprehensive molecular characterization of BL by sequencing DNA and RNA from a large BL cohort - including endemic, sporadic, pediatric and adult cases - in order to define the genetic and phenotypic features that drive these cancers. These data will be analyzed with an intent toward developing new therapeutic strategies that can be deployed worldwide. Methods: The goal is to collect 160 BL cases, of which 50% will be endemic, 38% sporadic (pediatric and adult) and 12% from HIV+ patients. For the discovery phase, each tumor requires case-matching normal DNA as well as treatment, outcome and other clinical information. The optimal source of tumor DNA and RNA is from frozen tissue with at least 50% tumor nuclei, but FFPE immobilization is also accepted. Accrual locations include Africa, Brazil, Europe and the US. The BLGSP has developed extensive standard operating procedures for tissue collection, pathology review and tissue processing to reduce the variation associated with these parameters in the interpretation of the results (see https://ocg.cancer.gov/programs/cgci/projects/burkitt-lymphoma). The project also established procedures that allow sharing of all clinical and sample information through the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov). Molecular characterization includes whole genome sequencing of tumor and normal DNA (80X and 40X coverage, respectively), RNA-sequencing (RNA-seq) and micro-RNA sequencing. These data will enable the BLGSP to identify chromosomal rearrangements, chromosomal copy number alternations, somatic mutations (single nucleotide, insertions, deletions), viral insertions, expression signatures, viral expressions and miRNA regulation of transcripts. Results: To date we have accrued 80 cases of BL of which 75% passed diagnostic pathology review. There was an additional 25% attrition at the tissue processing stage, either due to low quality nucleic acids or low percent tumor nuclei. We have completed sequencing for 45 cases, all but one of which have a MYC translocation involving one of the 3 Ig loci; one case has a MYC rearrangement by FISH analysis that is being characterized further. We have identified recurrent mutations in ID3, DDX3X, ARID1A, FOXO1, TP53, SMARCA4 and other genes. Most mutations are supported by the RNA-seq data, which is also useful in defining the pattern of EBV genome transcription. Preliminary unsupervised hierarchical clustering and principal component analysis of gene expression data defined sample clusters that do not correspond to mutation status or EBV infection, warranting further investigation. Some genes accumulated somatic mutations in a BL subtype-specific fashion. Discussion: BLGSP is an ongoing international collaborative project that will provide a comprehensive molecular portrait of BL subtypes when completed, with the potential to suggest new molecular targets for therapy that can eventually lead to effective treatments that are less toxic than the current regimens. Disclosures Casper: Janssen: Consultancy, Research Funding; Roche: Consultancy, Other: Travel, Accommodation, Expenses; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Up to Date: Patents & Royalties; GSK: Other: Travel, Accommodation, Expenses. Abramson:Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding.

Description: Introduction: Burkitt lymphoma (BL) is an aggressive B-lineage non-Hodgkin lymphoma that has traditionally been classified into 3 subtypes: "endemic", "sporadic", and HIV-associated. The highest incidence of BL - prompting the "endemic" classification - is observed in children in sub-Saharan Africa, where the distribution of the disease is closely associated with that of P. falciparum malaria. The tumor cells in most cases of BL from sub-Saharan Africa are infected with Epstein Barr virus (EBV). In contrast, a minority of BL tumors from Europe and North America - which are commonly described as "sporadic" - are EBV positive. Although excellent outcomes have been observed for patients with BL treated with multi-agent chemotherapy in Europe and North America, the outcomes of BL patients in sub-Saharan Africa are suboptimal. We hypothesized that improved understanding of the molecular heterogeneity that exists within BL, particularly the molecular features that distinguish cases from sub-Saharan Africa from those from Europe/North America, will enable more effective treatment strategies that can readily be implemented in low-resources settings. Methods: Tumor biopsies were collected from 19 pediatric patients who presented to the Uganda Cancer Institute with maxillofacial tumors histologically confirmed to be BL. Total RNA was extracted and polyA-selected sequencing libraries were prepared. Paired-end, 50-base pair sequencing was performed on the Illumina HiSeq 2500 platform at a depth of 100 million reads per sample. A publicly available RNA sequencing dataset from 28 BL cases from Europe and North America that were previously analyzed by both microarray (NEJM 2006; 354[23]: 2431-2442) and RNA sequencing (Nature 2012; 490[7418]: 116-120) was analyzed in parallel for comparison. RNA sequencing on the 28 sporadic BL tumors was performed on polyA-selected sequencing libraries with paired-end, 108-base pair sequence reads generated on the Illumina HiSeq 2000 platform. The reads from all 47 BL tumors were aligned to the human and EBV (GenBank ID KC207813.1) genomes using the STAR aligner. Tumors were deemed EBV positive if the ratio of mapped viral reads to human reads exceeded 0.001%, since the EBV genome is 0.005% the size of the human genome. Normalization and differential expression analysis were performed on aligned reads using the DESeq2 R package. Somatic variants were identified by the Genome Analysis Tool Kit. Analysis of alterative splicing was performed with the SGSeq R package. Results: All of the Uganda BL cases but only 70% of the previously published European/North American cases were obtained from children less than 18 years; 61% and 87% were from male patients, respectively. One half of patients in both groups presented with Ann Arbor stage I or II disease, while the remainder presented with higher stage disease. The median survival in the 19 Ugandan patients was less than one year. Sequence reads from 15 (79%) of the Ugandan tumors and 4 (15%) of the European/North American tumors aligned to the EBV genome. Although BL is reported to express only latency type I EBV genes, expression of latency type I, II, and III genes was detected. Among the 4 tumors from Uganda that did not contain EBV-derived reads, 1 was from a child who was HIV-seropositive, while the others were from HIV-seronegative patients. Unsupervised hierarchical clustering and principal component analysis of all annotated genes failed to separate EBV positive and EBV negative tumors. Cluster analysis of the Ugandan tumors revealed no association with clinical variables. Ongoing analyses are being performed to identify associations between differentially expressed genes and somatic mutations and alternative splicing that may distinguish the tumor subtypes. Conclusions: While the terms "endemic" and "sporadic" have traditionally been used to classify BL, this nomenclature is archaic and does not accurately capture the underlying biology of the tumor subtypes. We performed a comparative whole transcriptome analysis of BL tumors from patients in Uganda to a dataset of BL tumors from patients in Europe/North America to identify features that distinguish the two cohorts. While cluster analysis failed to clearly separate tumors by their EBV or clinical status, ongoing analyses are being performed to identify alterations that may contribute to their distinct gene expression profiles. Disclosures No relevant conflicts of interest to declare.

Description: Background: Endemic Burkitt Lymphoma (eBL) is the most common childhood cancer in many countries of sub-Saharan Africa (SSA). Reported overall survival (OS) rates in SSA are low at 30-50%, especially compared to survival of children with sporadic Burkitt Lymphoma treated in high-resource settings (OS 85-95%)(Patte, Auperin et al. 2007, Buckle, Maranda et al. 2016, Stanley, Westmoreland et al. 2016). The Burkitt Lymphoma (BL) project in Uganda was initiated as a collaboration between the Fred Hutchinson Cancer Research Center and Uganda Cancer Institute (UCI) in July 2012. The project provided resources for timely pathologic diagnosis, chemotherapy during stock-outs, case management to improve adherence, transportation, and standardized recording of care and clinical outcomes. We sought to determine OS and response to treatment in this patient population with eBL who received enhanced care through this demonstration project. Methods:Every child presenting to the UCI with suspected BL and enrolled in the BL project between July 2012 and December 2014 underwent diagnostic evaluation with a core needle biopsy of the tumor, abdominal ultrasound, and chest radiography. Patients with confirmed BL at enrollment, as determined by pathology review and physician assessment, were staged based on physical exam according to Ziegler. Most received first-line therapy consisting of cyclophosphamide, vincristine and methotrexate (COM) every two weeks for six planned cycles. Treatment response was evaluated ≤ 3 months from starting the 6th cycle of COM. Following completion of therapy, patients were followed monthly for three months, then every three months for up to a year. Follow-up data through March 26, 2015 was included. Kaplan-Meier methodology was used to estimate 1-year OS. Results:A total of 202 patients with suspected BL were followed by the BL project during this time period. Of these, 142 (70%) were confirmed to have BL. The remainder had other cancers or benign diseases (24%) or had inadequate diagnostic data (6%). The median age of patients with BL was 7 years and the majority were male (63%). Approximately half of patients had late-stage disease (49% Ziegler stages C, D or AR) and had a high LDH at presentation (54%). Of the 142 with BL, 78% initiated COM, 6% other chemotherapy, and 16% were not treated with chemotherapy (18 died in the first 40 days and 5 were exited). Among 110 patients who initiated COM, the treatment response after 6 cycles was complete response (CR) for 46%, partial response (PR) for 7%, stable (SD) or progressive disease (PD) for 2%, relapsed disease (RD) for 1%, but there was no response assessment within 3 months of the 6th cycle for 10%. The remaining patients who did not complete 6 cycles either switched to second line therapy (7%), abandoned treatment (9%), died (9%), exited the program (3%), or were censored (6%) within 6 months of starting treatment. Among the subset of 73 patients who completed six cycles of treatment, the responses after 6 cycles were as follows: CR 70%, PR 11%, SD or PD 3%, RD 1%, unknown 15%. At 1 year, OS for the entire cohort with confirmed eBL was 53% (95% CI 43%, 62%; Figure 1). Among the patients who initiated COM, survival at 1 year post treatment initiation was 60% (95% CI 49%, 70%; Figure 2). These data represent preliminary results of our ongoing analysis. An updated analysis, along with associations of baseline factors, including CNS status, anemia, thrombocytopenia, B symptoms, tumor lysis syndrome and HIV status, as well as other infections (malaria, Hepatitis B), with clinical outcomes will be presented. Conclusion:Survival ofpatients with eBL treated in a low-resource setting remains inferior compared to children treated for sporadic BL in higher resource settings. The BL project was able to provide pathologic diagnoses, assure access to chemotherapy, enhance supportive care, and reduce abandonment to less than 10%, but still only slightly more than half of patients with a confirmed diagnosis survived one year. Ongoing obstacles to improving outcomes are inaccurate diagnosis and lacking supportive care to allow more intensive therapies. Improved diagnostic capacity as well as the ability to provide more potent and potentially less toxic treatment modalities may help to address the poor survival of children with a disease that is so successfully treated in higher resource settings. Disclosures Casper: Up to Date: Patents & Royalties; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Janssen: Consultancy, Research Funding; GSK: Other: Travel, Accommodation, Expenses; Roche: Consultancy, Other: Travel, Accommodation, Expenses.