Deep-Sequencing Of The Molecular Landscape In Burkitt Lymphoma/Leukemia

In 2012, whole-genome, whole-exome and global transcriptome sequencing studies unraveled the molecular complexity of Burkitt lymphoma (BL). Amongst a set of up to 70 recurrently mutated genes (Love C. et al., Nat. Genet. 2012), essential affected oncogenic pathways were discovered including mutations in MYC and the transcription factor TCF3 (Schmitz R. et al., Nature 2012), as well as inactivating mutations of ID3 (Richter J. et al., Nat. Genet. 2012).

(1) To validate the mutation frequency and mutational landscape of selected candidate target genes (ID3, MYC, TCF3, and TP53) in an independent cohort of 60 adult patients with Burkitt Lymphoma/Leukemia. (2) To investigate the clonal composition and associations with BCL2 rearrangements using a quantitative next-generation sequencing assay.

The patient cohort included 33 cases with Burkitt Leukemia (33/60, 55.0%) and 27 cases with Burkitt Lymphoma (27/60, 45.0%). 36 (60.0%) of these were single-hit lymphomas only harboring MYC-rearrangements, whereas 24 (40.0%) cases were multiple-hit lymphomas harboring in addition at least one of the following gene rearrangements: BCL2, BCL6, and/or CCND1. The median age was 60.8 years (range 5.5 - 82.7). All cases were comprehensively characterized by cytomorphology, cytogenetics and FISH, including evaluation for MYC-, BCL2-, BCL6-, and CCND1-rearrangements. Four candidate genes were sequenced using a quantitative deep-sequencing NGS assay (median coverage of 633 reads per mutation). The lower limit of detection of mutations was set at 2%. Amplicon sequencing libraries were prepared using genomic DNA extracted from mononuclear cells. The following regions were investigated: ID3 (exons 1-2), MYC (exons 2-3), TCF3 (exons 18), and TP53 (exons 4-11), respectively.

In total, 112 mutations were detected in 39/60 (56.5%) patients. 9/112 (8.0%) were detected with a clone size of <10%. In 17 patients with single mutations, either MYC (n=7) or TP53 was detected mutated (n=10). Double mutations were seen in 15 cases and six cases harbored three and one case four gene mutations, respectively. Interestingly, all patients with ID3 mutations harbored additional mutations. In detail, 27 ID3 mutations were observed in 14/60 (40.0%) cases. These mutations included 17 missense, 6 nonsense, 1 deletion, 2 splice-site and one frame-shift events. Eight of the 14 ID3 mutated patients harbored two or more concomitant ID3 mutations. The mutations clustered around hotspot codons p.Leu64Phe (9/27) or p.Gln81* (4/27). Of the double-mutated cases 7/8 patients harbored at least one mutation in the hotspot region. Of note, 23/27 mutations were located within the helix-loop-helix domain. MYC, the predominantly recognized oncogene in BL, was mutated in 26/60 (43.3%) of cases and in total 50 distinct mutations were discovered, including 46 missense, 2 deletions, one splice-site, and one indel events. A high number of 9 (34.6%) patients harbored two or more MYC mutations. The mutations clustered around a recurrent codon p.His374 (4/60). TCF3 mutations were detected in 2/60 (3.3%) of cases, both of them also mutated in MYC and TP53. Finally, 33 TP53 mutations were found in 27/60 (45.0%) patients. Six patients harbored two mutations (with quite different clone sizes in two of the six cases). Furthermore, we observed a positive correlation for ID3 and MYC mutations (11/14 (78.6%) ID3 mutated cases harbored MYC mutations vs. 15/46 (32.6%); p=0.004). Further, we studied correlations of the type of lymphoma (single-hit vs. multiple-hit) and found ID3 mutations positively correlated with single-hit lymphoma (13/36 (36.1%) vs. 1/24 (4.2%) cases within multiple-hit patients; p=0.005). Next, we studied correlations with BCL2 rearrangements, which were present in 20/60 (33.3%) cases. Cases with BCL2 rearrangements were associated with significantly lower mutation frequencies in TP53 (5/20 (25.0%) vs. 22/40 (55.0%), p=0.032) and ID3 (1/20 (5.0%) vs. 13/40 (32.5%), p=0.023). Finally, within our cohort of 60 lymphomas, 11 (18.3%) patients presented as single-hit lymphoma and without any of the mentioned molecular mutations.

In this independent validation study we can confirm the high frequency of mutations in ID3, MYC, TCF3 and TP53 in adult BL. As our understanding of the genetic landscape is further increased novel therapeutic approaches seem possible in this disease.

Kohlmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Denzel:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.