Genetic features of precursor B‐cell phenotype Burkitt leukemia with IGH‐ MYC rearrangement

Abstract Background An atypical form of Burkitt leukemia/lymphoma (BL), BL with a phenotype of precursor B‐cells (preBLL), is listed in the WHO Classification. Recent reports suggested that preBLL and classical BL could be distinguished by the differences in IG‐MYC translocation architecture and an additional mutated genes profile. The characteristics of classical BL are IG‐MYC by aberrant somatic hypermutation or class switch recombination, and BL‐specific gene mutations such as MYC, ID3, and CCND3. Meanwhile, preBLL is characterized by IG‐MYC due to aberrant VDJ recombination and mutations in NRAS and KRAS. However, it is not clear whether all preBLL cases can be differentiated. This report investigated the molecular characteristics of an infant preBLL case, with a more advanced stage of maturity than typical preBLL. Case The patient showed BL‐like morphology with IGH‐MYC rearrangement. In the immunophenotyping, CD20 and surface immunoglobulin were negative, whereas other markers were consistent with BL. To evaluate the genetic contribution, we performed whole‐exome sequencing. The breakpoint analysis revealed the IG‐MYC occurred due to an aberrant VDJ recombination. Meanwhile, additional somatic mutations were detected in FBXO11, one of the mutant genes specific to BL. In the analysis of the specimen in complete remission, mutation in KRAS, frequently mutated in preBLL, was detected with low frequency, suggesting somatic mosaicism. Conclusion The present case showed the characteristics of both typical preBLL and classical BL. Because preBLL includes atypical cases such as the present case, further studies are required to elucidate preBLL features.


| INTRODUCTION
IGH and MYC translocation is one of the representative genetic abnormalities in Burkitt lymphoma/leukemia (BL). Generally, BL shows a mature B-cell phenotype. However, in the WHO Classification, a more immature phenotype of BL (preBLL) is listed as a phenotype of precursor B-cells, with expression of terminal deoxynucleotidyl transferase (TdT), and sometimes CD34, and absence of CD20 and surface immunoglobulin expression. 1 Recently, Wagener et al. reported that preBLL and classical BL could be distinguished from each other by differences in the translocation architecture of IGH-MYC. [2][3][4] They reported that aberrant V (variable), D (diversity), and J (joining) gene segments recombination resulting in IG-MYC characterized by N-sequences and loss of some bases at the breakpoint are features of preBLL. They also revealed a difference in mutation profile between preBLL and BL. PreBLL was associated with NRAS and/or KRAS mutations whereas BL was associated with MYC, ID3, and CCND3. Subsequently, Yoon et al. showed that IGH-MYC translocations due to the aberrant VDJ recombination were also detected in the TdT-negative cases, which indicated that these cases were at a more advanced maturation stage than those of Wagener et al. 5 Their cases were associated with mutations in MYC and/or TP53, which are often mutated genes in BL.  Figure S1). Morphological findings, negativity for CD34 and TdT, and positivity for cyμ and CD45 were suggestive of mature B-cell phenotype, whereas negative for CD20 and surface globulins were immature B-cell features (preBLL). Cerebrospinal fluid examination showed an increase in the number of dysmorphic cells (40/μl) with a high N/C ratio, nucleus with irregular shapes, and distinct nucleoli, and was cytologically determined as class V. Computed tomography revealed bilateral renal swelling, hepatomegaly, and splenomegaly. As the present case had both B-cell precursor and mature B-cell features of ALL, we considered that both lymphoma-oriented intensive block-type chemotherapy and ALLoriented chemotherapy, including maintenance therapy, should be adopted. Although the present case was infant ALL, we did not adopt infant-specific chemotherapy such as in MLL-10, 6 which did not include block-type treatment. Therefore, the present case was treated according to AIEOP-BFM ALL 2000 high-risk chemotherapy. 7 Despite suspending prednisolone for 2 days due to tumor lysis syndrome, the patient showed a good prednisolone response.
The patient achieved a complete remission (CR) after induction therapy. Considering that the patient was an infant, intensification of intrathecal chemotherapy was adopted to avoid the late complications of cranial radiotherapy. As of 38 months from diagnosis, the patient is alive without disease relapse.

| Identification of additional somatic mutations and IGH-MYC translocation architecture
To reveal the present case's genetic characteristics, we further performed whole-exome sequencing of tumor-normal pairs (detailed in Data S1). We filtered out the variants that were present in normal sample, and identified two somatic mutations in FBXO11 and one in TNIK (Table 1). Visual screening using the integrative genomics viewer (IGV) viewer (IGV 2.3.97) revealed that the two mutations in FBXO11 were located on different alleles ( Figure S2). Next, we focused on the breakpoint junction of the IGH-MYC translocation using the IGV. The breakpoint mapped to the 5 0 end of IGHJ4, with loss of some bases from the 5 0 end, and 1.7 kbp upstream of MYC ( Figure 1A). As the breakpoint was close to recombination signal sequences and nucleotides resembling N-sequences were added at the breakpoint, the translocation most likely occurred through an aberrant VDJ recombination.

| Identification of germline variants of cancerpredisposing genes
As the present case developed atypical leukemia in infancy, we further searched for germline pathogenic variants of cancer-associated genes (detailed in Data S1). As a result, a pathogenic variant of KRAS   G12R (Bio-Rad, Hercules, CA). The mutation allele frequency was 9.5% in the CR sample and 46% in the tumor sample, respectively.
The mosaicism could not be validated in any other organ due to the lack of available samples. The summary of the present case's characteristics is shown in Figure 1B, along with a comparison with typical preBLL and BL characteristics.

CONFLICT OF INTEREST
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

AUTHOR CONTRIBUTIONS
All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.