Identification of a Novel IGHCδ-BACH2 Fusion Transcript In B-Cell Lymphoma/Leukemia and the Expression Analysis of BACH2 Related with Other B-Cell Differentiation-Associated Genes In B-Cell Malignancies

Abstract 4450

In B-cell malignancies, the genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In the present study, we have identified BTB and CNC homology 2 (BACH2) as a novel translocation partner gene of the immunoglobulin heavy chain (IGH) locus, resulting in chimeric Cδ-BACH2 gene in a patient with MYC-IGH-positive highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). A 71-year-old male was diagnosed as having highly aggressive B-cell lymphoma/leukemia. SKY analysis revealed the representative karyotype of tumor cells as 45,X,-Y,der(3)t(3;X)(p21.2;q24),del(6)(q15),der(14)t(8;14)(q24;q32),del(16)(q22),der(20) t(3;20)(q21;p13). FISH and long-distance PCR analyses identified a fusion of MYC with Cγ. FISH analysis also detected a small IGH signal on del(6), and a VH on del(6)(q15). Genome copy number analysis using SNP-array detected an approximately 60Mb deletion in 6q15–25 region, and its centromeric breakpoint within BACH2 gene. The cDNA bubble PCR using BACH2 primers detected multiple PCR products, and sequence analysis has revealed that one of the products contained a sequence of the first exon of IGHCδ fused to 5’ untranslated region of BACH2 exon 2. Genomic fusion point of Cδ-BACH2, was within intron 1 of Cδ and intron 1 of BACH2. Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, indicating that whole coding region of BACH2 was fused to Cδ. This suggested that Cδ-BACH2 fusion cause constitutive activation of BACH2.

We next analyzed the expression levels of BACH2, MYC, PRDM1, and IRF4 genes in the patient using real-time PCR and compared them with those of several hematologic malignancy cell lines, including 14 non-Hodgkin's lymphoma (NHL), 10 multiple myeloma (MM), and 3 B-lineage acute lymphoblastic leukemia, and 3 EB-virus transformed B-cell lines from normal healthy volunteers. IGH-MYC-positive MM cell lines showed increased levels of MYC expression compared with the other cell lines. The MYC expression level in our patient was lower than those in IGH-MYC-positive MM cell lines; however, it was similar to 5 IGH-MYC-positive NHL cell lines [3 Burkitt lymphoma (BL) and 2 diffuse large B-cell lymphoma (DLBCL)]. On the other hand, BACH2 expression levels were higher in NHL cell lines, especially in BL cell lines, but not correlated with the IGH-MYC translocation status. Our IGH-BACH2-positive case also showed a higher level of BACH2 expression. The expression levels of IRF4 and PRDM1 were higher in MM cell lines than in other cell lines. PRDM1 is activated by MYC through IRF4, and the MYC is negatively regulated by activated PRDM1. In MM cell lines with IGH-MYC translocation, MYC was highly expressed regardless of high expression of PRDM1, indicating that MYC activated by IGH translocation could not be inhibited by PRDM1. Unexpectedly, the expression levels of PRDM1 and IRF4 were very low in NHL including our patient, suggesting that the regulation of MYC in NHL is different from that in MM.

BACH2 is a B cell-specific transcription repressor, and is specifically required for class switch recombination, somatic hypermutation, and germinal center formation. One of the target genes of BACH2 is PRDM1 at 6q21-q22.1 that is required for plasma cell differentiation. In this patient, deletion of 6q15–25 was found, indicating loss of one PRDM1 allele. It was reported that PRDM1 is inactivated by chromosomal alterations in 24% of activated B cell–like DLBCL, suggesting that PRDM1 acts as a tumor suppressor gene, and its inactivation may contribute to lymphomagenesis by blocking post–GC differentiation. The combination of BACH2 and MYC in double IGH translocations is unique and consistent with previous reports demonstrating that each partner gene found in double or multiple IGH translocations is exclusively specific to certain types of B-cell lymphoma. These results suggest that the promoter of IGHCδ and/or Eμ enhancer of IGH activate the expression of BACH2, and that BACH2 may act as oncogene in some cases with B-cell lymphoma. Although the IGH-BACH2 translocation is rare in NHL, our data suggest that the BACH2 plays a critical role in B-cell lymphomagenesis through not only IGH translocation but also activation by some other mechanisms.