Detection of Recurrent Genetic Mutations in Intravascular Large B-Cell Lymphoma By Comprehensive Genetic Analyses Using Peripheral Blood Circulating Cell-Free DNA

Background: Intravascular large B-cell lymphoma (IVLBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) characterized by the selective growth of tumor cells in the lumina of small vessels. The characteristics of the disease, such as the lack of remarkable lymphadenopathy and the presence of non-specific clinical symptoms, including documented fever of unknown origin, make precise diagnosis difficult; random organ biopsies from bone marrow, skin, and lung are still performed to detect tumor cells. Recently, many recurrent genetic mutations have been detected, especially in DLBCL, but the genetic background in IVLBCL has not been fully elucidated because of the difficulty in obtaining sufficient live tumor cells from diagnostic specimens for subsequent genetic analyses. Previously, we demonstrated that peripheral blood circulating cell-free DNA (cfDNA) in DLBCL is an alternative tumor DNA (tDNA) source for comprehensive genetic analyses (Suzuki Y, et al. ASH 2014 #1658). In this report, we demonstrate that the concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and that efficiently accumulating the tDNA in peripheral blood is useful for comprehensive genetic analyses to detect recurrent genetic mutations in IVLBCL.

Aims: Confirmation of recurrent genetic mutations in IVLBCL using plasma cfDNA.

Methods: The study population included 54 B-cell lymphoma patients (IVLBCL, N=21; DLBCL, N=21; follicular lymphoma, N=12) and healthy volunteer donors (N=14). In IVLBCL patients, primary bone marrow (BM) cells containing tumor cells were transplanted into NOD/Shi- scid IL2Rγ^null mice to generate a xenograft model (Shimada K, et al. Leukemia 2016), and the tumor cells from tumors formed in the spleen of the mouse model were purified using anti-CD19 antibody-conjugated magnetic beads to obtain tDNA. For genetic analyses using cfDNA and DNAs from purified tumor cells, whole-exome sequencing (WES) using the HiSeq 2000 (Illumina) and Sanger sequencing for reconfirmation of those mutations were performed.

Results: The cfDNA concentrations were significantly higher in DLBCL and IVLBCL than in normal donors (p=0.037 and 0.036, respectively), and the concentration was significantly higher in IVLBCL than in DLBCL (p<0.001). WES using DNA from whole BM cells (BM-DNA) with infiltration of IVLBCL tumor cells (10 to 20% by flow cytometry analysis, 2 patients) or tumor cells purified from the IVLBCL xenograft model and corresponding cfDNA indicated that mostly the same mutations were detected (more than 200 mutations in each patient). There was a tendency for a higher variant allele frequency in cfDNA than in BM-DNA. If the tumor cell ratio in BM was 5% or less, mutations that could be detected by cfDNA were not detected efficiently in BM-DNA. WES analysis to detect genetic mutations was feasible in all cfDNA samples from IVLBCL patients. The mutations confirmed frequently in IVLBCL were: CD79B (67%), MYD88 (52%), IRF4 (38%), SETD1B (52%), PRDM1 (33%), TOX (33%), ETV6 (57%), and TBL1XR1 (48%). Somatic hypermutations in PIM1 and IGLL5 genes were frequently confirmed (95% and 87%, respectively). Interestingly, abnormalities in genes related to immune escape such as CD58 and HLA-B were also detected in a significant number of patients.

Discussion: The concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and it appeared to be sufficient to carry out comprehensive genetic analyses to accumulate tDNA. Moreover, the WES analysis using cfDNA could detect genetic mutations more efficiently than that using corresponding BM-DNA. The profiling of recurrent genetic mutations in IVLBCL is partly similar to that in activated B-cell type DLBCL with mutations in NF-κB pathway-related factors, and, intriguingly, they resemble those in B-cell lymphomas that infiltrate into immunologically sanctuary organs, e.g., primary CNS, testis, and breast lymphomas. The present data suggest that genetic analysis using cfDNA in addition to random organ biopsies may become a powerful, safer, and painless strategy to predict the presence of IVLBCL in the clinical setting.