Clinical Significance of MYD88 Mutation in Patients with Diffuse Large Cell Lymphoma

Mutation of the MYD88 has recently been identified in activated B cell like diffuse large B cell lymphoma (DLBCL) and enhanced cell proliferation systems such as JAK-STAT and NF-kB signaling pathways. However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive and an automatic method utilizing guanine-quenching probes (QP) to detect mutation and investigated the relationship between MYD88 L265P mutation and clinical significance. We amplify a DNA fragment including the mutation to intend for by PCR and associate it with Q-probe with complementary sequence, using the temperature that Q-probe dissociates varying according to a conformity degree of the complementarity sequence. We judge it by detecting the fluorescence to be provided by dissociation. Results were obtained from 1ul of DNA solution(10ng) within 90 min by the method. Detected mutations were identical between QP method and allele-specific PCR (AS-PCR).Eighty-nine patients with a diagnosis of de novo DLBCL made between 1999 and 2014, and treated with CHOP or R-CHOP therapy. We retrospectively analyzed the outcome of 89 patients (age range; 21-88 and 59% were female). The median follow-up time was 4.4 y. Survival analyses were performed using the Kaplan-Meier method. None of the patients had a known history of human immunodeficiency virus infection. MYD88 L265P mutation was both assessed by Q-probe system that can detect low levels of mutant DNA and allele-specific TaqMan polymerase chain reaction assay. We performed the direct sequence method using 3130 Applied Biosystem Genetic Analyzer as antithesis. The cell-of-origin was determined based on immunohistochemical (IHC) stains for CD10, BCL-6 and MUM-1 by Hans' algorithm.

MYD88 L265Pmutation was detected in 25.8% (23/89) in various tissues of DLBCL. MYD88 mutations occurred more frequently in males (P<0.05), cases without B symptoms (P<0.05). MYD88 mutation was infrequent in DLBCL arising in lymph nodes (10.6%), but more frequently found in extranodal sites such as testes (83%, 5/6), nasal (75%,9/12), central nervous system (50%,2/4), and leg (100%,1/1). In agreement with recent studies, we found no mutated cases among gastric cases. As somatic mutations in MYD88 was reported to be the most frequent alterations found in non-GCB type, we further analyzed GCB or non-GCB type by IHC. MYD88 mutations were predominantly observed in the non-GCB type (74%, 17/23), compared with 26%, 6/23 in GCB type. Overall survival (OS) for 3 years were 84.2% and 70.2% in patients with wild-type MYD88 and in MYD88 mutation group (P=0.366), respectively. Progression-free survival (PFS) for 3 years, 76.9% and 64.3% in patients with wild type and in mutated group (P=0.156), respectively. However, all four cases with CNS relapse had this mutation, 2 originated from testis, and remained 2 from lymph nodes. Our results confirm the remarkable site-specific occurrence of MYD88 mutation. In addition, Q-probe system for detection of MYD88 mutation was very useful because of its sensitivity and in the case who obtained only a small amount of biopsy specimen. MYD88 L265Ppromotes survival of malignant lymphoid cells through several mechanisms. Further large scale study should be necessary for more understanding of biological and clinical significance of DLBCL patients with MYD88 mutation.