MYD88L265P and MYD88other variants show different molecular characteristics and prognostic significance in diffuse large B-cell lymphoma

Purpose This study aims to investigate the clinical and molecular differences between diffuse large B-cell lymphoma (DLBCL) patients with MYD88L265P and MYD88other. Methods DLBCL patients with MYD88 variations were collected from the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CHCAMS), and Suzhou Municipal Hospital from February 6th, 2007 to May 20th, 2022. Clinicopathological parameters and treatment outcomes between MYD88L265P and MYD88other were investigated. Results A total of 132 patients with MYD88 variations from a cohort of 475 DLBCL patients were included, among which, 78 were MYD88L265P, while 54 were MYD88other. MYD88L265P was more common in non-GCB subtype than MYD88other (83% vs. 60%, P = 0.004). Besides, MYD88L265P was significantly related to higher proportion of testicle/ central nervous system involvement (31% vs. 6%, P < 0.001), PIM1 mutation (71% vs. 39%, P < 0.001), and PIM1 hypermutation (28% vs. 11%, P = 0.018), compared with MYD88other. Compared with MYD88L265P, MYD88other were more likely to have higher percentage of advanced stage (60% vs. 42%, P = 0.044), extranodal site ≥ 2 (45% vs. 28%, P = 0.044), elevated LDH (55% vs. 35%, P = 0.033), positive CD10 expression (36% vs. 16%, P = 0.009), BCL-6 translocation (20% vs. 8%, P = 0.033), and NOTCH pathway gene alteration (24% vs. 13%, P = 0.040). In non-GCB DLBCL subtype, patients with MYD88other were significantly associated with worse progression free survival (PFS) than those with MYD88L265P when treated initially with R-CHOP/R-CHOP-like regimen (P = 0.010). Conclusion The findings of this study indicate that DLBCL patients with MYD88L265P and MYD88other are likely to be two subgroups with different clinical and molecular characteristics. The survival of patients with MYD88other is not superior than those with MYD88L265P, even poorer when focusing on the non-GCB subtype. Supplementary Information The online version contains supplementary material available at 10.1007/s00432-023-04714-1.


Introduction
Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphoid malignancy in adults, representing the most common entity in non-Hodgkin lymphomas (Alaggio et al. 2022). Despite about 60% DLBCL patients can be cured via standard front-line rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), most of those refractory and relapse patients will succumb to their disease (Flowers et al. 2010;Shi et al. 2022). With the development of large-scale high-throughput sequencing technologies, the underlying heterogeneity in DLBCL has been characterized at an unprecedented scale. Genomic aberration-based classification of DLBCL subtypes is becoming increasingly accepted. Several taxonomies such as the four subtypes (MCD, N1, EZB, BN2), five subtypes (Cluster 1-5) or (MYD88, BCL2, SOCS1/ SGK1, TET2/SGK1, and NOTCH2), and seven subtypes (MCD, N1, A53, BN2, ST2, EZB-MYC + , EZB-MYC − ) have been reported (Schmitz et al. 2018;Chapuy et al. 2018;Lacy et al. 2020;Wright et al. 2020). Genetic subtypes provide useful prognostic information and treatment reference for clinical oncologist. Of special note is MYD88 mutation is a pivotal genetic driver determining the genetic classification in all above mentioned taxonomies and is mostly deemed to be an unfavorable prognostic factor along with CD79B mutation.
MYD88 encodes an adapter protein that functions as an essential signal transducer in the interleukin-1 and Tolllike receptor signaling pathways (Iwasaki and Medzhitov 2010;Ishii and Akira 2006). MYD88 Leu 265 Pro (L265P) mutation, occurred in about 30% of activated B-cell-like (ABC) DLBCL, is the most common non-synonymous and gain-of-function driver mutation. In contrast, this mutation is rare in GCB cases (Ngo et al. 2011;Dubois et al. 2017;Rovira et al. 2016a). Other recurrent non-L265P variants of MYD88 were also identified in DLBCL. However, their role in DLBCL lacks adequate attention due to the low prevalence. Several studies have indicated the difference between MYD88 L265P and MYD88 other but lacking of direct and detailed comparison in clinical and genetic features. Besides, the samples are limited, which might cause insufficient analysis and biased conclusions. Moreover, the survival of DLBCL patients with MYD88 L265P and MYD88 other varies in different studies, which is still a matter of debate (Chapuy et al. 2018;Dubois et al. 2017;Rovira et al. 2016;Xie et al. 2022

Patient selection and data collection
This study retrospectively collected 475 patients diagnosed with de novo DLBCL by two experienced pathologists at the CHCAMS and Suzhou Municipal Hospital from February 6th, 2007 to May 20th, 2022. A total of 132 patients with MYD88 variations were included for analysis. The inclusion criteria were as follows: (1) patients were histologically diagnosed with DLBCL based on the World Health Organization classification of Tumors of Hematopoietic and Lymphoid Tissue (2008) (Sabattini et al. 2010); (2) patients were confirmed MYD88 variation via next generation sequencing (NGS) technology as part of routine procedure; (3) patients who provided informed consent and adequate tissue for molecular and genetic detection. The exclusion criteria were those cases without adequate tissue for molecular and genetic detection or the information of MYD88 variation was unavailable. For survival analysis, we excluded patients with primary mediastinal large B-cell lymphoma, primary testicular DLBCL, and primary central nerves system lymphoma because of their particular biological behavior, clinical characteristics, treatment management, and prognosis. Only 85 patients who were received R-CHOP or R-CHOPlike regimens and with well-documented progression free survival (PFS) (from the date of initial diagnosis to the date of disease progression, relapse or death from any causes), overall survival (OS) (from the date of initial diagnosis to death from any causes), and survival status data were eventually included. The screening flowchart is displayed in Fig. 1 Patients baseline clinical characteristics, molecular and genetic alteration information, and follow-up data were collected, including gender, age, Ann Arbor stage, Eastern Cooperative Oncology Group (ECOG) performance status (PS), number of extranodal involvement sites, lactate dehydrogenase (LDH) level, International Prognostic Index (IPI) score, bulky disease, B symptoms, primary site, testicle/central nervous system (CNS) involvement, relapse, bone marrow/peripheral blood involvement, cell of origin (COO) type, first-line therapy, protein expression of CD5, CD10, CD20, BCL-2, BCL-6, c-MYC, MUM-1, Ki-67, and PD-L1, genetic alteration of CD79, TP53, BCL-2, BCL-6, c-MYC, PIM1, SGK1, cell cycle-related genes (CDKN2A, CCND3), NOTCH pathway-related genes (NOTCH1, NOTCH2, DTX1), JAK-STAT pathway-related genes (SOCS1, STAT3, STAT6), PI3K pathway-related genes (ITPKB, PTEN), immune-related genes (B2M, CIITA, CD58), epigeneticsrelated genes (KMT2D, KMT2C, CREBBP, EP300, TET2, EZH2, MEF2B), RAS pathway-related genes (GNA13, RHOA, RAS, BRAF). Any gene occurred alteration in the pathway will be defined as "pathway gene alteration".

Detection of chromosome translocation
Interphase fluorescence in situ hybridization (FISH) was performed on three-micrometer-thick FFPE tumor tissues using Vysis LSI CMYC/BCL2/BCL6 Dual Color Break Apart Rearrangement Probe (Abbott Molecular, Abbott Park, IL, USA) according to the manufacturer's instructions. Assessment of FISH signals was performed using Zeiss Axio Imager M2 epifluorescence microscope (Carl Zeiss, Oberkochen, Germany). Fifty tumor cells were counted, and the percentage of tumor cells with split-signal over 15% indicated the translocation of c-MYC, BCL-2, or BCL-6.

Next-generation sequencing
Genomic DNA was extracted from the collected FFPE DLBCL tissues using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany). DNA concentration was quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). DNA fragmentation was conducted using Covaris S2 Ultrasonicator (Covaris, Woburn, MA,  . Fragments of 200-250 bp were selected by AMPure beads (Agencourt AMPure XP kit; Beckman Coulter, Brea, CA, USA). End repair, phosphorylation, adaptor ligation, hybridization with capture-probe baits, hybrid selection with magnetic beads, and polymerase chain-reaction amplification were subsequently processed. Two capture panels were adopted, one consisted of 112 commonly altered genes in lymphoma and hematologic malignancies, the other covering 413 frequently mutated genes in DLBCL. There were 101 overlapping genes between the two panels. Capturebased targeted sequencing was performed on a Next Seq500 Sequencer (Illumina, Hayward, CA, USA) with pair-end reads at Geneplus-Beijing (Beijing, China) or Burning Rock Biotech (Guangzhou, China). Detailed sequencing procedure was performed as our previous study described (Qin et al. 2020(Qin et al. , 2021Jiang et al. 2020a, b).

Statistical analysis
Chi-squared test or Fisher's exact test when appropriated was adopted for the comparisons between categorical variables. Kaplan-Meier survival curve and log-rank test were performed for comparing the PFS and OS. P-value below 0.05 was considered statistically significant. All statistics were achieved via R software, version 4.21 (https:// www.rproje ct. org/).

Discussion
MYD88 mutation is a widely accepted pivotal oncogenic driver in B-cell lymphomas, among which, the hotspot MYD88 L265P is mostly studied. In this study, the mutation frequency of MYD88 L265P is 59%, which is slightly lower than Dubois et al. reported as 64%. For MYD88 other , Dubois et al. identified the frequent variants were p.S243A, p.S219C, p.V217P, and p.M232T, while our results showed that p.S219C, p.L273P, and p.S243N were the top three frequent variants (Dubois et al. 2017). This reflects great complexity and heterogeneity of MYD88 other variants in different cohorts of DLBCL. We found that MYD88 L265P was more common in non-GCB DLBCL compared with MYD88 other (83% vs. 60%, P = 0.004), which is consistent with previous reports (Ngo et al. 2011;Rovira et al. 2016a). As we know, the ABC DLBCL is characterized with chronic B-cell receptor signaling and activation of NF-κB (Davis et al. 2010(Davis et al. , 2001Havranek et al. 2017;Compagno et al. 2009). MYD88 L265P has been identified as non-synonymous and gain-of-function driver mutation, which can promote cell survival via assembling IRAK1 and IRAK4 contained protein complex, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, and JAK kinase activation of STAT3 in ABC DLBCL (Ngo et al. 2011 (Ngo et al. 2011). Given that, patients with MYD88 other should be also brought to attention in clinical. Intriguingly, there were 17% and 40% of GCB DLBCL patients with MYD88 L265P and MYD88 other , respectively. Albeit GCB DLBCL is not typically characterized by constitutive NF-κB activation. However, classic and alterative NF-κB pathway can be activated in both ABC and GCB DLBCL. About 60% ABC DLBCL and 30% GCB DLBCL present nuclear localization of NFKB1/p50 (classical pathway) and NFKB2/p52 (alternative pathway) (Compagno et al. 2009). Besides, mutations of BCR/PI3K signaling intermediates (RHOA, GNA13, and SGK1) and NF-kB modifiers (CARD11, NFKBIE, and NFKBIA) were also found to be enriched in cluster 4 subtype of DLBCL, which is primary GCB DLBCL (Chapuy et al. 2018). Whether those GCB DLBCL with MYD88 alteration will be involved in NF-κB pathway remains to be explored in future study. The genomic background hidden behind MYD88 is extremely complicated. We found that patients with MYD88 variation also frequently occurred PIM1, CD79B, KMT2D, TP53, BCL2, PRDM1, IRF4, CREBBP, BCL6, BM2, TET2, EP300, and DTX1 alterations. This is partly in accordance with previously reported results (Dubois et al. 2017;Shen et al. 2020). We found that the CD10 expression, NOTCH pathway gene alteration, and BCL-6 translocation were more common in MYD88 other than MYD88 L265P . On the contrary, the PIM1 mutation and PIM1 hypermutation were more common in MYD88 L265P than MYD88 other . These provided some clues implying why MYD88 other is more likely related to GCB subtype. For example, CD10 and BCL6 are markers of germinal center B cell. DLBCL patients with CD10 + , or CD10-/BCL6 + /MUM1-was classified into GCB subtype according to COO algorism (Alizadeh et al. 2000). Besides, according to several large-scale genetics-based classification of DLBCL subtype articles, PIM1 mutation was less frequent in GCB DLBCL (Schmitz et al. 2018;Chapuy et al. 2018;Lacy et al. 2020;Wright et al. 2020;Reddy et al. 2017). However, the BCL6 translocation, NOTCH1 mutation, and NOTCH2 mutation were all reported to be more common in non-GCB subgroup, which still remains confusing on their preference in MYD88 other ( Schmitz et al. 2018). Given above, the intricate genomic variation underlies MYD88 variation type and their relationship with COO subtype requires persistent investigation.
No significant difference in PFS and OS between MYD88 L265P and MYD88 other was observed in the whole DLBCL group. However, when focusing on the non-GCB type, the PFS of patients with MYD88 other was significantly shorter than those with MYD88 L265P . Several studies have investigated the survival of DLBCL patients between MYD88 L265P and MYD88 other variants. In Dubois's study, he performed a survival analysis on 26 ABC DLBCL patients with MYD88 L265P and 9 ABC DLBCL patients with MYD88 other , who all received R-CHOP therapy. No statistical significance was found in OS and PFS (Dubois et al. 2017). Another study also investigated the PFS and OS between 39 patients with MYD88 L265P and 8 patients with MYD88 other . Their results suggested that the OS and PFS of patients with MYD88 L265P showed a trend to be inferior than that of patients with MYD88 other , but didn't achieve statistically significant (Rovira et al. 2016). The samples in the two above studies are limited, which might lead to insufficient statistical efficacy and bias results. Compared with the above two studies, larger samples including 42 patients with MYD88 L265P and 43 patients with MYD88 other were performed for survival analysis in this study. MYD88 other was indicated as an unfavorable factor in DLBCL patients especially in non-GCB subgroup. Why patients with MYD88 other showed not superior survival than those with MYD88 L265P remains to be discussed. We did find that patients with MYD88 other presented higher percentage of advanced stage, extranodal site ≥ 2, and elevated LDH than those with MYD88 L265P , which may provide some hints. Nevertheless, the genetic alteration and corresponding biological function difference between MYD88 L265P and MYD88 other may be the predominant factors influencing survival, which needs further study.
Altogether, this study highlights the clinical and genomic heterogeneity hidden behind the MYD88 L265P and MYD88 other variants. Furthermore, prognostic differences were revealed, most notably highlighting the survival of patients with MYD88 other is not superior than those with MYD88 L265P , even poorer when focusing on the non-GCB subtype. Finally, this study added the importance of MYD88 variants status to the current knowledge and provided reference for the individualized targeted therapy and management of DLBCL patients.