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MYD88 mutational status improves classification and prognostication in DLBCL
Introduction
Diffuse large B-cell lymphoma (DLBCL) is characterized by substantial heterogeneity in tumor biology and clinical behavior.1,2 Currently, rituximab, cyclophosphamide, dox- orubicin, vincristine, and prednisone (R-CHOP) is used as a ‘one-size-fits-all’ treatment. Unfortunately, a consider- able percentage of patients will experience chemorefracto- ry disease or relapse, resulting in a 5-year overall survival (OS) of approximately 60%.3 Particularly, patients with chemorefractory disease or an early relapse have a poor prognosis. For optimal counseling, DLBCL patients are categorized in risk groups according to the IPI.4 The IPI consists of clinical and biochemical parameters, but does not include tumor biological characteristics or provide any indication for precision medicine.5
The recently updated WHO classification of lymphoid neoplasms (2016) recognizes this heterogeneity by includ- ing selected drivers of lymphomagenesis for subclassifica- tion of DLBCL, i.e. the delineation of high-grade B-cell lymphomas (HGBL) with MYC and BCL2 and/or BCL6 rearrangements, and of Epstein-Barr virus-positive (EBV+) DLBCL.6 MYC, BCL2, and BCL6 rearrangements are found in respectively 4-14%, 20-30%, and ~20% of DLBCL.7-9 HGBL comprise approximately 5-10% of all DLBCL.9-11 It is thought that the combination of MYC-stimulated cell proliferation and anti-apoptotic effects of BCL2 in HGBL cause aggressive growth, relative resistance to therapy, and inferior OS.12 In addition, Asian studies showed a fre- quency of 1-14% EBV positivity in DLBCL and an associ- ation with inferior survival.13,14 EBV-associated viral pro- teins, such as latent membrane proteins (LMP)-1/2 and nuclear antigens, stimulate proliferation of B-cells via acti- vation of nuclear factor-kappa-B (NFκB), regulate immune evasion, and inhibit apoptosis.13
In the search for additional oncogenic drivers and to dis- criminate different molecular DLBCL subtypes, large next- generation-sequencing (NGS) studies have revealed specif- ic mutational profiles that reflect the dysregulation of dis- tinct intracellular pathways, including epigenetic regula- tion and NF-κB, Toll-like receptor (TLR), and B-cell recep- tor (BCR) signalling.1,2,15,16 Recurrent ‘hotspot’ mutations in MYD88 (L265P) and CD79B (Y196) belong to the most prevalent sequence alterations in DLBCL. By altering the toll/interleukin-1 receptor domain of MYD88, the L265P increases interaction and consecutive phosphorylation of downstream targets, potentially without external stimuli from the TLR.17 The connection of MYD88 with BCR sig- nalling within the so-called ‘My-T-BCR’ supercomplex facilitates activation of the NF-κB pathway via TLR9.2 Hotspot mutations, such as Y196, in the CD79B subunit of the BCR lead to increased BCR expression and inhibi- tion of feedback in the BCR signalling pathway by atten- uating downstream Lyn kinase. Therefore, CD79B muta- tions are thought to contribute to lymphomagenesis by enhancing chronic active BCR signalling.18
Both MYD88 and CD79B mutations are more prevalent in the so-called non-germinal center B-cell (GCB)-type DLBCL according to the cell-of-origin (COO) concept, originally developed on the basis of gene expression pro- filing.1,2,19 In addition, the prevalence of these mutations varies greatly among DLBCL originating at different anatomical sites. We recently described a high percentage of MYD88 L265P and CD79B Y196 mutations in intravas- cular large B-cell lymphomas (44% MYD88 and 26%
CD79B).20 A high frequency of these mutations has also been found in other extranodal DLBCL, such as primary cutaneous DLBCL, leg type,21 orbita/vitreoretinal DLBCL,22-24 primary breast DLBCL,25 and DLBCL present- ing at immune-privileged (IP) sites, i.e. primary testicular DLBCL (PTL)26 and primary central nervous system B-cell lymphoma (PCNSL).27-29 Several studies have shown that MYD88 mutations are associated with inferior OS in DLBCLs compared to wild-type MYD88.30, 31
Despite the increasing knowledge of the landscape of genetic drivers in DLBCL, the clinical implications of dif- ferent oncogenic driver mutations remain unclear,32 and the R-CHOP regimen is used as a uniform treatment. Since patients with chemorefractory disease or relapses after R-CHOP have a poor outcome, the global 5-year OS in DLBCL is approximately 60%.3 While HGBL patients have been recognized as a particularly unfavorable sub- group, prognostication for the remaining DLBCL is based on clinical and biochemical parameters that define the IPI as well as primary extranodal manifestations.4,5 In con- trast, the prognostic significance and interaction of muta- tions in MYD88 and CD79B with standard molecular aberrations (as designated by the WHO 2016) have not yet been conclusively elucidated. Therefore, the present study investigated whether the assessment of the mutational status of MYD88 and CD79B would improve the classifi- cation and prognostication of DLBCL.
Methods
Patient cohort
This retrospective study investigated a cohort of 250 primary DLBCL. DLBCL patients were diagnosed between 2000-2016 at the Amsterdam University Medical Center (AUMC), the Leiden University Medical Center (LUMC), and their affiliated hospitals. In all cases, diagnosis was centrally revised following the WHO classification 2008. A subset of this cohort was previously pub- lished without survival analysis.28,29 As our academic hospitals are tertiary referral centers, this cohort is enriched for IP locations. Formalin-fixed and paraffin-embedded (FFPE) tissue samples were obtained during standard diagnostic procedures. The study was performed in accordance with the Dutch Code for Proper Secondary Use of Human Tissue in accordance with the local institutional board requirements and the revised Declaration of Helsinki 2008 and was approved by the medical ethics commit- tees of both the AUMC (W15_213#15.0253) and the LUMC (B16.048). Patients were eligible in case tissue was available and MYD88 mutational analysis was successful.
Histopathologic and molecular characterization
In the majority, immunohistochemistry was performed for CD20, CD10, BCL6, MUM1, and BCL2. The Hans’ algorithm was used for the COO classification.33 The EBV status was assessed by EBER-ISH. MYC, BCL2, and BCL6 rearrangements were analyzed by FISH using break-apart probes. Antibodies and probes are depicted in the Online Supplementary Table S1.20,29 In the AUMC, DNA was isolated using the QIAamp DNA Micro kit (Qiagen) and mutational status of MYD88 and CD79B was established by allele-specific PCR, followed by mutation-specific primers and confirmed by Sanger sequencing, as described before.28, 29 In the LUMC, DNA isolation was automatically performed with the TPS robot (Siemens Healthcare Diagnostics), as presented previously.34 The Ampliseq Cancer Hotspot Panel V.2-V.4 (Thermo Fisher Scientific) was used for the detection of variants in MYD88 (exons
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