Anatomical pathologyDNMT1 is predictive of survival and associated with Ki-67 expression in R-CHOP-treated diffuse large B-cell lymphomas
Introduction
Diffuse large B-cell lymphoma (DLBCL) is an aggressive subtype of mature B-cell neoplasm1 where approximately 30–40% of the patients develop relapsed/refractory disease despite the use of the standard immunochemotherapy regimen R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).2 Gene expression profiling has identified at least two clinically and biologically distinct DLBCL subtypes: germinal centre (GC) B-cell-like DLBCL (GCB-DLBCL) and activated B-cell-like DLBCL (ABC-DLBCL) subtypes,3 with ABC-DLBCL patients exhibiting worse outcomes in response to the R-CHOP regimen.4, 5
Altered epigenetic regulation including methylation profiles is a recognised feature of DLBCL.6 Molecular subtypes of DLBCL (ABC- or GCB-DLBCL) exhibit distinct DNA methylation profiles,7 and a greater magnitude of DNA methylation changes from normal GC B cells is associated with worse outcomes in R-CHOP-treated DLBCL patients.8 DNA methylation of gene promoters at CpG sites and intragenic regions is mediated by DNA methyltransferases (DNMTs): DNMT1, DNMT3A, and DNMT3B. Of the DNMTs, DNMT3A and DNMT3B mediate de novo DNA methylation by establishing methylation of unmodified DNA, while DNMT1 maintains the methylation pattern by preferentially methylating the nascent unmethylated strand during cell replication and DNA synthesis.9
Studies using murine models have shown that Dnmt1 is required for GC formation and Dnmt1 hypomorphic mice show increased DNA damage in GC B cells.10 Moreover, Dnmt1 is required to sustain T-cell lymphomas by preventing apoptosis in a murine MYC-induced T-cell lymphoma model.11 In terms of DLBCL, DNMT1 protein was overexpressed in a series of DLBCL cases treated with CHOP-like regimens with concomitant expression of both DNMT1 and DNMT3B being correlated with treatment resistance.12 Priming of high-risk DLBCL patients with azacytidine, that mainly targets DNMT1 and DNMT3, followed by R-CHOP treatment has been shown to achieve a high complete remission rate,13 and decitabine (targets DNMT1 and DNMT3) has recently completed phase I trials (ClinicalTrials.gov identifier: NCT00109824 and NCT00275080) investigating the efficacy and safety of the drug in DLBCL.
However, it is unknown whether DNMT1 expression confers prognostic value in DLBCL patients treated with R-CHOP. In this study, we set out to investigate the expression pattern of DNMT1 in DLBCL cases and its correlation with clinico-demographic parameters and survival in a multi-centre series of R-CHOP-treated DLBCL patients (n = 230).
Section snippets
Patient samples
The R-CHOP-treated primary DLBCL cases (n = 230) have been described previously, with 40 cases from Spain, 33 cases from Malaysia,14 and another 157 cases from Denmark.15 Immunostaining was performed with other markers previously.16, 17, 18, 19 Clinico-demographic data of all patients are listed in Table 1. The cohort consisted of 133 males and 97 females (1.37:1 ratio) with median age of 64 years (20–91 years), and median overall survival (OS) and progression-free survival (PFS) (1–147 months
DNMT1 is highly expressed in germinal centres of lymphoid follicles
We set out to determine the optimal primary anti-DNMT1 antibody that produces specific staining by IHC, and four distinct antibody clones were tested on reactive tonsils. The polyclonal ab19905 and monoclonal clone 2B5 antibodies (both from Abcam) yielded strong and specific staining of the GCs, while weak or absent staining was observed in tonsils immunostained with the other two antibodies (clone 60B1220.1 from Abcam; clone 18/DNMT1 from BD Biosciences, USA) even when tested at low dilutions (
Discussion
In this study, by using a validated monoclonal anti-DNMT1-2B5 antibody, we showed that DNMT1 was frequently expressed in normal GC B cells and in primary DLBCL cases (n = 209, 90.9%). We observed that DLBCL cases negative for DNMT1 expression showed particularly poor survival with less than 30% of these patients achieving 5-year OS or PFS, and they formed the majority of the patients (n = 21 of 33) within the DNMT1 <20% frequency subgroup.
Epigenetic heterogeneity, characterised by DNA
Acknowledgements
The technical assistance of Jamaliah binti Lin from Department of Pathology, Universiti Sains Malaysia, is gratefully acknowledged.
References (59)
- et al.
The 2016 revision of the World Health Organization classification of lymphoid neoplasms
Blood
(2016) - et al.
Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy
Blood
(2011) - et al.
Molecular subtyping of diffuse large B-cell lymphoma: update on biology, diagnosis and emerging platforms for practising pathologists
Pathology
(2016) - et al.
The epigenetic basis of diffuse large B-cell lymphoma
Semin Hematol
(2015) - et al.
DNA methylation signatures define molecular subtypes of diffuse large B-cell lymphoma
Blood
(2010) - et al.
Variability in DNA methylation defines novel epigenetic subgroups of DLBCL associated with different clinical outcomes
Blood
(2014) - et al.
DNA methylation: a promising landscape for immune system-related diseases
Trends Genet
(2012) - et al.
DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation
Blood
(2011) - et al.
Low HIP1R mRNA and protein expression are associated with worse survival in diffuse large B-cell lymphoma patients treated with R-CHOP
Exp Mol Pathol
(2015) - et al.
Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray
Blood
(2004)
Ki67 assessment in breast cancer: an update
Pathology
Germinal centres in T-cell-dependent antibody responses
Immunol Today
The FOXO1 Transcription Factor Instructs the Germinal Center Dark Zone Program
Immunity
Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response
Blood
Laccaic acid A is a direct, DNA-competitive inhibitor of DNA methyltransferase 1
J Biol Chem
Identification of DNMT1 selective antagonists using a novel scintillation proximity assay
J Biol Chem
Developing new chemical tools for DNA methyltransferase 1 (DNMT 1): a small-molecule activity-based probe and novel tetrazole-containing inhibitors
Bioorg Med Chem
Novel drug targets for personalized precision medicine in relapsed/refractory diffuse large B-cell lymphoma: a comprehensive review
Mol Cancer
Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling
Nature
Essential role for Dnmt1 in the prevention and maintenance of MYC-induced T-cell lymphomas
Mol Cell Biol
DNA methyltransferase DNMT3b protein overexpression as a prognostic factor in patients with diffuse large B-cell lymphomas
Cancer Sci
Mechanism-based epigenetic chemosensitization therapy of diffuse large B-cell lymphoma
Cancer Discov
Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone
J Clin Oncol
Reciprocal expression of the endocytic protein HIP1R and its repressor FOXP1 predicts outcome in R-CHOP-treated diffuse large B-cell lymphoma patients
Leukemia
FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas
Leukemia
FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures
Oncotarget
TRPM4 expression is associated with activated B cell subtype and poor survival in diffuse large B cell lymphoma
Histopathology
Expression profiling shows differential molecular pathways and provides potential new diagnostic biomarkers for colorectal serrated adenocarcinoma
Int J Cancer
Claudin-1 expressions decrease in pterygium with respect to normal conjunctiva
Cutan Ocul Toxicol
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2021, EBioMedicineCitation Excerpt :Levels of K27ac within the TCF4 SEs are significantly correlated with expression level (r=0.6642, p<0.0005) (Fig. 2H). DNMT1 is a maintenance DNA methyltransferase whose downregulation has been implicated in EBV latency and is associated with worse outcomes in DLBCL [71–73]. We detected significantly lower (2- to 10-fold) DNMT1 expression in all BCL subtypes compared to all healthy control B-cell subsets (Fig. 2I-J).
DNMT1: A key drug target in triple-negative breast cancer
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DNMT1 is associated with cell cycle and DNA replication gene sets in diffuse large B-cell lymphoma
2018, Pathology Research and PracticeCitation Excerpt :DNMT1 has been implicated in the pathogenesis of several malignancies being overexpressed in hepatocellular carcinoma [27] and prostate cancer [28], and shown to promote growth of triple-negative breast cancer [29], survival of multiple myeloma cells [30] and MYC-induced T-cell lymphomas [31]. We previously showed that DNMT1 protein was frequently expressed in DLBCL patient cases [14] and in this study, we integrated GEP data of DNMT1-knockdown DLBCL cells (HT cell line) with that of primary DLBCL cases to uncover the putative roles of DNMT1 in DLBCL. While tumor-derived cell lines are important in vitro models most widely used in cancer research [32] that generally mirror primary tumors [33,34], several studies have found underlying genomic differences between cancer cell lines and primary tumors [35–38].