Krieken, J. Han van

Abstract Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a rare and aggressive lymphoma with molecular heterogeneity not well characterize. In this study, we performed next-generation sequencing analysis for a large number of DNA and RNA samples from patients with PT-DLBCL. DNA sequencing analysis identified ≥ 3 chromosomes with copy number variations (CNVs) and microsatellite instability as prognostic biomarkers, rather than MYD88 mutations and genetic subtypes. Remarkably, targeted RNA-seq analysis in 195 patients revealed that TP53 mutations with a ≥ 40% variant allele frequency had significantly adverse prognostic impact, and that a 150-gene expression signature subdivided PT-DLBCL into two distinct clusters, termed as testicular lymphoma tumor (TLT) and microenvironment (ME) subtypes. The TLT subtype featured upregulation of genes involved in B-cell receptor signaling, cell cycle, DNA damage and repair, higher frequencies of CNVs and MYD88 mutations, elder ages, larger tumor sizes, and significantly poorer survival. Genomic microRNA profiling analysis identified significantly differentially expressed microRNAs between 113 PT-DLBCL and 180 systemic DLBCL patients, and further subdivided the PT-DLBCL cohort by microRNA signatures. The subcohort with upregulation of 16 microRNAs associated with PT-DLBCL and testicular tissue expression had significantly better survival. This study revealed characteristic genetic, gene expression, and microRNA profiles and heterogeneity in PT-DLBCL.

Abstract Introduction: MYC+BCL2+ double expression (DE) is a biomarker for poor prognosis in diffuse large B-cell lymphoma (DLBCL), likely due to the cooperation between MYC and BCL2 oncoproteins in tumor cells. However, as generally MYC and BCL2 are separately assessed on two different slides by immunohistochemistry (IHC), MYC+ and BCL2+ results are from different cells. With the WHO-recommended cutoffs, ≥40% for MYC and ≥50% for BCL2 (sum < 100%), there is chance that the so-called DE comes from non-overlapping regions of the tumor (not single-cell DE). Moreover, tumor expression scores based on morphology assessment often vary among pathologists. In this study, we performed fluorescent multiplex IHC (fmIHC) using a MultiOmyxTM immunofluorescence platform (NeoGenomics Laboratories, Inc.) to determine single-cell MYC and BCL2 co-expression for prognostic analysis in DLBCL. Patients and Methods: Co-expression of MYC, BCL2, and 11 immunophenotypic markers by fmIHC was digitally quantified for 547 patients with DLBCL. Percentage of cells with MYC/BCL2 double or single expression was calculated for various cell types. Prognostic analysis was performed for de novo DLBCL cases, including 108 patients treated CHOP chemotherapy, and 356 patients treated with rituximab (R)-CHOP. Results: First, although MYC and BCL2 were expressed in multiple cell types, the vast majority (>90%) of MYC+BCL2+ DE cells were lymphoma cells. Frequencies of MYC and BCL2 co-expression were consistent with random distribution of independent MYC/BCL2 expression in positive cases. The activated B-cell-like subtype compared with the germinal center B-cell-like subtype had a significantly higher mean percentage of MYC+BCL2+ DE, but not single BCL2 or MYC expression, in PAX5+ cells. Second, in both the R-CHOP and CHOP cohorts, high percentages of DE in PAX5+ cells and in all DAPI+ cells were associated with significantly poorer survival of DLBCL patients. Very high percentage of single BCL2 expression in PAX5+ cells was also associated with poorer survival, however, only in a small number of patients in both cohorts. Only in the R-CHOP cohort, high single MYC expression in PAX5+ cells (in a significant proportion of the cohort) was associated with significantly poorer overall survival. In the R-CHOP cohort, eight cases had MYC/BCL2 double hit by FISH, including 3 cases with MYC/IGH fusions (DE) and 5 cases with MYC/non-IGH rearrangements (only one DE). The exclusion of these double hit cases did not impact the significance of DE and single MYC+ expression. Summary: True MYC+BCL2+ double protein expression at the single cell level is predominantly in lymphoma cells, and associated with significantly poor survival of DLBCL patients. Our results suggest high cutoffs for common MYC/BCL2 IHC to identify true double expressor lymphoma. Citation Format: Zijun Yidan Xu-Monette, Yong Li, Qingyan Au, Harry Nunns, Wenyu Shi, Alexandar Tzankov, Carlo Visco, Govind Bhagat, Eric Hsi, Xiaoxian X. Zhao, Karen Dybkaer, April Chiu, Wayne Tam, Youli Zu, Fredrick B. Hagemeister, Andrew Song, Michele Pellegrino, Heounjeong Go, Maurilio Ponzoni, Andrés Ferreri, Michael B. Møller, Benjamin Parsons, J. Han van Krieken, Miguel A. Piris, Jane N. Winter, Mingzhi Zhang, Bing Xu, Ken H. Young. Single-cell double expression of MYC and BCL2 proteins by fluorescent multiplex IHC is mainly in lymphoma cells with significant prognostic impact in large B-cell lymphoma while suggesting high cutoffs for routine IHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6418.

Abstract Purpose: Tumor-infiltrating B lymphocytes (TIL-B) have demonstrated prognostic and predictive significance in solid cancers. In this study, we aimed to distinguish TIL-Bs from malignant B-cells in diffuse large B-cell lymphoma (DLBCL) and determine the clinical and biological significance. Experimental Design: A total of 269 patients with de novo DLBCL from the International DLBCL R-CHOP Consortium Program were studied. Ultra-deep sequencing of the immunoglobulin genes was performed to determine B-cell clonotypes. The frequencies and numbers of TIL-B clonotypes in individual repertoires were correlated with patient survival, gene expression profiling (GEP) data, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex IHC at single-cell resolution. Results: TIL-B abundance, evaluated by frequencies of normal B-cell clonotypes in the immunoglobulin repertoires, remarkably showed positive associations with significantly better survival of patients in our sequenced cohorts. DLBCLs with high versus low TIL-B abundance displayed distinct GEP signatures, increased pre-memory B-cell state and naïve CD4 T-cell state fractions, and higher CD4+ T-cell infiltration. TIL-B frequency, as a new biomarker in DLBCL, outperformed the germinal center (GC) B-cell–like/activated B-cell–like classification and TIL-T frequency. The identified TIL-B–high GEP signature, including genes upregulated during T-dependent B-cell activation and those highly expressed in normal GC B cells and T cells, showed significant favorable prognostic effects in several external validation cohorts. Conclusions: TIL-B frequency is a significant prognostic factor in DLBCL and plays a crucial role in antitumor immune responses. This study provides novel insights into the prognostic determinants in DLBCL and TIL-B functions with important therapeutic implications.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2–targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. Implications: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

Key PointsPhenotypic and genotypic profiling of MDM2 in DLBCL. MDM2 as a negative regulator of p53 tumor suppressor function.