Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (oncovin), and ...prednisone R-CHOP); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.
•KMT2D and TP53 are predominantly mutated in rrDLBCL and remain clonally stable following therapy.•MS4A1 mutations are commonly acquired and undergo clonal expansion following treatment with rituximab-containing therapy.
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Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and is characterized by its genetic and clinical heterogeneity. Patients can develop DLBCL de ...novo or as a transformation from other lymphoid malignancies, most commonly follicular lymphoma. For patients with relapsed/refractory DLBCL (rrDLBCL), prognosis is extremely poor with 2-year overall survival of 20-40%. While numerous treatments are under investigation to improve patient outcomes, the success of these treatments has been limited as the genetic mechanisms underpinning treatment resistance are largely unknown. Identifying genomic alterations which contribute to relapse may improve salvage therapy for patients with rrDLBCL or allow patients to be stratified prior to frontline treatment.
Methods
To identify genomic alterations which contribute to R-CHOP resistance, we previously collected samples from patients enrolled in four clinical trials exploring candidate salvage therapies for patients with rrDLBCL as well as a retrospective rrDLBCL cohort, totalling 193 cases (133 de-novo DLBCL, 60 transformed). Plasma samples were collected from each patient upon relapse along with diagnostic tissue biopsies where available. A combination of exome sequencing and target-panel sequencing of lymphoma associated genes was performed on circulating tumour DNA and tissue biopsies (if available). Mutations implicated in R-CHOP resistance were identified through two complimentary strategies. First, the mutation frequency of recurrently mutated genes across de novo rrDLBCL samples was compared to a cohort of unrelated diagnostic DLBCL cases (n=1691) to identify genes enriched for mutations. Second, the genomic landscape and tumor clonal structure was compared prior to and following R-CHOP to identify mutations in each patient that underwent clonal expansion following therapy. Anti-CD20 antibody binding affinity of MS4A1 mutants was evaluated using flow cytometry on transfected CHO-S cells.
Results
We have identified five genes enriched for mutations in our rrDLBCL cohort relative to diagnostic DLBCL: KMT2D (Mutated in 49%, Q=0.0385), TP53 (47%, Q=1.07x10-9), FOXO1 (11%, Q=0.0727), NFKBIE (11%, Q=0.0385), and MS4A1 (8%, Q=0.0522). Consistent with its characterization as a poor prognostic marker, mutations in TP53 were typically present at diagnosis and remained stable following R-CHOP therapy for both de novo and transformed DLBCL (23/27 cases, 85%). Recurrent mutations affecting Arg248 of TP53 (6.8%, Q=0.0413) were also clonally stable and have previously been associated with poor overall survival across several cancer types. The histone methyltransferase KMT2D is dominated by nonsense and frameshift mutations which were stable or underwent clonal expansion following R-CHOP (17/19, 89%). Recurrent missense mutations in MS4A1 targeted the small loop and adjacent transmembrane domains of CD20, including several patients with a Tyr86 mutation. Transfected cells carrying Tyr86Cys or Leu66Arg mutations were not bound by rituximab or other anti-CD20 antibodies including obinituzumab and ofatumumab. Subclonal populations containing MS4A1 mutations underwent clonal expansion (6 cases) or were stable (1 case) following treatment, including one case with multiple MS4A1 mutations in distinct subclonal populations which both underwent clonal expansion. In another unique case, a series of ctDNA samples were available prior to and following R-CHOP and salvage therapy, where we again observed convergent evolution of two mutually exclusive clonal subpopulations containing MS4A1 mutations. The first subpopulation underwent clonal expansion following frontline therapy but was extinguished following salvage therapy, while the other subpopulation underwent clonal expansion following salvage therapy and harboured a transmembrane domain mutation.
Conclusion
Mutations in TP53 and truncating mutations in KMT2D are generally present prior to treatment and will be investigated as biomarkers of treatment failure. Additional mutations are not always present at diagnosis, but their emergence can be detected in ctDNA and relapsed tissue, specifically mutations in MS4A1. As mutations in MS4A1 attenuate rituximab binding and are recurrently associated with clonal expansion, they likely impart a selective advantage and lead to resistance against anti-CD20 antibodies.
Michaud:Epizyme: Employment. Daigle:Epizyme: Employment. Jain:Kite/Gilead: Consultancy. Kuruvilla:Roche: Honoraria; Astra Zeneca: Honoraria; Novartis: Honoraria; Merck: Honoraria; Karyopharm: Honoraria; Gilead: Honoraria; Celgene: Honoraria; BMS: Honoraria; Amgen: Honoraria; Seattle Genetics: Consultancy; Roche: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Gilead: Consultancy; Janssen: Research Funding; Roche: Research Funding; BMS: Consultancy; Abbvie: Consultancy; Seattle Genetics: Honoraria; Janssen: Honoraria. Assouline:Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau. Scott:NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting Institution, Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding. Johnson:BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding.
Abstract
A significant proportion of diffuse large B-cell lymphoma (DLBCL) patients treated with immunochemotherapy containing rituximab (R-CHOP) exhibit either primary or acquired treatment ...resistance. The advancement of therapeutics in the relapse setting has likely been encumbered by our limited understanding of the molecular features that underlie resistance to R-CHOP. Unfortunately, our knowledge of DLBCL genetics is mostly limited to analyses conducted on diagnostic tissue biopsies, which have not been exposed to the selective pressures imposed by therapy. Identifying genetic alterations that contribute to treatment resistance may reveal additional treatment options and lead to biomarkers allowing patients to be paired with appropriate treatments. Genetic subgroups are gaining popularity as a new strategy to implement precision medicine in DLBCL (1). The relevance of these and other biomarkers in the relapse setting remains unclear due to limited genetic exploration of relapsed and refractory DLBCL (rrDLBCL). Progress has been limited, in part, by the requirement of tissue biopsies collected after relapse. It is well established that quantitative genomic techniques such as digital PCR and targeted sequencing can be used to determine the proportion of tumor DNA in plasma from lymphoma patients (2). With a sufficiently broad panel, sequencing affords additional opportunities including the ability to identify subclonal structure and population dynamics over time. This presentation will discuss our recent analysis of a large collection of ctDNA primarily comprising DLBCL patients on various clinical trials (3). Targeted sequencing of these samples and comparison to exome data from a meta-cohort of previously characterized untreated DLBCL biopsies revealed six genes significantly enriched for mutations upon relapse. We found both TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations persisted in the dominant clone following relapse, suggesting a role in primary treatment resistance. By inferring subclonal dynamics, we observed recurrent patterns of clonal expansion and contraction following rituximab-based therapy, with MS4A1 mutations representing the only example of consistent clonal expansion. MS4A1 missense mutations within the transmembrane domains led to loss of CD20 expression in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. Our analysis nominates TP53 and KMT2D mutation status as novel prognostic factors that may facilitate the identification of high-risk patients prior to therapy. Moreover, we have demonstrated the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens. In certain scenarios whole-exome sequencing (WES) or whole-genome sequencing (WGS) can be successfully applied to ctDNA, thereby allowing the identification of mutations, structural variation, and copy number changes. Low-pass sequencing of shotgun libraries can also be used to ascertain course estimates of ctDNA levels as well as the copy number landscape (4). Given the importance of copy number and structural alterations in the inference of genetic subgroups, these methods may allow the exploration of these groups and their stability over time. Through a series of illustrative examples, this presentation will explore the benefits of each of these techniques in the study of tumor evolution and acquired treatment resistance in DLBCL.
References: 1. Morin RD, Scott DW. DLBCL subclassification: Divide and conquer? Blood 2020;135:1722–4. 2. Rossi D et al. The development of liquid biopsy for research and clinical practice in lymphomas: Report of the 15-ICML workshop on ctDNA. Hematol Oncol 2020;38:34–7. 3. Rushton CK et al. Genetic and evolutionary patterns of treatment resistance in relapsed B-cell lymphoma. Blood Adv 2020;4:2886–98. 4. Adalsteinsson VA et al. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors. Nat Commun 2017;8:1324.
Citation Format: Nicole Thomas, Laura K. Hilton, Neil Michaud, Kevin Bushell, Ryan Rys, Michael Jain, Lois Shepherd, Marco A. Marra, John Kuruvilla, Michael Crump, Koren Mann, Sarit Assouline, Christian Steidl, Mark S. Cragg, David W. Scott, Nathalie Johnson, Ryan D. Morin, Christopher K. Rushton, Sarah E. Arthur, Miguel Alcaide, Matthew Cheung, Aixiang Jiang, Krysta M. Coyle, Kirstie L. S. Cleary. Detecting and quantifying mutations associated with treatment resistance in aggressive lymphomas using ctDNA abstract. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA42.
Retinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to ...regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARα fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.
Retinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to ...regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARα fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.
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