CCND1 Translocation Research Articles

Cyclin-D1 rearrangement as a secondary event in the large cell transformation of splenic marginal zone lymphoma with a TP53 deletion.

IGH::CCND1 translocation has been implicated as a preliminary finding in mantle cell lymphoma (MCL) and plasma cell myeloma, with cyclin D1 over-expression by immunohistochemistry stain, without gene rearrangement, reported in other hematologic neoplasms. CCND1 rearrangement has been reported infrequently as a secondary event in high-grade B-cell lymphomas. We present the case of an elderly man who was found to have a splenic marginal zone lymphoma with a heterozygous TP53 deletion. Years later at the time of relapse, the patient acquired a CCND1 rearrangement, in addition to apersistent TP53 deletion. Sequencing of the two lymphomas demonstrated clonal relatedness of the two processes. To our knowledge, this is the first report of a splenic marginal zone lymphoma witha TP53 deletion at diagnosis, evolving into a large B-cell lymphoma with a CCND1 rearrangement.

Large B-cell lymphomas with CCND1 rearrangement have different immunoglobulin gene breakpoints and genomic profile than mantle cell lymphoma

Mantle cell lymphoma (MCL) is genetically characterized by the IG::CCND1 translocation mediated by an aberrant V(D)J rearrangement. CCND1 translocations and overexpression have been identified in occasional aggressive B-cell lymphomas with unusual features for MCL. The mechanism generating CCND1 rearrangements in these tumors and their genomic profile are not known. We have reconstructed the IG::CCND1 translocations and the genomic profile of 13 SOX11-negative aggressive B-cell lymphomas using whole genome/exome and target sequencing. The mechanism behind the translocation was an aberrant V(D)J rearrangement in three tumors and by an anomalous IGH class-switch recombination (CSR) or somatic hypermutation (SHM) mechanism in ten. The tumors with a V(D)J-mediated translocation were two blastoid MCL and one high-grade B-cell lymphoma. None of them had a mutational profile suggestive of DLBCL. The ten tumors with CSR/SHM-mediated IGH::CCND1 were mainly large B-cell lymphomas, with mutated genes commonly seen in DLBCL and BCL6 rearrangements in 6. Two cases, which transformed from marginal zone lymphomas, carried mutations in KLF2, TNFAIP3 and KMT2D. These findings expand the spectrum of tumors carrying CCND1 rearrangement that may occur as a secondary event in DLBCL mediated by aberrant CSR/SHM and associated with a mutational profile different from that of MCL.

Genomic and Transcriptomic Landscape of Hyper-Apobec Multiple Myeloma

Introduction APOBEC mutational signatures are one of the most important mutational signatures in newly diagnosed multiple myeloma (NDMM). APOBEC mutagenesis primarily works through two enzymes, APOBEC3A (A3A) and APOBEC3B (A3B). While these genes are expressed in most NDMM patients, their influence on APOBEC mutagenesis appear to be extremely heterogeneous, with some patients having no detectable mutations and others having high contribution (hyper-APOBEC). Methods To decipher the mechanism of APOBEC mutagenesis, we interrogated 752 low coverage long insert whole genomes, 723 whole exomes (WES) and 767 RNA sequencing (RNA-Seq) from NDMM patients enrolled in the CoMMpass study. Results APOBEC mutational activity was identified in 416/723 (57.5%) patients using WES. Of these, 41 (5.8%) had MAF/MAFB translocations. Overall, 48/723 (6.6%) patients were defined as hyper-APOBEC (13 without MAF/MAFB events). We used differential expression (DE) analysis between three groups: hyper-APOBEC with MAF/MAFB (HA_TRA: n=31), hyper-APOBEC without MAF/MAFB (HA_NORM: n=8), and non-hyper-APOBEC without MAF/MAFB (WT: n=510). 534 genes were significantly associated with hyper-APOBEC independently from MAF/MAFB events. Importantly, A3A and A3B expression were higher in hyper-APOBEC compared to other cases (logFC=2.38, logFC=1.58 respectively). Using Spearman's correlation, we identified 50 significantly correlated genes (r-squared>0.18; p<0.01) with A3B. Most of the genes belonged to the APOBEC-associated group defined by the DE and were enriched for cell cycle/proliferation activity, suggesting a link between highly proliferative disease and hyper-APOBEC. To validate this finding, we evaluated the link between these APOBEC-associated genes and hyper-APOBEC among ICGC breast cancers, a tumor known to have high prevalence of APOBEC mutagenesis. Overall, 35/50 of these genes showed similar correlation between hyper-APOBEC breasts and MM. Interestingly, most of these genes appear to be negatively controlled by E2F4 (DREAM complex), and positively controlled by E2F1, FOXM1, and MYBL2 (i.e. cell cycle genes). In line with mouse models (Roelofs et al., eLife, 2020), our data confirmed that highly proliferating tumors suppress the DREAM complex leading to induction of A3B. Overall, these data suggest a strong link between proliferation and A3B expression and mutagenesis. However, not all high proliferating tumors had high APOBEC mutagenesis. To better investigate this aspect, we compared 139 known MM genomic events to identify potential differences between the hyper-APOBEC group (HA) and the rest of the cohort (WT). HA_TRA and HA_NORM showed similarities, including a highly complex genomic profile with enrichment for 1q gain/amp (p < 0.02), 13q (p < 0.05) and 16q deletions (p<0.01). Interestingly, 5/13 (38.4%) of HA_NORM samples harbor CCND1 translocation. In terms of gene expression, both groups showed enrichment for GEP70 indicating a high-risk transcriptional profile apart from MAF/MAFB overexpression. Finally, HA_TRA and HA_NORM showed similar poor outcomes compared to WT. To further validate the association between APOBEC and complex genomic profile, we explored again the ICGC breast WGS, observing a strong correlation between homologous recombination deficiency (HRD) and hyper-APOBEC (p < 0.05). To validate the relationship between the DREAM complex and APOBEC, we pharmacologically inhibited FOXM1 (FDI6, 20uM) and E2F4/6 (HLM00074, 20uM) in 8226, U266 and MM1.S multiple myeloma cell lines and showed increased A3B levels upon 72h of E2F4/6 inhibition. Simultaneously, FOXM1 inhibition confirmed A3B downregulation. Conclusion Overall, our data support a model where, APOBEC genes are variably expressed in all NDMM, but only patients with high proliferation rate and high level of complexity acquire mutations (Figure 1).

Effective venetoclax-based treatment in relapsed/refractory multiple myeloma patients with translocation t(6;14).

Introduction: The selective Bcl-2 inhibitor venetoclax has shown promising therapeutic potential in multiple myeloma, particularly in cases associated with t(11;14) IGH::CCND1 translocation. However, the efficacy of venetoclax in myeloma patients with the t(6;14) IGH::CCND3 translocation remains less investigated. Methods: In this study, we conducted a retrospective analysis to investigate the efficacy of venetoclax-based therapy in relapsed/refractory myeloma patients with t(6;14) translocation. The treatment courses of three patients, that included previous therapies and responses to venetoclax, were assessed. Clinical data, laboratory results, and adverse events were analyzed to evaluate treatment outcomes. Results: Our findings demonstrated remarkable therapeutic responses in three consecutive patients with t(6;14) translocation-associated myeloma who received venetoclax-based therapy. Patient 1, a lenalidomide-bortezomib-daratumumab and alkylator treatment refractory patient, achieved sustained stringent complete remission (sCR) after combining carfilzomib-dexamethasone with venetoclax, which was his best response ever. Similarly, Patient 2, refractory to frontline bortezomib-thalidomide-dexamethasone therapy, attained CR following a transition to bortezomib-dexamethason-venetoclax treatment. Patient 3, who was immunomodulatory (IMID)-intolerant, showed a highly favorable response to venetoclax-dexamethasone therapy after his first relapse following autologous stem cell transplantation. No significant adverse effects were observed in any of the patients. Discussion: Our study provides compelling preliminary evidence for the efficacy of venetoclax in t(6;14) translocation-associated myeloma. The outcomes observed in our patients suggest that venetoclax-based therapy holds substantial promise as an effective treatment option for this specific genetic subgroup. Furthermore, the similarities in treatment response between t(11;14) and t(6;14) translocation subgroups highlight the importance of personalized approaches targeting specific genetic abnormalities to optimize therapeutic outcomes.

Utility of Measurable Residual Disease (MRD) Assessment in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) treatment advances have significantly improved disease-free remission, with greater focus in clinical trials being placed on measurable residual disease (MRD) as a marker of subclinical disease assessment. While this concept is used extensively in other haematological neoplasms, there is yet to be a consensus on the threshold for MRD in MCL that demonstrates prognostic and therapeutic significance, and in this context has yet to reach routine clinical practice.The historical long-term method for MCL MRD assessment has been real-time quantitative polymerase chain reaction (PCR), targeting the clonal immunoglobulin heavy locus (IGH) rearrangement or the IGH::CCND1 translocation rearrangement. A significant problem at present relates to identifying alternative assays for patients who do not have a suitable molecular target by this method.This article reviews existing techniques used in MRD assessment for MCL and describes novel methods which may overcome existing limitations, including next-generation sequencing modalities. The use of circulating tumour DNA is explored, with techniques such as CAPP-Seq and PhasED-Seq demonstrating promise in B-lymphoproliferative disorders, though application in MCL requires further study.The other aspect of practice using MRD is identifying therapeutic options which can address a subclinical molecular relapse. Developing suitable interventions that can alter the disease trajectory based on longitudinal MRD kinetics are needed to justify its incorporation into standard care.

Differential Phosphoproteomics Deciphers Physiopathology of High-Risk Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a rare adult-onset non-Hodgkin lymphoma of which genetic hallmark is CCND1 translocation with IG genes, although CCND2, CCND3 or CCNE translocations are alternatives. The median survival of patients with MCL has lately increased due to improved therapies. However, the identification of high-risk MCLs characterized by pleomorphic or blastoid appearance, high proliferation index and TP53 deletions/mutations, remains mandatory due to their highly aggressive features and dismal clinical course even with intensified regimens. Therefore, specific therapeutic approaches are still warranted for such patients, but clinical and biological data are scarce since high-risk MCLs represent only 10-20% of all MCLs. A valuable source of rare samples for clinical/translational research is archived collections of formalin-fixed paraffin-embedded (FFPE) tissue blocks which enable retrospective studies to be performed. However, FFPE samples remain challenging for omics studies, especially phosphoproteomics. Here we implemented an original process to overcome this technical roadblock and took full advantage of FFPE samples to investigate biological processes differentially involved in high-risk and classical MCL (cMCL) to highlight rationale for innovative targeted therapies for these aggressive forms. Material and methods: We employed an unbiased differential label-free phosphoproteomic approach using archived FFPE MCL samples. Briefly, archived FFPE lymph nodes conclusive for cMCL or blastoid MCL (bMCL) diagnoses were retrieved from the Biological Resource Center of Strasbourg University Hospital from 1996 to 2016. H&E slides were reevaluated, and areas enriched in tumoral cells were selected to be punched from FFPE blocks. Our original sample preparation procedure entailed 1/ xylene-based deparaffinization, 2/ protein extraction with high content SDS-based lysis buffer, 3/ sample reduction/alkylation followed by cleanup and protein digestion on carboxylate paramagnetic beads and Lys-C/trypsin proteolytic enzyme mix, 4/ phosphopeptide enrichment on the AssayMap Bravo platform (Agilent) using IMAC Fe(III)-NTA cartridges. All samples were then analyzed on a NanoElute coupled to a TimsTOF Pro mass spectrometer (Bruker). Label-free quantification was performed using MaxQuant with the human database downloaded from SwissProt in April 2022. Further bioinformatics analyses were conducted in Perseus and Prostar. Data were functionally annotated with Reactome Pathway Database and STRING for association networks. Results: For this monocentric study we retrieved 30 FFPE blocks of lymph node biopsies conclusive for MCL diagnosis, including 15 bMCL and 15 cMCL. Although FFPE samples represented a challenge to work with for phosphoproteomics, we extracted enough protein from three 2mm-punches to proceed to the phosphopeptide enrichment according to our original sample preparation procedure. Overall, we successfully identified and quantified 7,220 class I phosphosites, i.e., sites of phosphorylation for which the probability of the phosphate group localization is greater than 0.75. No significant difference was found considering neither the archived period length of FFPE blocks nor the MCL subtypes (bMCL or cMCL). Differential analysis of bMCL and cMCL identified 180 differentially phosphorylated phosphosites (p-value < 0.05) corresponding to 103 proteins. Among these phosphosites, 139 were more phosphorylated in bMCL. Functional annotation of their corresponding proteins highlighted an over-representation of proteins involved in cell cycle, DNA repair and RNA metabolism within bMCL differential phosphoproteome (Fig.1). In addition, the kinase enrichment analysis of these differentially phosphorylated proteins predicted an increased activity of cyclin-dependent kinases in bMCL compared to cMCL. Conclusion: Here we performed for the first time phosphoproteomics from archived FFPE MCL samples. Differential analysis revealed that high-risk MCLs are characterized by an increased phosphorylation of proteins involved in cell cycle and an increased activity of cyclin-dependent kinases. Further studies are mandatory to fully understand to which extent cyclin-dependent kinases could be suitable targeted therapeutic candidates for patients with high-risk MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinicopathological and molecular genetic features of cyclin D1-negative mantle cell lymphoma

Objective: To investigate the clinicopathological features and molecular genetics of cyclin D1-negative mantle cell lymphoma (MCL). Methods: The clinicopathological features and molecular genetics of CyclinD1-negative MCL diagnosed between January 2016 and July 2021 at the First Affiliated Hospital of Zhengzhou University were analyzed using immunohistochemistry and fluorescence in situ hybridization. Clinical information was collected and analyzed. Results: A total of five Cyclin D1-negative MCL cases from all 212 MCL patients (5/212, 2.4%)were included. There were three male and two female patients,age ranged from 59 to 70 years (median 64 years). All patients presented with nodal lesions. None of the patients had B symptoms but four had bone marrow involvement. Histopathologically, four cases were classic MCL and one case was pleomorphic variant type. All five cases were negative for Cyclin D1 but SOX-11 were positive in all cases. CD5 was positive in four cases and one case was weakly positive for CD23. CD10 and bcl-6 were negative in all cases. CCND1 translocation was identified in three cases and CCND2 translocation in one case by FISH analysis. However,CCND3 translocations were not found in the five cases. Conclusions: Cyclin D1-negative MCL are uncommon, its accurate diagnosis needs combined analysis with morphologic and immunophenotypic characteristics and genetic changes. It may be particularly difficult to distinguish from other small cell type B cell lymphomas. FISH analyses for CCND1/CCND2/CCND3 translocations and immunohistochemistry for SOX-11 are helpful to resolve such a difficult distinction.

The pathologic diagnosis of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell non-Hodgkin lymphoma usually characterized by t(11;14) (q13;q32), or CCND1 translocation and Cyclin D1 over expression. A very small subset of MCL may lack the t(11;14) (q13;q32) translocation and Cyclin D1 over expression, but show alternative translocations involving CCND2 and CCND3, and over expression of SOX11. In general, MCL has been considered a very aggressive and incurable lymphoma and patients with MCL usually have a poor prognosis. However, indolent variants, including in situ mantle cell neoplasm and the recently recognized leukemic non-nodal MCL do exist. In recent years, genome-wide molecular genetic studies have revealed a characteristic MCL genetic profile. This review will focus on the pathologic diagnosis of MCL using the traditional morphological and immunophenotypic strategies combined with cytogenetic characteristics and recently identified molecular profile. Morphological subtypes, immunophenotypic variants, recently recognized indolent variants, as well as MCL risk stratification will also be discussed.

Silencing Oncogene Translation Using Pateamine a Analogues As a Novel Therapeutic Strategy for c-Myc Driven Lymphoma

IntroductionThe oncogene c-Myc is a master transcription factor that is associated with pathogenesis and chemo-resistance in a vast array of human cancers including aggressive lymphoma. Although the therapeutic benefit of targeting c-Myc has been demonstrated in many preclinical studies, direct inhibitors of c-Myc have not been successfully developed for the clinic. On the other hand, translation of c-Myc is highly dependent on the eukaryotic translation initiation factor 4F (eIF4F), a complex comprised of the eIF4A, eIF4G, and eIF4E subunits. These results suggest that silencing the translation of c-Myc via inhibiting eIF4F or its subunits may be a valid alternative strategy for indirectly targeting the “undruggable” c-Myc oncogene. In the current project, we have investigated the pharmacological activity and mechanism of Pateamine A (PatA) analogues in lymphoma cells in vitro . PatA was originally purified as a marine natural product and demonstrated anti-proliferation activity. One of the analogues, Biotin-DMDA-PatA conjugate (B-DMDA-PatA) pulled down eIF4A, specifically inhibits de novo protein synthesis with much less effect on de novo RNA synthesis, and specifically inhibits cap dependent translation. DMDA-PatA acts by decreasing the interaction between eIF4A and eIF4G. Our hypothesis is the following: If PatA analogues preferentially inhibit the translation of oncogenes such as c-Myc, then PatA analogues will be highly effective for c-Myc driven cancers and potentially safe for animals and humans.MethodsMZ-735 is a recently developed and optimized PatA analogue, based on insights from DMDA-PatA. Both compounds were synthesized at the CPRIT Synthesis and Drug-Lead Discovery Laboratory at Baylor University. Cytotoxicity was evaluated in lymphoma cell lines using CellTiter-Glo (Promega®). The cells were exposed to the PatA analogues or the vehicle control on either continuous exposure or on a treatment (progressively decreasing exposure)-washout schedule. To evaluate the toxicity of PatA analogues to normal cells, cytotoxicity in peripheral blood mononuclear cells (PBMC) of healthy donors were tested. Immunoblot was used to study the molecular effects of MZ-735 on translation of oncogenes, particularly that of c-Myc, in lymphoma cell lines representing DLBCL, DHL, mantle cell lymphoma (MCL), cutaneous T cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL), and adult T cell lymphoma/leukemia (ATL). To further investigate the broad translational downregulation of poorly understood network of oncogenes by MZ-735, we are conducting in parallel proteomic and RNAseq studies in 3 cell lines representing DLBCL (no MYC translocation), DHL (with MYC and BCL2 translocation), and MCL (with CCND1 translocation). Finally, the efficacy and safety of MZ-735 will be investigated in xenograft mouse models of DHL.ResultsDMDA-PatA and MZ-735 were studied in 13 lymphoma cell lines. DMDA-PatA and MZ-735 exhibited concentration- and time-dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations (Figure 1A). The IC20 and IC50 values of each lymphoma cell lines are shown in Table 1. MZ-735 was more effective in B cell lymphoma than in T cell lymphoma cells. MZ-735 was significantly more potent than DMDA-PatA in all tested cell lines. Remarkably, a brief treatment by MZ-735 for as short as 3 hours followed by washout of the compound produced comparable cytotoxicity to that of continuous exposure of the compound without washout. Importantly, DMDA-PatA and MZ-735 did not show significant cytotoxic effect in PBMC from healthy donors (Figure 1B). MZ-735 markedly repressed the protein level of c-Myc in all tested lymphoma cell lines, and effectively suppressed the protein level of cyclin D1 in MCL cells (Figure 2). The expression of the MDM2 oncogene was suppressed in a way similar to C-MYC .ConclusionThese results suggest that the PatA analogue MZ-735 has potent pharmacological activity in lymphoma cell lines, and acts by suppressing the production of critical oncogenes, including C-MYC, CCND1, and MDM2 , but is not toxic to normal PBMC. MZ-735 may cause rapid and irreversible modification of the translation apparatus, which is not affected by drug washout, leading to rapid commitment to growth arrest and death of cancer cells. MZ-735 is a promising and novel treatment for C-MYC- and CCND1 -driven aggressive lymphoma. [Display omitted] DisclosuresO'Connor:Trillium Therapeutics: Research Funding; Celgene: Honoraria, Research Funding.

Molecular Characterization of Bone Marrow and Peripheral Blood Compartments from Patients with Plasma Cell Leukemia in the Mmrf Commpass Study

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma (MM) where neoplastic plasma cells (PCs) escape the bone marrow (BM) and circulate in the peripheral blood (PB). Traditionally, PCL is defined by the presence of &gt;20% circulating plasma cells (CPCs), however, recent studies have suggested that PCL be redefined as the presence of &gt;5% CPCs. The Multiple Myeloma Research Foundation CoMMpass study (NCT01454297) is a longitudinal, observational clinical study with 1143 newly diagnosed MM patients. BM-derived MM samples were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. When &gt;5% CPCs were detected by flow cytometry, PCs were enriched independently from both compartments, and T-cells were selected from the PB as a control for WGS and WES. This substudy within CoMMpass provides the largest, most comprehensively characterized dataset of matched MM and PCL samples to date, which can be leveraged to better understand the molecular drivers of PCL. At diagnosis, 813/1143 CoMMpass patients had flow cytometry data reporting the percent PCs in PB, of which 790 had &lt;5%, 17 had 5-20%, and 6 had &gt;20% CPCs. Survival analyses revealed that patients with 5-20% CPCs (median = 20 months) had poor overall survival (OS) outcomes compared to patients with &lt;5% CPCs (median = 74 months, p &lt; 0.001), and no significant difference in outcome was observed between patients with 5-20% and &gt;20% (median = 38 months) CPCs. Patients with 1-5% CPCs (median = 50 months, HR = 2.45, 95% CI = 1.64 - 3.69, p &lt; 0.001) also exhibited poor OS outcomes compared to patients with &lt;1% CPCs (median = 74 months), suggesting that patients with &gt;1% CPCs are a higher risk population, even if they do not meet the PCL threshold. Using a cutoff of &gt;5% CPCs, 23/813 (2.8%) patients presented with primary PCL (pPCL) at diagnosis. Of these patients, 7 (30%) were hyperdiploid (HRD), of whom 1 had a CCND1 and 1 had a MYC translocation; while 16 (70%) were nonhyperdiploid (NHRD), all of whom had a canonical immunoglobulin translocation (6 CCND1, 5 WHSC1, 3 MAF, 1 MAFA, and 1 MAFB). Of 124 patients with serial sample collections, 5 (4%) patients without pPCL had &gt;5% CPCs at progression, and thus relapsed with secondary PCL (sPCL). Of the 5 sPCL patients, 2 (40%) were NHRD with a CCND1 or MAF translocation; while 3 (60%) were HRD, 1 with a WHSC1 translocation. Median time to diagnosis of sPCL was 22 months (range = 2 - 31 months), and patients with sPCL (median = 22 months) and pPCL (median = 30 months) exhibited poor OS outcomes as compared to MM patients (74 months, p &lt; 0.001). Sequencing data was available for 15 pPCL and 5 sPCL samples. For 12 patients with WES, WGS, and RNAseq performed on their PCL tumor sample, an integrated analysis identified recurrent, complete loss-of-function (LOF) events in only CDKN2C/FAF1, SETD2, and TRAF3. Five pPCL patients had complete LOF of a gene involved in G1/S cell cycle control, including CDKN2C, CDKN2A, CDKN1C, and ATM. These LOF events were not observed in NHRD t(11;14) PCL patients, suggesting that CCND1 overexpression and LOF of genes involved in G1/S cell cycle control may represent independent drivers of PCL. Comparing WES and WGS data between matched MM and PCL tumor samples revealed a high degree of similarity in mutation and copy number profile. However, differential expression analysis performed for 13 patients with RNAseq data comparing their MM and PCL tumors revealed 27 up- and 39 downregulated genes (padj &lt; 0.01, FDR = 0.1) in PCL versus MM. Pathway analysis revealed an enrichment (p &lt; 0.001) for genes involved in adhesion and diapedesis, including upregulation of ITGB2, PF4, and PPBP, and downregulation of CCL8, CXCL12, MMP19, and VCAM1. The most significantly downregulated gene in PCL (log2FC = -6.98) was VCAM1, which plays a role in cell adhesion, and where loss of expression (TPM &lt; 0.01) was observed across all PCL samples. Upregulation of four S100 genes including S100A8, S100A9, S100A12, and S100P, which have been implicated in tumor growth, metastasis, and immune evasion, was also observed in PCL. Interestingly, a S100A9 inhibitor has been developed and may represent a novel treatment option for PCL patients. In summary, PCL was found to be associated with molecular events dysregulating G1/S cell cycle control coupled with subtle changes in transcription that likely occur in a subclonal population of the MM tumor. Disclosures Lonial: Genentech: Consultancy; GSK: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy.

Unravelling the Heterogeneity of Mantle Cell Lymphoma Ecosystem By Single Cell RNA Sequencing

Background: Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma that is incurable. MCL has a complex ecosystem of malignant B-cells and stromal and immune cells that play a supporting role for tumor growth, ultimately leading to the potential re-emergence of the disease. The tumor microenvironment has been reported as a crucial factor in MCL pathogenesis and progression. Thus, if we can identify the tumor microenvironment components and define the characteristics of malignant and non-malignant cells, this will pave the way for studying clonal evolution of MCL in vivo.Methods: Both pre- and post-treatment, fresh tumor biopsy samples of MCL were obtained. The cells were dissociated and re-suspended in PBS with >10% serum. A final concentration of 1,200 cells/uL were used for single cell sorting in the chromium system (10X Genomics, California). We sequenced the mRNA in the NextSeq 500 platform. All analysis was conducted using R-programming language (version 3.4).Results: From four MCL patients (L1-L4), we obtained 9,400 cells. Three of the four samples were collected through apheresis (i.e., L1-L3), and one sample (i.e., L4) from surgical biopsy of the involved lymph node. One patient had known TP53 mutated status (i.e., L1) and another patient had CCND1 translocation (i.e., L2). From the apheresis samples (L1-L3), the proportion of lymphocytes was 87%, 68% and 65%. We identified 10 defined clusters of cells based upon their gene expression from all four samples. Six of the 10 clusters were clonal B-cells with strong expression of CCND1, CD79A and CD79B. We also identified clonal T-cells (both CD8+ and CD4+) and monocyte/macrophage clusters. SOX11 expression was absent in one B-cell clone, indicating this clone may be SOX11-negative MCL. The monocyte-macrophage cluster demonstrated strong BCL2 expression, which was not expressed by the B-cells clones. CD19 expression was ubiquitous among the B-cell clones but weaker as compared with other B-cell markers. When the signaling was compared among the four samples, the chemo-resistant cells (sample L1) demonstrated upregulation of NOTCH1 signaling, DNA-damage repair, interferon-alpha response, MYC targets and the HIF1A pathway. Two cell clones did not express any canonical markers and were not identified as a defined cluster.Conclusions: We identified meaningful sub-populations of MCL that define the tumor microenvironment. There is considerable inter-tumor and intra-tumor heterogeneity of MCL at a single cell resolution, which indicates that developing a uniform treatment regimen may prove to be difficult. Ubiquitous expression of CD79A or CD79B may help guiding precision medicine such as the development of novel CAR-T cell therapy. Longitudinal follow up of the same patients may define clonal evolution of MCL and unravel the spatio-temporal interplay. [Display omitted] DisclosuresWang:AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Novartis: Research Funding; Acerta Pharma: Honoraria, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding.

Circulating Tumor DNA Dynamics during Therapy Predict Outcomes in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma with striking variability in tumor biology and clinical behavior across patients. Noninvasive blood-based biomarkers that monitor disease and treatment response may guide clinical decisions throughout disease course. Circulating tumor DNA (ctDNA) is a highly tumor-specific biomarker detectable in the blood of virtually all MCL patients. Within a prospective clinical trial, we studied Adaptive's next-generation sequencing assay to detect and quantify ctDNA in serum throughout therapy. Previously, we reported that ctDNA clearance after induction predicts both PFS and OS (Roschewski et al. ASCO 2018). Bortezomib (BZ) is a proteasome inhibitor shown to improve PFS when added to combination chemotherapy (Robak et al. N Eng J Med 2015). Herein, we analyze the clinical significance of ctDNA dynamics during therapy in patients treated with BZ + DA-EPOCH-R without consolidation.Methods: Untreated MCL patients received BZ monotherapy x 1 cycle then induction with BZ + DA-EPOCH-R up to 6 cycles. After induction, patients were randomized to observation vs. BZ maintenance x 18m. Patients had serum prospectively collected pre-Tx, after BZ monotherapy, before each cycle, after induction, and with each surveillance visit. Surveillance visits were paired with CT scans q4mos x 2y, q6mos in years 2-4, then annually. Pre-treatment specimens (FFPE or frozen cells) were analyzed for tumor-specific clonotypes. Tumor DNA was amplified using locus-specific primer sets for the Ig heavy-chain and light-chain loci, CCND1, and BCL2 translocations. Amplified products were sequenced, and patients without a high-frequency tumor clonotype were excluded. Levels of ctDNA at baseline, after BZ monotherapy, and after 1, 2 cycles of induction were analyzed.Results: 53 untreated MCL patients received BZ + DA-EPOCH-R between September 2005 and January 2016. After median potential f/u of 10y, median PFS is 29.3m and the 5-yr OS is 78.2%. BZ maintenance had no impact on PFS or OS. Of 52 patients w/available biopsies, 50 (96%) had a tumor-specific clonotype identified. Of 48 patients w/pre-treatment serum, 46 (96%) had detectable ctDNA. ctDNA was successfully tracked in 625 of 647 (97%) serum samples. The median level of baseline ctDNA was 742.98 cell equivalents/mL (range 0 to 101,008.58). Baseline ctDNA correlated with total metabolic tumor volume on PET scan (rs=0.74) but was not associated with PFS (p=0.45) or OS (p=0.22). Of 42 patients who received BZ monotherapy, 26 (62%) had decreases in ctDNA, 15 (36%) had increases, and 1 (2%) had no change. The median absolute decrease in ctDNA was 35 cell equivalents/ml (range -20,901 to +6,667) and the median relative decrease was 24% (range -100% to +530%). Notably, lower absolute ctDNA levels after BZ window were associated with improved PFS compared to patients with ctDNA above the median (34m vs 22m, p=0.02)(Figure 1). Clearance of ctDNA after 1 cycle of DA-EPOCH-R + BZ was strongly associated with a superior median PFS (76.4m vs 20.7m, p=0.0037)(Figure 2) and a trend towards superior 4-yr OS (92.3% vs 73.0%, p=0.23). Clearance of ctDNA after 2 cycles of DA-EPOCH-R + BZ was also associated with a superior median PFS (32.4m vs 21.4m, p=0.015) and a trend towards superior median OS (82.2m vs 73.2m, p=0.15).Conclusions: Circulating tumor DNA is detectable in nearly all patients with MCL. Dynamic changes that occur very early during therapy may predict clinical outcomes and may be an early readout for the activity of targeted agents. Given its broad applicability, ctDNA should be prospectively studied as part of response-adapted approaches in MCL.This work was supported by the Intramural Research Program of NCI and Adaptive Biotechnologies, Inc. [Display omitted] DisclosuresJacob:Adaptive Biotechnologies: Employment, Equity Ownership. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

High Frequency of β-Catenin Mutations in Mouse Hepatocellular Carcinomas Induced by a Nongenotoxic Constitutive Androstane Receptor Agonist

Activation of Wnt/β-catenin signaling is frequent in human and rodent hepatocarcinogenesis. Although in mice the tumor-promoting activity of agonists of constitutive androstane receptor (CAR) occurs by selection of carcinogen-initiated cells harboring β-catenin mutations, the molecular alterations leading to hepatocellular carcinoma (HCC) development by the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCP) in the absence of genotoxic injury are unknown. Here, we show that CAR activation per se induced HCC in mice and that 91% of them carried β-catenin point mutations or large in-frame deletions/exon skipping targeting Ctnnb1 exon 3. Point mutations in HCCs induced by TCP alone displayed different nucleotide substitutions compared with those found in HCCs from mice pretreated with diethylnitrosamine. Moreover, unlike those occurring in HCCs from diethylnitrosamine+TCP mice, they did not result in increased expression of β-catenin target genes, such as Glul, Lgr5, Rgn, Lect2, Tbx3, Axin2, and Ccnd1, or nuclear translocation of β-catenin compared with the control liver. Remarkably, in the nontumoral liver tissue, chronic CAR activation led to down-regulation of these genes and to a partial loss of glutamine synthetase-positive hepatocytes. These results show that, although chronic CAR activation per se induces HCCs carrying β-catenin mutations, it concurrently down-regulates the Wnt/β-catenin pathway in nontumoral liver. They also indicate that the relationship between CAR and β-catenin may be profoundly different between normal and neoplastic hepatocytes.

Tumor suppressor CD99 is downregulated in plasma cell neoplasms lacking CCND1 translocation and distinguishes neoplastic from normal plasma cells and B-cell lymphomas with plasmacytic differentiation from primary plasma cell neoplasms

AbstractCD99(MIC2) is a widely expressed cell surface glycoprotein and functions as a tumor suppressor involved in downregulation of SRC family of tyrosine kinase. CD99 expression is tightly regulated through B-cell development. The principal aims of this study were to investigate the clinical utility of CD99 expression (i) in distinguishing normal plasma cells from primary plasma cell neoplasms; (ii) in detection of minimal residual disease in primary plasma cell neoplasms; and (iii) in distinguishing plasma cell component of B-cell lymphomas from primary plasma cell neoplasms. We analyzed expression of CD99 by flow cytometry and immunohistochemistry in lymph nodes, peripheral blood, and bone marrow samples. CD99 showed stage-specific expression with highest expression seen in precursor B and plasma cells. In contrast to the uniform bright expression on normal plasma cells, CD99 expression on neoplastic plasma cells was lost in 39 out of 56 (69.6%) cases. Furthermore, 8 out of 56 samples (14%) showed visibly (>10-fold) reduced CD99 expression. Overall, CD99 expression was informative (absent or visibly dimmer than normal) in 84% of primary plasma cell neoplasm. In the context of minimal residual disease detection, CD99 showed superior utility in separating normal and abnormal plasma cells over currently established antigens CD117, CD81, and CD27 by principal component analysis. Preservation of CD99 expression was strongly associated with cyclin D1 translocation in myeloma (p < 0.05). B-cell lymphomas with plasma cell component could be distinguished from myeloma by CD99 expression. In summary, we established that tumor suppressor CD99 is markedly downregulated in multiple myeloma. The loss is highly specific for identification of abnormal cells in primary plasma cell neoplasms, and can be exploited for diagnostic purposes. The role of CD99 in myeloma pathogenesis requires further investigation.

Pleomorphic mantle cell lymphoma morphologically mimicking diffuse large B cell lymphoma: common cyclin D1 negativity and a simple immunohistochemical algorithm to avoid the diagnostic pitfall.

To characterize the clinicopathological and genetic features of pleomorphic mantle cell lymphoma (PMCL), which morphologically mimics diffuse large B cell lymphoma (DLBCL). We screened systematically 500 B cell lymphomas morphologically compatible with DLBCL using an immunohistochemical algorithm of three markers (CD5, cyclin D1 and SOX11). Ten cases of PMCL were identified for further study and, surprisingly, four (40%) of them were cyclin D1-negative. These 10 patients were mainly elderly males with advanced disease, and their median survival was only 11 months. All cyclin D1-positive PMCLs tested showed an IGH-CCND1 translocation, whereas one of the four cyclin D1-negative PMCLs had a translocation involving CCND2 and a high CCND2 mRNA level (P < 0.000001). The genomewide copy number profiles of both cyclin D1-positive and cyclin D1-negative PMCLs were similar to those of classical mantle cell lymphoma (MCL) reported previously, confirming the diagnosis. Secondary genetic alterations involved in oncogenic pathways of MCL were observed more frequently in these PMCLs, possibly decreasing the dependence on the driving CCND1 translocation and accounting for the common cyclin D1 negativity. Copy number gains of PIK3CA and CCDC50 were detected in all cyclin D1-negative PMCLs but in only 40% of the cyclin D1-positive PMCLs. These additional oncogenic signals may compensate for the common absence of CCND2 translocation in cyclin D1-negative PMCL. We demonstrate for the first time that cyclin D1 negativity is surprisingly common in PMCL morphologically mimicking DLBCL, and the use of a simple immunohistochemical algorithm can prevent misclassification and inappropriate treatment.

Cyclin D1 expression and polysomy in lymphocyte-predominant cells of nodular lymphocyte-predominant Hodgkin lymphoma.

Cyclin D1 protein expression in lymphocytes is classically associated with mantle cell lymphoma. Although increasingly recognized in other lymphoproliferative disorders, cyclin D1 expression and CCND1 gene abnormalities have not been well studied in nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). Using a double stain for CD20/cyclin D1, we quantified cyclin D1 expression in 10 cases of NLPHL and correlated those findings with SOX11 expression, CCND1 gene abnormalities, and clinical data. For comparison, we examined 5 cases of T cell-/histiocyte-rich large B-cell lymphoma (THRLBCL). All cases of NLPHL stained for cyclin D1 showed at least rare positivity in lymphocyte-predominant (LP) cells. In 4 cases, at least 20% of LP cells were positive for CD20/cyclin D1. Neither SOX11 expression nor CCND1 gene rearrangement was found in any of the cases, but fluorescence in situ hybridization showed a proportion of the large cells with 3 to 4 copies of nonfused IGH and CCND1 signals or 3 intact CCND1 break-apart signals. Further study with CCND1/CEP11 showed polysomy in 6 of 9 cases with cyclin D1 expression and 5 of 16 NLPHL not examined for cyclin D1. Two of 5 cases of THRLBCL showed rare positive staining for CD20/cyclin D1; 1 case showed polysomy with CCND1/CEP11. Results show that cyclin D1 may be expressed in LP cells without SOX11 expression or CCND1 translocation. Polysomy with increased copies of CCND1 may account for cyclin D1 expression in some cases. Cyclin D1 expression is not useful for distinguishing NLPHL from THRLBCL and has no apparent clinical significance in NLPHL.

Targeting Cyclin-Dependent Kinases in Synovial Sarcoma: Palbociclib as a Potential Treatment for Synovial Sarcoma Patients

BackgroundIn synovial sarcomas alterations in the cyclin D1-CDK4/6-Rb axis have been described. Also, β-catenin, a cyclin D1 regulator, is often overexpressed. Additionally, studies have shown that the t(X;18) translocation influences tumor behavior partly through cyclin D1 activation. We investigated how alterations in the cyclin D1-CDK4/6-Rb axis impact prognosis and studied effects of targeting this axis with the CDK4/6 inhibitor palbociclib.MethodsSynovial sarcoma samples (n = 43) were immunohistochemically stained for β-catenin, cyclin D1, p16, p21, p27, Rb, and phospho-Rb. Fluorescent in situ hybridization (FISH) was performed to detect CCND1 amplification or translocation. In 4 synovial sarcoma cell lines sensitivity to palbociclib was investigated using cell viability assays, and effects on the sensitive cell lines were evaluated on protein level and by cell cycle arrest.ResultsExpression of nuclear phospho-Rb and nuclear β-catenin in the patient samples was associated with poor survival. FISH showed a sporadic translocation of CCND1 in a subset of tumors. An 8-fold CCND1 amplification was found in 1 cell line, but not in the patient samples investigated. Palbociclib effectively inhibited Rb-phosphorylation in 3 cell lines, resulting in an induction of a G1 arrest and proliferation block.ConclusionsIn this series nuclear phospho-Rb and nuclear β-catenin expression were negative prognostic factors. In vitro data suggest that palbociclib may be a potential treatment for a subset of synovial sarcoma patients. Whether this effect can be enhanced by combination treatment deserves further preclinical investigations.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-016-5341-x) contains supplementary material, which is available to authorized users.

Identification of Initiating Trunk Mutations and Distinct Molecular Subtypes: An Interim Analysis of the Mmrf Commpass Study

The Multiple Myeloma Research Foundation (MMRF) CoMMpass trial (NCT145429) is a longitudinal study of 1000 patients with newly-diagnosed multiple myeloma from clinical sites in the United States, Canada, Spain, and Italy. Each patient receives a treatment regimen containing a proteasome inhibitor, immunumodulatory agent, or both. Clinical parameters are collected at study enrollment and every three months through the five-year observation period. To identify molecular determinants of clinical outcome each baseline and progression tumor specimen is characterized using Whole Genome Sequencing, Exome Sequencing, and RNA sequencing. This will be the first public presentation of the interim analysis seven cohort with 760 enrolled patients of whom 565 are molecularly characterized. This cohort of patients includes 14 patients with baseline and secondary samples along with 7 patients with characterized tumor samples from the bone marrow and peripheral blood.Although the median follow-up time for the cohort is only 260 days the patients on proteasome and IMiD based combinations are currently showing a PFS and OS benefit compared to those receiving combinations with each agent alone. From the raw mutational analysis we identified 24 significant genes that are recurrently mutated and the mutated allele is detectably expressed in all but one, DNAH5. Suggesting these mutations are likely contributing to myelomagenesis through an unconventional mechanism. Interestingly, DIS3 mutations are independent of KRAS, NRAS, and BRAF indicating a potential mechanistic link while PRKD2 mutations are associated with t(4;14). To identify events driving the initiation of myeloma we performed a detailed clonality analysis using a bayesian clustering method that corrects for copy number abnormalities and tumor purity to assign mutations into distinct clonal branches versus the initiating trunk mutations. On average 63.8% of mutations are trunk mutations and in 86.7% of patients at least one trunk mutation is associated with somatic hypermutation of an immunoglobulin gene as expected in a late stage B-cell malignancy. This identified many expressed trunk mutations that did not come out in the classic significance analysis like ATM, EGR1, and CCND1. To identify molecular subtypes we performed unsupervised clustering using a consensus clustering approach on independent discovery and validation cohorts, which identified 12 distinct subtypes, using a combination of silhouette score and cumulative distribution of consensus scores. This analysis identified two distinct groups associated with t(4;14) with mutations in FGFR3 and DIS3 being exclusive to one subgroup. In addition, this analysis separates patients with cyclin D translocations into three different groups, with one group having the second lowest PFS proportion. Three patients without CCND1 or CCND3 translocations were found to have IgH translocations targeting CCND2. The MAF subgroup was associated with the lowest OS and PFS proportion, and the three MAF/MAFB translocation negative patients in the subgroup all had MAFA translocations. The remaining 6 subgroups are associated with hyperdiploid copy number profiles and harbor the majority of the IgH-MYC translocation events. Two of the hyperdiploid groups are associated with a low level of NFKB activation compared to the remaining four, one of these is defined by the highest proliferation index but paradoxically the other has the second worst OS proportion. Another group is enriched with FAM46C and NRAS mutations. The genomic profiles of the paired tumors isolated from the peripheral blood and bone marrow are highly similar indicating these are not genetically distinct tumor compartments, at least in this subset of seven patients. Applying our bayesian clustering method to the serial samples resolved additional clonal clusters as mutations with similar cancer cell fractions at diagnosis clearly diverged at later timepoints. These analyses have identified tumor initiating mutations and new subtypes of myeloma, which are associated with distinct molecular events and clinical outcomes. DisclosuresJagannath:Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Merck: Honoraria; Janssen: Honoraria. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Vij:Takeda, Onyx: Research Funding; Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy. Zimmerman:Amgen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria. Niesvizky:Celgene: Consultancy, Speakers Bureau. Rifkin:Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lonial:Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding.

The clinic and pathologic significance of plasma cell myeloma with CCND1

目的探讨细胞周期蛋白D1(CCND1）基因阳性多发性骨髓瘤（MM）患者的临床及骨髓病理学特征。方法回顾性分析158例初诊MM患者资料。所有患者均行骨髓活检及免疫组织化学染色、流式细胞术、FISH检测。CCND1基因阳性患者中有24例同时行细胞遗传学检查。结果158例患者中CCND1基因阳性者31例，CCND1基因阴性者127例。31例CCND1基因阳性患者中，男19例，女12例，其中IgG型11例，IgA型4例，IgD型4例，IgM型1例，轻链型共6例，不分泌型5例；阳性检出率在IgD(57.1%）、不分泌型（50.0%）中较IgA（12.1%）、IgG型（14.1%）高（P值均<0.05），在κ和λ型中差异无统计学意义（15.9％对19.0%，P=0.627）。31例CCND1基因阳性患者中浆细胞形态为小细胞型者24例（77.4%），为成熟浆细胞型者6例（19.4%），为不成熟浆细胞型者1例（3.2%）；流式细胞术检查示均为CD38+CD138+CD19−CD45−，其CD56阳性率较阴性组低（35.5％对61.4%，P=0.009），CD20阳性率较阴性组高（29.0％对4.0%，P<0.001）；免疫组织化学染色示cyclinD1+者22例（71.0%）。结论IgD型和不分泌型MM患者CCND1基因阳性检出率较高，常常表现为小细胞型形态，伴CD20高表达和CD56表达减低，多表达cyclinD1。主要需与淋巴浆细胞淋巴瘤等具有浆细胞样分化特征的小B细胞淋巴瘤相鉴别。

Mantle cell lymphoma--a spectrum from indolent to aggressive disease.

Mantle cell lymphoma (MCL) is a distinctive lymphoma type generally characterized by the presence of CCND1 translocation and overexpression of cyclin D1. MCL usually presents with advanced stage and rapid clinical progression. The diagnosis is in most instances uncomplicated but cases with variant morphologies or immunophenotypes, especially cyclin D1-negative cases, may cause diagnostic difficulties. During the mantle cell lymphoma (MCL) session at the European Association of Haematopathology/Society for Hematopathology workshop 2014 held in Istanbul, Turkey, submitted cases illustrated interesting features such as unusual morphology or immunophenotypes. In several submitted cases of cyclin D1-positive MCL, CCND1 rearrangement could not be detected by t(11;14)(q13;q32) dual-color dual-fusion FISH but was suggested by CCND1 break-apart probes, and advantages and disadvantages of different FISH probes were highlighted. Three cyclin D1-negative MCL cases were submitted. These were identified by SOX11 immunohistochemistry and found to carry CCND2 translocations and/or to express high levels of cyclin D2 mRNA. Features associated with aggressive clinical course were presented including high expression of p53 protein and MYC aberrations. The need to integrate histological, immunophenotypic, genetic, and clinical data to arrive at the correct diagnosis was emphasized.