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

Abstract 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. Figure. Figure. Disclosures Wang: 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.

Download Full-text

Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Blood ◽

10.1182/blood-2019-126889 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1527-1527

Author(s):

Sara Rodríguez ◽

Cirino Botta ◽

Jon Celay ◽

Ibai Goicoechea ◽

Maria J Garcia-Barchino ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Somatic Mutations ◽

Advisory Committees ◽

Normal Cells ◽

Cell Clones ◽

B Cell Precursors ◽

Myd88 L265p

Background: Although MYD88 L265P is highly frequent in WM, by itself is insufficient to explain disease progression since most cases with IgM MGUS also have mutated MYD88. In fact, the percentage of MYD88 L265P in CD19+ cells isolated from WM patients is typically <100%, which questions if this mutation initiates the formation of B-cell clones. Furthermore, a few WM patients have detectable MYD88 L265P in total bone marrow (BM) cells and not in CD19+ selected B cells, raising the possibility that other hematopoietic cells carry the MYD88 mutation. However, no one has investigated if the pathogenesis of WM is related to somatic mutations occurring at the hematopoietic stem cell level, similarly to what has been shown in CLL or hairy cell leukemia. Aim: Define the cellular origin of WM by comparing the genetic landscape of WM cells to that of CD34 progenitors, B cell precursors and residual normal B cells. Methods: We used multidimensional FACSorting to isolate a total of 43 cell subsets from BM aspirates of 8 WM patients: CD34+ progenitors, B cell precursors, residual normal B cells (if detectable), WM B cells, plasma cells (PCs) and T cells (germline control). Whole-exome sequencing (WES, mean depth 74x) was performed with the 10XGenomics Exome Solution for low DNA-input due to very low numbers of some cell types. We also performed single-cell RNA and B-cell receptor sequencing (scRNA/BCRseq) in total BM B cells and PCs (n=32,720) from 3 IgM MGUS and 2 WM patients. Accordingly, the clonotypic BCR detected in WM cells was unbiasedly investigated in all B cell maturation stages defined according to their molecular phenotype. In parallel, MYD88p.L252P (orthologous position of the human L265P mutation) transgenic mice were crossed with conditional Sca1Cre, Mb1Cre, and Cγ1Cre mice to selectively induce in vivo expression of MYD88 mutation in CD34 progenitors, B cell precursors and germinal center B cells, respectively. Upon immunization, mice from each cohort were necropsied at 5, 10 and 15 months of age and screened for the presence of hematological disease. Results: All 8 WM patients showed MYD88 L265P and 3 had mutated CXCR4. Notably, we found MYD88 L265P in B cell precursors from 1/8 cases and in residual normal B cells from 3/8 patients, which were confirmed by ASO-PCR. In addition, CXCR4 was simultaneously mutated in B cell precursors and WM B cells from one patient. Overall, CD34+ progenitors, B-cell precursors and residual normal B cells shared a median of 1 (range, 0-4; mean VAF, 0.16), 2 (range, 1-5; mean VAF, 0.14), and 4 (range, 1-13; mean VAF, 0.26) non-synonymous mutations with WM B cells. Some mutations were found all the way from CD34+ progenitors to WM B cells and PCs. Interestingly, concordance between the mutational landscape of WM B cells and PCs was <100% (median of 85%, range: 25%-100%), suggesting that not all WB B cells differentiate into PCs. A median of 7 (range, 2-19; mean VAF, 0.39) mutations were unique to WM B cells. Accordingly, many clonal mutations in WM B cells were undetectable in normal cells. Thus, the few somatic mutations observed in patients' lymphopoiesis could not result from contamination during FACSorting since in such cases, all clonal mutations would be detectable in normal cells. Of note, while somatic mutations were systematically detected in normal cells from all patients, no copy number alterations (CNA) present in WM cells were detectable in normal cells. scRNA/BCRseq unveiled that clonotypic cells were confined mostly within mature B cell and PC clusters in IgM MGUS, whereas a fraction of clonotypic cells from WM patients showed a transcriptional profile overlapping with that of B cell precursors. In mice, induced expression of mutated MYD88 led to a moderate increase in the number of B220+CD138+ plasmablasts and B220-CD138+ PCs in lymphoid tissues and BM, but no signs of clonality or hematological disease. Interestingly, such increment was more evident in mice with activation of mutated MYD88 in CD34+ progenitors and B-cell precursors vs mice with MYD88 L252P induced in germinal center B cells. Conclusions: We show for the first time that WM patients have somatic mutations, including MYD88 L265P and in CXCR4, at the B cell progenitor level. Taken together, this study suggests that in some patients, WM could develop from B cell clones carrying MYD88 L265P rather than it being the initiating event, and that other mutations or CNA are required for the expansion of B cells and PCs with the WM phenotype. Disclosures Roccaro: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Download Full-text

Efficacy and Safety Results of a Phase 1 Study of 177 lu-DOTA-HH1 (Betalutin®) with and without HH1 Pre-Dosing for Patients with Relapsed CD37+ Non-Hodgkin B Cell Lymphoma (NHL)

Blood ◽

10.1182/blood.v126.23.5118.5118 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5118-5118

Author(s):

Arne Kolstad ◽

Ulf Madsbu ◽

Bjørg Bolstad ◽

Caroline Stokke ◽

Tore Bach-Gansmo ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Cell Lymphoma ◽

Safety Data ◽

Efficacy And Safety ◽

B Cell Malignancies ◽

Advisory Committees ◽

Mantle Cell ◽

Equity Ownership

Abstract Background: CD37 is an internalizing transmembrane antigen highly expressed by normal B cells and on most of B-cell malignancies, and represents an interesting therapeutic target for the treatment of B-cell NHL. 177Lu-DOTA-HH1 (Betalutin®) is a novel CD37-targeting antibody radionuclide conjugate in clinical development. It consists of a CD37-binding murine IgG1 antibody HH1 labelled with the short-ranged beta-emitter lutetium-177 (T½ = 6.7 days) chelated to DOTA. 177Lu-DOTA-HH1 is delivered in a ready-to-use formulation. Efficacy and safety data of patients (pts) receiving 177Lu-DOTA-HH1 with HH1 pre-dosing, as well as new efficacy and safety data from pts receiving 177Lu-DOTA-HH1 without HH1 pre-dosing will be presented. Methods: Pts with relapsed incurable CD37 positive NHL of follicular grade I-IIIA, marginal zone, mantle cell, lymphoplasmacytic and small lymphocytic subtypes and with platelet counts ≥ 150 x109/l were eligible for inclusion in the study. In a 3+3 study design pts received rituximab (375 mg/m2) day 1 and 8 in order to deplete normal B cells. On day 29 pre-dosing with HH1 (50 mg, cold CD37 antibody) was administered before 177Lu-DOTA-HH1 injection (Arm 1). In Arm 2 177Lu-DOTA-HH1 was administered without HH1 pre-dosing on day 29. The starting doses for Arm 1 and 2 were 10 MBq/kg b.w. and 15 MBq/kg b.w, respectively. Pts enrolment has been completed (n=13) in Arm 1 with the dose-limiting toxicity (DLT) observed at 20 MBq/kg bw and a dose expansion cohort is currently open for enrollment at 15 MBq/kg with HH1 pre-dosing. Arm 2 is currently open for enrollment. Tumour response was assessed by FDG PET/CT scans (Cheson 2007), and pts will be followed for 5 years. Results: Arm 1:A total of13 (M/F 11/2) pts, median age 68 years, follicular lymphoma (n=12), and mantle cell lymphoma (n=1) have been enrolled since the study start in December 2012. The range of prior therapies was 1 to 8, where 5 of 13 pts were refractory to rituximab. The most common toxicities observed were hematologic and all DLTs were reversible and manageable. At 20 MBq/kg (n=3) G 3/4 neutropenia and/or thrombocytopenia were observed in all pts and platelet transfusions were required in 2 pts. At 15 MBq/kg (n=6) DLTs were: 1 G 3 thrombocytopenia lasting >14 days and 1 G 4 neutropenia/ thrombocytopenia lasting >7 days. The median time to nadir for platelets and neutrophils was 40 and 49 days, respectively. No pts experienced febrile neutropenia. Serious AEs were reported in 5 pts: at 10 MBq/kg pneumonia (possibly related) and pulmonary embolism (PE) unrelated, in the same pt, with history of PE; thrombocytopenia requiring platelet transfusions (2 pts) and epistaxis in 1 of them (20 MBq/kg), possibly related; transient atrial fibrillation (2 pts) at 15 MBq/kg, possibly related. No secondary malignancies or other long term events have been observed. Best overall tumor response observed across all dose levels were 4 complete and 3 partial remissions, 2 stable disease and 4 progression of disease (one pt had confirmed transformed lymphoma at 3 months). The duration of response (complete and partial remissions) ranged from 6 to more than 21 months. One patient is still in remission after 2 years. The median response duration has not yet been reached. Arm 2: Inclusion in this arm is ongoing. Data on efficacy and safety will be presented and compared with the pts receiving pre-dosing. Conclusions: 177Lu-DOTA-HH1, which is a single dose ready-to-use formulation, has a predictable and manageable safety profile. Most AEs were hematological in nature, all transient and reversible. Promising efficacy and durable responses have been observed. 177Lu-DOTA-HH1 has the potential to be a novel therapy for B-cell malignancies. Disclosures Kolstad: Nordic Nanovector ASA: Membership on an entity's Board of Directors or advisory committees, Research Funding. Bolstad:Nordic Nanovector ASA: Employment. Bruland:Nordic Nanovector ASA: Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership. Hartvig Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Phase 1b/2a Open-Label, Multiple-Dose, Dose-Escalation Study To Evaluate The Safety and Tolerability Of Intravenous Infusion Of SNS01-T In Patients With Relapsed Or Refractory Multiple Myeloma, Mantle Cell Lymphoma, Or Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.1950.1950 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1950-1950 ◽

Cited By ~ 1

Author(s):

John A Lust ◽

Charles Barranco ◽

Saad Z Usmani ◽

Frits van Rhee ◽

Mehdi Hamadani ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Advisory Committees ◽

Large B Cell Lymphoma ◽

Mantle Cell ◽

Equity Ownership ◽

Pro Inflammatory Cytokines

Abstract Eukaryotic translation initiation factor 5A (eIF5A) has been implicated in the regulation of cell proliferation, apoptosis, and inflammation, and is the only known protein to be modified by hypusination. Hypusinated eIF5A, the predominant form of eIF5A in cancer cells, is involved in cell survival and activation of inflammatory pathways. In contrast, accumulation of the unhypusinated form of eIF5A is associated with apoptosis and mutants of eIF5A that cannot be hypusinated (e.g. eIF5AK50R) are pro-apoptotic. SNS01-T was designed to treat B-cell cancers and consists of two active components: a plasmid DNA expressing the pro-apoptotic eIF5AK50R under the control of a B cell-specific promoter, and an siRNA against an untranslated region of native eIF5A mRNA. When these two components are combined with linear polyethyleneimine (PEI), the nucleic acids are condensed into nanoparticles for protection from degradation in the blood and enhanced cellular delivery. The mode of action of SNS01-T is siRNA-mediated inhibition of hypusinated eIF5A and simultaneous over-expression of pro-apoptotic eIF5AK50R to induce cell death. In vitro cell studies and in vivo xenograft studies have demonstrated the efficacy of this approach. The safety and tolerability of intravenous administration of SNS01-T is being investigated in a first-in-human Phase1b/2a study in patients with relapsed or refractory multiple myeloma (MM), mantle cell lymphoma (MCL) or diffuse large B cell lymphoma (DLBCL). Eligible patients are being enrolled sequentially into four cohorts at increasing doses. Each patient receives an intravenous infusion of SNS01-T twice weekly for 6 consecutive weeks. Eligible patients must have been diagnosed with MM according to IMWG criteria, or with MCL or DLBCL with histologic confirmation. Patients also must have measurable disease, have relapsed or refractory disease after two or more prior treatment regimens, have a life expectancy of at least 3 months, and not be eligible to receive any other standard therapy known to extend life expectancy. The primary objective is to evaluate the safety and tolerability of multiple escalating doses of SNS01-T. Secondary objectives include analysis of pharmacokinetics, immunogenicity, pro-inflammatory cytokines, and therapeutic efficacy. The required 3 patients per cohort have completed the dosing schedule in cohorts 1 and 2 from a total of 10 patients enrolled (9 patients with MM and 1 with DLBCL). Of the ten patients enrolled, four completed the full treatment period, two did not complete dosing but were evaluable for safety, and four (three in cohort 1 and one in cohort 2) discontinued treatment after fewer than 8 doses and were not evaluable. There were no drug-related serious adverse events or dose limiting toxicities in either cohort 1 or 2. In cohort 1 (0.0125 mg/kg SNS01-T), two of three evaluable patients did not progress on treatment and were considered stable at week 3 and week 6, the end of the dosing regimen. The third patient progressed after receiving 10 of the 12 doses and was evaluable for safety. In cohort 2 (0.05 mg/kg), 3 patients (2 with MM and 1 with DLBCL) were evaluable for safety. Stabilization of serum monoclonal protein levels was observed in one MM patient of cohort 2. Two patients (1 with MM and 1 with DLBCL) progressed after receiving 8 of the 12 doses and were evaluable for safety. Results from ongoing pharmacokinetic studies, immunogenicity studies, and quantification of pro-inflammatory cytokines will be discussed. The planned dose levels for the third and fourth groups are 0.2 and 0.375 mg/kg, respectively. The results to date of this first-in-human clinical trial indicate that SNS01-T can be administered safely and the MTD has not yet been reached (Clinical Trials.gov Identifier: NCT01435720). Disclosures: Barranco: Senesco Technologies: Consultancy. Usmani:Celgene, Onyx, Millenium: Consultancy, Research Funding, Speakers Bureau. van Rhee:Jansen&Jansen: Research Funding. Thompson:Senesco Technologies: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Taylor:Senesco Technologies: stock options Other. Dondero:Senesco Technologies: Employment. Browne:Senesco Technologies Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Siegel:Celgene, Millenium, Onyx (same for all): Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Download Full-text

Single-Cell RNA-Seq Identifies Potentially Pathogenic B Cell Populations That Uniquely Circulate in Patients with Chronic Gvhd

Blood ◽

10.1182/blood-2019-130928 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 874-874

Author(s):

Jonathan C Poe ◽

Dadong Zhang ◽

Jichun Xie ◽

Rachel A. DiCioccio ◽

Xiaodi Qin ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Rna Seq ◽

Advisory Committees ◽

B Cell Subsets

While B cells are known to contribute to the pathogenesis of chronic graft-versus-host disease (cGVHD) in mice, it has been challenging to elucidate intrinsic mechanisms of tolerance loss in patients. To identify distinct and potentially targetable B-cell subsets in cGVHD, we employed single-cell RNA-Seq along with an unsupervised hierarchical clustering analysis, targeting 10,000 single B cells from each of eight patients who were >12 months post-allogeneic hematopoietic stem cell transplantation (HCT) and either had active cGVHD manifestations (n=4) or never developed cGVHD (n=4). Bioinformatics analysis of pooled cell data (using R with Seurat extension package) identified 6 major B cell clusters common to all patients (Figure 1A). "Intra-cluster" gene comparison (using R package DESeq2, false-discovery rate 0.05) revealed numerous differentially expressed genes between patient groups. The greatest number of differentially-expressed genes occurred in a cluster referred to herein as 'Cluster 6' (Figure 1A, in yellow with asterisk). Within Cluster 6, B cells from active cGVHD patients expressed significantly increased ITGAX (CD11c, Padj =0.007), TNFRSF13B (TACI, a receptor for BAFF, Padj =0.003), IGHG1 (IgG1, Padj =9.3e-06) and IGHG3 (IgG3, Padj =1.7e-12), along with 44 additional genes (to be discussed). Thus, Cluster 6 in cGVHD patients may represent a CD11cpos, BAFF-responsive B cell subset primed to undergo isotype switching in response to alloantigen. Flow cytometry analysis on PBMCs from an independent HCT patient cohort (n=10) confirmed that CD11cpos B cells were indeed significantly expanded in cGVHD (P < 0.01, Figure 1B), and revealed these B cells were also TACIpos, CD19high, forward scatter high (FSChigh) blast-like cells (Figure 1C). We found that these CD11cpos B cells had mixed expression of CD21, CD27, IgD and CD24 (Figure 1C). Remarkably, other recent studies on bulk patient B cells have suggested that similar CD11cposCD21negCD19highT-BETpos cells are critical drivers of humoral autoimmunity in diseases including systemic lupus erythematosus (SLE; Scharer et al. 2019; Rubtsova et al. 2017; Rubtsov et al. 2011). This subset now identified by single-cell RNA-Seq is consistent with a population of TACIhigh B cells that produced IgG in response to BAFF treatment ex vivo (Sarantopoulos 2009). Data suggest we have identified functionally distinct and potentially targetable B cell subpopulations. We are employing functional assays to determine whether the additional molecular pathways now elucidated account for our previous work showing greater ex vivo B cell survival rates and hyper-responsiveness to surrogate antigen (Allen et al. 2012, 2014), certain TLR agonists (Suthers et al. 2017), and NOTCH ligand (Poe et al. 2017). In addition to more deeply characterizing B-cell subsets in cGVHD, our single-cell RNA-Seq analyses identified several genes significantly altered across multiple B cell clusters in the cGVHD group, implicating more broad alterations of some genes in this disease. Among these is CKS2, a critical cell cycle regulator, which was significantly increased in cGVHD B cells (Padj 1.0e-10 to 0.018, depending on the cluster evaluated). Increased CKS2 expression was validated by qPCR analysis on B cells from a separate HCT patient cohort with or without cGVHD (P < 0.001, Figure 1D), suggesting that the majority of cGVHD B cells are primed to enter the cell cycle at multiple stages of differentiation when exposed to the proper stimuli. In summary, we used an unbiased approach to identify and further characterize an extrafollicular CD11cposTACIposCD19high B cell population in cGVHD patients that appears to be activated and undergoing active IgG isotype switching. This plasmablast-like B cell population is potentially amenable to therapeutic intervention to prevent pathogenic antibody production. Importantly, we also identify gene alterations across the cGVHD peripheral B cell compartment that potentially underpin promotion of hyperactivated B cells in this disease. Therapeutic strategies to target these pathways will also be discussed. This work was supported by a National Institutes of Health grant, R01HL129061. Disclosures Ho: Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Horwitz:Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Rizzieri:Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding.

Download Full-text

High-Dimensional Heterogeneity of Waldenström Macroglobulinemia within Its Immune Tumor Microenvironment

Blood ◽

10.1182/blood-2019-130011 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3975-3975

Author(s):

Jana Jakubikova ◽

Danka Cholujova ◽

Gabor Beke ◽

Zachary R Hunter ◽

Teru Hideshima ◽

...

Keyword(s):

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Effector Memory ◽

Advisory Committees ◽

Effector Memory Cells ◽

B Cell Precursors

Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Download Full-text

Dynamic Immune Surveillance in Durable Clinical Response to Combined BTK and BCL2 Inhibition in MCL at Longitudinal Single-Cell Resolution

Blood ◽

10.1182/blood-2021-152908 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1323-1323

Author(s):

Kevin Wang ◽

Maurizio Di Liberto ◽

Yang Hu ◽

Xiangao Huang ◽

Rachel M Koldej ◽

...

Keyword(s):

B Cells ◽

Nk Cells ◽

Single Cell ◽

Clinical Response ◽

Cd8 T Cells ◽

Immune Cells ◽

Research Funding ◽

Cell Lymphoma ◽

Immune Surveillance ◽

Mantle Cell

Abstract Combined inhibition of BTK with ibrutinib and BCL2 with venetoclax is one of the most promising therapies for B cell malignancies, especially mantle cell lymphoma (MCL), where durable complete remission continued after therapy cessation in some patients (Tam et al, NEJM 2018, Handunnetti ASH 2019). The MCL-intrinsic and extrinsic mechanisms underlying this deep and durable clinical response are unknown, nor have resistance mechanisms been identified. Since BTK is expressed mainly in B lineage cells and venetoclax inhibits BCL2 universally, we hypothesize that BTK inhibition selectively primes MCL cells for vulnerability to BCL2 inhibition while maintaining immune cell homeostasis, leading to differential elimination of MCL cells through immune surveillance. To test this, we undertook integrative longitudinal single-cell RNA-sequencing analysis (scRNA-seq) of PBMCs from sequential tissue and blood specimens (n=32) of 8 MCL patients before and during ibrutinib-venetoclax combination therapy, after therapy cessation or progression, as well as 4 treatment-naïve MCL patients and 4 normal subjects as controls. High dimensional analysis using a unique MCL RNA reference library that we built from bulk RNA-seq data of MCL cells from 57 patients reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling and proinflammatory pathways; C3 represents non-proliferating, long-lived MCL cells that accumulate as disease progresses; and C4 is highly proliferative, expanding with disease progression in untreated patients or on therapy. Integrative analysis of scRNA-seq and CBC with differential showed that homeostasis of all immune cells was maintained throughout ibrutinib-venetoclax therapy and after therapy cessation in 6 MCL patients with a complete response (CR). CD8+T and NK cells were functional, evidenced by the expression of cytotoxic genes such as GNLY, FGFBP2, and GZMH. In contrast, these genes were profoundly suppressed in CD8+T cells that were rapidly depleted on MCL progression after transient response in 2 patients. NK cells were also depleted on progression. In one patient, this was preceded by suppression of cytotoxic genes and loss of MHC-I and MHC-II in MCL cells. Exhaustion did not appear to be the cause. Rather, TSC22D3 upregulation suggests that inhibition of TCR-induced IL2 and IL2R expression and NF-kB activation may underlie the loss of CD8+T and NK cells in ibrutinib-venetoclax resistance. To determine whether BTK inhibition primes MCL cells for subsequent venetoclax killing while sparing other immune cells in a clinical response to ibrutinib-venetoclax, we found that BCL2 expression was higher in MCL cells than in other immune cells pre-therapy. Ibrutinib selectively downregulated the anti-apoptotic MCL1 and upregulated PMAIP1 encoding the pro-apoptotic NOXA in MCL cells, concurrent with TSC22D3 induction and inhibition of the NF-kB signaling pathway. As expected from the lack of BTK expression, CD4+ and CD8+T cells and NK cells expressed MCL1 over PMAIP1 in CR patients, but greater PMAIP1 in patients who developed progressive MCL. Collectively, these data support the hypothesis that BTK inhibition selectively primes MCL cells for venetoclax sensitivity. scRNA-seq analysis further showed that B cells emerging after therapy cessation in CR patients were nearly identical to the bone marrow B cells pre-therapy, as quiescent B cells in cluster 1 and activated B cells in cluster 2. None were detected in clusters 3 or 4. The restoration of B cell immunity confirms CR at the single-cell transcriptome level, speaking to the power of dual BTK and BCL2 inhibition, and supporting the role of immune surveillance in clinical response in targeted therapy. In summary, by unbiased longitudinal scRNA-seq analysis of sequential patient specimens from the ibrutinib-venetoclax clinical trial, we have provided the first evidence that priming of MCL cells for venetoclax vulnerability by BTK inhibition cooperates with immune surveillance to determine the depth and durability in combined targeting of BTK and BCL2 in lymphoma in humans. These findings parallel our longitudinal scRNA-seq analysis of dual BTK and CDK4/6 inhibition, suggesting priming of cancer cells and cooperation with immune surveillance underpin targeted therapy. Disclosures Koldej: CRISPR Therapeutics: Research Funding. Ritchie: CRISPR Therapeutics: Research Funding; Takeda: Research Funding; BMS: Research Funding; Novartis: Honoraria; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria. Martin: ADCT: Consultancy. Elemento: One Three Biotech: Consultancy, Other: Current equity holder; Owkin: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Janssen: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Champions Oncology: Consultancy. Tam: Beigene: Research Funding; Loxo: Honoraria; Beigene: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Abbvie: Research Funding; Janssen: Research Funding. OffLabel Disclosure: Venetoclax is a BCL2 inhibitor FDA-approved for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and acute myeloid leukemia (AML). It was used off-label in combination with ibrutinib in a phase II clinical trial in patients with mantle cell lymphoma.

Download Full-text

Mutations Affecting RNA Binding Proteins Are a Novel Feature of Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2019-129136 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1478-1478

Author(s):

Krysta M Coyle ◽

Prasath Pararajalingam ◽

Sarah E Arthur ◽

Nicole Thomas ◽

Miguel Alcaide ◽

...

Keyword(s):

Alternative Splicing ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Board Of Directors ◽

Research Funding ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cell Lymphoma ◽

Advisory Committees ◽

Mantle Cell

Objectives Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. We sought to extend our understanding of the molecular etiology of this malignancy using an integrative genomic analysis of diagnostic biopsies. Methods We performed exome sequencing on 51 frozen MCL tumors and analyzed these alongside previously published exome cohorts. We sequenced tumour genomes and matched constitutional DNA from 34 frozen MCLs, along with matched constitutional DNA, to more broadly identify the pattern of non-coding mutations. Based on mutations identified in this discovery cohort, we re-sequenced 18 recurrently-mutated genes in 212 archival MCLs, each having clinical follow-up data. We also performed RNA-seq on 110 of these cases and analyzed these data for alternative splicing and differential expression, including the differential splicing of HNRNPH1 in the context of recurrent intronic mutations. We investigated the functional and phenotypic effect of mutations and deregulated HNRNPH1 protein through ectopic expression of full-length HNRNPH1 and a mini-gene containing the exons and introns affected by mutations. Using custom droplet digital PCR (ddPCR) assays, we validated alternative splicing patterns in HNRNPH1 itself and other targets identified through re-analysis of available CLIP-seq data. Results In addition to confirming the prognostic association of TP53 and NOTCH1 mutations in MCL, we identified two additional genes associated with outcome: EWSR1 with poor outcome (HR = 5.6) and MEF2B with good outcome (HR = 0.2). By comparing mutation patterns to diffuse large B-cell lymphoma (DLBCL), we identified an MCL-specific missense hot spot in MEF2B, non-specific truncating mutations in EWSR1, and truncating mutations affecting the DAZAP1 C-terminus in both MCL and DLBCL. The DAZAP1 mutations are predicted to alter protein sub-cellular localization and disrupt protein-protein interactions. We also identified the focal recurrence of non-coding mutations surrounding a single exon of the HNRNPH1 gene that were largely restricted to MCL. These mutations affected a region bound by HNRNPH1 protein and disrupted the preferred binding motif of this protein. Intronic mutations were significantly associated with alternative splicing of the HNRNPH1 mRNA and appear to disrupt a negative regulatory loop that normally limits the level of HNRNPH1. Using cell-based assays, we have evaluated the role of HNRNPH1 in cell survival and proliferation. Our interrogation of alternative splicing events in downstream targets implicate HNRNPH1 as a master splicing regulator which may broadly perturb the transcriptome and proteome to favor lymphomagenesis in MCL. Conclusions We discovered three novel MCL-related genes with roles in RNA trafficking or splicing, namely EWSR1, DAZAP1, and HNRNPH1. Mutations in these RNA-binding proteins were identified in 49 of 291 (17%) samples analyzed. Our results improve the current understanding of the MCL mutational landscape, highlight the similarities and differences between MCL and DLBCL, and strongly implicate a role for aberrant regulation of RNA metabolism in MCL pathobiology. We elucidated a functional role for recurrent non-coding HNRNPH1 mutations specific to MCL and identified multiple downstream targets. We continue to explore putative trans targets of HNRNPH1, a novel oncoprotein in MCL. Disclosures Steidl: Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Bayer: Consultancy; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding. Connors:Bristol-Myers Squibb: Consultancy; Seattle Genetics: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Johnson:Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; 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. Scott:Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding.

Download Full-text

Tumor Microenvironment Molecular Signatures That Define Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2019-130512 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2762-2762

Author(s):

Krystle Nomie ◽

Nikita Kotlov ◽

Viktor Svekolkin ◽

Alexander Bagaev ◽

Qingsong Cai ◽

...

Keyword(s):

Tumor Microenvironment ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Immune Checkpoint ◽

Research Funding ◽

Cell Lymphoma ◽

Therapeutic Resistance ◽

Molecular Signatures ◽

Immune Infiltration ◽

Mantle Cell

Introduction The tumor microenvironment of mantle cell lymphoma (MCL), an aggressive and incurable subtype of B-cell lymphoma, is dynamic and complex. The MCL microenvironment provides a niche for the tumor by promoting survival, therapeutic resistance, and immune evasion. Although the intrinsic mechanisms underlying MCL pathogenesis have been well-studied, as demonstrated by our understanding of the important roles that B-cell receptor signaling, the PI3K/AKT/mTOR axis, and OXPHOS play in MCL survival and the development of therapeutic resistance, the extrinsic mechanisms regulated by the lymphoma microenvironment are less well-known. Methods Whole exome sequencing (WES; n = 42) and RNA-seq (n = 76) were performed on fresh peripheral blood or apheresis patient primary samples with an extremely high MCL tumor percentage as determined by flow cytometry versus biopsies with a more diverse cellular mixture, including MCL tumor microenvironment components such as macrophages, T-cells, NK cells, and monocytes. Our previously published MCL cohort was also analyzed in this study (Zhang et al., Science Translational Medicine, 2019). Joint WES and RNAseq mutation calling, expression analysis, and cell type deconvolution from the transcriptome were performed using the BostonGene automated pipeline. Results To characterize the cellular composition and functional state of the MCL tumor microenvironment as well as tumor properties, we created 26 separate molecular signatures related to various functional processes such as anti-tumor immune infiltration, immune checkpoint inhibition, immunosuppression, and stromal compartment represented by angiogenesis and mesenchymal stromal cells. We also utilized these 26 immune and stromal signatures in conjunction with PROGENy (Pathway RespOnsive GENes) analysis to create a signature-based model associated with sensitivity or resistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. In this model, increased T-cell, NK cell, and B-cell processes, in addition to p53 pathway activity, were associated with sensitivity to ibrutinib, demonstrating that the tumor microenvironment plays a critical role in the MCL response to this widely used FDA-approved agent. Moreover, initial analysis only identified one BTKC481S mutation in an ibrutinib-resistant MCL sample, again demonstrating that mutations in BTK are rare in ibrutinib-resistant MCL and that diverse mechanisms underlie the development of this resistance. For more in-depth analysis, we performed concurrent analysis of only the biopsy samples in conjunction with an additional previously published cohort (n = 123; Scott et al., JCO, 2017). Unsupervised clustering based on the activities of the proposed signatures produced 4 MCL types as follows: immune infiltration combined with increased stromal signatures (type MCL-A), high immune and checkpoint molecules expression with low stromal expression (type MCL-B), non-immune with increased stromal signature and tumor-promoting cytokines (type MCL-C), and lacking immune infiltration and stromal expression with highest content of malignant B cells (type MCL-D). Interestingly, the ibrutinib-resistant MCL samples primarily belonged to the MCL-C subtype (80%), whereas most of the ibrutinib-sensitive samples (70%) were assigned to subtype MCL-B (Chi-square test p-value = 0.01), which is prominent in anti-tumor immune infiltration without tumor-promoting stromal context, suggesting that ibrutinib may promote immune microenvironment effective action against MCL or work more effectively in an active immune environment. Conclusion The identified enriched immune cell signatures suggest that MCL cells may be sensitive to specific and novel immune checkpoint inhibitors and other immune activators. Ibrutinib sensitivity and resistance are clearly separated based on their tumor microenvironment portraits, suggesting that the tumor microenvironment has a prominent role in regulating ibrutinib activity and response. Furthermore, ibrutinib may alter the tumor microenvironment to promote anti-tumor activity, which requires further investigation. Disclosures Wang: MoreHealth: Consultancy, Equity Ownership; Celgene: Consultancy, Research Funding; Juno Therapeutics: Research Funding; Kite Pharma: Consultancy, Research Funding; Dava Oncology: Honoraria; Pharmacyclics: Consultancy, Honoraria, Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Aviara: Research Funding; BeiGene: Research Funding; BioInvent: Consultancy, Research Funding; VelosBio: Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Download Full-text

CD5 Negative Mantle Cell Lymphoma: Clinicopathologic Correlations and Outcome in 58 Cases

Blood ◽

10.1182/blood.v128.22.2964.2964 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2964-2964

Author(s):

Annapurna Saksena ◽

Yuan Miao ◽

Pei Lin ◽

Michael Wang ◽

C. Cameron Yin ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

P Value ◽

Clinicopathologic Features ◽

Advisory Committees ◽

Mantle Cell

Abstract Introduction: Mantle cell lymphoma (MCL) is a B-cell neoplasm that has a characteristic immunophenotype of being positive for CD5, B-cell antigens and cyclin D1. A small subset of cases of MCL can be negative for CD5, approximately 5% in the literature. The clinicopathologic features and prognosis of patients with CD5-negative MCL are poorly characterized. Here, we study a group of patients with CD5- MCL and compare them with a group patients with CD5+ MCL. Methods: From a total of 270 cases of MCL accessioned from 2004-2015, 58 CD5- cases (study group) and 212 CD5+ cases (control group) were identified. All cases of MCL were positive for cyclin D1 by immunohistochemistry and, in most patients, CCND1-IGH was shown FISH. Cases negative for CD5 were assessed by flow cytometry and/or immunohistochemistry. Fisher exact test was utilized to analyze differences between the CD5- and CD5+ groups. Patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate Cox proportional hazards model analyses for OS and PFS were performed (SPSS 22 software). A P-value of less than 0.05 was considered statistically significant. Results: The CD5- group included 39 men and 19 women with a median age of 66 years (range, 36- 88 years) at time of diagnosis. The CD5- and CD5+ groups shared overlapping clinicopathological features, but CD5- cases showed a lower percentage of men (P=0.006) than CD5+ cases. Treatment information was available for 50 patients. Twenty-nine (58%) patients were treated initially with R-Hyper CVAD therapy (rituximab, fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with high dose methotrexate and cytarabine). Seventeen (34%) patients were treated initially with less aggressive therapy: 7 with R-CHOP; 8 had other rituximab-based chemotherapy regimens; 2 received rituximab as a single agent. Four patients (8%) were observed without therapy. After induction, 34 patients achieved complete remission (CR), 5 patients achieved partial remission (PR), 6 patients showed no response (NR) or progressive disease (PD), and 5 patients lost follow-up. Ten patients also underwent stem cell transplantation (SCT): 5 patients received allogeneic SCT, the other 5 autologous SCT. With a median follow-up of 45.7 months (range, 2.0-174.3 months), 13 of 56 (23.2%) patients died, 43 of 56 (76.8%) patients were alive at last follow-up, and the rest of 2 patients lost follow up. The induction chemotherapy regimens and CR and PR rate were not significantly different between the CD5- and CD5+ groups (p>0.05). Survival analysis showed patients with CD5- MCL had a tendency for longer OS (Figure 1A, P=0.078). Further analysis showed that lack of CD5 expression predicted a superior OS in a few subsets of MCL patients defined with 1) normal WBC count (p=0.049); 2) Stage I/II disease (p=0.046); 3) Low/intermediate MIPI (p=0.041) and 4) Ki67≥30% (at a borderline p value of 0.05). Patients with CD5- MCL also showed a significantly longer progression-free survival (PFS) (Figure 1B, P=0.01). Absence of CD5 expression was associated with a better PFS in MCL patients with advanced disease (stage III-IV) (P=0.035), a normal leukocyte count (P=0.018), a normal serum lactate dehydrogenase level (P=0.046), classical morphology (P=0.029), and low/intermediate MIPI (p=0.0004). Multivariate Cox regression analysis revealed that MIPI was the only independent prognostic factor for both OS and PFS (P=0.026 and P=0.001 respectively) and CR/PR also predict a better OS (P=0.004) in CD5- MCL patients. Conclusion: The clinicopathologic features were similar between patients with CD5- MCL and those with CD5+ MCL, except that less men in the CD5- MCL group. Lack of CD5 expression was associated with a favorable PFS in MCL patients. Recognizing this subgroup of CD5- MCL has not only a diagnostic significance, but also a prognostic significance. Figure Figure. Disclosures Wang: Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Asana BioSciences: Research Funding; BeiGene: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Onyx: Research Funding.

Download Full-text

Transcriptomic Heterogeneity and Clonal Evolution Associated with Therapeutic Resistance in Mantle Cell Lymphoma Revealed By Single Cell RNA-Seq

Blood ◽

10.1182/blood-2019-129134 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5217-5217

Author(s):

Changying Jiang ◽

Shaojun Zhang ◽

Yuanxin Wang ◽

Rongjia Zhang ◽

Yang Liu ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Single Cell ◽

Research Funding ◽

Tumor Heterogeneity ◽

Cell Lymphoma ◽

Clonal Evolution ◽

Therapeutic Resistance ◽

Transcriptomic Profiling ◽

Pdx Model ◽

Mantle Cell

Introduction: Both inter- and intra-tumoral heterogeneity are obstacles to improving oncology clinical outcomes. Mantle cell lymphoma (MCL) is an extremely heterogeneous disease in clinical, pathological, genetic, and transcriptomic profiling. Furthermore, MCL patients frequently develop therapeutic resistance after frontline therapies. In this study, we performed longitudinal transcriptomic analysis on primary patient MCL specimens at single-cell resolution, aiming to understand the dynamic and complex cellular and molecular changes underlying therapeutic resistance and identify potential targets to overcome dual resistance to ibrutinib and venetoclax. Methods: Sequential single-cell transcriptome sequencing (scRNA-seq) was performed on patient specimens collected during the course of treatment(s) from 5 MCL patients (3 ibrutinib responders and 2 ibrutinib-venetoclax non-responders). Integrative computational approaches were employed to characterize the cellular and molecular basis of therapeutic resistance and clonal evolution. An orthotopic PDX model derived from one of the non-responders was established and used to validate the novel findings and to investigate the in vivo efficacies of multiple novel potential targets. Results: The 3 ibrutinib responders and 2 ibrutinib-venetoclax non-responders were highly heterogeneous in clinical and pathological profiling. To dissect the inter- and intra-tumor heterogeneity underlying the therapeutic resistance, we performed sequential scRNA-seq analysis of 21 specimens collected at baseline, during treatment, and/or at disease remission/progression. The scRNA-seq analysis revealed a high degree of inter- and intra-tumor heterogeneity with distinct cellular and transcriptomic profiling within and across ibrutinib-responders and ibrutinib-venetoclax non-responders. Unsupervised pathway enrichment analysis identified more than 15 cancer hallmarks significantly upregulated in ibrutinib-venetoclax non-responders. We tracked the clinical ibrutinib-induced lymphocytosis at a single-cell transcriptomic level in ibrutinib responders and disease-progression-associated clonal evolution in non-responders. Multiple actionable targets were identified, and targeting these showed effective anti-MCL activity in the orthotopic PDX model derived from one of the ibrutinib-venetoclax non-responders. Conclusions: This study demonstrates the potential of longitudinal single-cell transcriptomic analysis to reveal the molecular mechanisms underlying tumor heterogeneity, clonal evolution, disease progress, and therapeutic resistance, and to identify potential novel targets to circumvent therapeutic resistance in mantle cell lymphoma and other diseases. Disclosures Wang: Pharmacyclics: Honoraria, Research Funding; Juno Therapeutics: Research Funding; Celgene: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; Loxo Oncology: Research Funding; VelosBio: Research Funding; BioInvent: Consultancy, Research Funding; Dava Oncology: Honoraria; Aviara: Research Funding.

Download Full-text