A Somatic Variant in MYD88 (L265P) Revealed by Whole Genome Sequencing Differentiates Lymphoplasmacytic Lymphoma From Marginal Zone Lymphomas

Abstract Abstract 261 Lymphoplasmacytic (LPL) and marginal zone lymphoma (MZL) are distinct clinicopathological entities under the WHO classification system for B-cell lymphomas. Differentiation of LPL from MZL has been difficult due to overlapping clinical, morphological, histopathological, immunophenotypic, and cytogenetic features. We therefore sought to identify a molecular marker by which LPL could be differentiated from MZL. Using paired normal/tumor tissues from 10 LPL patients, whole genome sequencing was utilized to identify somatic variants. These studies identified a somatic variant at position 38182641 in chromosome 3p22.2 with a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P) in 10 of 10 LPL patients. MYD88 L265P is an oncogenically active mutation in DLBCL ABC cell lines via activation of IRAK1/4/TRAF-6/NF-κβ signaling, and is present in tumors from 29% of patients with ABC subtype of DLBCL, and 6% of patients with MALT lymphomas (Ngo et al, Nature 2011, 470:115–119). Further to these efforts, we performed Sanger sequencing of MYD88 in malignant cells obtained from 51 patients with LPL, 49 of whom had an IgM monoclonal protein and were therefore classified as Waldenstrom's Macroglobulinemia (WM), and 2 with an IgG monoclonal protein, along with 46 patients with MZL, which included 21 Splenic (SMZL), 20 Extranodal (EMZL), and 5 Nodal (NMZL) Subtypes, as well as B-cells from 15 healthy donors. Among LPL patients, the MYD88 L265P variant was found in malignant cells from 46/51 (90.1%) cases, which included 44 patients with WM, and 2 patients with IgG LPL. Expression of the MYD88 L265P variant was heterozygous in 42, and homozygous in 4 LPL patients. By comparison, only 3/46 (6.5%) patients with MZL (1 SMZL; 1 EMZL; 1 NMZL) exhibited the MYD88 L265P variant which was heterozygous (p<0.0001), and included 2 patients (1 SMZL, 1 NMZL) with extensive bone marrow involvement, a monoclonal IgM protein, and whose clinicopathological characteristics overlapped with LPL. By comparison, the MYD88 L265P variant was absent in CD19+ cells from all 15 healthy donors. The results of this study demonstrate that the MYD88 L265P mutation is widely expressed in patients with LPL, and can be used to differentiate LPL from MZL. Disclosures: Treon: Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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Chromoplexy and Chromothripsis Are Important Prognostically in Myeloma and Deregulate Gene Function By a Range of Mechanisms

Blood ◽

10.1182/blood-2019-130335 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3767-3767 ◽

Cited By ~ 1

Author(s):

Cody Ashby ◽

Eileen M Boyle ◽

Brian A Walker ◽

Michael A Bauer ◽

Katie Rose Ryan ◽

...

Keyword(s):

Board Of Directors ◽

Genome Sequencing ◽

Gene Function ◽

Research Funding ◽

Whole Genome ◽

Structural Variants ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Research Grant

Background: Structural variants are key recurrent molecular features of myeloma (MM) with two types of complex rearrangement, chromoplexy and chromothripsis, having been described recently. The contribution of these to MM prognosis, rapid changes in clinical behavior and punctuated evolution is currently unknown as is the mechanism by which they deregulate gene function. Methods: We analyzed two sets of newly diagnosed MM data: 85 cases with phased whole genome sequencing; and 812 cases from CoMMpass where long-insert whole-genome sequencing was available. Patient derived xenografts from five MM cases were used to generate epigenetic maps for the histone marks, BRD4, MED1, H3K27Ac, H3K4me1, H3K4me3, H3K9me3, H3K36me3 and H3K27me3. Results: In the 10X data the median number of structural events per case was 25 (range 1 - 182); with a median of 14 intra-chromosomal events (range 1 - 179; P<0.001) and 7 inter-chromosomal events (range 0 - 29). Structural events were seen most frequently on chromosomes 14 (64%), 8 (53%), 1 (44%) and 6 (42%). Complex chromosomal rearrangements involving 3 or more chromosomal sites were seen in 46%, 4 or more sites in 20%, 5 or more in 10% and 6 or more in 5% of samples. There were significantly more structural events in the t(4;14) subgroup compared to the t(11;14) subgroup. Significantly more events were also seen in the bi-allelically inactivated TP53 cases. Using an elbow test defined cutoff, we identified cases with high structural variant load in 10% of cases. Chromoplexy called by "Chainfinder" was seen in 18% of cases. Chromothripsis called by "Shatterseek" was seen in 9% of cases. Cases with a high structural load alone were not associated with an adverse outcome whereas cases with chromoplexy or chromothripsis were associated with adverse PFS and OS, p=0.001. A new high-risk subgroup comprising approximately 5% of cases was identified with chromoplexy, chromothripsis and a high structural load. Gene set enrichment analysis of cases with chromoplexy and chromothripsis showed an excess of MYC, E2F and G2M targets, and a reduction in RAS signaling. Interferon a and g responses, an excess of TP53 and reduction in TRAF3 mutations was associated predominantly with chromothripsis. How chromoplexy and chromothripsis are tolerated by the cell is unknown and the association with the cGAS/STING response is further being explored. To determine how chromoplexy may deregulate multiple genes we identified the full spectrum of structural variants to the immunoglobulin (Ig) and non-Ig loci. A range of genes are deregulated by Ig loci including MAP3K14 at a frequency of 2% confirming the importance of non-canonical NFkB signaling. A novel intra-chromosomal rearrangement to ZFP36L1 was upregulated in 10% of cases but was not prognostic. Gene upregulation by non-Ig super enhancers is frequent and targets include PAX5, GLI3, CD40, NFKB1, MAP3K14, LRRC37A, LIPG, PHLDA3, ZNF267, CENPF, SLC44A2, MIER1, SOX30, TMEM258, PPIL1, and BUB3. The topologically associating domain (TADs) containing super enhancers bringing about gene deregulation include TXNDC5, FOXO3, FCHSD2, SP2, FAM46C, CACNA1C, TLCD2 and PIK3C2G. These super enhancers frequently contain important MM genes, the coding sequence of which are disrupted by the rearrangement and could contribute to the clinical phenotype. Accurately reconstructing the structure of the complex rearrangements will allow us to identify the mechanism of gene deregulation and to distinguish between either gene stacking, receptor stacking or both. Conclusions: Upregulation of gene expression by super enhancer rearrangement is a major mechanism of gene deregulation in MM and complex structural events contribute significantly to adverse prognosis by a range of mechanisms as well as simple gene overexpression. Disclosures Boyle: Amgen, Abbvie, Janssen, Takeda, Celgene Corporation: Honoraria; Amgen, Janssen, Takeda, Celgene Corporation: Other: Travel expenses. Walker:Celgene: Research Funding. Thakurta:Celgene: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Davies:Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor; Janssen, Celgene: Other: Research Grant, Research Funding. Morgan:Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding.

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Pklr Intron Splicing-Associated Mutations and Alternate Diagnoses Are Common in Pyruvate Kinase Deficient Patients with Single or No Pklr Coding Mutations

Blood ◽

10.1182/blood-2018-99-117805 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3607-3607 ◽

Cited By ~ 3

Author(s):

Kimberly Lezon-Geyda ◽

Melissa J. Rose ◽

Melissa A. McNaull ◽

Christine M. Knoll ◽

Hassan M. Yaish ◽

...

Keyword(s):

Sequence Analysis ◽

Board Of Directors ◽

Pyruvate Kinase ◽

Genome Sequencing ◽

Research Funding ◽

Exon Skipping ◽

Whole Genome ◽

Coding Region ◽

Advisory Committees ◽

Exon 10

Abstract Pyruvate kinase (PK) catalyzes the second ATP-forming step in glycolysis. Erythrocytes lack mitochondria and are dependent on glycolysis for energy. Recessively inherited mutations in the pyruvate kinase (PKLR) gene are the most common defect in the glycolytic pathway associated with chronic nonspherocytic hemolytic (CNSHA) anemia. Symptomatology is variable, ranging from well compensated anemia to severe disease with lifelong transfusion dependence. Genetic analyses of PK-deficient patients have shown most carry missense, frameshift, and nonsense mutations that lead to qualitative or quantitative defects in pyruvate kinase. We studied CNSHA PK-deficient patients from 13 kindreds with one (n=8) or no (n=5) amino acid altering mutations identified in the PKLR gene. To search for potential splicing or regulatory mutations on the other allele, or an alternate red blood cell diagnosis, whole exome sequencing (WES) or whole genome sequencing (WGS) of genomic DNA from affected patients was performed. Five patients with no PKLR coding region mutations had other rare genetic variants predicted to be damaging or previously associated with hematologic disease, including a GATA1 mutation previously associated with PK deficiency, a KIF23 mutation, and 3 with pathogenic PIEZO1 mutations. DNA of patients from 5 other kindreds with single coding region mutations; 1) R486W; 2) G319D; 3) R510Q; 4) A392T; and 5) R510Q, had WGS performed. Genomic analyses did not identify deletional or structural variants in or around the PKLR locus. Haplotyping did not reveal any common shared alleles between the 5 kindreds. Detailed sequence analysis identified unique deep intronic mutations in the PKLR gene in affected members from all 5 kindreds: 1) intron 7 G>A, 2) intron 7 T>G, 3) intron 9 T>A, 4) intron 9 G>A, and 5) exon 7/intron 7 boundary G>A. Four of the 5 mutations were not found in the 1000 Genomes database, while the fifth was found at a frequency of 0.0006. The Scroogle algorithm predicted all 5 mutations would perturb normal mRNA processing; kindred 1) create a novel 3' acceptor splice site; 2) disrupt an intron splicing enhancer or create a 3' splice acceptor site, 3) and 4) create novel 5' donor splice sites, and 5) disrupt a wild type 5' donor splice site. Minigene assays were performed to examine whether these PKLR intron mutations influenced splicing in vitro. Each minigene contained the ANK1 erythroid promoter, a patient-specific PKLR fragment with a mutant intronic allele inserted into intron 2 of the HBG1 gene, and the HBG1 3'untranslated region and polyA signal. After transformation in K562 cells, minigene-specific RNA was harvested, RT-PCR performed, followed by shotgun subcloning of PKLR cDNA. Sequence analysis of plasmid DNA identified aberrant PKLR mRNA isoforms from all 5 minigenes including partial exon skipping, single or multiple exon skipping, and/or partial intron retention. Specifically, kindred 1) skip exon 8 or skip exons 7-9; 2) skip exons 7-8 or skip exons 7-9; 3) insert 38bp 5' of exon 10 or skip exon 10, 4) delete first 67bp of exon 10 or skip exon 10; 5) skip exon 7-8 or skip exon 7-9. In aggregate, these isoforms all to lead to frameshift, with premature chain termination predicted to trigger nonsense mediated decay. Splicing studies from primary patient reticulocyte RNA are ongoing. Three patients with heterozygous PKLR mutations, S8A, V134D, and V460M, had no obvious disease-associated variants detected on WGS. These variants, particularly V134D and V460M, could lead to a dominant negative phenotype. These results indicate that detailed investigation, including whole genome sequencing, lead to a specific hematologic diagnosis in most pyruvate-kinase deficient patients. They also show that in a subset of patients, intron mutations leading to aberrant splicing may be a genetic mechanism associated with pyruvate kinase deficiency. This may be an under recognized mechanism of genetic disease. Disclosures Glader: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Grace:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy; Agios Pharmaceuticals: Research Funding.

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Whole Genome Sequencing Identifies a Recurrent Mutation in Complement Factor I (CFI) in Primary Myelofibrosis (PMF)

Blood ◽

10.1182/blood-2021-154281 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1472-1472

Author(s):

Brooke Sadler ◽

Anna D Chorzalska ◽

Dennis M Bonal ◽

Gabe Haller ◽

Alissa Oakes ◽

...

Keyword(s):

Board Of Directors ◽

Genome Sequencing ◽

Research Funding ◽

Jak2 V617f ◽

Driver Mutations ◽

Whole Genome ◽

Complement Factor ◽

Advisory Committees ◽

Factor I ◽

Cd34 Cells

Abstract Background: The known driver mutations in PMF (including JAK2, CALR, and MPL) do not explain the highly inflammatory phenotype associated with this disease. Moreover, JAK2 inhibitors provide symptomatic relief in PMF, but they are not curative, indicating that our understanding of the molecular pathology of MPNs is incomplete. High-resolution insights into the mutational landscape of MPNs may inform new diagnostic and therapeutic approaches. Methods: We performed 60x whole-genome sequencing (WGS) on CD34+ hematopoietic stem/progenitor cells and matched in vitro-expanded CD3+ lymphocytes from 10 patients (pts) with PMF, of whom 5 had JAK2 V617F, 2 had MPL W515L, and 3 had CALR (del52 or ins5) mutations. Paired-end WGS was performed using BGISEQ-500 platform. Clean reads with Q20 and Q30 at 96.4% and 87.1%, respectively, were aligned to the human GRCh37 reference genome. 99.9% reads mapped successfully and 90.6% mapped uniquely. Mean sequencing depth for CD3+ and CD34+ cells were 72x and 83x, respectively (Fig A). Bioinformatic pipeline strategy is summarized on Fig B. We also examined complement activity in sera from 10 PMF pts using CH50 (a screening test for total complement activation) and C3 and C4 component activity. We further evaluated the effect of complement-neutralizing anti-C3 and anti-C5 (C3/C5 NeuAbs) on JAK/STAT and NF-κB signaling in mononuclear cells (MNCs) isolated from peripheral blood of these PMF pts. Results: ~3.4e6 SNPs were identified (n=10): 99.9% were represented in dbSNP, 98.1% were annotated in GnomAD, and 4,587 were novel. Among ~8.6e5 InDels, 91.9% were represented in dbSNP, 53.8% in GnomAD, and ~6.9e4, were novel. We identified 3,540 copy-number variations (CNVs) and 3,365 SVs. Rare non-synonymous variants (RNSV) were defined as SNVs with a maximum minor allele frequency <0.01 in any GnomAD population. RNSVs were further filtered based on clinical interpretation of their genomic variation in ClinVar. 78 unique RNSVs were identified (Fig C), after exclusion of RNSVs with benign, likely benign, or undetermined status. Known PMF drivers (JAK2 V617F, CALR, and MPL W515L) were present in both CD34+ and CD3+ cells, except in 2 samples JAK2 V617F was present only in CD34+ cells. This suggested WGS-detectable driver clonality in most but not all analyzed cases. All mutations except MPL were heterozygous. We further found known RNSVs in TP53, U2AF1, TCF12, as well as previously unreported in PMF pathogenic mutations in CD247 and OTUD6B. Among 11 RNSVs with conflicting classification in ClinVar, we observed mutations in BRCA2, TTN, APOB, ATM and CDH1 (previously linked to MPNs). Within this group of variants, we also detected a p.G119R mutation in Complement Factor I (CFI, rs141853578) in 2 samples. This RNSV was not previously reported in PMF. CFI G119R was detected in 1 sample with JAK2 and 1 with CALR driver mutation, in both CD34+ and CD3+cells as confirmed by Sanger sequencing (Fig D). CFI encodes a serine proteinase that regulates the complement pathway, and its deficiency is associated with severe inflammatory pathology. In the sera of different cohort of 10 pts with PMF, we observed an increase in CH50 levels (n=3) and depletion of C3 (n=3; Fig E), independent of the presence of specific driver mutations or treatment with ruxolitinib (RUXO) or hydroxyurea, suggesting that complement overactivation may contribute to the pathology of PMF in some pts. We further incubated PMF or healthy PBMCs in matching plasmas in the presence of C3/C5 NeuAbs either alone or in combination with RUXO. In PBMCs from healthy controls, STAT3 pTyr705 decreased upon treatment with RUXO alone or with C3/C5 NeuAbs, and NF-κB pSer536 decreased for all treatment conditions (Fig F). In contrast, in PMF, STAT3 pTyr705 did not consistently decrease in response to RUXO either alone or with C3/C5 NeuAbs. Notably, NF-κB pSer536 was unaffected by RUXO alone, and only treatment with RUXO + C3/C5 NeuAbs induced a decrease in NF-κB pSer536 (Fig F). Conclusions: Using WGS, we discovered an inactivating RNSV in CFI G119R in 20% of pts with PMF. In a separate cohort of PMF patients we detected elevated serum complement activation. Furthermore, PMF-specific dysregulation of STAT3 and NF-κB activation could be modulated by exposure to C3/C5 NeuAbs. This is a first, to our knowledge, report linking the overactivation of the complement cascade to inflammation-related pathogenesis of PMF. Figure 1 Figure 1. Disclosures Hobbs: Novartis: Consultancy; Bayer: Research Funding; Celgene/Bristol Myers Squibb: Consultancy; AbbVie.: Consultancy; Constellation Pharmaceuticals: Consultancy, Research Funding; Incyte Corporation: Research Funding; Merck: Research Funding. Oh: Abbvie: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Celgene Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Membership on an entity's Board of Directors or advisory committees; PharamaEssentia: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees. Di Paola: CSL Behring: Consultancy, Honoraria.

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Whole Genome Sequencing Identifies Recurring Somatic Mutations in the C-Terminal Domain of CXCR4, Including a Gain of Function Mutation in Waldenstrom's Macroglobinemia.

Blood ◽

10.1182/blood.v120.21.2715.2715 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2715-2715

Author(s):

Yang Cao ◽

Lian Xu ◽

Xia Liu ◽

Yangsheng Zhou ◽

Guang Yang ◽

...

Keyword(s):

Bone Marrow ◽

Whole Genome Sequencing ◽

Board Of Directors ◽

G Protein ◽

Genome Sequencing ◽

Somatic Mutations ◽

Surface Expression ◽

Whole Genome ◽

Advisory Committees ◽

Terminal Domain

Abstract Abstract 2715 Introduction: Waldenstrom's Macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells (LPC) in the bone marrow. Using paired normal/WM lymphoplasmacytic cell paired tissues and whole genome sequencing (WGS), we identified somatic mutations in the CXC chemokine receptor 4 (CXCR4) gene which were present in 16/55 (29%) WM patients. CXCR4 is a G-protein-coupled receptor, together with its ligand, the stromal cell- derived factor-1(CXCL12/SDF-1), play an important role in leukocyte and lymphocyte hematopoiesis and trafficking. Upon SDF-1 stimulation, CXCR4 is phosphorylated and interacts with b-arrestins, which then trigger extracellular signal-regulated kinase (ERK) MAPKs and chemotaxis. CXCR4 signaling is then terminated through receptor internalization which is mediated via phosphorylation of its C-terminal tail. Methods: Sanger sequencing was used to validate WGS results. To clarify the functional significance of one of the most common somatic mutation identified (C1013G), cloning by PCR was undertaken from CD19+ bone marrow cells from a WM patient with the C1013G CXCR4 (C1013G-CXCR4) mutation. Wild type (WT) and C1013G-CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy for WM cells into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by FACS using PE-conjugated anti-CXCR4 monoclonal antibody 12G5. CXCR4 internalization was studied by comparing CXCR4 surface expression before and after SDF-1 stimulation. Chemotaxis studies were performed using a transwell assay. The expression of phosphorylated ERK1/2 and total ERK2 was determined by western blot. Results: Validated somatic mutations in CXCR4 were all present in the C-terminal domain and included premature stop codons (C1013A; C1013G), and frameshift mutations. Importantly, the identified mutations are similar to those reported in the germline of patients with WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis) Syndrome, a dominant autosomal genetic disorder caused by tonic CXCR4 activation by impairment of CXCR4 internalization, and stimulation of G-protein-dependent responses, and chemotaxis. Consistent with such a role, SDF-1a stimulation showed decreased internalization of CXCR4 in C1013G-CXCR4 versus WT CXCR4 transduced BCWM.1 WM cells. SDF-1a stimulated ERK1/2 phosphorylation was also more robust C1013G-CXCR4 versus WT CXCR4 expressing cells. Lastly, C1013G-CXCR4 transduced cells displayed stronger migratory response toward SDF-1a versus WT CXCR4 expressing BCWM.1 cells. Conclusions: C-terminal domain somatic mutations are common in WM and overlap with germline mutation identified in WHIM syndrome. Moreover, the most common of these mutations (C1013G) confers gain of function including decreased CXCR4 internalization, more robust ERK ½ phosphorylation, and chemotaxis. The findings provide new insights into the pathogenesis of WM, and a framework for the study of CXCR4 inhibitors for WM therapy. Disclosures: Treon: Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees.

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Clinical-Grade Whole Genome Sequencing Reproduces FISH Cytogenetics and Provides Actionable Data in Newly Diagnosed Myeloma - a Pilot Study from the UK 100,000 Genomes Project

Blood ◽

10.1182/blood-2019-125145 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3062-3062

Author(s):

Oliver Lomas ◽

Sarah Gooding ◽

Karthik Ramasamy ◽

Angela Hamblin ◽

Maite Cabes ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Genome Sequencing ◽

Standard Of Care ◽

Whole Genome ◽

Clinical Grade ◽

Newly Diagnosed ◽

Fish Cytogenetics ◽

Advisory Committees ◽

Research Grants

Background Treatment decisions in Multiple Myeloma (MM) have been driven by a patient's age or ability to tolerate therapy, but as yet, not by their tumour genetics, even though understanding of the prognostic utility of genetic features continues to develop. The ability to identify genetic prognostic markers and potential therapeutic targets in a timely fashion within a health service is a vital step in delivering precision medicine to patients. The aims of this study were to assess the implementation of clinical grade whole genome sequencing (WGS) data at diagnosis to replicate, and ultimately replace, standard-of-care Fluorescence In Situ Hybridisation (FISH) cytogenetics; to provide markers for prognostication or MRD; and to identify actionable targets for clinical trials in precision therapy of MM. Methods Bone marrow aspirates from patients with newly-diagnosed myeloma were collected between June 2017 and April 2018 in a single tertiary hospital in the United Kingdom. The population comprised seven male and seven female patients with a mean age of 78 years. From the first-draw of bone marrow aspirate, standard-of-care FISH cytogenetic analyses were performed locally according to criteria from the International Myeloma Working Group (IMWG). From the remnant aspirate samples, CD138-positive plasma cells were enriched by magnetic bead-sorting and genomic DNA was extracted locally for WGS with a success rate of approximately 70%. Fourteen samples underwent successful plasma cell purification (78 - 99% morphological purity) with a yield of at least 0.5 μg DNA. To identify germline genomic variants, DNA was extracted from peripheral blood samples obtained simultaneously. WGS was performed at a centralised facility and mean coverage for germline samples was 35.1x and for plasma cell-enriched samples was 100.9x. Conventional cytogenetic FISH data were compared with genomic data for chromosome-level alterations. Identified somatic variants were automatically cross-referenced against publicly available databases that describe somatic mutations in cancer as well as a virtual panel of potentially actionable therapeutic targets including : NRAS, KRAS, BRAF, CDKN2C, FGFR3 and IDH2. Results In paired samples, WGS replicated all 13 translocation and chromosomal loss/gain events identified by FISH (Figure 1). Furthermore, three translocations involving the IGH locus suggested by FISH analysis were characterised by WGS. Using samples derived from surplus material, fast-track turnaround of 14 days was attainable. Five patients had no identifiable marker by FISH cytogenetics. In these patients, WGS found all five to possess somatic variants could be used as prognostic or potential Measurable Residual Disease (MRD) markers. Nine patients exhibited somatic variants in genes that may be subject to targetable therapy as determined by trials available on ClinicalTrials.gov: five NRAS, two KRAS, one BRAF and one FGFR3 mutation as of July 2019. Conclusion WGS assessment of newly diagnosed myeloma provides accurate, timely and actionable information beyond what is available from standard-of-care FISH. The application of WGS to myeloma diagnostics presents a number of advantages. Firstly, WGS can replicate and exceed existing myeloma FISH assessment of translocation and loss/gain events in a prompt turnaround time. Secondly, germline variants are deducted from tumour variants to provide a gold standard description of the somatic mutational landscape in the patient's disease. Thirdly, virtual panels of known somatic variants can be applied to the data as knowledge accumulates about the role of specific mutations on prognosis or therapeutic response. We demonstrate that centrally provided WGS and its analysis can be incorporated into routine local assessment of newly diagnosed myeloma. Therefore, these observations show that such technology has the potential to be rapidly scalable across existing hospital networks. Disclosures Gooding: Celgene Corporation: Research Funding. Ramasamy:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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T Cell Immunity Toward Viral- and Tumor-Antigens Is Preserved in MDS Patients

Blood ◽

10.1182/blood-2019-126792 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4225-4225

Author(s):

Hussein Hamad ◽

Wingchi K Leung ◽

Spyridoula Vasileiou ◽

Shivani Mukhi ◽

Quillan Huang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Healthy Donor ◽

Tumor Antigens ◽

Ex Vivo ◽

Advisory Committees ◽

Cell Immunity ◽

Healthy Donors ◽

T Cell Lines

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by bone marrow failure and a propensity to progress to acute myeloid leukemia (AML). A core component of the underlying pathogenesis in MDS is deregulation of inflammatory cytokines, such as tumor growth factor-β (TGFβ), which impact the function of immune cells and hence their capacity to mount anti-infective or anti-tumor responses. However, little is known about antigen-specific T cell function in patients with MDS. We hypothesized that virus-specific T cell (VST) function might be preserved in patients with MDS, and that the functional capacity of T cells reactive against tumor-associated antigens aberrantly overexpressed by clonal MDS cells such as Cyclin A1 (CCNA1) and Proteinase (PR3) might also be preserved and exploited for immunotherapeutic purposes. Following informed consent, we collected peripheral blood samples from 10 patients (pts) with MDS and 17 healthy donors. Most pts (9 out of 10) were transfusion dependent and 3 subsequently underwent an allogeneic HSCT. Table 1 summarizes the other clinical characteristics, karyotypic and mutational profile at the time of blood collection. Compared with T cells isolated from healthy donors, MDS patient-derived T cells had a similar CD4 to CD8 ratio (1.5-2.5:1 for healthy donors and 3:1 for MDS pts), but displayed a more exhausted profile at baseline (CD3+TIM3+: 1% in healthy donors and 5% in MDS pts) and produced higher levels of inflammatory cytokines [IFNγ (18±3pg/ml vs 36±16pg/ml, healthy donor vs MDS; p=0.12), and IL-8 (56±32 vs 704±446 pg/ml, p=0.01)]. Next, to assess the capacity of MDS pts to mount ex vivo functional virus-directed responses, we stimulated patient-derived PBMCs (n=5) with overlapping peptide libraries (pepmixes) spanning immunogenic AdV, CMV, EBV, BK and HHV-6 antigens. Similar to healthy donor-derived T cell lines (n=5, 3 specific for 4 viruses and 2 for 5 viruses), all 5 MDS patient-derived lines demonstrated specificity for one or more of the target viruses (1 for 5 viruses, 1 for 4, 2 for 3 and 1 for 1 virus) as observed by IFNγ ELISpot assay with comparable magnitude (range Adv: 43-730 vs 384-941 in healthy donors, CMV: 0-1599 vs 0-3002, EBV: 0-1486 vs 0-541, BK: 0-839 vs 38-275 and HHV6: 0-794 vs 5-407 SFU/2x105 cells, respectively). We next examined the feasibility of expanding autologous MDS-antigen directed T cell products (n=10) to determine whether an adoptive immunotherapeutic approach might be applicable for MDS treatment. Thus, we exposed patient-derived PBMCs to autologous dendritic cells (DC) loaded with pepmixes spanning 6 MDS-associated antigens (CCNA1, survivin, WT1, PRAME, PR3 and NYESO1). After 3 rounds of stimulation, the products obtained were predominantly CD3+ T cells (mean 88±1.3%) that were polyclonal (CD4: 46±5% and CD8: 41±4%) containing predominantly memory T cells (TEM: 36±6% TCM: 37±5% and Tnaïve =13±3%). Six lines (60%) showed specific recognition to at least one of the target antigens: 4 lines specific for PRAME, 1 for CCNA1, 1 for WT1 and 1 for NYESO1 (range 0-40, 0-184, 0-1386 and 0-179 SFU/2x105 cells, respectively by IFNγ ELIspot). T cell lines were capable of specifically secreting multiple effector cytokines in response to targets (TNFα: 12% and IFNγ: 16% in response to PRAME in a representative patient-derived T cell line). Furthermore, this line was capable of killing PRAME+ targets in a 4hr 51Cr release assay [60% specific lysis, E:T 20:1]. In conclusion, functional virus-directed T cell immunity in patients with MDS is preserved, potentially explaining the lower rates of viral reactivation seen in these patients compared with other infections. Moreover, T cells specific for MDS-expressed tumor antigens can also be successfully expanded ex vivo from patients. Taken together this raises the possibility of applying an adoptive immunotherapeutic approach for the treatment of MDS. Disclosures Ramos: Novartis: 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; Tessa Therapeutics: Research Funding. Leen:Allovir: Consultancy, Other: Cofounder, Ownership Interest; Marker Therapeutics: Consultancy, Other: Cofounder, Ownership Interest.

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Combining Gene Expression Profiling and Artificial Intelligence to Diagnose B-Cell Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2019-122405 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1476-1476

Author(s):

Victor Bobée ◽

Fanny Drieux ◽

Vinciane Marchand ◽

Vincent Sater ◽

Liana Veresezan ◽

...

Keyword(s):

Gene Expression ◽

Artificial Intelligence ◽

B Cell ◽

Board Of Directors ◽

Expression Profiling ◽

Marginal Zone ◽

Validation Cohort ◽

B Cell Lymphomas ◽

Advisory Committees ◽

Independent Validation

Introduction Non-Hodgkin B-cell lymphomas (B-NHLs) are a highly heterogeneous group of mature B-cell malignancies associated with very diverse clinical behaviors. They rely on the activation of different signaling pathways for proliferation and survival which might be amenable to targeted therapies, increasing the need for precision diagnosis. Unfortunately, their accurate classification can be challenging, even for expert hemato-pathologists, and secondary reviews recurrently differ from initial diagnosis. To address this issue we have developed a pan-B-NHL classifier based on a middle throughput gene expression assay coupled with a random forest algorithm. Material and Methods Five hundred ten B-NHL diagnosed according to the WHO criteria were studied, with 325 diffuse large B-cell lymphomas (DLBCL), 43 primary mediastinal B-cell lymphomas (PMBL), 55 follicular lymphomas (FL), 31 mantle cell lymphomas (MCL), 17 small lymphocytic lymphomas (SLL), 20 marginal zone lymphomas (MZL), 11 marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) and 8 lymphoplasmacytic lymphomas (LPL). To train and validate the predictor the samples were randomly split into a training (2/3) and an independent validation cohort (1/3). A panel of 137 genes was designed by purposely selecting the differentiation markers identified in the WHO classification for their capacity to provide diagnostic and prognostic information in NHLs. Gene expression profiles were generated by ligation dependent RT-PCR applied to RNA extracted from frozen or FFPE tissue and analyzed on a MiSeq sequencer. For analysis, the sequencing reads were de-multiplexed, aligned with the sequences of the LD-RTPCR probes and counted. Results were normalized using unique molecular indexes counts to correct PCR amplification biases. Results In DLBCL, unsupervised gene expression analysis retrieved the expected GCB, ABC and PMBL signatures (Fig A). These tumors also showed higher expressions of the KI67 (proliferation), CD68 and CD163 (tumor associated macrophages), and PD-L1/2 (immune response) markers. We also observed that the dual expression of MYC and BCL2 at the mRNA level significantly associates with inferior PFS and OS, independent from the International Prognostic Index and from the GCB/ABC cell-of-origin signature, validating the capacity of the assay to identify these highly aggressive lymphomas (Fig C). Overall, low-grade lymphomas were characterized by a significant T cell component. FLs associated with the GCB (BCL6, MYBL1, CD10 and LMO2) and Tfh (CD3, CD5, CD28, ICOS, CD40L, CXCL13) signatures. Other small B-cell lymphomas tended to overexpress activated B-cell markers (LIMD1, TACI, IRF4,FOXP1...), and the expected CD5, CD10, CD23 and CCND1 differential expressions in SLL, MCL and MZL were correctly retrieved (Fig B). Surprisingly, our analysis revealed that the Ie-Ce sterile transcript, expressed from the IGH locus during IgE isotype switching, is almost exclusively expressed by FLs, constituting one of the most discriminant markers for this pathology. We next trained a random forest classifier to discriminate the 7 principal subtypes of B-NHLs. The training cohort comprised 162 DLBCLs (ABC or GCB), 28 PMBL, 35 FLs (grade 1-3A), 21 MCLs, 12 SLLs, and 25 NHLs grouped into the MZL category (13 MZLs, 8 MALT and 4 LPLs). The independent validation series comprised 90 DLBCLs classified as GCB or ABC DLBCLs by the Lymph2Cx assay, 15 PMBLs, 12 FLs (grade 1-3A), 10 MCLs, 5 SLLs and 14 MZLs (7 MZL, 3 MALT and 4 LPL). The RF algorithm classified all cases of the training series into the expected subtype, as well as 94.5% samples of the independent validation cohort (Fig D). For ABC and GCB DLBCLs, the concordance with the Lymph2Cx assay in the validation cohort was 94.3%. Conclusion We have developed a comprehensive gene expression based solution which allows a systematic evaluation of multiple diagnostic and prognostic markers expressed by the tumor and by the microenvironment in B-NHLs. This assay, which does not require any specific platform, could be implemented in complement to histology in many diagnostic laboratories and, with the current development of targeted therapies, enable a more accurate and standardized B-NHL diagnosis. Together, our data illustrate how the integration of gene expression profiling and artificial intelligence can increase precision diagnosis in cancers. Figure Disclosures Oberic: Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees. Haioun:Miltenyi: Honoraria; Takeda: Honoraria; Servier: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Salles:Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Amgen: Honoraria, Other: Educational events; BMS: Honoraria; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Epizyme: Consultancy, Honoraria. Tilly:roche: Membership on an entity's Board of Directors or advisory committees; servier: Honoraria; merck: Honoraria; Roche: Consultancy; Celgene: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Karyopharm: Consultancy; BMS: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Jardin:celgene: Honoraria; roche: Honoraria; amgen: Honoraria; Servier: Honoraria; janssen: Honoraria.

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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.

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Results of a Phase I Trial of Lenalidomide, Rituximab (R2) and Ixazomib for Frontline Treatment of High Risk Follicular and Indolent Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2020-136370 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-2

Author(s):

Brian T. Hill ◽

Deepa Jagadeesh ◽

Alex V. Mejia Garcia ◽

Robert M. Dean ◽

Omer N. Koc ◽

...

Keyword(s):

High Risk ◽

Phase I ◽

Board Of Directors ◽

Research Funding ◽

Marginal Zone ◽

Proteasome Inhibitors ◽

Marginal Zone Lymphoma ◽

Aggressive Lymphoma ◽

Advisory Committees ◽

Frontline Treatment

INTRODUCTION: Lenalidomide and Rituximab (R2) is an effective frontline treatment regimen for patients (pts) with indolent B-cell lymphoma including follicular lymphoma (FL). Recent phase III data from the RELEVANCE trial comparing R2 to traditional chemoimmunotherapy showed that this regimen is generally well-tolerated and has favorable clinical efficacy [61% overall response, 53% CR rate, 77% 3-year progression free survival (PFS) (Morschhauser, et al)]. Proteasome inhibitors such as bortezomib disrupt NF-KB signaling and have shown clinical activity in indolent NHL. Although randomized trials have failed to demonstrate clinical benefit of adding bortezomib to standard chemoimmunotherapy regimen bendamustine + rituximab (BR) for frontline treatment of FL, the addition of proteasome inhibitors to lenalidomide is a mainstay of treatment for plasma cell neoplasms due to synergistic antitumor effect. The oral proteasome inhibitor ixazomib has less potential for dose-limiting neuropathy than bortezomib, making it an attractive option to incorporate into the R2 regimen. We sought to investigate the safety and efficacy of the addition of ixazomib to R2 for FL and indolent B-cell NHL through a phase I clinical trial of this combination for patients with high risk disease. METHODS: Adult (age ≥ 18) pts with untreated FL or other indolent lymphoma, adequate organ function and performance status were enrolled. To be enrolled, FL patients were required to have stage 2, 3 or 4 disease, with high tumor burden by GELF criteria and/or FLIPI score of 3-5. During 3 x 3 dose escalation, ixazomib was given at a dose of 2 mg (n=3), 3 mg (n=3) or 4 mg (n=12) PO on days 1, 8, and 15 with lenalidomide 20 mg PO on days 1-21 every 28 days. Rituximab was administered at standard dosing on days 1,8,15,21 for cycle 1, once every 28 days for cycles 2-6 and then once every 2 months for cycles 7-12. Treatment was continued for 12 cycles and no maintenance therapy was specified per protocol. All pts received low dose aspirin for venous thromboembolism prophylaxis and acyclovir for prevention of VZV reactivation. Response assessments by CT were performed after cycle 3 and 6 and by PET/CT at the end-of-treatment (cycle 12). RESULTS: 20 pts were enrolled and 18 were eligible for treatment [15 FL (14 grade 1-2, 1 grade 3A), 2 splenic marginal zone lymphoma and 1 nodal marginal zone lymphoma].The median age of treated pts was 61 (range 40-83) years old. 55% of patients were female. Stage at diagnosis was II (n =2), III (n = 4) and IV (n = 12). For FL pts, FLIPI scores at enrollment were low (n=2), intermediate (n = 5) and high risk (n=8) and FLIPI-2 scores were low (n=3), intermediate (n = 2) and high risk (n=10). There were no dose limiting toxicities during 3 x 3 dose escalation. Grade (G) 1/2 and G3/4 treatment-related hematologic adverse events (AEs) included neutropenia (6%, 28%), thrombocytopenia (16.7%, 5.6%) and anemia (16.7%, 0%). The most common treatment-related AEs included nausea/vomiting (44% G1, 11% G2), diarrhea, (50% G1, 22% G2, 5% G3), rash (33% G1, 6% G2, 11%G3), peripheral neuropathy (22% G1, 6% G2), myalgia/arthralgia (17% G1, 17% G2), and infection (33% G2, 17% G3). There was one pulmonary embolism and no cases of febrile neutropenia. As of June, 2020, median follow-up among living pts was 21 months. 4 pts discontinued treatment due to disease progression; 2 with transformation to aggressive lymphoma. Of the transformed cases, one subject died on study due to progression disease and one developed CNS disease on study treatment but proceeded to autologous stem cell transplant. The best overall response rate was 61.2% [55.6% CR, 5.6% PR): 22.2% had stable disease and 16.7% had disease progression. 18-month Kaplan-Meier estimates of PFS and overall survival were 71% and 94%, respectively (Figure). CONCLUSION: R2 can safely be combined with at the target dose of 4 mg of ixazomib for treatment-naïve indolent NHL patients. Non-hematologic AEs were generally consistent with known toxicity of each component of therapy. CR rate and PFS were was similar to the outcomes reported in the RELEVANCE trial despite enrolling high risk patient. R2 may serve as backbone for future studies of novel treatment combinations for high risk FL after thorough evaluation for occult transformation to aggressive lymphoma. Disclosures Hill: Abbvie: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding. Jagadeesh:MEI Pharma: Research Funding; Regeneron: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Debiopharm Group: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees. Caimi:Celgene Corp: Other: Incyte Corporation - Ownership - Pharmacyclics, Inc. - Ownership - Celgene Corp. - Other, Speakers Bureau; ADC Therapeutics: Research Funding; Genentech: Research Funding. Smith:Takeda: Research Funding; Celgene: Research Funding. OffLabel Disclosure: Ixazomib is off-label for treatment of NHL

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Whole Genome Copy Number Variation Analysis of Chronic Lymphocytic Leukemia (CLL) Cells From Early-Intermediate Stage, High Risk CLL Patients Prior to First Treatment Reveals New Loss of Heterozygosity and Duplication Events in the CLL Genome.

Blood ◽

10.1182/blood.v114.22.1265.1265 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1265-1265

Author(s):

Steven A. Schichman ◽

Annjanette Stone ◽

Maria Winters ◽

Weleetka Carter ◽

Lori Frederick ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Homozygous Deletion ◽

Chromosome 12 ◽

Whole Genome ◽

Advisory Committees ◽

Hemizygous Deletion ◽

Number Variation ◽

Trisomy 12

Abstract Abstract 1265 Poster Board I-287 Introduction Fluorescence in situ hybridization (FISH), in combination with other markers, is used as a prognostic tool for CLL patients at diagnosis. The presence or absence of trisomy 12 and deletions at 13q, 11q, and 17p helps to predict disease progression and to stratify patients for therapeutic decisions. We hypothesized that whole genome single nucleotide polymorphism (SNP)-based copy number variation (CNV) analysis would capture all of the information in current CLL FISH panels and would reveal new CNV features in the CLL genome. Patients and Methods Nineteen early-intermediate clinical stage, untreated CLL patients aged 29 to 77 were determined to be at high risk for disease progression by FISH, IgVH mutation status, ZAP-70, and CD38 prognostic markers. CLL cells and normal cells were separated by magnetic bead selection from patient peripheral blood samples with absolute lymphocyte counts that ranged from 7.4 to 162 × 109/L. CNV analysis was performed on purified genomic DNA from the CLL cells and from normal cells for each patient in order to distinguish acquired CNVs in malignant cells from polymorphic CNVs in the human genome. We used the Illumina human660w-quad beadchip, a SNP-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV. CNV data was analyzed using CNV partition (Illumina Genome Studio software) and PennCNV. Results 100% concordance is found between del(13q), del(11q), and del(17p) FISH abnormalities and loss of heterozygosity (LOH) at 13q, 11q, and 17p by CNV analysis. All three patients with trisomy 12 by FISH show copy number(CN)=3 of chromosome 12 by CNV analysis. Of 15 patients with del(13q) by FISH, 12 out of 15 have regions of hemizygous deletion on 13q that vary from ∼830 Kb to ∼38 Mb. The smallest region of LOH is located within 13q14.3. Three out of 15 patients show homozygous deletion within 13q14.3. One of these 3 patients has copy-neutral LOH of the entire 13q arm with an embedded 835 Kb segment of homozygous deletion at 13q14.3. Two patients have large discontinuous segments of LOH on 13q, indicating complex interstitial deletion events. Two out of 5 patients with del(13q) as a sole FISH abnormality show additional CNV events in the CLL genome. One of these patients has copy neutral LOH at 2q33.1-telomere(tel). One other patient with sole del(13q) FISH shows LOH events at 10q23.31-23.33 and at 15q15.1. Five out of six patients with del(11q) by FISH have either 13q LOH (n=4) or chromosome 12 CN=3 (n=1) without any other CNV events detected in the CLL genome. One patient with trisomy 12 and del(11q) by FISH has three additional acquired CNV abnormalities in the CLL genome: LOH at 7p15.2-tel, LOH at 11p13, and CN=3 at 3q24-tel. In contrast to patients with del(11q), del(13q), and trisomy 12, patients with del(17p) by FISH have numerous acquired CNV abnormalities in the CLL genome. These include LOH events at 1p34.3-p34.2, 2q34-q36.3, 3p21.31-tel, 4p13, 4p15.1-tel, 15q11.2-q14 and 15q14-q15.3, 16p13.3-tel, 16p13.11, 16p13.2, 18p11.21-tel, 20p11.21-tel, and 20q13.2-q13.31. CN=3 at 2p12-tel is detected in 2 out of 5 patients with 17p hemizygous deletion. One out of 5 patients with 17p hemizygous deletion shows CN=3 at 10q22.2-tel. One other patient also with 17p hemizygous deletion shows CN=3 at 22q12.2-tel. Conclusions Whole genome CNV analysis by SNP-based microarrays greatly expands our ability to detect acquired genomic events in CLL cells. These events include hemizygous deletion, homozygous deletion, copy-neutral LOH, and CN=3 duplication. Detection of copy-neutral LOH is not possible by FISH or array comparative genomic hybridization technology. The current study reveals a high number of acquired CNV events in earlier stage, untreated CLL patients with 17p hemizygous deletion. This observation, indicative of genomic instability, is consistent with the known poor prognosis of del(17p) patients. The new somatic CNV abnormalities detected in CLL cells may help to discover additional genes or signaling pathways involved in CLL initiation and progression. In addition, the new CNV markers may be used in larger clinical studies to improve CLL prognosis and patient stratification for therapy. Disclosures Shanafelt: Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding.

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