scholarly journals The Genomic Landscape of Waldenström Macroglobulinemia Reveals Sustained Germinal Center Activity and Late-Developing Copy Number Aberrations

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2394-2394
Author(s):  
Kylee H Maclachlan ◽  
Tina Bagratuni ◽  
Efstathios Kastritis ◽  
Bachisio Ziccheddu ◽  
Sydney X. Lu ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Loss Of Heterozygosity ◽  
Germinal Center ◽  
Copy Number ◽  
Research Funding ◽  
Advisory Committees ◽  
Copy Number Aberrations ◽  
Genomic Landscape ◽  
Center Activity ◽  
Over Time

Abstract The genomic landscape of Waldenström Macroglobulinemia (WM) is characterized by recurrent somatic mutations in MYD88, with a lower incidence of mutations affecting CXCR4, ARID1A, CD79B and the NFKB signaling pathway (Hunter et. al. Blood 2014). We aimed to characterize the relationship between single base substitutions (SBS), mutational signatures, copy number aberrations (CNA) and structural variants (SV) in WM. We performed whole genome sequencing (WGS) on 14 primary samples from WM patients at various clinical stages, including IgM monoclonal gammopathy (n=1), smoldering (n=5), newly diagnosed (n=7) and relapsed WM (n=1). We identified a median of 2806 clonal SBS per sample (IQR 1870-3079), and 12/14 (85%) samples harbored MYD88 mutations. To investigate which mutational processes are involved in shaping the genomic landscape of WM we performed a mutational signature analysis. Four previously reported SBS signatures were detected: SBS1 and SBS5 (aging), SBS9 (germinal center; GC) and SBS8, with the contribution of age-related signatures SBS1/SBS5 being directly correlated with age at presentation (R 2=0.44, p=0.014). The GC signature SBS9 demonstrated sustained GC activity, as evidenced by the same proportion of mutations attributable to SBS9 at both the clonal and subclonal level (24%). At the immunoglobulin loci, we observed evidence of clustered SBS84 (AID), reflecting somatic hypermutation, with SBS84 accounting for 30% of signature contribution from subclonal mutations. Overall, these data suggest that, similarly to MM and other hematological malignancies, the interaction between WM and the GC is sustained over time. We have previously demonstrated that SV and complex events are critical in the pathogenesis and clinical outcomes of multiple myeloma. In contrast, in this WM WGS cohort, we found a low prevalence of complex SV, with no chromothripsis detected, and a single chromoplexy event found in 3 patients (21%), all of whom had progressed to symptomatic WM. To explore WM CNA features in a larger cohort, we examined the WGS data together with 38 MYD88-mutated WM samples for which targeted sequencing was available (MSK-IMPACT-Heme 400 gene panel). In this combined dataset (n=52), GISTIC analysis identified significantly deleted regions at 6q16.1, 7q34, 17p13.1 (TP53) and 21q22.2, along with significant amplification at 6p22.1 (HLA-A). To better characterize the HLA loci using the loss of heterozygosity in human leukocyte antigen (LOHHLA) tool (McGranahan et. al. Cell 2017) we found the presence of HLA-specific loss of heterozygosity in 1 sample, while 4 samples had HLA CN >2.5 (all from patients who progressed to symptomatic WM). CNA analysis demonstrated that while some samples harbored typical CNA features, others had minimal changes, with MGUS / smoldering WM samples having less CNA compared with those who progressed to symptomatic WM. The 2 MYD88 wild type WGS contained a clonal gain affecting chromosome 12, which is typically an early event in chronic lymphocytic leukemia. Molecular time analysis (the corrected ratio between duplicated and non-duplicated clonal mutations within large chromosomal gains [Maura et al. Nat Comm 2019]) demonstrated that these 2 chromosomal gain events occurred early in cancer development (relative timing <0.5), while multiple other CNA changes occurred later in the disease course (timing >0.5) and tended to be subclonal. This data suggests that, while MYD88-mutations are central to WM clone establishment and can be observed in precursor disease, CNA may contribute to later phases and disease progression. In summary, WGS in WM allows the demonstration that germinal center activity is sustained over time. CNA in WM are not random in distribution, with specific loci being significantly amplified or deleted, and a potential role for HLA CNA. In contrast to MYD88 mutations, which are carried by stable precursor patients, the subclonal status and late molecular time of most CNA changes suggest a late role in cancer progression. Disclosures Kastritis: Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Diamond: Sanofi: Honoraria; Medscape: Honoraria. Kazandjian: Arcellx: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy. Dogan: Roche: Consultancy, Research Funding; Seattle Genetics: Consultancy; EUSA Pharma: Consultancy; Peer View: Honoraria; Takeda: Consultancy, Research Funding; Physicians' Education Resource: Honoraria. Lesokhin: Trillium Therapeutics: Consultancy; Serametrix, Inc: Patents & Royalties; Genetech: Research Funding; Iteos: Consultancy; bristol myers squibb: Research Funding; Behringer Ingelheim: Honoraria; pfizer: Consultancy, Research Funding; Janssen: Honoraria, Research Funding. Landgren: Janssen: Other: IDMC; Janssen: Honoraria; Celgene: Research Funding; Amgen: Honoraria; Janssen: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Palomba: Rheos: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; Notch: Honoraria, Other: Stock; Priothera: Honoraria; Kite: Consultancy; Novartis: Consultancy; Magenta: Honoraria; WindMIL: Honoraria; BeiGene: Consultancy; Juno: Patents & Royalties. Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Takeda: Honoraria; BeiGene: Honoraria.

scholarly journals Mytype: A Capture Based Sequencing Approach to Detect Somatic Mutations, Copy Number Changes and IGH Translocations in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5588-5588
Author(s):  
Venkata Yellapantula ◽  
Malin Hultcrantz ◽  
Even H Rustad ◽  
Heather J. Landau ◽  
Christine Iacobuzio-Donahue ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Somatic Mutations ◽  
Physician Education ◽  
Nucleotide Polymorphisms ◽  
Advisory Committees ◽  
Copy Number Aberrations ◽  
Synonymous Mutations

Abstract Introduction At diagnosis, Multiple Myeloma (MM) is traditionally classified into two clinical and prognostic subgroups groups on the basis of initiating cytogenetic abnormalities: IGH translocations and hyperdiploidy. Currently, these events are clinically ascertained by Fluorescent In-Situ Hybridization (FISH). In recent years, comprehensive genome profiling studies have shown that MM pathogenesis is defined by a spectrum of acquired somatic lesions, many of which are biologically and clinically relevant. To this effect, targeted gene sequencing approaches are becoming routine in the upfront diagnostic settings. Here we present myTYPE, a MM-specific targeted next generation sequencing panel to identify germline and somatic substitutions, indels, Copy Number Aberrations (CNA) and IGH translocations. Methods A multiplex bait panel was designed to capture the exons of 120 genes implicated in MM pathogenesis, entire IGH locus as well as genome wide representation of single nucleotide polymorphisms (SNPs) (1 in 3Mb) to enable detection of arm level copy number events and recurrent focal events. These 120 genes were selected on the basis of 1) frequently mutated and driver genes in MM 2) genes in important signaling pathways, e.g the NFKB pathway 3) treatment targets and candidate genes for drug resistance, e.g. cereblon.To validate the efficacy of the assay, 16 constitutional bone marrow samples and 18 tumor samples were sequenced using myTYPE. For validation, 6/18 tumor/normal pairs sequenced using myTYPE were subject to WGS and remaining 12/18 tumor samples were subject to FISH. After sequencing, we obtained an overall median target coverage of 815x. Results After alignment, substitutions and indels were called using Caveman, Pindel and Strelka. CNAs were identified using Facets and IGH translocations were identified using Delly along with a modified version of BRASS. Below is a description of the genomic abnormalities captured by the myTYPE assay. SNVs and Indels For the 6 tumor/normal pairs sequenced using myTYPE and WGS, we obtained a total of 21 (median = 3) non-synonymous mutations using myTYPE. When limiting the WGS calls to myTYPE targets, we recovered 20/21 non-synonymous mutations identified by myTYPE. These involved SNVs and indels in key MM related drivers including NRAS, KRAS, FAM46C and TP53 among others. For the mutations identified by both myTYPE and WGS, there was a high correlation between the variant VAFs, R2= 0.99 and as expected is better in capturing subclonal mutations. IGH rearrangements and Copy Number Aberrations (CNA) Next we compared myTYPE and WGS results for recurrent CNAs in MM. We specifically looked at deletions of 1p, 13p, 16q, 17p and gains of 1q, 11q and found a 100% concordance of these aberrations identified by both assays. The remaining 12 samples sequenced using myTYPE also had orthogonal FISH. myTYPE identified a total of 7 IGH rearrangements, 4 of which are also reported by FISH. Three additional t(11;14) translocations were uniquely identified by myTYPE in cases that remained clinically uncharacterized. FISH was also used to probe deletions in 17q, 13q, 1p and 1q gain. All aberrations identified by FISH were also identified in myType. Additionally, 13q- in four samples and 1p- in one sample were uniquely identified by myTYPE. Conclusion In summary, we present a targeted assay capable of identifying somatic mutations, CNAs and IGH translocations of prognostic and diagnostic relevance in MM. When compared to conventional assays currently used in clinical practice, myTYPE identified at least one disease defining alterations in all samples screened. Evaluation of sensitivity and specificity will require larger clinical cohorts. Importantly, myTYPE enables comprehensive profiling, large sample multiplexing and short turn around times which renders it as an optimal assay for utilisation in the upfront clinical setting. Disclosures Korde: Amgen: Research Funding. Mailankody:Juno: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Physician Education Resource: Honoraria. Landgren:Pfizer: Consultancy; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy.

scholarly journals Clonal Tracking By Whole Genome Sequencing Permits Comprehensive Mapping of the Genomic Landscape in Pre- and Post-Gene Therapy Sickle Cell Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 559-559
Author(s):  
Alyssa H. Cull ◽  
Michael Spencer Chapman ◽  
Marioara Ciuculescu ◽  
Emily Mitchell ◽  
Myriam Armant ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Board Of Directors ◽  
Sickle Cell ◽  
Research Funding ◽  
Phylogenetic Trees ◽  
Insertion Site ◽  
Advisory Board ◽  
Advisory Committees ◽  
Genomic Landscape

Abstract Recent advances in clonal stem cell tracking strategies have enabled interrogation of unperturbed human hematopoiesis. Whole genome sequencing (WGS) can be used to map the clonal dynamics of hematopoietic stem and progenitor cells (HSPCs) by employing spontaneous somatic mutations as unique clonal tags (Lee-Six et al., Nature, 2018). These tags allow for retrospective analysis of individual stem cell clones and the construction of phylogenetic trees mapping out stem cell relatedness, with mutations being acquired in a near-linear fashion over the course of an individual's life. The unprecedented level of information obtained in these studies is particularly well-suited to understanding genomic changes in gene therapy trials aimed at curing diseases such as sickle cell disease (SCD). In addition to mapping relatedness between stem cells, sequencing data can be used to better define mutational signatures for HSPC clones that have been successfully gene-modified as well as those that lack an integrated copy of the therapeutic vector. Given this method's ability to identify low frequency mutations in individual HSPC clones, mutations with extremely low variant allele frequencies can be detected much more readily than through traditional bulk sequencing approaches, something that is particularly relevant given recent safety concerns in some SCD gene therapy trials. In this study, we have mapped the clonal dynamics of HSPCs obtained from pre- and post-gene therapy samples from 4 SCD patients who have undergone autologous gene therapy performed using a BCL11A shmiR lentivirus vector (NCT 03282656, 12-36 months follow-up). HSPCs from mobilized peripheral blood (pre-gene therapy), bone marrow aspirates (both pre- and post-gene therapy) or unmobilized peripheral blood (post-gene therapy) were expanded as single clones and 1508 individual colonies were then sequenced using WGS to an average sequencing depth of 12.3x. Initial results indicate that the mean mutation burden per cell in a pre-gene therapy sample is elevated for some patients compared to what would be expected based on patient age in similar studies. In pre-gene therapy samples, the structure of the phylogenetic trees appeared to be highly polyclonal, indicating that there were no significant clonal expansion events prior to gene therapy. In one patient where we undertook extensive profiling, approximately 15-20 excess mutations per HSPC were observed across the entire genome 24 months after transplantation, presumably acquired as a consequence of gene therapy and/or reconstitution post-transplantation, which is equivalent to approximately one year of normal ageing without a transplantation intervention. However, no clonal expansions or driver mutations were identified at this 24 month follow-up timepoint, suggesting that no strong selective advantage or pre-leukemic events were present prior to or following the gene therapy protocol. Extending this approach to a wider range and larger number of patients will allow for comprehensive mapping of the genomic landscape and clonal evolution of stem cells in sickle cell patients and will also set the stage for improved assessment of safety and potential leukemia-initiating events in the context of gene therapy. Disclosures Esrick: bluebird bio: Consultancy. Williams: bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding; BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding. Campbell: Mu Genomics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Kent: STRM.bio: Research Funding.

scholarly journals BAX-Mutated Clonal Hematopoiesis in Patients on Long-Term Venetoclax for Relapsed/Refractory Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Piers Blombery ◽  
Ella R Thompson ◽  
Xiangting Chen ◽  
Tamia Nguyen ◽  
Mary Ann Anderson ◽  
...  
Keyword(s):  
Advisory Committee ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Clonal Hematopoiesis ◽  
Eliza Hall Institute ◽  
Hall Institute ◽  
Over Time

Venetoclax (Ven) is an effective element of treatments for chronic lymphocytic leukemia (CLL) with high response rates observed in the upfront and relapsed/refractory (R/R) settings. In addition to inducing apoptosis in CLL cells, Ven also induces apoptosis within normal and malignant myeloid lineage populations (accounting for its efficacy in the treatment of acute myeloid leukemia). We investigated the effects of Ven outside the target tumor compartment in patients (pts) with CLL receiving long-term continuous Ven and make the novel observation of the development of BAX-mutated clonal hematopoiesis in this heavily pre-treated patient group. 92 pts with CLL receiving continuous non time-limited Ven have been treated at our institutions on clinical trials. Of these, 41 had sufficient (>6 mo) follow up (median 70; range 14-95 mo) and suitable samples available for further analysis. 38/41 (93%) pts had received previous treatment with alkylators and/or fludarabine. In order to assess the non-CLL compartment in these 41 pts we identified those with peripheral blood or bone marrow aspirate samples taken during deep response to Ven demonstrating either minimal (<5%) or no CLL involvement by flow cytometry (sensitivity 10-4). We initially performed unique molecular index (UMI)-based targeted next generation sequencing of apoptosis pathway genes as well a panel of 60 genes recurrently mutated in lymphoid and myeloid malignancy. From these 41 pts we identified mutations in the apoptosis effector BAX in samples from 12 (29%). 20 different BAX mutations were observed across these 12 pts at variant allele frequencies (VAF) consistent with their occurrence in the non-CLL compartment. Mutations included frameshift, nonsense, canonical splice site and missense mutations occurring in key structural elements of BAX consistent with a loss-of-function mechanism (Fig 1A). Interestingly, an enrichment of missense and truncating mutations predicted to escape nonsense mediated decay were observed at the C-terminus of the BAX protein affecting the critical α9 helix. Mutations in this region have previously been shown in cell lines to cause aberrant intracellular BAX localization and abrogation of normal BAX function in apoptosis (Fresquet Blood 2014; Kuwana J Biol Chem 2020). For comparison, NGS targeted sequencing for BAX mutations was performed on samples from cohorts of pts with (i) myeloid or lymphoid malignancy (n=80) or (ii) R/R CLL treated with BTK inhibitors (n=15) after a similar extent of preceding chemotherapy. Neither of these cohorts had previous exposure to Ven. BAX mutations were not detected in any samples from these pts. Longitudinal sampling from pts on Ven harboring BAX mutations in the non-CLL compartment was performed to further understand compartment dynamics over time (in 9 pts over 21-93 months of follow up). Multiple pts demonstrated a progressive increase in VAF of single BAX mutations over time to become clonally dominant within the non-CLL compartment and with observed VAFs consistent with their presence in the myeloid compartment. Mutations in other genes implicated in clonal hematopoiesis and myeloid malignancy including ASXL1, DNMT3A, TET2, U2AF1 and ZRSR2 were also detected in these pts samples. Targeted amplicon single cell sequencing (Mission Bio) demonstrated the co-occurrence of clonally progressive BAX mutations within the same clones as mutations in DNMT3A and ASXL1 as well as the existence of further BAX mutations at low VAF outside these dominant clones which remained non-progressive over time (Fig 1B). In addition, fluctuations in the presence and VAF of myeloid-disease associated mutations was noted with Ven exposure. In aggregate these data are consistent with the existence of a selective pressure within the myeloid compartment of these pts and an interplay of BAX with other mutations in determining survival and enrichment of these clones over time with ongoing Ven therapy. In summary, we have observed the development of BAX-mutated clonal hematopoiesis specifically in pts with CLL treated with long-term Ven. These data are consistent with a multi-lineage pharmacological effect of Ven leading to a survival advantage for clones harboring BAX mutations within the myeloid compartment during chronic Ven exposure. Finally, our data support the further investigation of BAX mutations as a potential resistance mechanism in myeloid malignancies treated with Ven. Disclosures Blombery: Invivoscribe: Honoraria; Amgen: Consultancy; Janssen: Honoraria; Novartis: Consultancy. Anderson:Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Seymour:Celgene: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Mei Pharma: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Nurix: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tam:Janssen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; BeiGene: Honoraria. Huang:Servier: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.; Genentech: Research Funding. Wei:Janssen: Honoraria, Other; Walter and Eliza Hall Institute: Patents & Royalties; AMGEN: Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Other: Advisory committee; Pfizer: Honoraria, Other: Advisory committee; Macrogenics: Honoraria, Other: Advisory committee; Abbvie: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Genentech: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Speakers Bureau; Astra-Zeneca: Honoraria, Other: Advisory committee, Research Funding. Roberts:Janssen: Research Funding; Servier: Research Funding; AbbVie: Research Funding; Genentech: Patents & Royalties: for venetoclax to one of my employers (Walter & Eliza Hall Institute); I receive a share of these royalties.

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 ◽  
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.

Progressive but Previously Untreated CLL Patients with Greater Array CGH Complexity Exhibit A Less Durable Response to Chemoimmunotherapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2406-2406
Author(s):  
Neil E. Kay ◽  
Jeanette Eckel Passow ◽  
Esteban Braggio ◽  
Scott Van Wier ◽  
Tait Shanafelt ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Progression Free Survival ◽  
Advisory Committees ◽  
Free Survival ◽  
Genomic Complexity ◽  
Duration Of Response ◽  
Gains And Losses

Abstract Abstract 2406 The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. Disclosures: Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 722-722 ◽  
Author(s):  
Jonathan J Keats ◽  
Gil Speyer ◽  
Legendre Christophe ◽  
Christofferson Austin ◽  
Kristi Stephenson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Copy Number ◽  
Interim Analysis ◽  
Research Funding ◽  
Molecular Subtypes ◽  
Bayesian Clustering ◽  
Clustering Method ◽  
Advisory Committees

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

Five-Year Experience with Single-Agent Ibrutinib in Patients with Previously Untreated and Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 233-233 ◽  
Author(s):  
Susan M. O'Brien ◽  
Richard R. Furman ◽  
Steven E. Coutre ◽  
Ian W. Flinn ◽  
Jan Burger ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Grade 3 ◽  
Over Time

Abstract Background: Ibrutinib (ibr), a first-in-class, once-daily Bruton's tyrosine kinase inhibitor, is approved by the US FDA for treatment of patients (pts) with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) including pts with del17p. The phase 1b/2 PCYC-1102 trial showed single-agent efficacy and tolerability in treatment-naïve (TN; O'Brien, Lancet Oncol 2014) and relapsed/refractory (R/R) CLL/SLL (Byrd, N Engl J Med 2013). We report efficacy and safety results of the longest follow-up to date for ibr-treated pts. Methods: Pts received 420 or 840 mg ibr QD until disease progression (PD) or unacceptable toxicity. Overall response rate (ORR) including partial response (PR) with lymphocytosis (PR-L) was assessed using updated iwCLL criteria. Responses were assessed by risk groups: unmutated IGVH, complex karyotype (CK; ≥3 unrelated chromosomal abnormalities by stimulated cytogenetics assessed by a reference lab), and in hierarchical order for del17p, then del11q. In the long-term extension study PCYC-1103, grade ≥3 adverse events (AEs), serious AEs, and AEs requiring dose reduction or discontinuation were collected. Results: Median age of the 132 pts with CLL/SLL (31 TN, 101 R/R) was 68 y (range, 37-84) with 43% ≥70 y. Baseline CK was observed in 41/112 (37%) of pts. Among R/R pts, 34 (34%) had del17p, 35 (35%) del11q, and 79 (78%) unmutated IGVH. R/R pts had a median of 4 prior therapies (range, 1-12). Median time on study was 46 m (range, 0-67) for all-treated pts, 60 m (range, 0-67.4) for TN pts, and 39 m (range, 0-67) for R/R pts. The ORR (per investigator) was 86% (complete response [CR], 14%) for all-treated pts (TN: 84% [CR, 29%], R/R: 86% [CR, 10%]). Median progression-free survival (PFS) was not reached (NR) for TN and 52 m for R/R pts with 60 m estimated PFS rates of 92% and 43%, respectively (Figure 1). In R/R pts, median PFS was 55 m (95% confidence intervals [CI], 31-not estimable [NE]) for pts with del11q, 26 m (95% CI,18-37) for pts with del17p, and NR (95% CI, 40-NE) for pts without del17p, del11q, trisomy 12, or del13q. Median PFS was 33 m (95% CI, 22-NE) and NR for pts with and without CK, and 43 m (95% CI, 32-NE) and 63 m (95% CI, 7-NE) for pts with unmutated and mutated IGVH, respectively(Figure 2). Among R/R pts, median PFS was 63 m (95% CI, 37-NE) for pts with 1-2 prior regimens (n=27, 3 pts with 1 prior therapy) and 59 m (95% CI, 22-NE) and 39 m (95% CI, 26-NE) for pts with 3 and ≥4 prior regimens, respectively. Median duration of response was NR for TN pts and 45 m for R/R pts. Pts estimated to be alive at 60 m were: TN, 92%; all R/R, 57%; R/R del17p, 32%; R/R del 11q, 61%; R/R unmutated IGVH, 55%. Among all treated pts, onset of grade ≥3 treatment-emergent AEs was highest in the first year and decreased during subsequent years. With about 5 years of follow-up, the most frequent grade ≥3 AEs were hypertension (26%), pneumonia (22%), neutropenia (17%), and atrial fibrillation (9%). Study treatment was discontinued due to AEs in 27 pts (20%) and disease progression in 34 pts (26%). Of all treated pts, 38% remain on ibr treatment on study including 65% of TN pts and 30% of R/R pts. Conclusions: Single-agent ibrutinib continues to show durable responses in pts with TN or R/R CLL/SLL including those with del17p, del11q, or unmutated IGVH. With extended treatment, CRs were observed in 29% of TN and 10% of R/R pts, having evolved over time. Ibrutinib provided better PFS outcomes if administered earlier in therapy than in the third-line or beyond. Those without CK experienced more favorable PFS and OS than those with CK. Ibrutinib was well tolerated with the onset of AEs decreasing over time, allowing for extended dosing for 65% of TN and 30% of R/R pts who continue treatment. Disclosures O'Brien: Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Furman:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Coutre:Janssen: Consultancy, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. Burger:Pharmacyclics, LLC, an AbbVie Company: Research Funding; Gilead: Research Funding; Portola: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Roche: Other: Travel, Accommodations, Expenses. Sharman:Gilead: Research Funding; TG Therapeutics: Research Funding; Acerta: Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding. Wierda:Abbvie: Research Funding; Genentech: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Luan:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, Expenses. James:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment. Chu:Pharmacyclics, LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership.

Analysis of Outcomes of Patients with Relapsed/Refractory Acute Myeloid Leukemia Treated in Randomized Clinical Trials

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4000-4000 ◽  
Author(s):  
Raffi Tchekmedyian ◽  
Paul Elson ◽  
Aaron T. Gerds ◽  
Navneet Majhail ◽  
Hetty E. Carraway ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Phase Ii ◽  
Research Funding ◽  
Randomized Clinical Trials ◽  
Statistical Significance ◽  
Phase Iii ◽  
Advisory Committees ◽  
The Past ◽  
American Society ◽  
Over Time

Abstract Introduction The major reasons for failure to achieve cure in the majority of AML patients (pts) are primary refractoriness of disease to initial chemotherapy or failure to maintain complete remission (CR) that has been achieved (relapse). There is no uniformly accepted standard treatment for relapsed or refractory (RR) AML, with most available therapies regarded as palliative or as a bridge to allogeneic transplantation. While the past two decades have witnessed trials of several investigational therapies in RR AML, data regarding the effectiveness of these interventions remains unclear. We studied the impact of experimental drugs in RR AML pts by undertaking a comprehensive analysis of all phase 2 and 3 randomized clinical trials (RCTs) reported in the past 3 decades. Methods We searched PubMed, Embase, Cochrane Controlled Trials Register electronic databases, ClinicalTrials.gov and conference abstracts from the American Society of Hematology (ASH), American Society of Clinical Oncology (ASCO) and European Hematology Association (EHA) websites covering a period from 1988 to 2015. Key words used during this search included "refractory" or "relapsed" or "AML" or "phase II" or "phase III" or "randomized". Only double-arm, phase II with a sample size of at least 50 pts and phase III RCTs conducted in RR AML pts were included. Two reviewers independently extracted data on study methods, participants, therapies, and outcomes from all eligible trials: differences in how to classify agents in RCTs were resolved by discussion. The primary outcomes examined in the experimental arms (EAs) and standard arms (SAs) included CR rates, disease-free survival (DFS), refractory disease rates, treatment-related mortality (TRM) rates and overall survival (OS). Odds ratios (OR) were used to summarize differences between EAs and SAs. The DerSimonian and Laird random-effects model was used to compare them and to assess the overall impact of time. Results Of 5500 included pts, 40.5% were treated on 21 double-arm, phase II trials, 51% on 10 phase III trials and 6.6% analyzed through 4 retrospective studies. There was no change in CR rates in either EAs (p=.21) or SAs (p=.15) over time (Figure 1). The CR rates in EAs tended to be higher than in SAs [OR=1.24; 95% CI, 1.02-1.50, p=.03). Rates of disease refractoriness to salvage regimens in both EAs (p=.70) and CAs (p=.31) did not change over time and these rates were not significantly different between treatment arms [OR=0.82; 95% CI, 0.62-1.08, p=.16]. TRM rates tended to decrease over time but the change was not significant in either group [p=.24 for SAs and p=.33 for EAs]. TRM rates were higher in SAs compared to CAs but did not reach statistical significance [OR=1.21; 95% CI, 0.97-1.50, p=.09]. Over time, there was no significant change inDFS in either group (p=.32 for CAs and p=.58 for EAs). DFS rates did not differ between EAs and SAs [OR=1.01; 95% CI, 0.86-1.19, p=.89] (Figure 2). OS tended to remain stable over time in both groups [p=.85 for SAs and p=.66 for EAs]. While OS tended to be higher in SAs, it did not reach statistical significance [OR=0.93; 95% CI, 0.83-1.05, p=.27]. Conclusions: These findings indicate a lack of significant or clinically meaningful improvement in disease outcomes, including OS, in RR AML pts treated within RCTs over the past 3 decades. Greater efforts need to be directed towards designing RCTs using novel statistical approaches and directed agents based on recent discoveries of targetable mutations. Disclosures Carraway: Amgen: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Advani:Pfizer Inc.: Consultancy, Research Funding; Blinatumomab: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Mukherjee:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

The Genomic Landscape of Childhood and Adult Acute Erythroid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 39-39 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Ji Wen ◽  
Manja Meggendorfer ◽  
Catherine Carmichael ◽  
John K Choi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Transcription Regulation ◽  
Research Funding ◽  
Complex Karyotype ◽  
Advisory Committees ◽  
Nih3t3 Cells ◽  
Genomic Landscape

Abstract Introduction: The genetic basis of several acute myeloid leukemia (AML) subtypes remains poorly characterized, such as that of acute erythroid leukemia (AEL, AML M6) which is currently subclassified by morphology alone, and is associated with poor outcome. Here we sought to perform a definitive genomic analysis of AEL and translate these findings into faithful experimental models and novel therapeutic approaches. Methods: We studied 151 AEL cases (19.2% pediatric, 4.6% young adult, 21.9% adult and 54.3% older adult). Diagnosis of AEL was centrally confirmed and subclassified according to WHO 2008 and revised 2016 criteria. Whole exome and/or genome sequencing, RNA-sequencing and SNP array analysis were performed on all cases and in 2 AEL cell lines (TF-1 and Hel). Genomic data were compared to those from non-M6 childhood and adult AML from TARGET (n=192) and TCGA (n=197) studies. The functional effects of fusion transcripts and mutated genes were examined in IL-3 dependent Ba/F3 cells, NIH3T3 cells for focus formation assays and/or mouse lineage negative hematopoietic stem cells (lin- HSC) for colony forming and transplantation assays. Avatars of human AEL were established in immunocompromised NSGS and MISTRG mice for preclinical studies. Results: a) Genomic landscape of AEL. We identified 2,250 non-synonymous clonal and subclonal somatic mutations in 1,723 genes with a mean of 16.4 per case (range 2-88) and with missense and frameshift mutations accounting for 47.1% and 22.5% of all mutations, respectively. 78 genes were recurrently mutated in at least 3 cases. In frame fusions were detected in 31% of childhood and 27.5% of adult cases, and were more frequent in cases with complex karyotype. 124 potential driver genes were identified by statistical analysis or known pathogenic role in cancer, 9 of which were recurrent novel targets of mutation, most commonly involving chromatin modification (60.3%), cell cycle/tumor suppression (TP53, 33.8%), DNA methylation (28.5%), transcription regulation (26.5%), splicing (15.9%), NPM1 (11.9%), Ras (11.3%), JAK-STAT signaling (9.9%), the cohesin complex (8.6%), ALK/NTRK1 (4.6%) and PI3K signaling (3.3%). Overall, 33% of cases harbored a mutation in signaling genes amenable to inhibition by tyrosine kinase/Ras inhibitors. Mutations in TP53 and DNA methylation genes were significantly more frequent in adults while mutations in transcription regulators and Ras pathway were more frequent in children. Splicing mutations correlated with MDS phenotype and PI3K alterations with therapy-related AEL. Based on the co-occurrence and exclusivity of mutations 7 main distinct AEL genetic subtypes were defined: 1) pediatric AEL with NUP98-rearrangements (3.3% of all cases); 2) adult complex karyotype AEL with TP53 mutations (33.8%); 3) AEL with MLL-rearrangements (12.6%); 4) NPM1-mutated AEL (11.9%); 5) DNA-methylation/splicing mutated AEL (17.8%); 6) splicing/Ras/transcription regulation mutated AEL (21.2%) and 7) Other (8.6%) (Fig.1A). Mutations of chromatin modifiers occurred independently of karyotype, age and subtype (Fig.1B). NUP98-fusions and mutations in PTPN11, UBTF and GATA1 were more frequent in pediatric AEL compared to non-M6 AML. Among adults, mutations in TP53 and MLL were more frequent in AEL while FLT3, NPM1, DNMT3A and IDH1 were more in frequent in non-M6 subtypes. A complex karyotype, therapy-related AEL, TP53 mutations and NUP98-rearrangements were associated with poor outcome. b) Functional AEL modeling and therapeutic translations. Expression of NUP98-JARID1A in lin- HSC resulted in sustained self-renewal and development of an aggressive transplantable leukemia. At least three classes of signaling pathway mutations are targetable in AEL. ALK mutations in the extracellular MAM domain transformed Ba/F3 cells which were sensitive to crizotinib in vitro. Mutations in the tyrosine kinase domain of NTRK1 transformed NIH3T3 cells and were sensitive to entrectinib in vitro. Targeting of JAK-STAT, mTOR and PI3K pathways were examined in xenografts and sensitivity to JAK2 inhibitor ruxolitinib was confirmed in vivo. Conclusions: We provided the first comprehensive landscape of genomic alterations in AEL and defined distinct genomic groups with unique patterns of mutation occurrence compared to non-M6 AML. Finally, we showed that several pathogenic pathways are amenable to inhibition by approved targeted compounds. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Wei:Novartis: Honoraria, Research Funding. Loh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding.

scholarly journals Characterization of Residual CLL Following Long-Term Bruton Tyrosine Kinase Inhibitor Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2620-2620
Author(s):  
Shanmugapriya Thangavadivel ◽  
Alexander Pan ◽  
Xi Chen ◽  
Chen Song ◽  
Claire Snyder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Advisory Committees ◽  
Time Points ◽  
Bruton Tyrosine Kinase ◽  
Over Time

Abstract Introduction: The development of Bruton tyrosine kinase inhibitors (BTKi) and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL). Ibrutinib and other second generation BTKi as monotherapies generally do not produce minimal residual disease negative (MRD-) complete remissions even with extended therapy. The reason for lack of continued elimination of CLL to a MRD- status over time is unknown, and we hypothesized that biological differences in the CLL cells or immune microenvironment might make them resistant to elimination. Methods: Samples were obtained from patients on continuous ibrutinib who hadn't relapsed at time points of 3 years on treatment and 5 years on treatment; and pre-ibrutinib. Isolated CLL cells were subject to B-cell receptor (BCR) sequencing using NEBNext Immune Sequencing Kit by New England Biolabs (NEB, Inc., USA). In a separate cohort, 10X VDJ+5'-sequencing was performed on peripheral blood mononuclear cells. Flow cytometry and ELISA were used to identify alterations in immune cell subtype and identify immune profiles associated with MRD positive (MRD+) status. Results: To identify the clonal pattern in MRD+, we performed deep sequencing of the BCR repertoire on samples from 13 patients with 3 time points each. We found that dominant clones tended to remain constant, but new clones appeared in later time points (Figure 1). MiXCR (v3.0.5) was used with default parameters to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of unique productive and nonproductive CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. Two out of three patients (patients 1 and 3) showed significant change in the clone over time. In patients 1 and 2, we saw that heavy chain clones emerge at later time points. In patient 3 alone, we observed that at 5 years there are two dominant clones. Our findings suggest that each patient shows a diverse repertoire of CLL clones and that the dominant clone does not change significantly across time points. To identify cell populations based on gene expression patterns, we performed 10X VDJ+5'-seq. Based on the expression of known markers, we identified CLL cells and other immune cell subtypes. We identified differentially expressed genes (DEGs) for CLL cells in each time points. Over time, we observed upregulation of CD79a, LTB, TAGLN2, and LGALS, genes typically associated with leukemic cell survival. Suggesting differential expression of pro-survival genes contribute to continued presence of MRD over time. T cells are known to be dysfunctional in CLL and have not previously been extensively studied in the setting of long term BTKi. We performed flow cytometry to determine the repertoire and function of T cells at 3 and 5 years of ibrutinib therapy. We found that the percentage of CD3+ T cells increases at later time points in all the 8 patients (p&lt;0.05). Although T cell numbers increase, we do see skewing of these cells towards a terminally differentiated phenotype (p&lt;0.05). We also observed significant increases in NK cells across time points (p&lt;0.05), albeit non-functional due to high expression of inhibitory receptor KLRG1 in 7 out of 8 patients (p&lt;0.05). Although overall the number of immune cells increase in long time ibrutinib therapy, they exhibit exhausted or non-functional phenotypes. Conclusion: Extended ibrutinib treatment yields a subset of patients who become MRD- whereas a large majority remain MRD+. Our findings suggest that BCR repertoire in CLL MRD might change in long term ibrutinib therapy and induce necessary genes for its survival in the microenvironment. Although T cells and NK cells are non-functional at later time points, better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells. Differentiating the biology of why certain patients attain MRD- status on BTK inhibitor is of high interest as it could provide rationale for therapy discontinuation or add on approaches. Figure 1 Figure 1. Disclosures Rogers: AbbVie Inc.: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy, Research Funding; Innate Pharma: Consultancy; Pharmacyclics LLC: Consultancy; Janssen Pharmaceuticals, Inc: Research Funding; ovartis Pharmaceuticals Corporation: Research Funding. Bhat: Beigene: Consultancy; AstraZeneca: Consultancy; Aptitude Health: Honoraria; Onclive: Honoraria. Kittai: Bristol-Meyers Squibb: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Woyach: AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

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