Clonal Hematopoiesis Driven By MDM4 Amplification Defines a Canonical Route Towards Secondary MDS/AML in Fanconi Anemia Patients

Abstract Introduction Fanconi anemia (FA) is the most frequent inherited DNA-repair disease in human, driving hematopoietic stem cell (HSC) failure in children and a major predisposition to poor-prognosis myelodysplastic syndrome (MDS) and acute leukemia (AML) in children or young adults. MDS/AML secondary to FA have a dismal prognosis in this frail population with a high chemotherapy-related toxicity. How bone marrow (BM) cells progress to myeloid malignancies in a background of cell intrinsic genomic instability and stem cell exhaustion is still poorly understood. Here we aimed to identify the molecular and functional determinants of BM progression to MDS/AML in FA patients. Methods We studied a cohort of 335 FA patients, representing virtually all FA patients seen in France from 2002 to 2020. We performed longitudinal clinical studies (cytopenia, BM morphology and staging, HSCT, survival), somatic genomics (karyotype, myeloid cancer gene panel, aCGH, WES, WGS), expression analysis by RNAseq on clonal cells, and functional studies (gene modulation in HSPCs, transgenic MDM4 mice, CFU and competitive engraftment experiments). Paired clonal BM and skin fibroblasts samples were available for 62 MDS/AML FA patients; WES and WGS files from age-matched non FA MDS/AML were used as controls. Results 98 out of 335 patients (29%) experienced clonal evolution, first seen at a median age of 13y, including 51 (15%) with blastic evolution (>5% BM blasts, median age 16y). Unbalanced chromosomal translocations rather than point mutations underlaid clonal evolution in comparison to age-matched, sporadic (non-FA) AML cases. The most prominent driver lesion was chromosome 1q duplication (1q+), found in 52% of the clonal FA patients, while other recurrent lesions were gain of 3q (3q+/EVI1; 40%), translocations/del/mut involving the RUNX1 gene (35%), monosomy 7/7q- (31%), and signaling gene mutations (18%). Based on longitudinal studies and ranking models, we evidenced that 1q+ occurred early, yielding preleukemic clonal hematopoiesis, whereas 3q+, -7/del7q, RUNX1 and signaling mutations occurred later along with BM transformation. Regarding genomic instability, WGS analysis of FA AML cells revealed a unique mutational signature that shares features with BRCA-related solid cancers [homologous recombination deficient (HRD)-type substitution signature, accumulation of small/intermediate-size deletions and large structural variants (SV)]. SV breakpoint analysis identified microhomology-mediated end joining (MM-EJ, also known as Alt-EJ) as the preferential DNA repair mechanism in the FA context. Specifically, a fragile site in the 1q pericentromeric repeated region underlaid 1q+ translocations. Next, we found that the MDM4 oncogene, a negative modulator of p53 response located in the minimal 1q duplicated region, was overexpressed in 1q+ but not in clonal non-1q FA cells. We hypothesized that 1q+ may attenuate the FA-associated p53 pathway hyperactivation through increased gene dosage of MDM4. Consistently, RNA-seq of patient cells before and after clonal progression showed p53 pathway activation before clonal evolution and subsequent p53 downregulation along with 1q+. When evaluated in vitro by CFU assay, lentiviral overexpression of MDM4 rescued clonogenicity defect of HSCPs from both FA patients and Fanc-/- mice, at the same level as TP53 knockdown. We produced a transgenic mouse bearing a duplicated Mdm4 locus and showed that MdM4 overexpression conferred an advantage to FA-like HSPCs in competitive transplant experiments, modeling clonal hematopoiesis. Exposure of 1q+ FA cells to Mdm4 inhibitors raised therapeutic potential. Conclusions The somatic genomic landscape of FA MDS/AML reveals a unique FA mutational signature, characterized by structural rearrangements and copy number abnormalities rather than point mutations. Our results define a canonical oncogenic route towards secondary MDS/AML in FA patients, in which the early modulation of the p53 pathway through 1q+/MDM4 oncogene overexpression plays a pivotal role, raising novel monitoring and therapeutic prospects for the FA patients. Disclosures Sebert: BMS: Consultancy; Abbvie: Consultancy. Dalle: Jazz Pharmaceuticals: Honoraria. Socie: Alexion: Research Funding. Peffault De Latour: Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Amgen: Consultancy, Other, Research Funding; Jazz Pharmaceuticals: Honoraria; Alexion, AstraZeneca Rare Disease: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding.

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The Impact of gain1q on Mutational Structure and Clonal Evolution in a Uniformly Treated High-Risk Series of Patients at First Relapse

Blood ◽

10.1182/blood-2021-152123 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2683-2683

Author(s):

John R Jones ◽

Charlotte Pawlyn ◽

Niels Weinhold ◽

Timothy Cody Ashby ◽

Brian A Walker ◽

...

Keyword(s):

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Suppressor Gene ◽

Tumour Suppressor Gene ◽

Mutational Load ◽

Advisory Committees ◽

The Impact ◽

Support Research

Abstract Introduction In Multiple Myeloma (MM) the emergence of treatment resistant clones is a characteristic feature of relapse and this is particularly so for high-risk cases. A key driver event mediating progression, risk status and relapse is gain(1q) (1q+). We report on the impact of 1q+ on the genetic profile seen at first relapse in a uniformly treated, newly diagnosed series of 56 patients enrolled to the NCRI Myeloma XI Trial. Methods We included 56 high risk patients, defined as relapse within 30 months of maintenance randomisation (median 19 months, range 8-51). Of the 56 patients, 30 received lenalidomide maintenance and 26 were observed. Whole exome sequencing was conducted at presentation and relapse to a median depth of 122x for tumour samples and 58x for controls. Libraries were prepared using the SureSelectQXT sample prep kit and SureSelect Clinical Research Exome kit. MuTect was used to determine gene variants and SciClone clustering was undertaken to map mutation variant allele frequencies. MANTA was used to determine translocations and Sequenza for copy number aberrations. Clonal structure and mechanisms of clonal evolution were assessed using kernel density estimation of the cancer clonal fraction for all mutations. Wilcoxon matched-pairs signed rank tests (2-sided) were used to determine the significance between paired data sets, including mutational load. Fishers exact test was used to determine the difference between two nominal variables. Results We looked at mutational, structural and clonal evolution events in all patients based on 1q+ status at relapse. At diagnosis, 34% (19/56) patients had evidence of 1q+, increasing to 46% (26/56) at relapse, with all patients harbouring 1q+ at presentation having the lesion at relapse. There was a significantly higher non-synonymous mutational load at relapse in patients with 1q+, 107 vs 126 (p=0.047), compared to those without 1q+, 36 vs 44 (p=0.140). Twenty two genes known to be significant in MM and mutations within the genes known to be important in IMiD mechanism of action were reviewed. Of the patients with 1q+, 92% (24/26) had at least one mutation during the course of the disease, compared to 77% in those without 1q+ (p=0.15). The impact on tumour suppressor gene regions including deletions of chromosome 1p, 13, 14 and 17p was analyzed. Of the patients with 1q+, 77% (20/26) of patients had a deletion of one of these regions during the disease course, compared to 57% (17/30) of patients without 1q+ (p=0.16). At relapse a change in the profile of these lesions was noted in 23% (6/26) patients with 1q+, compared to 20% (6/30) patients without 1q+ (p=1). Translocations involving MYC (t MYC) were also determined and found in 27% (7/26) of patients with 1q+ and 27% (8/30) of patients without (p=1). As with 1q+, t MYC was always preserved at relapse. Mechanisms of evolution leading to relapse were established for all patients. Branching and linear evolution predominated, noted to be the mechanism leading to relapse in 88% (23/26) patients with 1q+ and 83% (25/30) without (p0.71). Stable evolution was noted in the remaining patients. 1q+ occurring as a new event at relapse was associated with branching or linear evolution in all patients (n=7), consistent with a change in clonal structure. Conclusion These data reveal that 1q+ is conserved throughout the disease course, suggesting it imparts a survival advantage and treatment resistant phenotype to the clone(s) containing it. The presence of 1q+ is associated with a significant increase in mutational load at relapse and a greater incidence of tumour suppressor gene structural deletions, mechanisms that may contribute to clonal evolution and therapeutic escape. Disclosures Jones: BMS/Celgene: Other: Conference fees; Janssen: Honoraria. Pawlyn: Celgene / BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Weinhold: Sanofi: Honoraria. Walker: Sanofi: Speakers Bureau; Bristol Myers Squibb: Research Funding. Cairns: Merck Sharpe and Dohme: Research Funding; Amgen: Research Funding; Takeda: Research Funding; Celgene / BMS: Other: travel support, Research Funding. Kaiser: AbbVie: Consultancy; Seattle Genetics: Consultancy; BMS/Celgene: Consultancy, Other: Travel support, Research Funding; Amgen: Honoraria; Karyopharm: Consultancy, Research Funding; Pfizer: Consultancy; Janssen: Consultancy, Other: Educational support, Research Funding; GSK: Consultancy; Takeda: Consultancy, Other: Educational support. Cook: Pfizer: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria. Drayson: Abingdon Health: Current holder of individual stocks in a privately-held company. Jackson: oncopeptides: Consultancy; takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; J and J: Consultancy, Honoraria, Speakers Bureau; celgene BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; amgen: Consultancy, Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau. Davies: BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees.

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Next Generation Sequencing May be Helpful in Designing Novel Combinations Among Heavily Pretreated Myeloma Patients, Refractory to All Available Approved Anti-Myeloma Drugs

Blood ◽

10.1182/blood-2019-127541 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3197-3197 ◽

Cited By ~ 1

Author(s):

Guldane Cengiz Seval ◽

Isinsu Kuzu ◽

Seher Yuksel ◽

Gulsah Kaygusuz ◽

Klara Dalva ◽

...

Keyword(s):

Bone Marrow ◽

Next Generation Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Advisory Committees ◽

Off Label ◽

Heavily Pretreated ◽

Generation Sequencing ◽

Support Research

Introduction: Multiple genetic alterations that occur at diagnosis or relapse are not only prognostic characteristics of multiple myeloma (MM) but also provide evidence for clonal evolution. Uncovering and dissecting true driver events in MM might provide rational for new potential targets and therapeutic approaches. However, whereas genetic diagnostics in MM namely FISH and gene expression profiling are well-established prognostic tools, individual mutation profiling has not yet been adopted for this purpose. Herein, we aimed to analyze the Next Generation Sequencing (NGS) platform results investigating mutational profiling of patients with relapsed and refractory MM (RRMM). Also, the clinical results of those who had a targetable mutation and were treated "off label" will be presented. Material and Methods: A total of 14 consecutive patients with MM referred to our center between November 2018 and May 2019 were studied. Plasma cells were isolated from bone marrow samples using Selection Kit microbead specific for EasySepTM Human CD138 marker (StemCell Technologies). DNA extracted form magnetic bead enriched cells, bone marrow aspiration smears for bone marrow involved, from FFPE tissue samples for extramedullary-involved cases. NGS method was performed on llumina Miseq platform (USA) by using QIAseq targeted DNA panel (12)- Human myeloid neoplasm panel, covers all exons and exon-intron junctions of 141 target genes. For the data analysis QCI Analyze Universal 1,5.0 was performed. The PCL analysis was performed on CD138 and Ki67 double immune stained paraffin sections, and the quantification was done by using 3DHistech digital pathology platform. Results: We obtained 16 samples of DNA from 12 heavily pretreated and two newly diagnosed myeloma patients. Female/male: 5/9 with a median age of 57 years (range, 39-87) patients had received a median four lines (range; 1-13) of treatment. Out of a panel of 141 genes, 59 mutations in 26 genes were detected (Figure-1). Among these recurrent genomic abnormalities, concomitant missense protein coding alterations were detected in all patients. The PTEN mutation was the most frequently detected, followed by mutations of RAS/MAPK pathway genes. The hotspots of mutation in KRAS codon 61 and NRAS included codons 61 and 13 as well as codon 600 in BRAF. In addition, we detected novel ie myeloproliferative and myelodysplasia associated mutations previously un-described in myeloma. A diverse range of recurrent gains and losses were detected in our cohort. Two patients at diagnosis also carried mutations of PTEN and KRAS. Based on these results three patients were able to obtain off-label approval for treatment with Everolimus (for PTEN) (Patient-1) or Trametinib (for KRAS) (Patient 5 & 6) in combination with Pomalidomide (EvoPomDex) w/wo Daratumumab or Tra-PomDex. Patient-1 had extensive extramedullary disease (EMD) in the skin, which responded completely to Dara-EvoPomDex combination. Complete disappearance of initial lesions (presented in Figure-2) and VGPR duration was only two months. Subsequent refractoriness and appearance of new lesions lead to death of the patient, one year from the initiation of EMD. Patient-6, also presented with EMD, was treated with TraPomDex as the seventh treatment line. TraPomDex treatment was well tolerated, the most significant adverse event diarrhea, infections and pancytopenia. Her biochemical response was a transient VGPR, which was lost during interruption of treatment due to infection. She also died four months after initiation of TraPomDex. Patient-5, plasma cell leukemia, has been on Tra-PomDex for a month and his response is PR yet. Conclusion: The detection of mutations can improve our ability to treat multiple refractory patients who have ran out of all therapeutic options. Though the responses observed among such very heavily pretreated patients are not durable, they are highly promising. Also, of additional importance is detection of age-related cumulative mutations belonging to background bone marrow precursors. Detection of sub-clonal mutations is very helpful in depth analysis of clonal response to treatment and clonal evolution. In the coming years, the identification of actionable mutations in myeloma opens the way for targeted therapy. Acknowledgement:This study is supported by Ankara University Research Grants (Project: 14A0230003) and Turkish Academy of Sciences. Disclosures Ozcan: Takeda: Honoraria, Other: Travel support, Research Funding; Bayer: Research Funding; AbbVie: Other: Travel support, Research Funding; Sanofi: Other: Travel support; Abdi Ibrahim: Other: Travel support; Celgene Corporation: Other: Travel support, Research Funding; Janssen: Other: Travel support, Research Funding; Archigen: Research Funding; Roche: Other: Travel support, Research Funding; Jazz: Other: Travel support; MSD: Research Funding; Novartis: Research Funding; Amgen: Honoraria, Other: Travel support; BMS: Other: Travel support. Beksac:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: trametinib and everolimus for myeloma patients

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Patterns of Clonal Evolution Assessed By Whole Exome Sequencing during Progression from MDS to AML Are Related to Therapy

Blood ◽

10.1182/blood.v128.22.4309.4309 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4309-4309

Author(s):

María Abáigar ◽

Jesús M Hernández-Sánchez ◽

David Tamborero ◽

Marta Martín-Izquierdo ◽

María Díez-Campelo ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Clonal Expansion ◽

Driver Mutations ◽

Advisory Committees ◽

Disease Evolution ◽

Mutational Burden ◽

Whole Exome ◽

Leukemic Phase

Abstract Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

Blood ◽

10.1182/blood-2020-137775 ◽

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.

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Profiling T-Cell Receptor Diversity and Dynamics during Lymphoma Immunotherapy Using Cell-Free DNA (cfDNA)

Blood ◽

10.1182/blood-2020-141655 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 49-50

Author(s):

Navika D Shukla ◽

Alexander F. M. Craig ◽

Brian Sworder ◽

David M. Kurtz ◽

Charles Macaulay ◽

...

Keyword(s):

T Cell ◽

Cell Therapy ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

T Cell Therapy ◽

Car T Cell ◽

Car T ◽

Tcr Repertoire ◽

Support Research

Background: Characterization of T-cell receptor (TCR) diversity and dynamics is increasingly critical to understanding therapeutic immune responses targeting tumors. Current TCR profiling methods generally require invasive tissue biopsies that capture a single snapshot of immune activity or are limited by the sheer diversity of the circulating TCR repertoire. In theory, T-cells with the greatest turnover could best reflect pivotal immune dynamics from both circulating and tissue-derived compartments, including non-circulating tissue-resident memory T-cells (Trm). To noninvasively capture such responses in the blood, we developed and benchmarked a high-throughput TCR profiling approach using plasma, optimized for the fragmented nature of cfDNA and the non-templated nature of rearranged TCRs. We then applied this method for residual disease monitoring in mature T-cell lymphomas (TCL) without circulating disease and for characterizing immune dynamics after anti-CD19 chimeric antigen receptor (CAR19) T-cell therapy of B-cell lymphomas with axicabtagene ciloleucel. Methods: We developed SABER (Sequence Affinity capture & analysis By Enumeration of cell-free Receptors) as a technique for TCR enrichment and analysis of fragmented rearrangements shed in cfDNA and applied this method using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq). We used SABER to profile a total of 381 samples (300 cfDNA and 81 PBMC samples) from 75 lymphoma patients and 18 healthy controls. After mapping sequencing reads (hg38) to identify candidate rearrangements within TCR loci, unique cfDNA fragments were resolved by a novel strategy to define consensus of unique molecular identifiers clustered by Levenshtein distances, followed by CDR3-anchoring for enumeration of final receptor clonotypes. SABER thus leverages information from fragmented TCRs, a critical requirement for cfDNA, to make V gene, CDR3, and J gene assignments after deduplication-mediated error-correction. We benchmarked SABER against established amplicon-based TCR-β targeted sequencing (LymphoTrack, Invivoscribe) and repertoire analysis methods (MiXCR; Bolotin et al, 2015 Nature Methods) when considering both cfDNA and PBMC samples from healthy adults and TCL patients. We assessed SABER performance for tracking clonal molecular disease in patients with mature TCLs from both cellular and cell-free circulating compartments (n=9). Malignant TCL clonotypes were identified in tumor specimens using clonoSEQ (Adaptive Biotechnologies). Finally, we evaluated TCR repertoire dynamics over time in 66 DLBCL patients after CAR19 T-cell therapy. Results: SABER demonstrated superior recovery of TCR clonotypes from cfDNA compared to both amplicon sequencing (LymphoTrack, Invivoscribe) and hybrid-capture methods when enumerating receptors using MiXCR (Fig. 1A). When applied to blood samples from TCL patients, SABER identified the malignant clonal TCR-β rearrangement in 8/9 (88.9%) cases, with significantly improved detection in cfDNA (p=0.015, Fig. 1B). Specifically, tumoral TCR clonotype was detectable only in cfDNA in 6 cases (75%), cfDNA-enriched in 1 case (12.5%), and detectable only in PBMCs in 1 case (12.5%). We applied SABER to monitor TCR repertoire dynamics in cfDNA after CAR T-cell therapy of patients with relapsed/refractory DLBCL and observed increased T-cell turnover and repertoire expansion (greater total TCR-β clonotypes) (Fig. 1C). As early as 1-week after CAR19 infusion, TCR repertoire size was significantly correlated both with cellular CAR19 T-cell levels by flow cytometry (p=0.008) as well as with retroviral CAR19 levels in cfDNA (p=2.20e-07) suggesting faithful monitoring of CAR T-cell activity (Fig. 1D). TCR repertoire size one month after infusion was significantly associated with longer progression-free survival (HR 0.246, 95% CI 0.080-0.754, p=0.014). Conclusions: SABER has a favorable profile for cfDNA TCR repertoire capture when compared to existing methods and could thus have potential broad applicability to diverse disease contexts. Given the higher abundance of lymphoma-derived TCRs in cfDNA than intact circulating leukocytes, SABER holds promise for monitoring minimal residual disease in T-cell lymphomas. This approach also holds promise for monitoring T-cell repertoire changes including after CAR T-cell therapy and for predicting therapeutic responses. Disclosures Kurtz: Genentech: Consultancy; Foresight Diagnostics: Other: Ownership; Roche: Consultancy. Kim:Corvus: Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Elorac: Research Funding; Forty Seven Inc: Research Funding; Galderma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Horizon Pharma: Consultancy, Research Funding; Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa-Kirin Pharma: Research Funding; Medivir: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; miRagen: Research Funding; Neumedicine: Consultancy, Research Funding; Portola: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Solingenix: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Trillium: Research Funding. Mackall:Lyell Immunopharma: Consultancy, Current equity holder in private company; BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy. Miklos:Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding. Diehn:Varian Medical Systems: Research Funding; Illumina: Research Funding; Roche: Consultancy; AstraZeneca: Consultancy; RefleXion: Consultancy; BioNTech: Consultancy. Khodadoust:Seattle Genetics: Consultancy; Kyowa Kirin: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

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ENESTnd Update: Continued Superiority of Nilotinib Versus Imatinib In Patients with Newly Diagnosed Chronic Myeloid Leukemia In Chronic Phase (CML-CP)

Blood ◽

10.1182/blood.v116.21.207.207 ◽

2010 ◽

Vol 116 (21) ◽

pp. 207-207 ◽

Cited By ~ 18

Author(s):

Timothy P. Hughes ◽

Andreas Hochhaus ◽

Giuseppe Saglio ◽

Dong-Wook Kim ◽

Saengsuree Jootar ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Molecular Response ◽

Newly Diagnosed ◽

Advisory Committees ◽

Free Survival ◽

Superior Efficacy ◽

Rate Of Progression

Abstract Abstract 207 Background: Results from the phase 3, international, randomized ENESTnd trial have demonstrated the superior efficacy of nilotinib over imatinib with significantly higher rates of major molecular response (MMR), complete cytogenetic response (CCyR), and with significantly lower rates of progression to AP/BC on treatment. Here, we present data with a median follow-up of 18 months. Methods: 846 CML-CP patients were randomized to nilotinib 300 mg twice daily (bid) (n=282), nilotinib 400 mg bid (n=281), and imatinib 400 mg once daily (n=283). Primary endpoint was MMR (≤ 0.1% BCR-ABLIS) rate “at” 12 months, as previously presented. Key secondary endpoint was durable MMR at 24 months. Other endpoints assessed at 24 months include progression to AP/BC (with and without clonal evolution), event-free survival, progression-free survival, and overall survival (OS). Results: With a median follow-up of 18 months, the overall best MMR rate was superior for nilotinib 300 mg bid (66%, P < .0001) and nilotinib 400 mg bid (62%, P < .0001) compared with imatinib (40%). Superior rates of MMR were observed in both nilotinib arms compared with the imatinib arm across all Sokal risk groups (Table). The overall best rate of BCR-ABLIS ≤ 0.0032% (equivalent to complete molecular response, CMR) was superior for nilotinib 300 mg bid (21%, P < .0001) and nilotinib 400 mg bid (17%, P < .0001) compared with imatinib (6%). The overall best CCyR rate was superior for nilotinib 300 mg bid (85%, P < .001) and nilotinib 400 mg bid (82%, P=.017) compared with imatinib (74%). The superior efficacy of nilotinib was further demonstrated using the 2009 European LeukemiaNet (ELN) 12-month milestone in which fewer patients had suboptimal response or treatment failure on nilotinib 300 mg bid (2%, 3%) and nilotinib 400 mg bid (2%, 2%) vs imatinib (11%, 8%). Rates of progression to AP/BC on treatment were significantly lower for nilotinib 300 mg bid (0.7%, P=.006) and nilotinib 400 mg bid (0.4%, P=.003) compared with imatinib (4.2%). The rate of progression on treatment was also significantly lower for nilotinib when including clonal evolution as a criteria for progression (Table). There were fewer CML-related deaths on nilotinib 300 mg bid (n=2), and 400 mg bid (n=1) vs imatinib (n=8). Estimated OS rate (including data from follow-up after discontinuation) at 18 months was higher for nilotinib 300 mg bid (98.5%, P=.28) and nilotinib 400 mg bid (99.3%, P=.03) vs imatinib (96.9%). Both drugs were well-tolerated. Discontinuations due to adverse events or laboratory abnormalities were lowest for nilotinib 300 mg bid (7%) compared with nilotinib 400 mg bid (12%) and imatinib (9%). With longer follow up there has been minimal change in the occurrence of AEs. Minimum 24-month follow-up data for all patients will be presented. Conclusions: With longer follow-up, nilotinib was associated with a significantly lower rate of progression to AP/BC on treatment and lower rates of suboptimal response or treatment failure vs imatinib. Nilotinib resulted in fewer CML-related deaths and a higher OS rate vs imatinib. Nilotinib induced superior rates of MMR, CMR, and CCyR vs imatinib in patients with newly diagnosed CML-CP. Taken together, these data support nilotinib as a new standard of care for patients with newly diagnosed CML. Disclosures: Hughes: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Meyers Squibb: Honoraria, Research Funding; Ariad: Honoraria. Hochhaus:Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Saglio:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Kim:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. le Coutre:Novartis: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Reiffers:Novartis: Research Funding. Pasquini:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Clark:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Honoraria, Research Funding. Gallagher:Novartis Pharma AG: Employment, Equity Ownership. Hoenekopp:Novartis Pharma AG: Employment. Haque:Novartis: Employment. Larson:Novartis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Kantarjian:Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding.

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A Randomized Study of Low Dose Oral Clofarabine 10 Mg Versus 20 Mg (flat dose) Daily × 5 for Patients with Higher-Risk Myelodysplastic Syndrome (MDS)

Blood ◽

10.1182/blood.v120.21.3851.3851 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3851-3851

Author(s):

Stefan Faderl ◽

Guillermo Garcia-Manero ◽

Farhad Ravandi ◽

Jorge E. Cortes ◽

Zeev Estrov ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Randomized Study ◽

Patient Characteristics ◽

Median Number ◽

Infectious Complications ◽

Advisory Committees ◽

Platelet Recovery ◽

Flat Dose ◽

Secondary Mds

Abstract Abstract 3851 Clofarabine (CLO) is a nucleoside analog with activity in myeloid malignancies. We have previously reported an overall response rate (ORR) of 43% (CR rate 25%) in patients (pts) with higher-risk MDS treated with oral CLO at doses between 20 mg/m2 and 40 mg/m2 daily × 5 (S Faderl et al. J Clin Oncol 2010, 28: 2755). However, myelosuppression and infectious complications were frequent. We therefore developed a trial based on a Bayesian randomization design of CLO 10 mg vs 20 mg (flat dose) orally daily × 5 days with the objective to maintain reasonable efficacy and minimize toxicities. Cycles were repeated every 4 to 8 weeks for up to a total of 12. Pts were eligible if they had MDS with ≥ 5% blasts (including RAEB-t by FAB) or IPSS intermediate-2 and high-risk, and CMML. Hematopoietic growth factor support prior to and during the study was permitted. Thirty-two pts (19 RAEB [59%], 7 RAEB-t [22%], 2 MDS/MPN [6%], 4 CMML [13%]) were randomized. Patient characteristics were similar between the groups and are summarized in Table 1. Table 1: Patient Characteristics Characteristic 10 mg 20 mg N 16 16 Median age, yrs (range) 67.5 (43–87) 72.5 (54–84) Median WBC, ×109/L (range) 4 (1.2–121.3) 4.8 (0.4–47) Median blood blasts, % (range) 0.5 (0–25) 3.5 (0–32) Median marrow blasts, % (range) 12 (4–28) 12 (2–24) Secondary MDS, N (%) 9 (56) 4 (25) Median N prior therapies (range) 1 (0–3) 1 (0–4) Prior hypomethylator therapy, N 14 (88%) 13 (81%) Cytogenetics 5 (31) 6 (38) Diploid, N (%) 4 (25) 4 (25) -5/5q- and/or -7/7q- 4 (25) 2 (13) IM/not done Seven pts (22%) responded: 3 CR and 4 CR without platelet recovery (CRp). Four pts (25%) in the 10 mg group and 3 pts (19%) in the 20 mg group responded (differences not significant). Median remission duration was 6.5 months for all pts (6.5 months [10 mg]; 4.4 months [20 mg], p=.942). The median number of cycles was 2 in each treatment group with a range of 1–4 (10 mg) and 1–12 treatment cycles (20 mg), respectively. One pt in each group died. Median survival for the whole group was 8.5 months (8.2 months [10 mg]; 8.5 months [20 mg], p=.9). Clofarabine at both 10 mg and 20 mg orally daily × 5 has a comparable CR/CRp rate as do higher doses. Myelosuppression does still occur but prolonged myelosuppression has been rare. Even lower doses including at different schedules may still warrant further study. Disclosures: Faderl: Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Clofarabine in MDS. O'Brien:Avila: Research Funding; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding; Gilead Sciences: Consultancy, Research Funding; Celgene: Consultancy; Cephalon: Consultancy; CII Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Genentech BioOncology: Research Funding; Genzyme: Consultancy; GlaxoSmithKline: Consultancy; MorphoSys: Consultancy; Novartis: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Seattle Genetics, Inc.: Consultancy; Sigma Tau Pharmaceuticals: Consultancy; Talon: Research Funding; The Medal Group: Speakers Bureau. Kantarjian:Genzyme: Research Funding.

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Transition of Chronic Myeloid Leukemia to Chronic Myelomonocytic Leukemia As a Tool to Observe Development of Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v126.23.5223.5223 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5223-5223

Author(s):

Jamshid S Khorashad ◽

Srinivas K Tantravahi ◽

Dongqing Yan ◽

Anna M. Eiring ◽

Hannah M. Redwine ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Clonal Evolution ◽

Chronic Myelomonocytic Leukemia ◽

Imatinib Treatment ◽

Advisory Committees ◽

Clonal Architecture ◽

Myelomonocytic Leukemia

Abstract Introduction. Development of abnormal Philadelphia (Ph) negative clones following treatment of chronic myeloid leukemia (CML) patients with imatinib has been observed in 3 to 9% of patients. Here we report on a 77 year old male diagnosed with CML that responded to imatinib treatment and subsequently developed chronic myelomonocytic leukemia (CMML). He achieved major cytogenetic response within 3 months but this response coincided with the emergence of monocytosis diagnosed as CMML. Five months after starting imatinib treatment the patient succumbed to CMML. We analyzed five sequential samples to determine whether a chronological order of mutations defined the emergence of CMML and to characterize the clonal evolution of the CMML population. Materials and Method. Five samples (diagnostic and four follow up samples) were available for analysis. CMML mutations were identified by whole exome sequencing (WES) in CD14+ cells following the onset of CMML, using CD3+ cells as constitutional control. Mutations were validated by Sequenom MassARRAY and Sanger sequencing and quantified by pyrosequencing. Deep WES was performed on the diagnostic sample to determine whether the mutations were present at CML diagnosis. To determine the clonal architecture of the emerging CMML, colony formation assays were performed on the diagnostic and the next two follow-up samples (Samples 1-3). More than 100 colonies per sample were plucked for DNA and RNA isolation. The DNA from these colonies were tested for the presence of the confirmed CMML mutations and the RNA was used for detection of BCR-ABL1 transcript using a Taqman real time assay. Results. Four mutations were identified by Sequenom and WES throughout the patient's time course [KRASG12R, MSLNP462H, NTRK3V443I and EZH2I669M ]. Sequenom did not identify these at diagnosis while deep WES did. Clones derived from colony formation assay revealed three distinct clones present in all samples analysed. Clone 1 had only KRASG12R, clone 2 had KRASG12R, MSLNP462H, and NTRK3V443I, and clone 3 had all four mutations. All clones containing any of these four mutations were BCR/ABL1 negative. Analysis of clonal architecture indicated that KRASG12R was acquired first and EZH2I669M last, while MSLNP462H and NTRK3V443I were acquired in between. These CMML clones increased proportionately as clinical CML metamorphosed into clinical CMML after initiation of imatinib therapy. Consistent with the colony data, pyrosequencing revealed that the ratio between the mutants remained largely stable throughout the follow up period. Conclusion. This case illustrates how targeted therapy impacts clonal competition in a heterogeneous MPN. While the CML clone was dominant in the absence of imatinib, it was quickly outcompeted by the CMML clones upon initiation of imatinib therapy. The clonal architecture analysis, in combination with in vivo kinetics data, suggest that the KRASG12R mutation alone was able to produce a CMML phenotype as clones with just KRASG12R remained at a relatively stable ratio during follow up. Unexpectedly, acquisition of additional mutations, including EZH2I669M as the last mutational event identified in this patient, did not increase clonal competitiveness, at least in the peripheral blood. These data show that clonal evolution may not invariably increase clonal fitness, suggesting that factors other than Darwinian pressures contribute to clonal diversity in myeloproliferative neoplasms. Disclosures Deininger: Gilead: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Dynamic Changes in Clonal Clearance with Decitabine Therapy in AML and MDS Patients

Blood ◽

10.1182/blood.v126.23.689.689 ◽

2015 ◽

Vol 126 (23) ◽

pp. 689-689

Author(s):

John S. Welch ◽

Allegra Petti ◽

Christopher A. Miller ◽

Daniel C. Link ◽

Matthew J. Walter ◽

...

Keyword(s):

Bone Marrow ◽

Complete Remission ◽

Clinical Response ◽

Exome Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Cells ◽

Advisory Committees ◽

Clonal Hematopoiesis ◽

Clinical Responses

Abstract To determine how AML subclonal architecture changes during decitabine treatment, and whether specific mutations might correlate with sensitivity vs. resistance to decitabine, we performed exome sequencing at multiple time points during single agent decitabine therapy. We enrolled 69 patients with either AML (age ≥ 60, or with relapsed/refractory disease, N = 45) or MDS (N = 24) on a phase I clinical trial. All subjects were treated with decitabine 20 mg/m2 on days 1-10 of 28 day cycles. With a median follow-up of 13.7 months, the intention to treat clinical response (complete remission with or without complete count recovery: CR/CRi) is 40%, with survival correlating with response (median survival - CR/CRi: 583 days; partial response/stable disease (PR/SD): 260 days; progressive disease (PD) or failure to complete cycle 1: 36 days, p < 0.0001). We performed exome sequencing on unfractionated bone marrow cells at diagnosis (day 0), cycle 1 day 10, cycle 1 day 28, cycle 2 day 28, and, when possible, during remission and at clinical relapse/progression. We have completed sequencing analysis for the first 34 cases (outcomes: 5 CR, 15 CRi, 3 PR, 8 SD, and 3 PD). Several important themes have emerged, as follows: 1) We correlated mutation status at diagnosis with clinical response. All six patients with TP53 mutations obtained clinical CR or CRi, and exome analysis demonstrated near complete elimination of the TP53- associated founding clones by the end of cycle 2 (p < 0.03). Long-term outcomes were similar in these patients compared with other patients who achieved CR/CRi: four patients relapsed after 8, 9, 10, or 17 cycles; 1 patient is doing well post-transplant; and one patient died of an infectious complication after cycle 2. No other mutations were significantly associated with clinical response or with consistent mutation clearance. 2) We observed a reduction in blast counts, which preceded mutation elimination in fourteen cases with CR, CRi or PR. This suggests that decitabine may induce morphological blast differentiation in vivo prior to mutation elimination. 3) In eight of nine cases with a clinical response followed by relapse, clinical progression was associated with expansion of a pre-existing subclone. We have not yet observed any recurrent mutations that reliably predict whether a subclone will contribute to relapse. Intriguingly, in two of these cases, the relapse-associated subclone was detectable at diagnosis and was eliminated more slowly than the founding clone mutations, suggesting that this subclone harbored intrinsic decitabine-resistance. 4) Complete remission can occur with concomitant non-malignant, clonal hematopoiesis. In three cases with a CR, a new clonal population was selected for during the remission. In two of these cases, there were no shared mutations between the founding clone and the emergent, non-malignant, clonal hematopoiesis, suggesting that these clones were unrelated. 5) Mutational architecture is generally stable, but differential chemo-sensitivity can be detected even between subclones in the same patient. In ten cases with PR or SD, we observed minimal shifts within the mutational burden over the course of eight weeks, suggesting that "clonal drift" is a relatively slow process. However, in four cases with SD, what appeared clinically to be simple persistent disease was in fact a dynamic elimination of one subclone, and its replacement by a different subclone. Similarly, in three cases with CRi, we observed rapid clearance of a subclone with slower clearance of the founding clone, again suggesting differential chemo-sensitivity among subclones. 6) Finally, we correlated pharmacologic markers with clinical outcomes. We observed no correlation between steady-state plasma decitabine levels and clinical responses. Using Illumina 450k methylation arrays, we observed a correlation between response and the extent of decitabine-induced hypomethylation in total bone marrow cells that persisted on cycle 1 day 28 (p < 0.01), but not on cycle 1 day 10 (p < 0.1). In summary, these data reveal that response to decitabine is associated with morphologic blast clearance before mutations are eliminated, that relapse is associated with subclonal outgrowth that may be identified early in the treatment course, and that TP53 mutations may be predictive of rapid clinical responses, although, like most responses to decitabine, these are not necessarily durable. Disclosures Off Label Use: Decitabine treatment of AML.. Uy:Novartis: Research Funding. Oh:CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Abboud:Novartis: Research Funding; Gerson Lehman Group: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Merck: Research Funding; Teva Pharmaceuticals: Research Funding. Cashen:Celgene: Speakers Bureau. Schroeder:Celgene: Other: Azacitidine provided for this trial by Celgene; Incyte: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy.

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Clonal Hematopoiesis Prevalence Increases throughout Treatment of Newly Diagnosed Multiple Myeloma Patients

Blood ◽

10.1182/blood-2021-152025 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1091-1091

Author(s):

Tarek H. Mouhieddine ◽

Chidimma Nzerem ◽

Robert A. Redd ◽

Andrew Dunford ◽

Matthew Joseph Leventhal ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Hematologic Malignancy ◽

Newly Diagnosed ◽

Advisory Committees ◽

Clonal Hematopoiesis ◽

First Time ◽

Time Point

Abstract Background: Recent studies have identified clinical and genomic factors contributing to worse clinical outcomes in patients with multiple myeloma (MM). Clonal hematopoiesis (CH) reflects the presence of somatic driver mutations in the blood or marrow of otherwise asymptomatic individuals. Using a variant allele frequency (VAF) cutoff of 2%, we recently reported CH in 21.6% of MM patients at the time of autologous stem cell transplant (ASCT) and found it was associated with shorter overall survival (OS) and progression-free survival (PFS) in those who did not receive maintenance therapy with an immunomodulatory drug (IMiD). However, this finding was based on a single tertiary center and only included MM patients who received ASCT. Methods: We studied a larger cohort of 986 newly diagnosed MM cases. Whole-exome sequencing (WES) data of peripheral blood and bone marrow samples of 986 MM patients (523 transplanted and 463 non-transplanted) from the Multiple Myeloma Research Foundation (MMRF) Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass, NCT0145429) study were analyzed. Both peripheral blood and tumor samples were analyzed to filter out myeloma mutations that could be contaminating the peripheral blood. Given the lower depth of coverage compared to prior targeted sequencing studies, small clones with a VAF below 2% were not detected. Altogether, the WES samples had a total depth of coverage of 117.68X. All data were analyzed using R version 3.5.0 (R Core Team). Results: Among the total cohort, 113 CH mutations were detected in 101/986 (10.24%) patients. CH was detected in 42/523 (8.03%) transplanted patients, compared to 59/463 (12.74%) non-transplanted patients. The most commonly mutated genes were DNMT3A, TET2, ASXL1, PPM1D, and TP53. The median age of the cohort was 63 years (range: 27 - 93), 60% were male, and median follow-up was 3.9 years (95% CI: 3.7 - 4.0). The presence of CH was associated with age (69 vs. 62 years, P < 0.001). As expected, the median age of transplanted patients was lower (60 vs. 67 years) than in the non-transplanted group, which likely explains the higher prevalence of CH detected in the non-transplanted group. CH was associated with recurrent bacterial infections (P = 0.01) and increased cardiovascular disease (P = 0.006), but not with cerebrovascular disease (P = 0.74) or coagulopathies (P = 0.65). There was a trend towards worse PFS in non-ASCT patients with CH who were not treated with IMiDs (1.8 years) compared to non-CH IMiD-treated patients (2.7 years) (P < 0.001). A CH effect on PFS was not detected in ASCT patients. OS was not different in those with or without CH in both ASCT and non-ASCT groups. 8 (0.8%) patients developed a second hematologic malignancy. CH at the time of MM diagnosis was not associated with an increased risk of developing a second hematologic malignancy (P = 0.58). To determine whether CH clones emerged or evolved during treatment, we examined serial samples from 52 patients (36 ASCT patients and 16 non-transplanted patients) with sequential samples. The median time between the first and second time point was 3.1 years (range: 1.0 - 5.4 years). At the first time point, only 3/52 (5.8%) patients had CH, but that number increased to 13/52 (25.0%) at the second time point. Five out of the 13 (38%) were non-transplanted patients. All but 1 patient were exposed to IMiDs. The most common emerging mutated gene was DNMT3A, found in 7 patient samples at the second time point, compared to 2 patients at the first time point. Conclusion: Using WES in a large cohort of newly diagnosed MM patients, we detected CH in 10.2% (VAF ≥ 2%) of patients. CH and non-IMiD treatment confers a shorter PFS in non-transplanted MM patients. However, throughout IMiD-based treatment, MM patients tend to acquire and/or expand previously undetected CH clones, particularly DNMT3A. The clinical significance of this clonal expansion during therapy is yet to be elucidated, and for now, this observation does not yet change clinical management. Figure 1 Figure 1. Disclosures Steensma: Novartis: Current Employment. Ebert: Deerfield: Research Funding; GRAIL: Consultancy; Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Skyhawk Therapeutics: Membership on an entity's Board of Directors or advisory committees. Soiffer: NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees; Gilead, USA: Other: Career Development Award Committee; Rheos Therapeutics, USA: Consultancy; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Precision Biosciences, USA: Consultancy; Jazz Pharmaceuticals, USA: Consultancy; Jasper: Consultancy; Takeda: Consultancy. Sperling: Adaptive: Consultancy. Getz: Scorpion Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; IBM, Pharmacyclics: Research Funding. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

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