Analysis of Clonal Evolution of AML Using Simultaneous Single-Cell DNA/RNA Analysis

Background Acute myeloid leukemia (AML) was defined by an increase of immature myeloid cells, or blasts that exceed ≥20% in bone marrow or peripheral blood. Many lines of evidence suggest that the development of AML is shaped by clonal evolution through multiple rounds of positive selection driven by newly acquired mutations, ultimately leading to an increased blast count. This process has been analyzed in detail in the case of progression from myelodysplastic syndromes (MDS) to secondary AML (sAML), which is invariably accompanied by expansion of cells that acquired new driver alterations, generating clonal substructures in many cases (Walter et al. NEJM. 2012, Makishima et al. Nat. Genet. 2015). However, it has not been fully elucidated how these newly acquired mutations contribute to increased blast cells that define AML. Results In order to understand how driver mutations contribute to the phenotype of blasts, we first focused on the driver mutations that have known to be enriched in sAML, including those in IDH1/2, NPM1, FLT3,NRAS, KRAS, PTPN11, CBL and WT1, and compared BM blast count (BC) and mutant cell fraction (MCF) of each driver mutation in 27 cases with sAML. Compared with BC, IDH1- or IDH2-mutated cells exhibited a larger MCF in most cases, suggesting that newly acquired IDH1/2 mutations contribute clonal expansion but only a part of the expanded cells undergo differentiation block and the remaining cells can differentiate into mature cells. Of interest, we observed lower MCFs than BC in approximately half of the cases with signaling pathway mutations, including FLT3 and RAS pathway (NRAS, KRAS, PTPN11 and CBL) mutations, in which MCFs for signaling pathway mutation accounted for less than 2/3 of BC, which was also observed in de novo AML cases. In fact, signaling pathway mutations in two representative cases were confirmed to account only for 30.4% and 3.4% of blast cells, using ddPCR of the blast cells collected as the CD45dim SSClow fraction, which were confirmed to show a blast morphology. These results suggest a possibility that the presence of mutant cells might affect the phenotype of the surrounding unmutated cells. Thus, to investigate the mechanism of such non-cell autonomous effects of mutations on blast cell morphology, we developed an advanced single-cell sequencing platform that enables simultaneous measurements of both mutations and gene expression profiles at a single-cell level and applied this to the analysis of immature (CD34+ Lin-) BM cells from 2 sAML cases with multiple RAS pathway mutations showing disproportionately small MCF compared to BC, in which gene expression of mutated and unmutated cells were evaluated separately. The same BM faction in 13 healthy donors was also analyzed as normal control. In single-cell mutation analysis, multiple RAS pathway mutations in both cases represented independent clones. As expected, cells carrying each RAS pathway mutation at sAML showed an immature myeloid phenotype. However, most of the cells, even carrying MDS mutations alone, also exhibited an immature myeloid phenotype similar to the RAS pathway mutated cells, although the latter cells showed upregulated RAS signaling compared with the former cells. Cells solely carrying MDS mutations in MDS phase showed multi-lineage differentiation, which was no longer observed in those cells in sAML phase. This was in contrast to another case who acquired MYC amplification on sAML progression, where nearly all cells having MYC-amplification showed an immature myeloid phenotype, whereas the remaining MDS clones lacking MYC-amplification retained multilineage differentiation even at the sAML phase. These results suggest that RAS mutants might have a non-cell autonomous effect on the surrounding cells including those hematopoietic cells lacking those mutations and other stromal cells, preventing their differentiation to mature cells, although we cannot exclude another possibility that altered BM microenvironment could influence the phenotype of both mutated and unmutated cells. Conclusions Although an acquisition of new mutations is essential for the progression of MDS to sAML, our results suggest that the blast cell phenotype may not solely be determined by cell-intrinsic effects of such mutations, but non-cell autonomous effects of mutated cells (and possibly also of an altered BM microenvironment) may have a role in increased blast count and therefore AML progression. Disclosures Inagaki: Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Ogawa:KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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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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Molecular Characterisation of Participants in the Phazar Trial Reveals Prognostic Impact of Mutations in Advanced-Phase-MPN

Blood ◽

10.1182/blood-2020-139520 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 40-41

Author(s):

Charlotte K Brierley ◽

Alba Rodriguez-Meira ◽

Matthew Bashton ◽

Angela Hamblin ◽

Rachel S Fletcher ◽

...

Keyword(s):

Board Of Directors ◽

Chromosomal Aberrations ◽

Research Funding ◽

Poor Prognosis ◽

Clonal Evolution ◽

Molecular Profiling ◽

Driver Mutation ◽

Driver Mutations ◽

Advisory Committees ◽

Advanced Phase

Advanced phase myeloproliferative neoplasms (AP-MPN) are associated with a very poor prognosis. The Phase Ib PHAZAR study set out to assess the safety & tolerability of oral ruxolitinib (RUX) in combination with 5-azaciditine (AZA) in patients (pts) with advanced-phase-MPN, defined as blast count >10%. The study included an observational arm for pts not suitable for the trial intervention. The clinical results of this study are presented in a separate abstract. Here we evaluate the molecular characteristics of PHAZAR pts and correlate with clinical features, outcome and therapy response. Driver mutation (JAK2/CALR/MPL) allele burdens were quantified using targeted next-generation sequencing (NGS) and non-driver mutation analysis was performed using an ISO accredited Illumina TruSeq Custom Amplicon Panel, including 32 gene mutation hotspots & exons (~36,000 bp, 287 amplicons). SNP karyotyping was performed using the Illumina InfiniumOmniExpress-24v1-3 BeadChip assay. Data analysis was performed using R v4.0. Clinical data were censored in February 2020, and NGS sequencing data were available for 24 interventional trial and 13 observational cohort participants. 11/13 observational pts received best supportive care, while 2/13 were treated with high-dose chemotherapy. All pts had a mutation in ≥1 targeted gene. 16% of pts were 'triple-negative' for MPN driver mutations, while 59%, 16% & 8% carried canonical mutations in JAK2, CALR & MPL respectively. 89% carried additional non-driver mutations, with a median of 2 (range 0-4) detected per pt (Fig 1A). Mutations in epigenetic regulators were detected in 21/37 pts (57%) (TET2, 38%; EZH2, 19%; ASXL1, 14%; PHF6, 5%; SETBP1, 3%) while 8/37 (22%) carried mutually exclusive spliceosomal mutations (SRSF2, 8%; U2AF1, 8%; SF3B1, 5%). 10/37 (27%) were TP53 mutant. High molecular risk (HMR) mutations (ASXL1, EZH2, IDH1/2, SRSF2, TP53, U2AF1 Q157) were detected in 24/37 (65%), and >1 HMR mutation in 7/37 (19%). SNP karyotyping data were available for 42 pts (n=29 interventional, n=13 observational). 4/42 (10%) were wild-type, while 90% harboured >=1 chromosomal aberrations (median 4, range 0-16). Of these, 21 were recurrent in 3+ samples. 9 frequently recurrent events in >=5 samples included gains at 1q, 3q26, 17q21and losses of 5q, 6q12, 17p13, 19q13, 20q, and multiple losses and gains on chromosome 21q. 5 pts demonstrated evidence of chromothripsis. The presence of TP53 mutation was associated with a higher number of chromosomal aberrations (median of 3 vs 6.5, p=0.02). Concerning clinical correlation, baseline driver mutation status did not impact on OS nor likelihood of achieving a durable response (DR, defined as having achieved a minimum of 6 months of complete or partial remission or stable disease as per published criteria (Cheson Blood 2006, Mascarenhas Leuk Res 2012)). The presence of >=3 additional mutations significantly impaired OS regardless of trial arm (1 yr OS 12% vs 55%, p=0.02), as did the presence of HMR mutations (1 yr OS 22% vs 73%, p=0.008) and TP53 mutations in isolation (1 yr OS 13% vs 55%, p=0.05). The presence of HMR mutations reduced the likelihood of achieving a DR (p=0.02). Pts with losses of >=1 chromosomal arms (other than 5q-) had a poor prognosis (1yr OS 27% vs 58%, p=0.05), while no pt with chromothripsis (n=5) survived to a year (1yr OS 0% vs 53%, p=0.002). Mutational profiling of serial samples on therapy were available for 5 pts who achieved a remission during AZA and RUX therapy. One pt achieved a CMR but developed clonal evolution and emergence of a new ETV6 mutant clone at relapse. The other 4 cases demonstrated no change in clonal abundance during remission. This supports the hypothesis that response to AZA is mediated by alteration of subclonal contributions or prevention of further clonal evolution, rather than elimination of founder clones. AP-MPN continues to confer a very poor prognosis and more effective therapies are urgently required. Genetic and molecular profiling of this prospective trial cohort demonstrates the high mutational burden and structural variants seen in this disease. Initial serial sample profiling demonstrates that molecular responses to AZA and RUX are rare and, where they occur, are not sustained. Incorporation of molecular profiling into trial design may help inform which patients are more likely to benefit from the intervention - e.g. those without evidence of chromothripsis at trial entry. Disclosures Harrison: Gilead Sciences: Honoraria, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Incyte Corporation: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Promedior: Honoraria; Roche: Honoraria; Sierra Oncology: Honoraria. Drummond:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint Medicine Corporation: Research Funding. Knapper:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mead:Gilead: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau.

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Single Cell DNA Sequencing Identifies Combinatorial Mutation Patterns and Clonal Architecture in Myeloid Malignancies

Blood ◽

10.1182/blood-2019-128763 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 913-913 ◽

Cited By ~ 1

Author(s):

Linde A Miles ◽

Robert L Bowman ◽

Tiffany R Merlinsky ◽

Aik Ooi ◽

Pedro Mendez ◽

...

Keyword(s):

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Clonal Diversity ◽

Single Cell Level ◽

Cell Level ◽

Advisory Committees ◽

Parallel Acquisition ◽

Ras Pathway ◽

Clonal Architecture

Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplasia (MDS), identified mutations with different allele frequencies. Recent studies of clonal hematopoiesis (CH) discovered a subset of MM disease alleles, while other alleles are only observed in overt MM. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. Although bulk sequencing informs prognostication, it cannot distinguish which mutations occur in the same clone and cannot offer definitive evidence of mutational order. Delineation of clonal architecture at the single cell level is key to understanding how the sequential/parallel acquisition of somatic mutations contributes to myeloid transformation. In order to elucidate the clonal structure of MM, we designed a custom single cell 109 amplicon panel of the most frequently mutated amplicons in 50 MM genes using the Mission Bio Tapestri v2 platform. Viable cells were sorted from 90 samples from 78 patients with CH, AML, and MPN/post-MPN AML followed by single cell amplification/sequencing. Mutation calls were filtered based on read depth, quality, and alleles genotyped per cell. We reconstructed a random distribution of clones by permuting genotype calls across cells and generated empirical p values for each clone. To identify dominant clones, we used a Poisson test to determine clones were significantly enriched compared to the mean clone size. Clones with significant p-values (p <0.05) were used to generate plots of clonal architecture of each sample (Figure 1A). Despite significant clonal complexity, the majority of MM patients (80%;72/90) present with one (51/90; 56.7%) or two (21/90; 23.3%) dominant clones. These data show there are specific genotypic combinations which lead to clonal dominance with increased fitness relative to other clones and/or suppression of minor clones by dominant clone(s). We next investigated whether specific molecularly defined AML subtypes had increased clonal complexity. FLT3-ITD mutant AML samples had a significantly greater number of clones (p < 0.002) compared to AML samples with multiple epigenetic modifier mutations. Similar findings were not observed when comparing AML samples with epigenetic mutations to RAS pathway mutant samples. We next investigated whether specific mutations were likely to co-occur/be mutually exclusive at a single cell level. We observed evidence of oligoclonality in CH, including parallel acquisition of DNMT3A mutations and clones with multiple mutations in the absence of progression to MM. By contrast, in MM the dominant clone(s) almost always harbored multiple epigenetic modifier mutations, suggesting cooperative epigenetic remodeling in myeloid transformation. Mutations in signaling effectors (FLT3-ITD/TKD; RAS/RAS) were mutually exclusive. We observed distinct FLT3-mutant clones in FLT3-mutant AML patients and parallel acquisition of different RAS pathway mutations. We used this data to develop clonal architecture trees in each patient, giving us a definitive picture of mutational acquisition and transformation at a single cell level. We calculated a Shannon diversity score and observed an increase in clonal complexity with disease evolution; CH samples had the lowest clonal diversity and FLT3-ITD AML patients the highest clonal diversity (Figure 1B). We extended our findings by combining cell surface marker assessment and single cell mutational analysis. Patient samples were stained with an antibody cocktail of 6 oligo-conjugated antibodies with barcode tags prior to single cell sequencing, which allowed simultaneous acquisition of single cell immunophenotypic and genotypic data. This allows us to identify distinct populations of stem/progenitor cells with distinct clonal/mutational repertoires (Figure 1C). Additional data will be presented with this novel approach, which allows us to combine an assessment of stem/progenitor cell frequency with genetic data. This includes studies of CD34+ and CD34- AML, which show striking differences in mutational representation in different stem/progenitor compartments. In summary, our studies of clonal architecture at a single cell level provide us novel insights into the pathogenesis of myeloid transformation and give us new insights into how clonal complexity contributes to disease progression. Disclosures Ooi: Mission Bio: Employment, Equity Ownership. Mendez:Mission Bio: Employment, Equity Ownership. Carroll:Janssen Pharmaceuticals: Consultancy; Incyte: Research Funding; Astellas Pharmaceuticals: Research Funding. Papaemmanuil:Celgene: Research Funding. Viny:Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Levine:Roche: Consultancy, Research Funding; Amgen: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Gilead: Consultancy; Celgene: Consultancy, Research Funding.

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

Blood ◽

10.1182/blood-2018-99-110540 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4118-4118

Author(s):

Makhdum Ahmed ◽

Hui Guo ◽

Shaojun Zhang ◽

Lalit Sehgal ◽

Preetesh Jain ◽

...

Keyword(s):

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Single Cell ◽

Research Funding ◽

Cell Lymphoma ◽

Clonal Evolution ◽

Advisory Committees ◽

Cell Clones ◽

Mantle Cell

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

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Mutations in Driver Genes and Changes in Clonal Dynamics Are Associated with Shorter Time to Treatment in MBL Cases

Blood ◽

10.1182/blood.v126.23.5264.5264 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5264-5264

Author(s):

Santiago Barrio ◽

Juhi Ojha ◽

Charla Secreto ◽

Kari G. Chaffee ◽

klaus Martin Kortum ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Germ Line ◽

Clonal Evolution ◽

Clonal Expansion ◽

Driver Mutations ◽

Driver Gene ◽

Driver Genes ◽

Advisory Committees ◽

Recurrent Mutations

Abstract Introduction: Monoclonal B cell lymphocytosis (MBL) is an asymptomatic expansion of clonal CD19+/CD5+ B cells with less than 5x109/L cells in the peripheral blood and without other manifestations of chronic lymphocytic leukemia (CLL). Approximately 1% of MBL evolves to CLL requiring therapy per year; thus it is critical to develop more precise tools to identify which MBL will progress to CLL and require treatment. Patients and Methods: In this study, we performed targeted deep sequencing (TDS) on 49 high-count MBL individuals (median B-cell count 3.7x109/L; range 0.8-4.9x109/L) and explored the mutation status of 20 driver genes recurrently mutated in CLL. We analyzed the clonal evolution in 45 of these 49 MBLs by screening 2-4 sequential samples (average time between samples 56 months, range 10-119 months). At last follow-up, 19 cases (39%) had progressed to Rai>0, and 10 cases (20%) required treatment. Tumor and germ line DNAs were isolated from sorted CD5+/CD19+ and CD5-/CD19- cell populations, respectively. Overall, 154 samples from 49 MBL cases (105 tumor and 49 germ line) were screened using semiconductor sequencing technology. The latter genetic information was integrated with relevant clinical and biological parameters, and we evaluated the effect of driver mutations and clonal expansion on time to CLL progression and time to treatment (TTT). Results and Discussion: Our cohort consisted in 17 women and 32 men, with a median age of 66 years (range: 44-80). Five cases presented secondary diseases, including melanoma, lung and bladder cancer. Clinical and biological parameters were collected, including IGHV mutation status (mutated 66%, unmutated 34%), ZAP70 and CD49 expression (25% each). At presentation, 46% of cases had del(13q), 27% trisomy 12, 6% del(11q), and 4% del(17p). Overall, we found somatic non-synonymous mutations in 23 of 49 MBLs (47%) at the initial time point including 22% of cases with more than one mutated driver gene. The average depth of coverage was 730x, thus allowing the identification of small subclonal mutations. Recurrent mutations were found in most of the drivers: CHD2, DDX3X (8% of cases), FBXW7, NOTCH1, SF3B1 (6% each), ATM, BCOR, BIRC3, BRAF, KRAS, MED12, MYD88 and ZMYM3 (4% each). Furthermore, ITPKB, POT1, SAMHD1 and XPO1 were mutated in only one case, whereas no mutations were found in HIST1H1E, RIPK1 and TP53. In 4 individuals, we found two mutations in the same gene (BRAF, DDX3X, KRAS and SAMHD1). Genes that are known to be associated with disease progression in CLL were either mutated with significantly lower incidence (NOTCH1, SF3B1) or not mutated (TP53). Mutations were detected on average 45 months (range 9-73) prior to progression to CLL Rai>0 indicating the early origin of most driver gene mutations in the MBL/CLL continuum. The presence of driver mutations in MBL was associated with shorter TTT (median TTT: present: 96 months vs. not present: not reached, HR: 5.52, 95% CI: 1.2-26.2, P =0.015). Next, we looked at clonal expansion of driver mutations over time (defined as >2-fold change in the allelic frequency of driver mutations between time points). Of 20 MBLs with mutations at baseline who had sequential samples available, 10 cases showed clonal expansion. Seven out of 10 MBLs who required therapy showed clonal expansion, which was detected on average 15 months (range 6-30 month) prior to treatment. Finally, the detection of clonal expansion was significantly associated with reduced TTT (median TTT: clonal expansion: 21 months vs. no clonal expansion: 84 months, HR: 7.79, 95% CI: 1.94-31.3, P <0.001). Conclusion: We have confirmed the existence of recurrent mutations in most CLL putative driver genes at the premalignant MBL stage many years before progression to CLL. Furthermore, the early identification of driver mutations and its clonal expansion predicts a shorter TTT. Of note, clonal evolution under selective pressure has recently been linked to the onset of CLL progression after therapy. In this study, we characterized the clonal dynamics in the pre-malignant stages of the disease and underlined its impact on clinical outcome. Despite the relatively small size of the cohort, these findings suggest that the sequential monitoring of MBL individuals with a simple and reliable technique, such as TDS, will be at least of prognostic use and thus its incorporation in the disease stratification and clinical management should be further tested. Disclosures Fonseca: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Applied Biosciences: Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Binding Site: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Onyx/Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding.

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High Clonal Complexity of Resistance Mechanisms Occurring at Progression after Single-Agent Targeted Therapy Strategies in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2020-137411 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 15-16

Author(s):

Ella R Thompson ◽

Tamia Nguyen ◽

Yamuna Kankanige ◽

Mary Ann Anderson ◽

Sasanka M. Handunnetti ◽

...

Keyword(s):

Single Cell ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Single Cell Analysis ◽

Clonal Evolution ◽

Single Agent ◽

Resistance Mechanisms ◽

Lymphocytic Leukemia ◽

Advisory Committees

Progression of chronic lymphocytic leukemia (CLL) on venetoclax (VEN) and BTK inhibitors (BTKi) is associated with acquired genomic variants in BCL2/MCL1/BCL2L1 and BTK/PLCG2, respectively, in some patients. We aimed to assess the clonal structure and evolution of resistance in patients (pts) with progressive disease treated with single agent VEN or BTKi (or both as sequential monotherapies) using next generation sequencing (NGS) and single cell sequencing. Seven pts with CLL and 1 with mantle cell lymphoma (MCL) with disease progression on VEN, ibrutinib (IBR) or zanubrutinib (ZANU) were identified from patients treated at our institutions. Pts were selected on the basis of multiple known resistance mechanisms from previous analysis of mutations (muts) and copy number changes detected using clinical bulk NGS targeting genes of interest including BCL2, MCL1, BCL2L1, BAX, BAK1, BTK, PLCG2, CXCR4, as well as TP53 and SF3B1. Of the 8 pts selected for single cell analysis, all had disease that was relapsed/refractory to chemotherapy prior to receiving either VEN (3 pts), BTKi (2 pts) or sequential VEN-BTKi (3 pts). 6,520-16,378 individual cells from 9 samples (8 pts) were analyzed (total 103,388 cells) using a custom panel targeting pt-specific muts on the Tapestri platform (Mission Bio). A summary of genomic abnormalities detected across the cohort is presented in Figure 1. We first evaluated the relationship between genomic resistance mechanisms within the context of single agent (VEN or BTKi) as well as sequential VEN-BTKi treatment. In CLL pts treated with a single agent, all BCL2 muts in VEN pts and BTK muts in IBR or ZANU pts were identified in different subclones consistent with an oligoclonal pattern of disease progression with independent clonal acquisition of resistance mechanisms. Both pts who received ZANU (either as a single agent or sequentially) harbored the BTK L528W mut (previously described as enriched in ZANU progressors; Handunnetti ASH 2019) in independent clones from BTK C481 muts. In pts who received sequential VEN-BTKi treatment, clones were observed that harbored established or novel dual genomic resistance mechanisms within the same cell (BTK mut/MCL1 amp in CLL, BTK/BAX muts in MCL). However, this was not observed in all clones or for all pts, suggesting the presence of further undetected resistance mechanisms (genetic or other). Given the unique ability of single cell sequencing to resolve mut context within a clonal hierarchy, we next assessed this phenomenon within our cohort utilizing other muts known to be present in these tumors. Analysis of TP53 muts exemplified the diversity of clonal patterns observed, with resistance muts being detected subclonally to parental TP53 muts in some pts and independently of TP53 muts in others. In addition, further evolution of resistant clones was observed through the development of TP53 muts within clones harboring acquired resistance muts, consistent with continued clonal evolution within the resistant disease compartment. In one pt, post-resistance clonal evolution was identified through the clonal acquisition of a CXCR4 mut within a BTK mutated population. Finally, to understand the contribution of BTK zygosity and gender to BTKi resistance (given its location on the X-chromosome), we performed single cell analysis on a disease specimen from a female pt with progressive MCL harboring multiple BTK mutations following treatment with sequential VEN-BTKi. Analysis revealed four clonally independent heterozygous BTK muts inferring the sufficiency of a single mutant allele to drive resistance in this context. Interestingly, this pt also harbored a BCL2 mut and a BAX mut, the latter co-occurring with a BTK mut (BCL2 not assessable). This pt therefore represents the first description of BCL2 or BAX muts occurring in a pt with progressive MCL on VEN and the first of a BTK L528W mut in MCL progressing on ZANU. In summary, these data highlight the significant clonal complexity of CLL progression on VEN and BTKi. Our data show that disease progression in this context is consistently oligoclonal with separate clones harboring distinct identifiable resistance mechanisms. These data have pt-specific implications for the potential utility of cycling back to previously efficacious targeted therapies as well as providing a strong rationale for the early use of disease-appropriate combination targeted therapies. Disclosures Anderson: Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Handunnetti:AbbVie: Other: Travel expenses; Roche: Honoraria; Gilead: Honoraria. Yeh:Novartis: Honoraria; Gilead: Research Funding. Tam:BeiGene: Honoraria; Janssen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Seymour:Morphosys: Consultancy, Honoraria; Mei Pharma: Consultancy, Honoraria; Gilead: Consultancy; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: 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; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nurix: Honoraria. 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. Blombery:Amgen: Consultancy; Novartis: Consultancy; Invivoscribe: Honoraria; Janssen: Honoraria.

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Analysis of Mechanisms Underlying Clonal Evolution of AML By a New Single-Cell Sequencing Platform

Blood ◽

10.1182/blood-2019-126444 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 542-542

Author(s):

Ryosaku Inagaki ◽

Masahiro Marshall Nakagawa ◽

Yasuhito Nannya ◽

Qi Xingxing ◽

Lanying Zhao ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Single Cell ◽

Research Funding ◽

Clonal Evolution ◽

Clonal Expansion ◽

Ras Pathway ◽

Sequencing Platform ◽

Single Cell Sequencing ◽

Equity Ownership

Background Leukemic cell populations are highly heterogeneous in terms of both gene mutations and gene expression, which is shaped by acquisition of multiple mutations and expansion of adapted clone. This evolutional process is clinically important because it is observed in the contexts of treatment resistance and relapse as well as leukemic transformation, and molecular mechanisms involved in clonal selection can be exploited as a therapeutic target. Nevertheless, direct analysis of such mechanisms in patients' cells is hampered by technical difficulties to characterize both clonal structure and gene expression at a single-cell resolution. On this issue, we have recently developed a new method which enables simultaneously detection of mutations and whole transcriptome information at single-cell level by extensively modifying an existing single cell RNA-seq (Nakagawa et al. ASH abstract 2018). The aim of this study is to understand heterogeneity of clones and to clarify mechanisms behind clonal expansion in AML by longitudinal analysis using our novel single-cell sequencing platform. Results In order to estimate clone frequencies and select samples to be analyzed by single-cell sequencing, we first sequenced bulk bone marrow cells from patients with AML. Of interest, we found that AML samples frequently harbored multiple clones having different Ras pathway mutations, most frequently involving NRAS, which exhibited dynamic change in their clone size during the course of AML. These are interesting targets of the analysis of mechanism of clonal evolution of AML. Thus, three patients having multiple (n=3-5) Ras pathway mutations were investigated by sequencing their bone marrow Lin-, CD34+ cells using the newly established single-cell method, which successfully separated distinct clones having distinct mutations, where all of detected Ras pathway mutations were present in independent clones as expected. In order to examine these independent clones with Ras pathway mutations might show equal or heterogenous cellular phenotypes, proliferation or differentiation statuses as determined from transcriptome data was analyzed for all detected NRAS mutated clones. Among the NRAS mutated clones, some showed significant increase in proliferation-associated gene expression signature (calculated as proliferation score) compared with NRAS wild type clones, and no NRAS mutated clones showed decrease of the score, which is consistent with pro-proliferative function of Ras pathway. Interestingly, such increase in proliferation showed considerable heterogeneity among clones, where some NRAS mutated clones showed greatly increased proliferation scores compared to other NRAS mutated clones. Differentiation statuses of NRAS clones also showed heterogeneity among clones. In order to examine whether this inter-clone proliferation difference correlates with clone dynamics, we then analyzed longitudinal bone marrow samples for a patient who showed different proliferation between clones. The NRAS mutated clone with highly increased proliferation compared with wild type clone (NRAS p.G12S) had undergone rapid expansion in 3 months (cell frequency 0.08 to 0.74) in spite of continuous azacitidine treatment, while the NRAS mutated clone with less increase in proliferation (NRAS p.G12D) had showed regression (cell frequency 0.72 to 0.14). To investigate the mechanism of this therapy-resistant clonal expansion, we compared transcriptome data of these clones. Unlike the regressed clone, the expanded clone uniquely exhibited increase in expression of genes in PI3K/AKT pathway and unfolded protein response (UPR) pathway, one of cellular stress response pathway. UPR is recently reported to responsible for the promoted survival and competitive advantage in mouse hematopoietic stem cells with Nras mutations (Liu et al. Nat. Cell Biol. 2019). Our data suggest that the enhanced UPR pathway contributes to clonal expansion also in human AML with Ras pathway mutations. Conclusions Using a newly developed single-cell sequencing platform, we have successfully characterized gene expression profiles associated with clonal evolution of AML with Ras pathway mutations. Simultaneous measurement of both mutations and transcriptomes at a single-cell level will help understand the mechanism of clonal evolution of AML. Disclosures Inagaki: Sumitomo Dainippon Pharma Co., Ltd.: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Yoda:Chordia Therapeutics Inc.: Research Funding. Ogawa:RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy.

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Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Blood ◽

10.1182/blood-2021-150191 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3-3

Author(s):

Alba Rodriguez-Meira ◽

Haseeb Rahman ◽

Ruggiero Norfo ◽

Wei Wen ◽

Agathe Chédeville ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Wild Type ◽

Advisory Committees ◽

Tp53 Mutations ◽

Molecular Landscape ◽

Mutant Cells

Abstract In myeloid malignancies, presence of 'multi-hit' TP53 mutations is associated with lack of response to conventional therapy and dismal outcomes, particularly when found in combination with a complex karyotype. Therefore, it is crucial to understand the biological basis of TP53-mutant driven clonal evolution, suppression of antecedent clones and eventual disease transformation to inform the development of more effective therapies. Myeloproliferative neoplasms (MPN) represent an ideal tractable disease model to study this process, as progression to secondary acute myeloid leukemia (sAML) frequently occurs through the acquisition of TP53 missense mutations. To characterize tumor phylogenies, cellular hierarchies and molecular features of TP53-driven transformation, we performed single-cell multi-omic TARGET-seq analysis (PMID: 33377019 & 30765193) of 22116 hematopoietic stem and progenitor cells (HSPCs) from 35 donors and 40 timepoints (controls, MPN in chronic phase, pre-AML and TP53-mutated sAML; Figure1a). TARGET-seq uniquely enables single-cell mutation analysis with allelic resolution with parallel transcriptomic and cell-surface proteomic readouts. We invariably identified convergent clonal evolution leading to complete loss of TP53 wild-type alleles upon transformation, including parallel evolution of separate TP53 "multi-hit" subclones in the same patient (n=4/14) and JAK2-negative progression (n=2/14). Complex clonal evolution driven by chromosomal abnormalities (CAs) was present in all patients and TP53 multi-hit HSPCs without CAs were rarely observed. Subclones with recurrent CA such as monosomy 7 showed upregulation of RAS-associated transcription and preferentially expanded in xenograft models. Together, these data indicate that TP53 missense mutation, loss of TP53 wild-type allele and cytogenetic evolution are collectively required for leukemic stem cell (LSC) expansion. Integrated transcriptomic analysis of sAML samples (Figure1b) revealed three major populations: (1) a TP53-mutant cluster (Figure1c) characterized by an erythroid signature (e.g. KLF1, GATA1, GYPA; an unexpected finding as no cases showed diagnostic features of erythroid leukemia), (2) an LSC TP53-mutant cluster (Figure1d) and (3) a TP53-WT preleukemic cluster (Figure1e). The LSC cluster showed dysregulation of key stem cell regulators, from which we derived a novel 48-gene LSC score with prognostic impact in an independent AML cohort (HR=3.13; Figure1f). Importantly, this score was predictive of outcome irrespective of TP53 status for both de novo and sAML, demonstrating its broader potential clinical utility. TARGET-seq analysis uniquely allowed us to characterize rare TP53-WT preleukemic cells (preLSCs), which were almost exclusively confined to the immunophenotypic lineage-CD34+CD38-CD90+CD45RA- HSC compartment. PreLSC from sAML samples presented increased stemness, increased quiescence, aberrant inflammatory signaling and differentiation defects (Figure1g) as compared to HSCs from normal or MPN donors, both at the transcriptional and functional levels through in vitro long-term and short-term cultures. This indicates cell-extrinsic suppression of residual TP53-WT hematopoiesis. Longitudinal analysis of TP53-heterozygous mutant HSPCs at different stages of disease evolution (Figure1a) revealed that aberrant inflammatory signalling (e.g. BST2, IFITM1, IFITM3) in the genetic ancestors of TP53 "multi-hit" LSCs, but not the presence of TP53-mutations alone, was predictive of subsequent transformation. In a mouse model system, TP53-mutant cells challenged with sustained inflammatory stimuli acquired a mean 3-fold competitive advantage in WT: TP53 R172H/+chimeras. This indicates that pro-inflammatory cues from the tumour microenvironment promote fitness advantage of TP53-mutant cells whilst supressing antecedent clones. In summary, we present a comprehensive single-cell multi-omic analysis of the genetic, cellular and molecular landscape of TP53-mediated transformation, providing unique insights into the evolution of chronic hematological malignancies towards an aggressive acute leukemia (Figure1h). Since TP53 is the most commonly mutated gene in human cancer, we anticipate these findings will be of broader relevance to many other cancer types. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment. Drummond: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison: Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mead: Abbvie: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau.

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Altered Immunophenotypes on Leukemic and/or Monocytic Cells from Acute Myeloid Leukemia Highly Predict for Nucleophosmin Gene Mutation

Blood ◽

10.1182/blood-2019-131733 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2687-2687

Author(s):

Sergio Matarraz ◽

Pilar Leoz ◽

Xavier Calvo ◽

Luis García Alonso ◽

Rosa Ayala Bueno ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Cell ◽

Monocytic Differentiation ◽

Advisory Committees ◽

Monocytic Cells ◽

Blast Cells ◽

Cell Expression ◽

Acute Myeloid

Introduction. Nucleophosmin gene mutation (NPM1mut) occurs in around 30% of acute myeloid leukemia (AML) patients, frequently linked with favourable prognosis in the absence of FLT3-ITDmut, which occurs in around 40% of NPM1mutAML. Therefore, more expeditious diagnostic approaches may contribute to early diagnosis and prognostic stratification of these patients. Herein, we investigated the association of immunophenotypic features of leukemic and monocytic cells with the presence of NPM1mut in AML. Methods. A total of 404 bone marrow (BM) samples from newly-diagnosed AML patients according to WHO 2017 classification were retrospectively studied by 8-color flow cytometry, including 225 AML with NPM1mut and 179 cases wild type gene (NPM1wt). Information on FLT3-ITD could be obtained from 397/404 cases. Thus, FLT3-ITDmut was present in 85/397 (21%), being concomitant with NPM1mutin 62/85 AML cases (73%). Logistic regression analysis was used to identify predictive phenotypes for the presence of NPM1mut. Results. Overall, blast cell with immunophenotypic features of monocytic differentiation (corresponding to FAB M4 and M5 AML subtypes) were observed among 135/225 (60%) and 69/179 (38.5%) AML patients with NPM1mutand NPM1wt, respectively (p<0.001). In the remaining AML cases without monocytic differentiation (FAB M0, M1 and M2; n=200), both immature/myeloid blasts and remaining monocytic cells were immunophenotypically characterized. Among AML with monocytic blast cell differentiation, altered monocytic phenotypes were more frequent among NPM1mut vs. NPM1wtcases (98% vs. 36%) (p<0.001). In detail, these phenotypic alterations consisted of asynchronous expression of CD300e prior CD14 (79% vs. 5% NPM1wtcases, respectively; p<0.001) and/or CD35 prior CD14 (84% vs. 30%), which were observed independently of FLT3-ITD. Of note, coexistence of the latter two asynchronous monocytic patterns (CD300e prior CD14 and CD35 prior CD14) on monocytic blast cells was specific for NPM1mut (64% vs. 0% NPM1wtpatients; p<0.001). Noteworthy, in AML cases without blast cell monocytic differentiation, remaining monocytic cells showed similar asynchronous phenotypic patterns, which were also more frequent among NPM1mut cases (78% vs. 23% of NPM1wtcases, respectively; p <0.001). Thus, remaining monocytic cells from NPM1mut AML cases also showed a higher frequency of asynchronous CD300e prior CD14 (64% vs. 8% of NPM1wt cases; p<0.001) and CD35 prior CD14 expression (58% vs. 20% of NPM1wt cases, respectively; p<0.001). In addition, aberrant CD9 blast cell expression was found in a significant proportion of all AML cases studied (124/222, 56%). However, altered CD9 was more frequent on (either monocytic or immature/myeloid) blast cells from AML cases with NPM1mut (76% vs. 46% NPM1wtcases; p<0.001), but not in AML with only-FLT3-ITDmut(39% vs. 46% of NPM1wtFLT3-ITDwt; p>0.05). In turn, aberrant CD25 expression on blast cells was otherwise linked to FLT3-ITDmut (61% vs. 20% of FLT3-ITDwtcases; p<0.001), being more frequent in AML with FLT3-ITDmutNPM1wt and double mutated AML cases vs. AML with FLT3-ITDwtNPM1mut and FLT3-ITDwtNPM1wt (79% and 47% vs. 20% and 20% of cases, respectively; p<0.001). In addition, overall, FLT3-ITDmut was associated with a significantly higher proportion of immature (i.e. CD34+) blasts, as compared to FLT3-ITDwt cases (median of 10% vs. 0.3% CD34+ blasts; p<0.001). In multivariate analysis, baseline detection of monocytic-lineage blast cells with asynchronous expression of CD300 prior CD14 -C-index= 0.954, odds ratio (OR), 78.8; 95% confidence interval (CI), 13.1-471; p<0.001- and CD35 prior CD14 (OR, 24.5; 95% CI, 4.8-123; p<0.001) and detection of these asynchronous patterns on remaining monocytic cells from AML without monocytic differentiation (C-index= 0.816, OR, 14.7; 95% CI, 6.4-34; p<0.001 and, OR, 2.5; 95% CI, 1.2-5.3; p=0.01, respectively) showed the highest predictive value for NPM1mut in AML. In turn, CD25 aberrant blast cell expression was the only immunophenotypic parameter with predictive value for FLT3-ITDmut (OR, 6.4; 95% CI, 2.9-14.5; p<0.001). Conclusions. Detection of specific aberrant immunophenotypic patterns among blast cells and/or remaining monocytic cells from AML patients is highly predictive for NPM1mut, which may contribute to early diagnosis and follow-up of these patients. Disclosures Díez-Campelo: Celgene Corporation: 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.

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Mutational Landscape of MDS Patients with HMA Failure Revealed By the Correlative Analysis from Inspire Trial

Blood ◽

10.1182/blood-2021-152048 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1517-1517

Author(s):

Koichi Takahashi ◽

Anna Jonasova ◽

Selina M. Luger ◽

Aref Al-Kali ◽

David Valcarcel ◽

...

Keyword(s):

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Gene Mutations ◽

Standard Of Care ◽

Driver Mutations ◽

Advisory Committees ◽

Ras Pathway ◽

Oncogenic Ras ◽

Survival Difference

Abstract Background: Hypomethylating agents (HMA) are well established standard of care for patients (pts) with higher-risk MDS (HR-MDS). However, approximately half of the pts do not respond to HMA therapy and most of the responders eventually lose response (HMA failure). There is no standard of care for pts after HMA failure and median overall survival (OS) post HMA failure is around 6 months (Jabbour et al. 2015). While the mutational landscapes and their role in prognosis are increasingly becoming apparent in pts with HR-MDS at diagnosis, mutational profiles at the time of HMA failure and their impact on clinical outcomes are not well understood. Here, using samples collected from a global Phase 3 trial randomizing HR-MDS pts post HMA failure to I.V. rigosertib (RGS) or physician's choice (PC) (INSPIRE: NCT02562443), we analyzed the landscape of driver mutations in HR-MDS after HMA failure and investigated the association with the clinical outcomes. Since RGS is a non-ATP-competitive small molecule RAS mimetic (Athuluri-Divakar 2016), the study also offered an opportunity to test the hypothesis whether HR-MDS pts with oncogenic RAS pathway mutations benefit from RGS. Methods: HR-MDS pts after HMA failure were randomized 2:1 to RGS or PC. All pts failed to respond to or progressed on prior HMA therapy. Bone marrow samples or peripheral blood samples were collected at the time of trial screening. Genomic DNA was sequenced by the targeted capture deep sequencing of 295 genes (median 500x). Results: 372 pts were enrolled in INSPIRE trial (248 to RGS and 124 to PC). The median age of the trial participants was 73 (range: 40-85). All pts were previously treated and with an HMA with the median duration of prior HMA therapy of 6.7 months. 64% and 28% of the pts were classified as IPSS-R very high risk or high risk, respectively, at the time of randomization. Among the 372 participants, DNA sequencing of pre-treatment samples was performed in 188 pts (51% of the participants, N = 122 in RGS arm, N = 66 in PC arm). The most frequently identified driver mutations involved ASXL1 (36%) followed by RUNX1 (24%), TET2 (23%), STAG2 (22%) and TP53 (21%). Mutations in splicing pathway genes were found in 36% of the pts. Oncogenic RAS pathway mutations were detected in 15% of the pts (NRAS = 3 %, KRAS = 2%, CBL = 4%, NF1 = 5%, PTPN11 = 3%, and 1% had multi-hit mutations). Compared to the previously untreated MDS pts (N = 446, Papaemmanuil et al. Blood 2013), mutations in ASXL1 (36% vs. 18%, P < 0.0001), BCOR (9% vs. 3%, P = 0.002), CEBPA (4% vs. 0.2%, P = 0.001), NF1 (5% vs. 0.9%, P = 0.003), RUNX1 (25% vs. 11%, P < 0.0001), STAG2 (22% vs. 5%, P < 0.0001), TP53 (21% vs. 6%, P < 0.0001), and IDH1/2 (13% vs. 7%, P =0.016) were significantly more enriched in pts with HMA failure, whereas mutations in SF3B1 (7% vs. 37%, P < 0.0001), TET2 (23% vs. 35%, P = 0.006), and splicing pathway genes (36% vs. 68%, P < 0.0001) were significantly less frequent in HMA failure pts. These results are consistent with the high-risk profiles of HMA failure pts. Frequency of oncogenic RAS pathway mutations were similar between HMA failure and previously untreated MDS pts (15% vs. 13%, P = 0.612). Correlation analysis between the types of HMA failure and gene mutations showed that TP53 mutations were significantly enriched in pts who relapsed after initial response to HMA (P = 0.001), whereas oncogenic RAS pathway mutations were significantly enriched in pts who progressed during the HMA therapy (P = 0.03). Overall, RGS did not significantly improve the overall survival (OS) of HMA failure pts compared to PC. Survival difference between RGS arm and PC arm was not observed in any subgroups stratified by the gene mutations. The only subgroup that showed improved OS with RGS compared to PC was pts with RAEB-t (median OS 7.5 vs. 3.9 months, P = 0.049). Of note, among pts with oncogenic RAS pathway mutations, no survival difference was observed between RGS and PC arms. Conclusion: High-risk gene mutations, such as TP53, ASXL1, RUNX1, and STAG2 (Ogawa. Blood 2019). were significantly enriched in MDS pts with HMA failure, suggesting their role in HMA resistance and disease progression. Identifying the mutations present de novo and with HMA failure offers the opportunity to determine prognosis based on these mutations as well as potential strategies to target these mutations with new medical entities. Disclosures Takahashi: GSK: Consultancy; Celgene/BMS: Consultancy; Novartis: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Luger: Syros: Honoraria; Agios: Honoraria; Daiichi Sankyo: Honoraria; Jazz Pharmaceuticals: Honoraria; Brystol Myers Squibb: Honoraria; Acceleron: Honoraria; Astellas: Honoraria; Pfizer: Honoraria; Onconova: Research Funding; Celgene: Research Funding; Biosight: Research Funding; Hoffman LaRoche: Research Funding; Kura: Research Funding. Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution. Diez-Campelo: BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Rigosertib for MDS patients after HMA failure

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