Clonal Evolution in a Murine CALM-AF10 Leukemia: Evidence of Functional Heterogeneity of Leukemia Stem Cells

Abstract Myeloid leukemia is caused by acquired genetic changes in haematopoietic stem cells. The combination of stepwise acquisition of genetic changes together with selection of the fittest clones results in great genetic and clonal heterogeneity. We used a CALM-AF10-driven retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) to study clonal hierarchy and clonal evolution starting with a primary leukaemia (Fig 1: Leu7) which developed after 131 days and had B220 marker expression on 4% of its cells. Limiting dilution assays (LDAs) showed that the leukemia stem cell (LSC) frequency of Leu7 was 1:2339 (95% confidence interval: 1:794-1:6885). Whole exome sequencing (WES) and analysis of the variant allele fraction of somatic mutations revealed that Leu7 was composed of a main clone (Fig 1: grey) with two subclones (blue and red). Half a million leukemic cells from Leu7 were transplanted into 4 sublethally irradiated recipients, which all developed secondary leukemias after a latency of 19 days (Leu7Sec1 to 4). All secondary leukemias showed similar B220 expression levels to Leu7, and all showed an expansion of the blue subclone. When again half a million cells each of one of the secondary leukemias (Leu7Sec2) were transplanted into 4 recipients, the expansion of the blue subclone continued, the red subclone vanished and, surprisingly, the proportion of B220 expressing cells increased to between 16 to 26%. LDAs showed that the LSC frequency of Leu7Sec2 had not changed. However, several of the leukemias from the LDAs had greatly varying latencies (27 to 193 days) and B220 marker expression (2 to 85%). Four of these tertiary LDA leukemias (Leu7Sec2Ter5 to 8), which each arose from a single LSC, were analysed more closely using WES. Leu7Sec2Ter5 showed a similar latency (27 days) and B220 expression levels like Leu7SecTer1 to 4 and also had the expansion of the blue subclone. Leu7Sec2Ter6 had a long latency of 69 days and a very low B220 expression. Leu7Sec2Ter6 was driven by a new, third subclone (pink), and both the blue and the red subclone disappeared. Very interestingly, Leu7Sec2Ter7 and Leu7Sec2Ter8 had a very long latency of 193 days, and showed an expansion of a subclone (green) of the red subclone. The B220 expression was high (37%) to very high (85%) in these two leukemias. Taken together, these observations paint an interesting picture with the blue subclone outcompeting the red subclone, as leukemias arising from the red subclone only appear after a long latency and in leukemias initiated by a single LSC, when there is no blue subclone LSC present. As the four leukemias (Leu7Sec2Ter5 to 8), which each were derived from a single LSC, showed striking differences in latency and surface marker expression, it can be concluded that this variation in phenotype is an intrinsic property of an individual LSCs most likely a consequence of the distinct combination of somatic mutations present in the individual LSCs. These observations also suggest that distinct LSCs with different properties might be present in a single human leukemia. Figure 1 Figure 1. Disclosures Browett: Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

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Impact of Eltrombopag on Expansion of Clones with Somatic Mutations in Refractory Aplastic Anemia

Blood ◽

10.1182/blood.v126.23.300.300 ◽

2015 ◽

Vol 126 (23) ◽

pp. 300-300 ◽

Cited By ~ 1

Author(s):

Bhumika J. Patel ◽

Bartlomiej Przychodzen ◽

Michael J. Clemente ◽

Cassandra M. Hirsch ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Aplastic Anemia ◽

Board Of Directors ◽

Deep Sequencing ◽

Somatic Mutations ◽

Clonal Evolution ◽

Clonal Expansion ◽

Quantitative Detection ◽

Sequencing Analysis ◽

Advisory Committees ◽

Before And After

Abstract Despite documented success of immunosuppressive therapy (IST) in the treatment of aplastic anemia (AA), a significant minority of patients remain refractory, most responses are incomplete, and allogeneic stem cell transplantation is not available for older patients or those with significant comorbidities. Until introduction of the cMpl agonist eltrombopag, anabolic steroids were the most commonly used salvage drugs. At least theoretically, engaging growth factor receptors with eltrombopag has the potential to promote the evolution or expansion of mutant clones and thereby increase the rate of progression to secondary MDS, a feared complication of AA occurring in 10-20% of patients. Recently we and others reported detection of clonogenic somatic mutations typical of MDS in patients with AA and PNH. Subsequent study demonstrated that mutations characteristic of sMDS can be found in some patients at presentation of AA and may constitute risk for subsequent progression to MDS. As the risk of MDS evolution was a prominent concern when filgrastim was more widely used in management of AA, now similar questions have been raised regarding use of eltrombopag, be it as salvage therapy or to complement IST. Recently, one of our primary refractory patients receiving eltrombopag progressed to AML. This clinical observation led to investigation of the impact of eltrombopag on evolution and clonal expansion using deep sequencing of a cohort of patients with AA. DNA from bone marrow cells was sequenced before and after initiation of eltrombopag to evaluate clonal expansion or evolution using a targeted multi-amplicon deep sequencing panel of the top 60 most commonly mutated genes in MDS. Among 208 AA patients treated at Cleveland Clinic, we identified 13 patients (median age 68 yrs.) who were treated with eltrombopag for IST-refractory AA; median duration of treatment was 85 wks. The overall response rate, defined as sustained improvement in blood counts and transfusion independence after 12 weeks of therapy, was 46% (6/13), while 38% (5/13) of patients showed stable disease with intermittent transfusions (one of whom underwent HSCT). Among the two non-responders, one patient developed a PNH clone and another progressed to AML (see below). Expansion of PNH granulocytes after eltrombopag treatment was observed in 2 patients. Two patients had chromosomal abnormalities at initial diagnosis, one with t (10; 18) in 2 metaphases, and one with an extranumeral Y chromosome. Use of next generation sequencing (NGS) allows for the quantitative detection of clonal events. We hypothesized that serial analysis by NGS before and after eltrombopag therapy may provide clues as to potential effects of this drug on clonal evolution. Sequencing analysis before eltrombopag treatment revealed the presence of a sole clonal mutational event in 3/13 cases, including CEBPA, EZH2, and BCOR. In the patient with a CEBPA mutation, the mutation persisted during treatment with minimal clonal expansion evidenced by a change in VAF from 53% to 65%. In the second patient, NGS results revealed the initial presence of an EZH2 mutation. A post eltrombopag sample clearly identified acquisition of additional clonal events in genes highly associated with advanced disease and clonal evolution (RUNX1 and U2AF1), as well as slight expansion of a persistent EZH2 clone from 2 to 8%. The third patient harbored a BCOR mutation which expanded markedly, increasing from 8% to 21%, and was accompanied by a hematological response. Sequencing results after eltrombopag treatment revealed the acquisition of new somatic mutations in 5/13 (38%) cases: 2 new CEBPA mutations, 1 new BCOR mutation, and, as discussed, one case with an initial EZH2 mutation in which RUNX1 and U2AF1 mutations were later discovered. In the 5th patient, evolution to AML was observed and accompanied by a large DNMT3A and U2AF1 clone that was absent on initial evaluation. In conclusion, we did observe occasional expansion of clones with potentially leukemogenic mutations during treatment with eltrombopag. At our institution a case control study of patients with refractory aplastic anemia without treatment with eltrombopag is ongoing; ideally a prospective trial would be needed to confirm results. Our results suggest that the initial detection of certain somatic mutations (CBL, SETBP1 and RUNX1) associated with post-AA MDS may contraindicate use of eltrombopag in AA. Disclosures Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

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Clonal Dynamics of Refractory Aplastic Anemia in Patients Treated with Eltrombopag

Blood ◽

10.1182/blood.v128.22.3892.3892 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3892-3892 ◽

Cited By ~ 2

Author(s):

Bhumika Patel ◽

Bartlomiej P Przychodzen ◽

Michael J. Clemente ◽

Cassandra M. Hirsch ◽

Caner Saygin ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutations ◽

Hematopoietic Cell Transplantation ◽

Chromosomal Abnormalities ◽

Clonal Evolution ◽

Cleveland Clinic ◽

Clonal Expansion ◽

Case Control ◽

Control Cohort ◽

Advisory Committees

Abstract Despite documented success of immunosuppressive therapy (IST) in the treatment of AA, a minority of patients remain refractory, most responses are incomplete, and use of hematopoietic cell transplantation (HCT) is limited in older patients or those with significant comorbidities. While the introduction of the cMpl agonist eltrombopag (EPG) as salvage therapy or in conjunction with IST has revolutionized treatment for refractory AA. It may be effective in improving primary response rates to IST, engaging growth factor receptors with agonistic therapeutics (such as EPG) and also has the potential to promote evolution/expansion of mutant clones, thereby increasing the rate of progression to secondary myelodysplastic syndromes (MDS), a serious complication of AA occurring in 10-20% of patients. Clonogenic somatic mutations typical of MDS in patients with AA and PNH may increase the risk of progression to MDS. DNA from marrow samples of primary refractory AA patients was subjected to analysis before and after initiation of EPG to evaluate clonal expansion or evolution using a targeted multi-amplicon deep NGS panel of all ORFs of the top 60 most commonly mutated genes in MDS. In addition to the EPG treatment group, a case control cohort matched for age and duration from AA diagnosis to last clinical follow up (who did not receive EPG), was studied. Among 210 AA patients treated at Cleveland Clinic, we identified 26 who were treated with EPG for IST-refractory AA; median duration of treatment was 56 wks. The overall response rate after 12 weeks of therapy was 58% (15/26), while 31% of patients (8/26) showed stable disease with intermittent transfusions (one of whom underwent HCT). In 3 non-responders, one developed PNH, one had refractory AA/PNH, and one progressed to AML (see below). Expansion of PNH granulocytes after EPG treatment was observed in 23% of patients (6/26). In addition, 15% (4/26) had atypical subclonal chromosomal abnormalities. Prior to EPG, at least a single somatic event was found in 31% of patients (8/26), with 2 patients harboring 2 mutations. Events included CEBPA, EZH2, BCOR/BCORL1, ASXL1, U2AF1/2, TET2, and DNMT3A mutations. Following EPG therapy, acquisition of new somatic mutations was observed in 23% of cases, including RUNX1, U2AF1, BCOR, RIT1, and CEBPA. In cases with pre-existing clones, 6 clones expanded (e.g., BCOR or ASXL1 from VAF of 8 to 21% and 9 to 29%, respectively) despite clinical hematologic response, while in 2 cases clones disappeared (e.g., U2AF2 and BCORL1). In 54% of cases (14/26), we found detectable levels of a PNH clone at the time of diagnosis. Six of those cases had PNH clonal expansion post-EPG treatment, of which two developed clinically significant PNH clonal burden requiring eculizumab therapy. In the case-control cohort, 26 AA patients who received IST but were not treated with EPG, were followed for comparable time periods, and no evidence of progression to MDS was recorded. One patient was noted to have trisomy 15 on cytogenetics at diagnosis. "MDS type" molecular mutations were present in 10 patients similar to EPG cohort. Among these patients, 3 had persistent clones of U2AF1, DNMT3A, and STAT3 over one year without acquisition of any new molecular mutations. . PNH granulocytes expanded in 50% of AA cases, decreased in 30% and stayed stable in 20%. Thus, we did not observe any difference in expansion of PNH clones between those treated and untreated with EPG (p=0.73). Unlike for PNH clones, accounting for both new evolution and expansion of preexisting molecular mutations, the frequency of these clonal events was significantly higher in the EPG treated group (p=0.009). In conclusion, we observed occasional expansion of clones with potentially leukemogenic mutations during treatment with EPG in pts with AA. While higher rates of MDS evolution were not observed in this cohort of EPG treated patients, we found that serial evaluation of somatic mutations can inform clonal evolution and can potentially be used as abiomarker for evaluation of risk for post-AA MDS. Continued use of EPG in such patients should be judicious. Disclosures Carraway: Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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Analysis of Clonal Hierarchy Shows That Other Ancestral Events May Precede Evolution of Del(5q) in Myeloid Neoplasms

Blood ◽

10.1182/blood.v124.21.4605.4605 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4605-4605

Author(s):

Naoko Hosono ◽

Hideki Makishima ◽

Bartlomiej P Przychodzen ◽

Thomas LaFramboise ◽

Chantana Polprasert ◽

...

Keyword(s):

Board Of Directors ◽

Tp53 Mutation ◽

Somatic Mutations ◽

Clonal Evolution ◽

Common Mutation ◽

Advisory Committees ◽

Tp53 Mutations ◽

Clonal Architecture ◽

Myeloid Neoplasms ◽

Associated Lesions

Abstract The molecular pathogenesis of myeloid neoplasms characterized by 5q deletion (del(5q)) has not been completely elucidated. While some pathomorphologic features including e.g., megakaryocytic and erythroid dysplasia, have been associated with specific genes within minimal common deleted regions (CDR), genes responsible for clonal advantage and expansion have not been identified. It is not clear if haploinsufficiency of one or multiple genes within del(5q) is responsible for clonal evolution or whether mutations in those genes or other genes located in other genomic areas are present. Moreover, with the recognition of intra-tumor diversity and hierarchical clonal architecture, it may be possible to establish whether del(5q) or other lesions, including common somatic mutations, constitute the ancestral event in the pathophysiologic cascade. We performed a comprehensive mutational screen in 124 patients with del(5q), including 59 patients studied by whole exome sequencing (WES) and 65 by targeted deep NGS of genes within the deleted area and the other most commonly mutated genes as previously determined in WES cohorts. To identify pathogenic genes, those most consistently found to be haploinsufficient in del(5q) were matched for the presence of mutations in diploid cases. For the purpose of this study haploinsufficiency was quantitated based on the number of cases with del(5q) showing <60% expression of the corresponding genes. E.g.,HDAC3 in 81%, PPP2CA in 62% and RPS14 in 14% of cases with del(5q). For all somatic mutations, we also describe the clonal composition based on deep sequencing in serial samples and analyses of variant allelic frequency. Finally, we compare the clonal size for individual mutations with that of del(5q). The latter was accomplished by calculation of clonal size based on allelic imbalance for informative SNPs present within deleted regions in heterozygous configurations in germ line samples. The average deviation from the ideal 50/50 distribution in tumor samples allowed for precise calculation of the proportion of cells in the sample affected by the deletion. Using this approach, there was a good correlation to the size of del(5q) clone by FISH (r=.94) Our results demonstrate that 10/14 genes were haploinsufficient within the CDR, but only 2 hemizygous somatic mutations were identified. However, 12 mutations in 7 genes (MATR3, SH3TC2, CSNK1A1, PDGFRB, CD74, FAT2 and G3BP1) were present with the area corresponding to the CDR in diploid cases. TP53 mutations were more commonly associated with del(5q) (73%, vs. 27% in diploid 5, p<.001) and were particularly frequent in patients affected with 2 commonly retained regions (CRR1;5q11.1-5q14.2 and CRR2; 5q34-qter), where they were found in 81% of cases (30/37) vs. 19% (7/30) among CDR deletions (p<.001). In lower-risk MDS, mutations were detected in 11% of deletion cases, whereas they were only found in 5% of diploid chr5 (p<.0001). In higher-risk MDS, TP53 mutation were found in 42% of del(5q) vs. 4% of diploid chr5 (p<.0001). Similarly, 45% patients with concomitant -7/del(7q) and del(5q) had TP53 mutations. The most common mutation associated with del(5q) was TP53, while mutations of FLT3, NRAS or TET2 were significantly mutually exclusive (p=0.03, 0.04 and 0.03; respectively). Next we determined the earliest somatic event by comparing of clonal size of the associated lesions. Del(5q) was present in 17-98% of tumor cells. We identified three theoretical possibilities as to the clonal architecture of del(5q) myeloid neoplasms: i) Tumors in which driver somatic mutations precede del(5q) (35%), ii) those in which del(5q) appears to precede any other somatic mutation (6%) and iii) the succession cannot be determined because of very expanded clones of similar size (“clonal saturation”) i.e., these cases were not informative. For cases in which del(5q) was a secondary lesion, TP53 was the ancestral event 64% of the time, and DNMT3A 27% of the time. The TP53 mutation was detected as a secondary event in 1 of 2 samples in which del(5q) was found to be ancestral. In sum, our results suggest that del(5q) is not universally an ancestral event. The TP53 mutation is the most common mutation in del(5q) and may also serve as ancestral event. While UPD17p and hemizygocity for TP53 can be found in 33% of TP53 mutant cases, most of the detected TP53 mutations were likely to heterozygous, and therefore the clonal size was not overestimated. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

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A Network Of Epigenetic Regulators Guide Developmental Hematopoiesis In Vivo

Blood ◽

10.1182/blood.v122.21.1174.1174 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1174-1174

Author(s):

Katie L Kathrein ◽

Hsuan-Ting Huang ◽

Abby Barton ◽

Zachary Gitlin ◽

Yue-Hua Huang ◽

...

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Large Scale ◽

Chromatin Domain ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marker Expression

Abstract Long-term hematopoietic stem cells (HSCs) are capable of self-renewal and differentiation into all mature hematopoietic lineages. This process is regulated by transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, we have undertaken the first large-scale in vivo reverse genetic screen targeting chromatin factors. We have designed and injected antisense morpholinos to knockdown expression of 488 zebrafish orthologs of conserved human chromatin factors. The resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC marker genes, c-myb and runx1, which are expressed in the developing blood stem cells. Morphants were categorized into five groups based on HSC marker expression, ranging from no change to mild, intermediate, or strong reduction in expression or an increase in expression. 29 morpholinos caused a complete or near complete knockdown of HSC marker expression, while 4 were found to increase HSC marker expression. As ubiquitous knockdown of chromatin factors could interfere with vascular development and the establishment of proper arterial identity, a crucial upstream event for HSC formation, we subsequently analyzed morphants with the most robust HSC phenotypes using two vascular markers: kdr for overall vasculogenesis and ephrinb2a for arterial formation. We found that of the 29 morpholinos that caused reduced marker expression, only 9 showed reduced overall vascular or arterial marker staining, suggesting that the majority of morphants with HSC phenotypes are specific to HSC formation. For the 4 morphants with increased HSC marker expression, vasculature appeared normal. These factors likely function as potent negative regulators of HSC development. Several genes known to be essential for HSC self-renewal and maintenance were identified in the screen. For example, knockdown of Mll or Dot1, which are also present in leukemia fusion proteins, fail to specify HSCs, as indicated by a nearly complete reduction in expression of the HSC markers in embryos tested. Of the remaining hits, many represent factors with no previous function ascribed in hematopoiesis. By incorporating protein interaction data, we have defined a handful of complexes necessary for HSC specification, including the SWI/SNF, ISWI, SET1/MLL, CBP/P300/HBO1/NuA4, HDAC/NuRD, and Polycomb complexes. As chromatin factors associated with the same complex likely share target binding sites, we analyzed 34 published ChIP-seq datasets in K562 erythroleukemia cells of chromatin factors tested in the screen, including hits from our screen: SIN3A, CHD4, HDAC1, TAF1, and JARID1C associated with the HDAC/NuRD complex and RNF2, SUZ12, CBX2, and CBX8 from the Polycomb complexes. We ranked triplet combinations of these factors together with all other groups of three factors based on the percent overlap of target genes. The HDAC/NuRD and PRC1/2 complex combinations predicted from our screen fell within the top 20% of all possible combinations of 3 factors, suggesting that our screen has identified chromatin factors that function in distinct complexes to regulate hematopoietic development. Our work has been compiled into a web-based database that will be made publicly available upon publication. Within this database, users can search by gene names and aliases, chromatin domain names and human or zebrafish genes. All experimental data, including experimental design, materials, protocols, images, and all further analyses of the 33 most robust morphants is included. Our large-scale genetic analysis of chromatin factors involved in HSC development provides a comprehensive view of the programs involved in epigenetic regulation of the blood program, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders and leukemia. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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Characterization of an Acute Myeloid Leukemia Murine Model Driven By MLL/AF9: Effect of Retroviral Insertion Sites and Somatic Mutations on Gene Expression

Blood ◽

10.1182/blood-2021-154012 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4329-4329

Author(s):

Hui Mei Lee ◽

Niloofar Zandvakili ◽

Rhea Desai ◽

Peter J. Browett ◽

Purvi M Kakadia ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Board Of Directors ◽

Murine Model ◽

Somatic Mutations ◽

Fusion Gene ◽

Advisory Committees ◽

Leukemia Development ◽

Insertion Sites ◽

Secondary Leukemias

Abstract The MLL/AF9 fusion is found in approximately 30% of MLL-rearranged leukemias and has an intermediate prognosis. Genomically well-characterized murine leukemia models enable us to understand leukemogenesis. We generated a retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) using the MLL/AF9 fusion gene. Fifteen of 20 mice transplanted with syngeneic bone marrow transduced with a MLL/AF9 carrying retrovirus developed leukemia after a median latency of 149 days. Half a million leukemic bone marrow (LBM) cells from two of these primary leukemias, MA03-P and MA86-P, were transplanted into irradiated recipient mice to establish secondary leukemias, MA03-S (n=3) and MA86-S (n=4). Half a million LBM cells from these secondary leukemias were further transplanted into irradiated recipient mice to generate tertiary leukemias, MA03-T (n=3) and MA86-T (n=4). The latency of the leukemias shortened from 141 days in MA03-P to 18 and 22 days in MA03-S and MA03-T, respectively. Similarly, MA86-P had a latency of 98 days, and the latency was reduced to about 28 days in MA986-S and MA986-T. We used retroviral insertion sites (RISs) to track leukemia clones during serial transplantation. We identified 5 RISs in MA03-P. One RIS, RIS#1-03 at chromosome 7:4602500-4609499 accounted for 52.5% of the total RIS-related reads in MA03-P, while the other four RISs were each represented by fewer than 5% of the reads. Only RIS#1-03 was detected in all of the MA03 secondary and tertiary leukemias , indicating that the cells with RIS#1-03 were the dominant clone in MA03 leukemias. Two RISs were detected in MA86-P: RIS#1-86 at chromosome 19:41338500-41341999 and RIS#2-86 at chromosome 10:127106000-127109499 at 46.7% and 2.5%, respectively . RIS#1-986 was contained in the dominant clone as only this RIS was subsequently detected in the secondary and tertiary MA86 leukemias. The relatively long latency to leukemia development in our MLL/AF9 model was most likely due to the requirement of cooperating somatic mutations. We performed whole exome sequencing on DNA from LBM (n=15) and DNA from their corresponding germline (n=2). An average of 4.5 of single nucleotide variants (SNVs) and 11.4 indels affecting protein coding sequences were found in the MA03 family of leukemias (n=7) which, among others, mutated genes involved in tyrosine kinase pathways such as Epha5 and Pik3r1. We identified an average of 14.8 (SNVs) and 0.5 indels per exome in the MA86 leukemias (n=8). Transcription regulator (Brd1) and tumor suppressor genes (Stk11 and Trp53) were affected by somatic changes in the MA86 family. RNA sequencing was performed on LBM (n=15) and healthy bone marrow (HBM) (n=8). Principal component analysis (PCA) on the expression profiles showed that LBM samples clustered together. Differential gene expression analysis identified genes such as Six1, Eya1 and Bcor which had been reported in previous studies to be essential for leukemogenesis in MLL/AF9 murine model. We also observed downregulation of genes such as Gata2, Btg1, Ifitm1, which had been implicated in other types of leukemias. We next investigated the effect of the RISs and somatic mutations on gene expression. RIS#1-903 was in intron 1 of Ppp6r1. A reduction in fragments per kilobase of transcript per Million mapped reads (FPKM) of Ppp6r1 was observed in MA03 family leukemias compared to leukemias of the MA86 family which did not have RIS#1-03 and showed no difference to HBM samples (MA03: 87.71±1.5; MA86: 132.1±5.1; HBM: 77.56±1.7, p< 0.001). We then determined the expression of Tm9sf3 as it is located 600bp away from RIS#1-986. The FPKM of Tm9sf3 was significantly higher in LBM (both of MA903 and MA986 leukemias) than in HBM (LBM: 146.0±12.7; HBM: 64.66±2.8, p<0.001). In MA86 leukemias which all have RIS#1-86, the FPKM of Tm9sf was two fold higher than in MA03 leukemias without RIS#1-86 (MA86: 189.3±4.4; MA03:97.59±1.7, p < 0.001). In contrast, none of the somatic mutations had a significant effect on the expression of any of the mutated genes. In conclusion, we have established a MBMTLM driven by the MLL/AF9 fusion gene. This well-characterized model provides insights to further understand leukemia development and drug testing. Moreover, we demonstrated that RISs can have an impact on gene expression. Future work on whether Ppp6r1 and Tm9sf3 identified by our RIS analysis are drivers in MLL/AF9 leukemias is warranted. Disclosures Browett: MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

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Haploinsufficiency and Deletions of G3BP1 on Chromosome 5q Result in Induction of TP53

Blood ◽

10.1182/blood.v124.21.784.784 ◽

2014 ◽

Vol 124 (21) ◽

pp. 784-784

Author(s):

Naoko Hosono ◽

Mahfouz Reda ◽

Bartlomiej P Przychodzen ◽

Chantana Polprasert ◽

Latifa Zekri ◽

...

Keyword(s):

Growth Inhibition ◽

Board Of Directors ◽

Cell Lines ◽

Lower Risk ◽

Chromosomal Instability ◽

Somatic Mutations ◽

Clonal Evolution ◽

Loss Of Function ◽

Advisory Committees ◽

Low Expression

Abstract Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. The extent of individual defects vary, which may account for observed clinical diversity. Del(5q) pathogenesis has been related to haploinsufficiency of genes contained in the common deleted regions (CDR), including RPS14, miR-145/146a and SPARC. Driver mutations or pathogenic microdeletions were not identified for these genes, suggesting that multiple genes must function in combination to promote clonal evolution and phenotypic heterogeneity. Hence, we performed a comprehensive analysis of somatic mutations in genes located on chromosome 5 (chr5), both in patients with diploid 5q and in those with del(5q), to clarify the role of germline and somatic mutations in disease pathogenesis. In parallel, expression analysis was performed to correlate haploinsufficiency with the frequency of mutational events, in particular for diploid 5q cases. Applying SNP-array karyotyping to samples from 146 patients with del(5q), the lesion was identified in 5q31.1q33.1. Two retained regions (CRRs) were also observed in q11.1q14.2 (CRR1) and q34qter (CRR2). Lower-risk MDS is frequently affected by CDR, while in higher-risk MDS and secondary AML CRR1/2 are commonly co-involved. Using whole exome sequencing, we identified 11 hemizygous mutations located within the deleted area in del(5q) (N=59), while in cases diploid for 5q (N=330), 243 heterozygous mutations were found. One of the mutations discovered on chr5q afflicted a gene G3BP1 (5q33.1), located within the CDR and present in 2 patients. Both were missense mutations (one heterozygous and the other homo/hemizygous). A mutant case showed good responses to lenalidomide even though diploid 5. In addition, other somatic mutations of driver genes including TET2, CUX1 and EZH2 were concomitantly observed. Whole transcriptome sequencing demonstrated hemizygous loss of G3BP1 resulting in haploinsufficiency. G3BP1 was haploinsufficient in 48% of RAEB as well as low-risk MDS cases with del(5q). Overall, defective G3BP1 is associated with shorter overall survival (P<.001) in AML, consistent with the reports that del(5q) is a worse prognostic factor in myeloid neoplasms with aggressive phenotype. G3BP1 is a nuclear RNA-binding protein and is ubiquitously expressed in bone marrow, CD34+ progenitors and leukemic cell lines. Furthermore, G3BP1 binds to TP53 and its expression leads to the redistribution of TP53 from the nucleus to the cytoplasm. Similar to RPS14, haploinsufficient of G3BP1 resulted in TP53 up-modulation. Moreover, low expression of G3BP1 in diploid 5q cases was indeed associated with higher TP53 expression. Next, we generated haploinsufficient G3BP1 cell lines using shRNA transduction. Decreased expression of G3BP1 led to growth inhibition and impaired colony formation by transduced cells lines and hematopoietic progenitor cells, respectively. Knockdown of G3BP1 in K562 cell line increased TP53 in the nucleus, and when treated with CPT11, DNA-damaged induced G1-arrest was more prominent in knockdown cells. Furthermore, after knockdown of G3BP1 in TP53-null HL60 cells, we observed increased aneuploidy, suggesting that the loss of function of G3BP1 and TP53 may result in chromosomal instability. Most significantly, G3bp1-/+ mice showed lower blood counts and defective, dysplastic hematopoiesis, similar to lower-risk MDS. As previously described, TP53 defects are associated with advanced disease but recently it became apparent that TP53 may be one of the most common somatic lesions found in the context of del(5q). We stipulate that loss of TP53 function might overcome TP53 tumor suppressor effects and induce leukemic evolution in the defective G3BP1 status. In our cohort, TP53 mutations were more frequently present in high-risk phenotype with G3BP1 haploinsufficient expression. In conclusion, novel somatic mutations of G3BP1 suggest that it could be a candidate gene associated with the clonal evolution of del(5q). Loss of function or low expression of G3BP1 has been shown to up-modulate TP53 and result in dysplasia and growth inhibition, hallmarks of early stages of MDS. Additional events constitute loss of function of TP53, resulting in chromosomal instability, which is associated with leukemogenesis. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

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High Burden of Clonal Hematopoiesis in First Responders Exposed to the World Trade Center Disaster

Blood ◽

10.1182/blood-2019-129276 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3720-3720

Author(s):

Sakshi Jasra ◽

Orsi Giricz ◽

Rachel Zeig-Owens ◽

David Goldfarb ◽

Angelica Barreto-Galvez ◽

...

Keyword(s):

Stem Cells ◽

Dna Replication ◽

Board Of Directors ◽

Research Funding ◽

Somatic Mutations ◽

First Responders ◽

Fragile Sites ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis

Introduction The World Trade Center (WTC) disaster exposed first responders to high levels of aerosolized carcinogens (Lioy et. al. Env. Health Perspect 2002). Clonal hematopoiesis is associated with exposure to smoking and genotoxic stimuli (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM 2015). We sought to determine its incidence in WTC-exposed first responders. We also assessed the effect of WTC particulate matter (WTC-PM) on genome integrity in vitro, and in murine studies. Methods Deep targeted sequencing was performed on blood collected from 481 first responders (429 WTC-exposed firefighters, 52 WTC-exposed emergency medical service workers) and 52 non-exposed first responders. Samples were analyzed for 237 genes mutated in hematologic malignancies and interpreted using reference databases. Non synonymous somatic mutations were annotated and analyzed. Results In the WTC-exposed cohort, 57 individuals with 66 somatic mutations of expected pathogenic potential were identified (overall prevalence 11.9%). In the non-exposed cohort, only one pathogenic mutation was found in the IDH2 gene (overall prevalence 1.9%). There was a strong association between increasing age and prevalence of mutations in the WTC-exposed cohort (Fig 1A). DNMT3A (16/66), TET2 (7/66), SF3B1 and SRSF2 (3/66 each) were the most common genes identified in the WTC-exposed cohort (Fig 1B). Median VAF was 12% and missense mutations were most frequent alteration. Aging, smoking, DNA repair and alkylating agent exposure related mutational signatures were observed with a cytosine to thymine (C→T) transition being most common. Next, we assessed the effect of WTC-PM on genome integrity and replication in vitro. WTC-PM that was collected in the first three days after 9/11 was used in concentrations mimicking exposure levels. Lymphocytes exposed to WTC-PM demonstrated a significant increase in phosphorylated H2AX foci accumulation, suggesting a DNA damage response (Fig 2). Since common fragile sites (CFSs) detect basal levels of stress in the cell, and activate DNA damage response (DDR), we profiled DNA replication dynamics at CFS-FRA16D at very high resolution using the single molecule analysis of replicated DNA (SMARD) assay. Treatment with WTC-PM significantly altered replication at two common fragile sites (regions 1 and 2 of FRA16D, Fig 3A) with replication pausing being observed at multiple sites (Fig 3B-I, white rectangles). Striking increase in replication initiation was seen, characterized as dormant origins activated to rescue replication pausing (Fig 3E, J). These alterations were accompanied by a corresponding increase in replication speed, conditions that lead to DNA replication errors and mutagenesis (Fig 3F, K). Next, we treated mice with WTC-PM via the oropharyngeal route to mimic first responder exposures, and then harvested and analyzed their bone marrow compartments. Significant expansion of hematopoietic stem cells (Kit+, Sca1+, Lineage-ve, KSL) was seen in WTC-PM treated mice (Fig 4A,B). Whole genome sequencing of sorted stem cells showed a significant increase in non-synonymous SNPs, deletions and indels in the WTC-PM treated samples when compared to control (Fig 4C-E). These genomic alterations were found to occur at low VAF throughout the whole genome, demonstrating widespread genotoxic effects of WTC-PM on hematopoietic stem cells in vivo (Fig 4F). Discussion We report a high burden of mutations in 11.9% (57/481) WTC-exposed first responders compared to the non-exposed cohort (1.9%, 1/52). The frequency of the somatic mutations was many fold higher than in previous studies (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM, 2015). In the 50-59 year age group, 10% of WTC-exposed individuals carried somatic mutations, compared to the frequency of 2.5% reported by Jaiswal et. al. for the same age group. Despite deeper sequencing performed in our study, the median VAF in our study was 12%, indicating that the difference in technique did not bias our study towards increased detection of small, subclinical clones when compared to previous studies. Furthermore, we demonstrate that WTC-PM can perturb DNA replication and increased genomic instability in vivo, potentially leading to higher burden of clonal hematopoiesis in WTC-exposed first responders. These results demonstrate adverse environmental exposures can be associated with a high rate of clonal hematopoiesis. Disclosures Landgren: Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Fletcher:Genoptix/Neogenomics: Employment. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Steidl:GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Will:Novartis Pharmaceuticals: Research Funding. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

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Association of MHC Class I Chain-Related Gene a (MICA) Polymorphisms with Allogeneic Hematopoietic Cell Transplantation Outcomes in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-112745 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2075-2075

Author(s):

Sagar S. Patel ◽

Betty K. Hamilton ◽

Lisa Rybicki ◽

Dawn Thomas ◽

Arden Emrick ◽

...

Keyword(s):

Board Of Directors ◽

Binding Affinity ◽

Myeloid Leukemia ◽

Research Funding ◽

Somatic Mutations ◽

Hematopoietic Cell Transplantation ◽

Advisory Committees ◽

Post Transplant ◽

Transplant Outcomes ◽

Nkg2d Receptor

Abstract Background MHC class I chain-related gene A (MICA) is a polymorphic ligand of the natural killer (NKG2D) receptor on immune effector cells. The activating NKG2D receptor controls immune responses by regulating NK cells, NKT cells and γδ-T cells. Dimorphisms at sequence position 129 of the MICA gene confers varying levels of binding affinity to NKG2D receptor. MICA previously has been associated with post-allogeneic hematopoietic cell transplantation (alloHCT) outcomes including graft-versus-host-disease (GvHD), infection, and relapse. However, it is unclear how MICA interacts with cytogenetic and somatic mutations in regards to these outcomes in acute myeloid leukemia (AML). Methods We conducted a single center, retrospective analysis of adult AML patients in first or second complete remission (CR1, CR2), who underwent T-cell replete matched related or unrelated donor alloHCT. Analysis was limited to those who had MICA data available for donors and recipients. In addition to cytogenetic risk group stratification by European LeukemiaNet criteria (Döhner H, et al, Blood 2016), a subset of patients had a 36-gene somatic mutation panel assessed prior to alloHCT by next-generation sequencing. Dimorphisms at the MICA-129 position have previously been categorized as weaker (valine/valine: V/V), heterozygous (methionine/valine: M/V), or stronger (methionine/methionine: M/M) receptor binding affinity. Fine and Gray or Cox regression was used to identify the association of MICA and outcomes with results as hazard ratios (HR) and 95% confidence intervals (CI). Results From 2000 - 2017, 131 AML patients were identified meeting inclusion criteria. Median age at transplant was 54 years (18-74), with 98% Caucasian. Disease status at transplant included 78% CR1 and 22% CR2. Cytogenetic risk stratification showed 13% of patients as favorable, 56% as intermediate, and 31% as adverse-risk. The five most common somatic mutations were FLT3 (15%), NPM1 (14%), DNMT3A (11%), TET2 (7%), and NRAS (6%). 60% of patients had a related donor. A myeloablative transplant was performed in 84% of patients and 53% had a bone marrow graft source. The most common conditioning regimen used was busulfan/cyclophosphamide (52%). 12% of patients were MICA mismatched with their donor. The distribution of donor MICA-129 polymorphisms were 41% V/V, 53% M/V, and 6% M/M. In univariable analysis, donor-recipient MICA mismatch tended to be associated with a lower risk of infection (HR 0.49, CI 0.23-1.02, P=0.06) and grade 2-4 acute GvHD (HR 0.25, CI 0.06-1.04, P=0.06) but was not associated with other post-transplant outcomes. In multivariable analysis, donor MICA-129 V/V was associated with a higher risk of non-relapse mortality (NRM) (HR 2.02, CI 1.01-4.05, P=0.047) (Figure 1) along with increasing patient age at transplant (HR 1.46, CI 1.10-1.93, p=0.008) and the presence of a TET2 mutation (HR 6.00, CI 1.77-20.3, P=0.004). There were no differences between the V/V and the M/V+M/M cohorts regarding somatic mutational status, cytogenetics and other pre-transplant characteristics and post-transplant outcomes. With a median follow-up of 65 months for both cohorts, 45% vs. 49% of patients remain alive, respectively. The most common causes of death between the V/V and the M/V+M/M cohorts was relapse (38% vs. 62%) and infection (31% vs. 8%), respectively. Conclusion While previous studies have demonstrated associations of somatic mutations and cytogenetics with survival outcomes after alloHCT for AML, we observed mutations in TET2 and the V/V donor MICA-129 polymorphism to be independently prognostic for NRM. Mechanistic studies may be considered to assess for possible interactions of TET2 mutations with NK cell alloreactivity. The weaker binding affinity to the NKG2D receptor by the V/V phenotype may diminish immune responses against pathogens that subsequently contribute to higher NRM. These observations may have implications for enhancing patient risk stratification prior to transplant and optimizing donor selection. Future investigation with larger cohorts interrogating pre-transplant AML somatic mutations with MICA polymorphisms on post-transplant outcomes may further elucidate which subsets of patients may benefit most from transplant. Disclosures Nazha: MEI: Consultancy. Mukherjee:Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; BioPharm Communications: Consultancy; Bristol Myers Squib: Honoraria, Speakers Bureau; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; LEK Consulting: Consultancy, Honoraria; Aplastic Anemia & MDS International Foundation in Joint Partnership with Cleveland Clinic Taussig Cancer Institute: Honoraria. Advani:Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy; Novartis: Consultancy. Carraway:Novartis: Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau. Gerds:Apexx Oncology: Consultancy; Celgene: Consultancy; Incyte: Consultancy; CTI Biopharma: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy. Majhail:Incyte: Honoraria; Anthem, Inc.: Consultancy; Atara: Honoraria.

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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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The Application of Machine Learning to Improve the Subclassification and Prognostication of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-139013 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-28

Author(s):

Hassan Awada ◽

Arda Durmaz ◽

Carmel Gurnari ◽

Ashwin Kishtagari ◽

Manja Meggendorfer ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Somatic Mutations ◽

Cross Validation ◽

Steering Committee ◽

Advisory Board ◽

Advisory Committees ◽

Genomic Features ◽

Acute Myeloid

Genetic mutations (somatic or germline), cytogenetic abnormalities and their combinations contribute to the heterogeneity of acute myeloid leukemia (AML) phenotypes. To date, prototypic founder lesions [e.g., t(8;21), inv(16), t(15;17)] define only a fraction of AML subgroups with specific prognoses. Indeed, in a larger proportion of AML patients, somatic mutations or cytogenetic abnormalities potentially serve as driver lesions in combination with numerous acquired secondary hits. However, their combinatorial complexity can preclude the resolution of distinct genomic classifications and overlap across classical pathomorphologic AML subtypes, including de novo/primary (pAML) and secondary AML (sAML) evolving from an antecedent myeloid neoplasm (MN). These prognostically discrete AML subtypes are themselves nonspecific due to variable understanding of their pathogenetic links, especially in cases without overt dysplasia. Without dysplasia, reliance is mainly on anamnestic clinical information that might be unavailable or cannot be correctly assigned due to a short prodromal history of antecedent MN. We explored the potential of genomic markers to sub-classify AML objectively and provide unbiased personalized prognostication, irrespective of the clinicopathological information, and thus become a standard in AML assessment. We collected and analyzed genomic data from a multicenter cohort of 6788 AML patients using standard and machine learning (ML) methods. A total of 13,879 somatic mutations were identified and used to predict traditional pathomorphologic AML classifications. Logistic regression modeling (LRM) detected mutations in CEBPA (both monoallelic "CEBPAMo" and biallelic "CEBPABi"), DNMT3A, FLT3ITD, FLT3TKD, GATA2, IDH1, IDH2R140, NRAS, NPM1 and WT1 being enriched in pAML while mutations in ASXL1, RUNX1, SF3B1, SRSF2, U2AF1, -5/del(5q), -7/del(7q), -17/del(17P), del(20q), +8 and complex karyotype being prevalent in sAML. Despite these significant findings, the genomic profiles of pAML vs. sAML identified by LRM resulted in only 74% cross-validation accuracy of the predictive performance when used to re-assign them. Therefore, we applied Bayesian Latent Class Analysis that identified 4 unique genomic clusters of distinct prognoses [low risk (LR), intermediate-low risk (Int-Lo), intermediate-high risk (Int-Hi) and high risk (HR) of poor survival) that were validated by survival analysis. To link each prognostic group to pathogenetic features, we generated a random forest (RF) model that extracted invariant genomic features driving each group and resulted in 97% cross-validation accuracy when used for prognostication. The model's globally most important genomic features, quantified by mean decrease in accuracy, included NPM1MT, RUNX1MT, ASXL1MT, SRSF2MT, TP53MT, -5/del(5q), DNMT3AMT, -17/del(17p), BCOR/L1MT and others. The LR group was characterized by the highest prevalence of normal cytogenetics (88%) and NPM1MT (100%; 86% with VAF>20%) with co-occurring DNMT3AMT (52%), FLT3ITD-MT (27%; 91% with VAF <50%), IDH2R140-MT (16%, while absent IDH2R172-MT), and depletion or absence of ASXL1MT, EZH2MT, RUNX1MT, TP53MT and complex cytogenetics. Int-Lo had a higher percentage of abnormal cytogenetics cases than LR, the highest frequency of CEBPABi-MT (9%), IDH2R172K-MT (4%), FLT3ITD-MT (14%) and FLT3TKD-MT (6%) occurring without NPM1MT, while absence of NPM1MT, ASXL1MT, RUNX1MT and TP53MT. Int-Hi had the highest frequency of ASXL1MT (39%), BCOR/L1MT (16%), DNMT3AMT without NPM1MT (19%), EZH2MT (9%), RUNX1MT (52%), SF3B1MT (7%), SRSF2MT (38%) and U2AF1MT (12%). Finally, HR had the highest prevalence of abnormal cytogenetics (96%), -5/del(5q) (68%), -7del(7q) (35%), -17del(17p) (31%) and the highest odds of complex karyotype (76%) as well as TP53MT (70%). The model was then internally and externally validated using a cohort of 203 AML cases from the MD Anderson Cancer Center. The RF prognostication model and group-specific survival estimates will be available via a web-based open-access resource. In conclusion, the heterogeneity inherent in the genomic changes across nearly 7000 AML patients is too vast for traditional prediction methods. Using newer ML methods, however, we were able to decipher a set of prognostic subgroups predictive of survival, allowing us to move AML into the era of personalized medicine. Disclosures Advani: OBI: Research Funding; Abbvie: Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Immunogen: Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Other: advisory board; Takeda: Research Funding. Ravandi:Abbvie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; BMS: Consultancy, Honoraria, Research Funding. Carraway:Novartis: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; BMS: Consultancy, Other: Research support, Speakers Bureau; Abbvie: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Jazz: Consultancy, Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kantarjian:Sanofi: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Research Funding; BMS: Research Funding; Abbvie: Honoraria, Research Funding; Aptitute Health: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Adaptive biotechnologies: Honoraria; Ascentage: Research Funding; Amgen: Honoraria, Research Funding; BioAscend: Honoraria; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria. Kadia:Pfizer: Honoraria, Research Funding; Novartis: Honoraria; Cyclacel: Research Funding; Ascentage: Research Funding; Astellas: Research Funding; Cellenkos: Research Funding; JAZZ: Honoraria, Research Funding; Astra Zeneca: Research Funding; Celgene: Research Funding; Incyte: Research Funding; Pulmotec: Research Funding; Abbvie: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Amgen: Research Funding. Sekeres:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

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