Invariant Patterns of Clonal Succession Determines Specific Phenotypic and Clinical Features of Myelodysplastic Syndromes (MDS)

Abstract MDS arises through stepwise acquisitions of multiple mutations. Mutation configuration (bi-allelic vs. mono-allelic), specific distributions (hot spot), timing of acquisitions/ranks within clonal hierarchies, and combinatorial spectra, can all affect the pathogenesis and clinical phenotype of this disease. We performed integrative analyses of these processes to determine which relationships are deterministic vs. random. We analyzed 1,809 clinically annotated MDS patients. Deep targeted NGS was applied to a panel of the 36 genes frequently mutated in myeloid neoplasms. Copy number alterations (CNA) were evaluated by combining karyotyping, microarray, and digital CNA analysis. With a mean coverage of 862x, after removing SNPs and errors, we identified 3,971 somatic mutations. Frequently mutated genes/CNAs were TET2 (27%), SF3B1 (23%), ASXL1 (19%), del(5q) (16%), SRSF2 (14%), DNMT3A (11%) and del(7q) (10%), each present in >10% of cases. DNMT3AMT affected the canonical site (R882) in 17% (35/203) of DNMT3AMT cases, were truncating in 39% (79/203) and were other missense mutations in 44% (89/203). Bi-allelic alterations of EZH2 and TP53 most commonly involved a mutation paired with a copy number deletion or UPD. In contrast, RUNX1 and TET2 commonly involved multiple mutations of the gene. Examining intragenetic relationships, we assumed that the impact of individual mutations depends on their clonal hierarchical position. To discriminate 1st hits ("dominant") from subsequent "secondary mutations", we used a stringent binominal distribution algorithm to compare the expected vs. observed VAFs using read counts (Figure 1). A mutation with the largest VAF in a sample was defined as "dominant". Mutations with overlapping 95% CI of expected VAFs were "co-dominant", and those with non-overlapping 95% CIs were "secondary". These assumptions were validated by Pyclone (concordance rate > 95%). Accordingly, 1,474 (36%) and 1,372 (35%) mutations were dominant and secondary, respectively. SF3B1, DNMT3A, U2AF1, and TP53 were more likely to be dominant, ASXL1, CBL, ETV6, and KRAS were more likely to be secondary. For example, SF3B1 mutations were dominant in 17% (303/1809) of patients and secondary in 2% (39/1,809), p<.0001, q<.01. 78 common combinations were identified among these different types of mutations. For instance, compared to U2AF1S34 mutations, U2AF1Q157 mutations were more associated with ASXL1 mutations and del(7q), and less with DNMT3A and BCOR mutations (p<.01). Compared to TET2 mono-allelic mutations, TET2 bi-allelic mutations co-existed more with ZRSR2 and SRSF2 mutations, but less with del(5q). DNMT3AR882 mutations at 77% (27/35) were more likely to be dominant than truncating or other missense DNMT3A mutations at 51% (40/79) and 45% (47/98), p=.0046, q=.07. We also evaluated the impact of different types of mutations and their combinations on disease phenotypes and survival. Many relationships were identified between mutations in different genes and bi-allelic vs. mono allelic hits. Among frequently correlated dominant/secondary pairs, pairs of dominant EZH2 mutations and secondary RUNX1 or ASXL1 mutations were associated with higher-risk subtypes (q=.08) (Figure 2), whereas patients with dominant SF3B1 mutations and secondary JAK2 mutations had myeloproliferative features and lower-risk subtypes (q<.001; q=.09). DNMT3AR882 mutations associated with worse overall survival than did truncating and other missense DNMT3A mutations. Five pairs of dominant and secondary mutations significantly affected survival (p<.01, q<.1). We hypothesized that if a fraction of MDS originates from clonal hematopoiesis of indeterminate potential (CHIP), the compositions of dominant mutations in MDS should be similar to those in CHIP. MDS patients with dominant mutations of TET2, DNMT3A, and ASXL1 were defined as "CHIP-derived MDS" and had more secondary TET2 and ZRSR2 mutations than "De novo MDS". In conclusion, we report a comprehensive analysis of various mutational scenarios and the resultant clinical features. Most significantly, our results demonstrate how invariant patterns of evolution evolve with certain dominant hits dictating high probabilities of specific secondary events. Such patterns coincide with unique combinations of clinical properties that arise with hits along specific pathways. Disclosures Nazha: MEI: Consultancy. Sekeres:Celgene: 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; Opsona: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

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Mutational Type and Configuration of an Individual Gene May Differentially Impact the Clinical and Phenotypic Features

Blood ◽

10.1182/blood-2019-128893 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2992-2992

Author(s):

Yasunobu Nagata ◽

Hideki Makishima ◽

Cassandra M Kerr ◽

Bhumika J. Patel ◽

Hassan Awada ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Somatic Mutations ◽

The Other ◽

Missense Mutations ◽

Advisory Committees ◽

Different Types ◽

Equity Ownership ◽

The Impact

Myelodysplastic syndromes (MDS) arise in older adults through the stepwise acquisition of multiple somatic mutations. The genetic heterogeneity that results includes mutations of diverse genes and their combinations, clonal hierarchy, genetic configuration (e.g., bi-allelic or compound heterozygous, hemizygous lesions), specific positions within a gene including canonical hotspots vs. other positions, and types of mutation (truncations vs. missense), all of which could differentially affect pathogenesis. Given the binary status (e.g. mutated vs. wild-type) used in many clinical analyses, the true impact of specific types of mutations may be obscured and their specific roles underestimated. Deep targeted NGS was carried out for a panel of the 36 most frequently mutated genes in 1,809 MDS patients (low-risk MDS (n=839) vs. high-risk MDS (n=607), MDS/MPN (n=212), and sAML n=151). Copy number alterations (CNA) were also evaluated by combining karyotyping, microarray, and digital copy number analysis. With a mean coverage of 862x, after removing SNPs and errors, 3,971 somatic mutations were identified, the most common (>10% of cases) being TET2, SF3B1, ASXL1, del(5q), SRSF2, complex karyotype, and del(7q). For the purpose of this proof of concept analysis we focused on illustrative genes (TP53, RUNX1, TET2, and EZH2) affected by 2 recurrent hits. Bi-allelic TET2 or TP53 mutations were found in 15% (271/1,809) and 4% (72/1,809) of patients, respectively. TET2 and RUNX1 were most likely biallelic, whereas TP53 and EZH2 were most often affected by mutations and somatic deletion. Comparing the distribution of canonical vs. other types of mutations in genes, DNMT3A mutations affected the canonical site (R882) in 17% (35/203) of patients, were truncating in 39% (79/203) and missense in 44% (89/203) have also been found; deletions affecting the DNMT3A locus are rare. Within U2AF1, U2AF1Q157 are more frequent than U2AF1S34 (54% vs. 35%). Next, we checked correlation between these different types of mutations of one gene. 78 significant combinations were found. For instance, U2AF1Q157 mutations more commonly accompanied ASXL1 mutations and del(7q) and less frequently DNMT3A and BCOR mutations, trisomy8 and del(20) when compared to U2AF1S34 mutations [ASXL1 mutations 53% (42/80) in U2AF1Q157 vs. 16% (8/49) in U2AF1S34, P < .0001]. TET2 Bi-allelic mutations were more commonly associated with ZRSR2 and SRSF2 mutations, and less frequently del(5q) when compared to TET2 mono-allelic mutations [SRSF2 mutations 29% (80/276) in TET2-bi vs. 15% (34/227) in TET2-mono, P = .003]. In addition, patients with SRSF2 missense mutations were more likely to have RUNX1 bi-allelic mutations than those with SRSF2 in-frame mutations. We evaluated the impact of different types of mutations and combinations of them on disease phenotypes and survival. We then evaluated the impact of different types of mutations and their combinations on clinical phenotypes including dichotomous morphological (MDS vs. MDS/MPN) features, progressive (low- vs. high risk) subtypes. EZH2 bi-allelic alterations were more commonly associated with myleoproliferative features` compared to EZH2 mono-allelic alteration (q=.016). TET2 bi-allelic alterations and truncating mutations were found more frequently in higher-risk subtypes than TET2 mono-allelic and missense mutations (q<.001). In survival analyses, patients with DNMT3AR882 mutations had a poorer prognosis than those with truncating and the other missense mutations [P = .033, HR 1.86 (1.05-3.3)]. Next, using the PyClone bioanalytic pipeline, we recapitulated for each patient the clonal hierarchy and defined "dominant" vs. "secondary" mutations. DNMT3AR882 mutations were likely to be dominant/founder lesions compared to truncating or the other missense mutations: 77% (27/35) for R882 vs. 51% (40/79) for truncating vs. 45% (47/98) for the other missense, p=.0046. Specific dominant and secondary mutational pairs also differentially affected survival compared to the reverse configuration (q<.1) including EZH2 and RUNX1 or BCOR and U2AF1 or RUNX1 and BCOR. In conclusion, we report a comprehensive analysis of various types and configurations of lesions of individual commonly affected genes. Our results indicate that establishment of clinical or phenotypic correlations requires consideration of the type, rank and configuration of somatic mutations. Disclosures Mukherjee: McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy. Nazha:Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

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Distinct Clinical and Biological Implications of Various DNTMT3A Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.2872.2872 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2872-2872

Author(s):

Suresh Kumar Balasubramanian ◽

Mai Ali ◽

Taha Bat ◽

Bhumika Patel ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Clinical Features ◽

Bone Marrow Failure ◽

Missense Mutations ◽

Negative Consequences ◽

Wild Type ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Significant Difference

Abstract DNMT3A, a member of the DNA methyltransferases family along with DNMT1 and DNMT3B, is located on chromosome 2p23. Recurrent somatic mutations in DNMT3A are typically heterozygous and found mostly in non-CBF AML, less frequently in MDS and MPN. DNMT3A mutations are reported with other common myeloid mutations including NPM1, FLT3 and IDH1/2. The most canonical DNMT3A mutations are missense alteration in the R882 codon, accounting for >60% of all DNMT3A mutations and they imply dominant negative consequences. Overall, DNMT3A mutations carry a poor prognosis compared to the AML or MDS with wild type (WT) DNMT3A, although data within different subgroups (e.g., incorporating cytogenetic profiles) are conflicting. We hypothesized that molecular consequence of R882 mutations will differ from those of other somatic alterations of DNMT3A and may also result in distinct clinical features and outcomes. To test this theory, we analyzed a cohort of 1174 patients with myeloid neoplasias including 32% AML, 33% MDS, 13% MDS/MPN, 6% MPN and 16% other bone marrow failure disorders. These cases were subjected to multiamplicon targeted deep NGS including all ORFs of DNMT3A and other recurrently mutated genes. After application of various bioanalytic algorithms, confirmatory sequencing and thus stringent exclusion of all artifacts and germline alterations, we identified 140 somatic mutant cases (12% of the cohort), including 89 missense mutations (53 at R882, 19 at R693 and 17 other non-canonical missense alterations) and 51 truncations/frame shifts (all heterozygous). There was an age-related increase in the incidence of DNMT3A mutations, with the peak occurrence at 35-40 yrs. of age. Mutations in DNMT3A were most common in AML (54% in primary (p) AML, 8% in secondary (s) AML) followed by MDS (28%), MDS/MPN (4%), MPN (3%) and other bone marrow failure disorders (3%). Mutation in the R693 codon and truncating mutations were most commonly associated with MDS (p=.013) and sAML (p=.0013) whereas mutation occurring in codon R882 and other non-canonical missense mutations were frequently associated with pAML (p=.00001). For the whole cohort, DNMT3A mutations were most frequently associated with NPM1 (21% vs 8%, p=.014), FLT3 (24% vs. 2%, p=.0001), and IDH1/2 (26% vs. 8%, p=.001), compared to wild type DNMT3A. However, PRC2 complex mutations were less likely to occur in the context of DNMT3A mutations (6% vs. 24%, p=.0006). Canonical R882 mutation was commonly associated with FLT3 (p=.03) mutations, while truncating mutations were not (p=.03). Analyses of clonal hierarchy by ranking of VAF values demonstrated that 53% of DNMT3A mutations were dominant (mean VAF 39%, range 5-93%) (n=74/140). When DNMT3A mutations were dominant, IDH 1/2 (14%), TET2 (9%), ASXL (5%), PRC2 complex (3%) and BCOR (3%) mutations were common secondary events. In subgroup analyses, 55% of mutations in the R693 codon were dominant compared to 45% in R882 and 47% in truncating mutations. TET2 mutations were the most common associated secondary hits in dominant R693 mutations (n=10) compared to truncating (n=24) and R882 mutations (n=23) (40% vs. 8% vs. none, p=.0001). When DNMT3A mutations are secondary (mean VAF 34%, range 1-60%), as in 47% of our cases (n=66/140), then the common first hits were TET2 (10%), U2AF1 (8%) and cohesin complex (RAD21, SMC3, STAG2) mutations (6%). Dominant DNMT3A mutations correlated with MDS/MPN (60%, p=.007), while secondary DNMT3A mutations correlated with sAML (73%, p=.001). DNMT3A mutant myeloid neoplasms showed worse survival (p<.0001) compared to WT cases. Among different subgroups, there was significant difference in OS between R882, R693, truncating and other non-canonical missense mutations (p=.013). The R882 mutations had worse survival compared to other DNMT3A mutations (p=.003). Non-canonical mutations (truncating and other missense) vs. canonical mutations (R882 and R693) had better survival (p<.04). Survival for mutant R882 DNMT3A was worse compared to truncating mutations (p=.005) while there was no difference between R693 and truncating mutations. Among AML cases, R882 mutations vs. other mutations had worse survival (p=.01) while in MDS and MDS/MPN there was no significant difference in OS. DNTMT3A mutations often occur as founder lesion in AML. Our study shows that different types of mutations other than canonical R882 alterations may have a differential impact on OS and distinct clinical features. Disclosures Carraway: Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

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MYC Rearrangements in Multiple Myeloma Are Complex, Can Involve More Than Five Different Chromosomes, and Correlate with Increased Expression of MYC and a Distinct Downstream Gene Expression Pattern

Blood ◽

10.1182/blood.v130.suppl_1.65.65 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 65-65

Author(s):

Aneta Mikulasova ◽

Cody Cody Ashby ◽

Ruslana G. Tytarenko ◽

Michael Bauer ◽

Konstantimos Mavrommatis ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Copy Number ◽

Chromosomal Rearrangement ◽

Hot Spot ◽

Chromosomal Rearrangements ◽

Advisory Committees ◽

Equity Ownership ◽

Downstream Gene Expression

Abstract Introduction: The proto-oncogene MYC (locus 8q24.21) is a key transcription factor in multiple myeloma (MM) resulting in significant gene deregulation and impacting on many biological functions, including cell growth, proliferation, apoptosis, differentiation, and transformation. Chromosomal rearrangement and copy number change at the MYC locus are secondary events involved in MM progression, which are thought to lead to aggressive disease. Current analyses of the MYC locus have not been large and have reported rearrangements in 15% of new-diagnosed MM. However, more recent studies using advanced genomic techniques suggest that the frequency of MYC rearrangements may be much higher, and that a full reassessment of the role of MYC in MM pathogenesis may be critical. In this study, we analyzed 1280 MM patients to provide a better understanding of the role of this important genomic driver in MM pathogenesis. Methods: In total, 1280 tumor normal pairs of CD138 sorted bone marrow plasma cells and their germline control samples were analyzed by: 1. Targeted sequencing of 131 genes and 27 chromosome regions (n=100) with 4.5 Mb captured region surrounding MYC ; 2. Exome sequencing (n=461) with 2.3 Mb captured region surrounding MYC ; 3. Whole genome sequencing (n=719). Normalized tumor/germline depth ratio in targeted-sequencing cases and MANTA were used for detection of somatic copy number and structural variants. Expression analysis was performed using RNA-seq or microarrays. Results: MYC translocations were found in 25% (323/1280) of patients and occurred most frequently as inter-chromosomal translocations involving 2-5 chromosomes (90%, 291/323). Of the remaining cases, 5% (17/323) of the translocations involved inversion of chromosome 8 and 5% (15/323) were complex, affecting more than 5 chromosomal loci. The proportion of MYC translocations involving 2, 3, 4, and 5 loci was 62% (200/323), 23% (74/323), 8% (26/323) and 3% (8/323), respectively. Using abnormal rearranged cases (29/100), we found copy number imbalances >14.2 kb in size associated with a MYC translocation in 76% (22/29). Another 7% (2/29) of cases with translocations showed complex intra-chromosomal rearrangement. A region of 2.0 Mb surrounding MYC was identified as a translocation breakpoint hot-spot incorporating 96% of breakpoints. This region also contained two hotspots for chromosomal gain and tandem duplications. MYC rearrangements were not randomly distributed across the spectrum of MM with an excess being seen in hyperdiploidy (76% of rearranged samples, P <0.0001). Importantly, 67% (207/308) of cases with a MYC translocation involving 5 or less chromosomes had one of the commonly known super-enhancers involved in the translocation. Gene expression analysis was used to explore the impact of these events on downstream gene expression patterns. The results showed that inter- and/or intra-chromosomal rearrangements were associated with a significantly (P <0.0001) higher MYC expression (4.1-fold). In patients where rearrangements were associated with additional copies of MYC there was higher expression of MYC in comparison to cases with a translocation but lacking copy number gain (P=0.04). To identify downstream genes deregulated by MYC rearrangements we compared gene expression between those with and without a translocation, independently of hyperdiploidy. Genes that showed >2-fold change in expression (P <0.01) included MYC and the non-protein coding oncogene PVT1 that is located next to MYC . Genes with significantly lower levels of expression were involved in B-cell biology including CD79A and AHR, or were associated with cell proliferation, migration, adhesion, apoptosis and/or angiogenesis (FGF16, ADAMTS1, FBXL7, HRK, PDGFD, and PRKD1) . Conclusions: This study confirms the central role of MYC in the pathogenesis of clinical cases of MM, and as such defining it as a critical therapeutic target. We will be able to target MYC better if we understand how it is deregulated and in this respect we show that the MYC locus rearrangements are complex and it is a hot-spot for heterogeneous inter- as well intra-chromosomal rearrangements, including complex rearrangements involving >5 chromosomes. These events lead to increased MYC expression consistent with it being a driver of disease progression, particularly in the hyperdiploid subset of MM. Disclosures Mavrommatis: Celgene Corporation: Employment. Trotter: Celgene Corporation: Equity Ownership; Celgene Institute for Translational Research Europe: Employment. Davies: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Morgan: Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria.

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Characterization of the "Immune Evasion" Phenotype of Richter Syndrome and the Implications for Immune-Checkpoint Inhibitor Therapy

Blood ◽

10.1182/blood-2019-127800 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4290-4290

Author(s):

Clare Gould ◽

Jennifer Lickiss ◽

Yamuna Kankanige ◽

Satwica Yerneni ◽

John Markham ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Board Of Directors ◽

Immune Checkpoint ◽

Copy Number ◽

Research Funding ◽

De Novo ◽

Advisory Committees ◽

Immune Checkpoint Molecules ◽

Checkpoint Inhibitor Therapy

Richter syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) to a high-grade B-cell lymphoma and is associated with an aggressive clinical course and poor prognosis. Conventional treatment options for RS are generally associated with low response rates and limited durability making this entity an area of significant unmet therapeutic need. Immune checkpoint inhibitor therapy has shown promise in the treatment of some aggressive lymphoma subtypes. In RS, modest benefits have been reported in small phase two trials of anti-PD-1 monotherapy and in combination with ibrutinib, however larger scale studies are lacking (Ding et al Blood, 2017; Jain et al Blood, 2016). We sought to characterise the immune-evasion phenotype of RS focussing on potential genetic biomarkers which may inform the selection of patients who are most likely to benefit from immune-directed therapies. We first assessed the gene expression of immune-checkpoint molecules given their potential clinical relevance and ability to be targeted by available therapeutic agents. Given immunohistochemical (IHC) assessment of immune-checkpoint molecules is recognized to be associated with high inter-observer variability and there is a high correlation between gene expression of immune-checkpoint molecules and IHC, we performed gene expression quantification using the Nanostring nCounter Human Immunology V2 panel (Nanostring Technologies, USA). Nanostring analysis was performed on samples from 17 patients with histologically confirmed RS (DLBCL subtype) and compared to 73 cases of de novo (non-transformed) DLBCL. Significant differences in the gene expression of checkpoint molecules was observed between RS and DLBCL biopsies, including higher expression of LAG3, PD1 and TIGIT in RS (p=0.0001, logFC 1.9; p=0.0017, logFC 1.1 and p=0.0437, logFC 0.7 vs DLBCL, respectively). PD-L2 and TIM3 gene expression were both significantly lower in RS compared to DLBCL (p = 0.0059, logFC 0.8; p = 0.012, logFC 0.8). PDL1 and CTLA4 gene expression did not significantly differ between RS and DLBCL. We next assessed the gene expression of T- and NK- cell markers (including CD3, CD4, CD8, FOXP3 and CD56) and the ratios of these markers to malignant B-cells (CD19). We observed no significant difference between RS and DLBCL, consistent with a similar relative quantity of immune cell infiltration between the two entities. Significantly higher gene expression of CD39, a marker of CD8+ T-cell exhaustion, was observed in RS than DLBCL (p = 0.031; logFC 0.5). Additional immune-related genes were next assessed, including those involved in antigen presentation (e.g. B2M, HLA molecules, TAP), immunosuppressive cytokine generation (e.g. ARG1, IDO1) and apoptosis resistance (e.g. FAS) which showed no significant differences in expression between RS and DLBCL. To assess whether these findings were consistent across other transformed lymphoma subtypes, we compared RS to a cohort of transformed follicular lymphoma (tFL, n=16) and transformed marginal zone lymphoma (tMZL, n=25). LAG3 expression was significantly higher in RS compared to both tFL and tMZL (p=0.0002, logFC 2.7; p=0.019, logFC 1.7). PD1 expression was also significantly higher in RS than tFL but not tMZL (p=0.0045, logFC 1.7; p=0.39, logFC 0.4). Given the established association of copy number amplifications involving immune checkpoint molecules (e.g. PD-L1/PD-L2 on 9p24.1) representing a potential predictive biomarker of response in other lymphomas, we performed hybridization-based NGS with whole genome copy number assessment to evaluate immune checkpoint gene loci in the three cohorts. No significant focal amplifications were detected in RS samples with overexpressed immune-checkpoint molecules. In contrast, three patients in the DLBCL/transformed cohort had focal copy number amplifications involving PD-L1. No copy number amplification of LAG3 was observed in either RS or DLBCL. In summary, we have observed significantly increased gene expression of LAG3, PD1 and TIGIT in RS compared to de novo DLBCL. Combined with increased gene expression of the exhausted cytotoxic T-cell marker CD39, these data provide a strong biological rationale for pursuing LAG3 inhibition either alone or in combination with other immune checkpoint blockade to enhance anti-tumour T cell responses in this difficult-to-treat entity. CG/JL/YK co-first authors Disclosures Gould: NovoNordisk: Other: Travel funding - domestic flights to attend education, May 2018. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Neeson:Roche Genetech: Research Funding; Allergan: Research Funding; Juno/Celgene: Research Funding; Compugen: Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Seymour:Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding. Dickinson:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Blombery:Invivoscribe: Honoraria; Novartis: Consultancy; Janssen: Honoraria.

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The BMP/SMAD Pathway Is a Key Mediator of Leukemic Transformation of TP53-Mutant Post-MPN AML

Blood ◽

10.1182/blood-2021-152036 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 626-626

Author(s):

Bing Li ◽

Wenbin An ◽

Hua Wang ◽

Aishwarya Krishnan ◽

Shoron Mowla ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Sequencing Analysis ◽

Erythroid Progenitors ◽

Leukemic Transformation ◽

Advisory Committees ◽

Smad Pathway ◽

The Impact

Abstract Leukemic transformation (LT) after an antecedent myeloproliferative neoplasm (MPN) carries a dismal prognosis. As such, there is a pressing need for new mechanistic insights into LT as well as novel therapeutic approaches. Mutational inactivation of TP53 is the most common somatic mutation in LT. However, the impact of TP53 allelic state on the ability to potentiate LT, as well as the pathways involved in this process, have largely remained unresolved. To investigate the role of Tp53 alterations in LT, we generated an allelic series of mouse models with Jak2V617F/+ combined with conditional Tp53 knockout and point mutant alleles (all crossed to Rosa-CreERT2); Jak2V617F/+(J VF) , Jak2V617F/+-Tp53fl/+(J VFP +/-), Jak2V617F/+-Tp53fl/fl (J VFP -/-), Jak2V617F/+-Tp53R172H/+(J VFP R172H/+), Jak2V617F/+-Tp53R172H/fl (J VFP R172H/-). After tamoxifen-induced recombination, mice transplanted with J VF, J VFP +/- and J VFP R172H/+ cells developed an MPN phenotype, whereas all the recipients of J VFP -/- and J VFP R172H/- bone marrow initially developed an MPN phenotype followed by transformation to acute leukemia with significantly impaired survival, and changes in blood counts and organ weights, compared to other genotypes (Fig 1A/B). Histopathology of J VFP -/- and J VFP R172H/- mice was consistent with pure erythroleukemia (PEL; Fig 1C). Analysis of stem and progenitor compartments demonstrated that the MEP (Megakaryocyte Erythroid Progenitors) compartment was significantly expanded in the bone marrow and spleen of both J VFP -/- and J VFP R172H/- mice, compared to other genotypes, at both the MPN and PEL stages of disease, consistent with erythroid-biased hematopoiesis (Fig 1D). Given we observed sequential MPN->AML progression, we hypothesized that additional genetic/biological events were required to promote LT. Sparse whole genome sequencing analysis revealed that transformation to PEL was associated with the development of recurrent copy number alterations (CNA) . Importantly, CNAs were restricted to the MEP compartment and not identified in the GMP compartment (Fig 1E), suggesting that MEPs might represent the leukemia initiating population with capability of acquiring additional genomic instability. Consistent with this hypothesis, mice transplanted with MEPs, but not GMPs from J VFP -/- and J VFP R172H/- mice at the MPN stage developed PEL. Further, single-cell RNA sequencing of J VF and J VFP -/- (at both MPN and PEL stage) demonstrated that the gene-expression signature of the leukemic population was most similar to that of erythroid progenitors and erythroblasts, and that by copy number inference analysis, CNAs were restricted to the leukemic population. We identified 617 genes up-regulated in both J VFP -/- and J VFP R172H/- leukemic MEPs when compared to J VF MEPs using RNA-seq. Pathway analysis demonstrated increased expression of Bone morphogenetic protein (BMP) pathway genes in both J VFP -/- and J VFP R172H/- leukemic mice (Fig 1F). Importantly, similar observations were made in human PEL samples as well. To investigate the function of this pathway, leukemic MEPs from J VFP -/- and J VFP R172H/- mice were transduced with an shRNA-targeting Bmp2 or a control and injected into lethally irradiated recipient mice. Mice injected with Bmp2-shRNA MEPs demonstrated leukemic regression and restoration of normal hematopoiesis as evidenced by significant reductions in leukocytosis (p<0.05) and increased HGB (p<0.05) and an increase in PLT count (p<0.05/p<0.01) (Fig 1G). Finally, as compared to mice injected with leukemic MEPs with control shRNA, mice injected with Bmp2-shRNA had significantly longer survival (p<0.05) (Fig 1H). Thus, downregulation of Bmp2 results in attenuation of the leukemic phenotype. Using novel models, we have identified that bi-allelic, but not mono-allelic Tp53 alteration is required for LT of MPN. The leukemia initiating population arises within the MEP compartment and is characterized by recurrent CNAs acquired in a specific hematopoietic compartment. Moreover, the BMP/SMAD pathway is upregulated in leukemic MEPs and plays a functional role in LT. Collectively, our data yields novel biological insights into the process of leukemic transformation mediated by Tp53 alterations. Data on selective therapeutic targeting of p53-mutant PEL will be presented at the meeting. Figure 1 Figure 1. Disclosures Xiao: Stemline Therapeutics: Research Funding. Lowe: Oric Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Mirimus, Inc: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Faeth Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; PMV Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Gilead: Honoraria; Amgen: Honoraria; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Janssen: Consultancy; Astellas: Consultancy. Rampal: Pharmaessentia: Consultancy; Abbvie: Consultancy; Kartos: Consultancy; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Disc Medicine: Consultancy; BMS/Celgene: Consultancy; Novartis: Consultancy; CTI: Consultancy; Sierra Oncology: Consultancy; Stemline: Consultancy, Research Funding; Blueprint: Consultancy; Memorial Sloan Kettering: Current Employment.

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A New Gene Expression Profile Signature CRLF2 Overexpression Based Identifies Novel Adult "Triple Negative" Acute Lymphoblastic Leukemia Subgroups

Blood ◽

10.1182/blood-2018-99-118062 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5284-5284

Author(s):

Anna Ferrari ◽

Silvia Vitali ◽

Valentina Robustelli ◽

Andrea Ghelli Luserna Di Rora ◽

Simona Righi ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Triple Negative ◽

Lymphoblastic Leukemia ◽

Gene List ◽

Advisory Committees ◽

The Impact ◽

Stat Pathway

Abstract Background: The heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL patients (pts) that doesn't have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as "triple negative" (Ph-/-/-) ALL. CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Clustering and biological characterization of Ph-/-/- ALL (that represents 61% of adult B-ALL; Roberts KG, J Clin Oncol. 2016), considering CRLF2 overexpression event, in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) were performed on 55 Ph-/-/- ALL, 29 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized with the Expression Console Software. Successively we cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in the top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (TruSight Pancancer-Illumina; MLPA and/or dMLPA-MRC-Holland; SNP Array-Affymetrix; 454 Junior-Roche and PCR). Results: Clustering our Ph-/-/- gene expression data using the impact of the 10 single genes in our cohort, we could identify a defined 2-clusters-subdivision (Gr1 and Gr2; Fig 1A). The Gr2 is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) overexpression and it represents 14.1% of all B-ALL. The Gr2 GEP is similar to Ph+ one. Fusion copy number alteration and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mutations or deletion), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. Molecular characterization shed light on a very heterogeneous scenario especially in the group 1, suggesting the need of a more discerning clustering for this group. In spite of the small number of cases is required, preliminary Gr1 subclustering discerns MLLr and ZNF384 gene expression subgroups. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upregulated in the group1 and CDK6 in the Gr2. CRLF2 and CD200 immunoblotting and CD200 immunohistochemistry preliminary analyses suggest that protein expression of CRFL2 and CD200 are higher in Gr2 in comparison to Gr1. Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. 3C-up represents 14.1% of all B-ALL and it is characterized by a) high co-expression of three main genes: CRLF2, CTGF and CD200; b) IKZF1 deletion; c) JAK-STAT pathway mutations/fusions/deletions. Gr1 represents 46.9% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identify new potential therapeutic targets with available drug (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; tyrosine kinase inhibitors already effective on Ph+ and Ph-like) to test. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Figure. Figure. Disclosures Cavo: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: 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; GlaxoSmithKline: Honoraria, 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, Speakers Bureau. Martinelli:Novartis: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Ariad/Incyte: Consultancy; Amgen: Consultancy.

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NGS-Based Copy Number Analysis in 1,185 Patients with Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.955.955 ◽

2016 ◽

Vol 128 (22) ◽

pp. 955-955 ◽

Cited By ~ 2

Author(s):

Ryunosuke Saiki ◽

Yusuke Shiozawa ◽

Tetsuichi Yoshizato ◽

Kenichi Yoshida ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Board Of Directors ◽

Deep Sequencing ◽

Copy Number ◽

Research Funding ◽

De Novo ◽

Driver Genes ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Targeted Deep Sequencing ◽

De Novo Aml

Abstract Background Copy number alteration (CNA) is a hallmark of cancer genomes and has been implicated in the development of human cancers, including myeloid neoplasms. We developed a novel, next-generation sequencing-based platform for highly sensitive detection of CNAs with a single exon resolution, which was applied to sequencing data from 1,185 patients to delineate a comprehensive landscape of CNAs in myeloid neoplasms. Materials and Methods We enrolled 1,185 patients with different myeloid neoplasms including myelodysplastic syndromes (n = 607), myelodysplastic/myeloproliferative neoplasms (n = 80), de novo acute myeloid leukemia (AML) (n = 136), secondary AML (sAML) (n = 226), and unknown myeloid malignancies (n = 136). Whole-exome sequencing (WES) was performed on samples from 260 patients, while samples from 925 patients including pre-transplantation peripheral blood samples provided by Japan Marrow Donor Program were subjected to targeted deep sequencing. Eight cases were serially evaluated before and after progression tosAML. RNA baits for targeted deep sequencing were designed to cover 69 driver genes in myeloid neoplasms and 1,158 single-nucleotide polymorphisms (SNPs)for assessment of allelic imbalance. In WES, allelic imbalance was examined using allele frequencies of SNPs within coding regions. Focal CNAs were defined as CNAs whose lengths relative to the chromosomal arms were below 10%. Results To obtain a landscape of CNAs in coding regions, a comprehensive copy number analysis was performed on 260 patients including 136 with de novo AML and 124 with myeloid neoplasms with myelodysplasia, all of whom were studied by WES. A total of 755 CNAs (502 deletions and 253 amplifications) were identified, where 52% of the patients harbored at least one alteration. Using GISTIC 2.0 algorism, we identified 21 significantly altered regions involving known or putative driver genes (Figure 1): losses of 7q22.1 (CUX1), 12p13.2 (ETV6), 13q14 (RB1),17p13.1(TP53), and 17q11.2 (NF1), and gains of 3q26-27 (EVI1), 8q24.21 (MYC), 11q13.5-14.1(PAK1), 11q23.3 (MLL),11q24-25 (ETS1), 13q12.2 (FLT3),21q22.2 (ETS2 and ERG). We next compared the frequencies of CNAs between de novo AML and myeloid neoplasms with myelodysplasia. While chromosomes 7, 12, and 17 were commonly affected, deletions of 13q14 were significantly enriched in myeloid neoplasms with myelodysplasia (Odds ratio [OR]: 5.07, P = 0.040), and amplifications of 11q24-25 (OR: 5.54, P = 0.028), and 21q22.2 (OR: 6.10, P = 0.020) in de novo AML, suggesting a specific role of these events in each disease entity. In addition, serial sampling revealed trisomy8, deletions of 7q and 12p were recurrently acquired during leukemic transformation in patients withmyelodysplasia. Taken together, many driver genes in myeloid neoplasms were frequently targeted by CNAs includingmicrodeletions. Based on these finding, we sought to obtain a more detailed landscape of CNAs in a larger cohort. We combined copy number profiles of patients studied by targeted deep sequencing and those by WES. Of total, 1,691 CNAs (1,096 deletions and 595 amplifications) were detected, where 39% of the cases harbored at least one alteration. Microdeletionsor focal amplifications were frequently found in the significantly altered regions revealed by WES: microdeletionsof ETV6 (n = 10), NF1 (n = 8), CUX1 (n = 5), TP53 (n = 5), and amplifications of FLT3 (n = 7), ETS1 (n = 3), ETS2 (n = 3), and ERG (n = 3), validating the result obtained from a cohort studied by WES. We also identified known driver genes in myeloid neoplasms were recurrently affected with focal CNAs: microdeletions of RUNX1, BCOR, ASXL2, DNMT3A, and ZRSR2, and amplifications of GNAS, RIT1, CSF3R, and BCL11A. Among them, DNMT3A and ASXL2, located within 500 kb in chromosome 2, tended to be co-deleted (3 out of 4 cases). Focal deletions of TP53 were often affected with homozygous deletions or were accompanied by gene mutations, implying bi-allelic inactivation. High amplifications were also observed in regions including ETS1, MLL, FLT3, MYC, and PAK1, which suggest a critical role in the pathogenesis of myeloid malignancy. Conclusion We obtained the landscape of CNAs in myeloid neoplasms based on the sequencing data of 1,185 patients. Collectively, our results indicated that CNAs targeted a specific set genes including well-known drivers of myeloid malignancies, indicating a critical role inleukemogenesis. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

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Therapy Biosimulation Using the Cellworks Computational Omics Biology Model (CBM) Is Predictive of Individual Acute Myeloid Leukemia (AML) Patient Probability of Clinical Response (CR) and Overall Survival (OS): Mycare-023

Blood ◽

10.1182/blood-2021-154023 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 689-689

Author(s):

Scott C Howard ◽

Drew Watson ◽

Michael Castro ◽

Shweta Kapoor ◽

Prashant Ramachandran Nair ◽

...

Keyword(s):

Clinical Response ◽

Board Of Directors ◽

Likelihood Ratio ◽

De Novo ◽

Disease Network ◽

Advisory Committees ◽

Treatment Benefit ◽

De Novo Aml ◽

The Impact ◽

Better Than

Abstract Background: Although some genomic biomarkers have been integrated into therapeutic decision-making for the management of AML, the complete remission and cure rates have significant margin for improvement. Except for a few targeted therapies, genomic assessments offer limited guidance on treatment. Nevertheless, comprehensive molecular profiling of AML discloses a complex and heterogeneous disease network that impacts the efficacy of individual chemotherapeutics differently in individual patients. The Cellworks Computational Omics Biology Model (CBM) was developed using artificial intelligence heuristics and literature sourced from PubMed to generate a patient-specific protein network map. The Cellworks Biosimulation Platform uses the CBM to model each patient's unique cancer and predict personalized responses to standard AML drugs, identify novel drug combinations for treatment-refractory patients and optimize treatment selection to improve outcomes. Methods: A prospectively designed study involving observational data from 416 de novo AML patients was used to test the hypothesis that biosimulation using the Cellworks Biosimulation Platform predicts clinical response to individual drugs and estimates likelihood of response and survival better than physician prescribed treatment (PPT) alone. Cytogenetic and molecular data obtained from clinical trials including AMLSG 07-04, Beat AML, TCGA and PubMed publications was used to create personalized in silico models of each patient's AML and generate a Singula™ biosimulation report with a Therapy Response Index (TRI) to determine the efficacy of specific chemotherapeutic agents. The impact of specific AML agents on each patient's disease network was biosimulated to determine a treatment efficacy score by estimating the effect of chemotherapy on the cell growth score, a composite of cell proliferation, viability, apoptosis, metastasis, DNA damage and other cancer hallmarks. The mechanism of action of each drug was mapped to each patient's genome and biological consequences determined response. Multivariate logistic regression models for clinical response and likelihood ratio tests were used to assess the contribution of the Cellworks Biosimulation Platform beyond PPT. Similarly, multivariate Cox proportional hazards models were used to test the hypothesis that the Cellworks Biosimulation Platform is predictive of overall survival (OS) and provides predictive information beyond PPT alone. Scoring quantifies the benefit of each drug used to treat each patient's AML. Kaplan-Meier curves, associated log rank tests, and median OS are provided for patients predicted by predefined low and high treatment benefit groups. Results: The TRI Score, scaled from 0 to 100, predicted complete response (CR) (likelihood ratio χ 12 = 52.54, p < 0.0001). Specific leukemia therapies generated a variable likelihood of benefit for individual patients. Notably, Cellworks biosimulation was able to predict treatment benefit or failure better than PPT alone (likelihood ratio χ 12 = 14.86, p < 0.0001). The use of therapy biosimulation to select therapy is estimated to increase the odds of CR by 19% per every 25 units of the TRI Score. TRI was also a significant predictor of OS (likelihood ratio χ 12 = 80.41, p < 0.0001) and provides predictive information above and beyond PPT alone (likelihood ratio χ 12 = 58.70, p < 0.0001 ). Inclusion of the Cellworks Biosimulation Platform is estimated to reduce the hazard ratio for death above and beyond PPT alone by 16% per every 25 units of the TRI Score. Furthermore, predictiveness curves suggest that approximately 25% of de novo AML patients had low probabilities of CR resulting in lower OS and could benefit substantially from inclusion of drugs and combinations identified by biosimulation into frontline management. Conclusions: By predicting the impact of aberrations and copy number alterations on drug response, the Cellworks Biosimulation Platform can improve treatment outcomes for AML patients. The Cellworks TRI predicts response and OS beyond PPT alone, and the Cellworks Biosimulation Platform provides individualized, networked-based alternate treatment options for patients predicted to be non-responders to standard care. Disclosures Howard: Sanofi: Consultancy, Other: Speaker fees; Cellworks Group Inc.: Consultancy; Servier: Consultancy. Watson: Cellworks Group Inc.: Consultancy, Other: Advisor; CellMax Life: Consultancy, Other: Advisor; AlloVir: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioAi Health: Consultancy, Membership on an entity's Board of Directors or advisory committees. Castro: Cellworks Group Inc.: Current Employment; Bugworks: Consultancy; Guardant Health Inc.: Speakers Bureau; Exact sciences Inc.: Consultancy; Caris Life Sciences Inc.: Consultancy; Omicure Inc: Consultancy. Kapoor: Cellworks Group Inc.: Current Employment. Nair: Cellworks Group Inc.: Current Employment. Prasad: Cellworks Group Inc.: Current Employment. Rajagopalan: Cellworks Group Inc.: Current Employment. Alam: Cellworks Group Inc.: Current Employment. Roy: Cellworks Group Inc.: Current Employment. Sahu: Cellworks Group Inc.: Current Employment. Lala: Cellworks Group Inc.: Current Employment. Basu: Cellworks Group Inc.: Current Employment. Ullal: Cellworks Group Inc.: Current Employment. Narvekar: Cellworks Group Inc.: Current Employment. Ghosh: Cellworks Group Inc.: Current Employment. Sauban: Cellworks Group Inc.: Current Employment. G: Cellworks Group Inc.: Current Employment. Agrawal: Cellworks Group Inc.: Current Employment. Tyagi: Cellworks Group Inc.: Current Employment. Suseela: Cellworks Group Inc.: Current Employment. Raju: Cellworks Group Inc.: Current Employment. Pampana: Cellworks Group Inc.: Current Employment. Patel: Cellworks Group Inc.: Current Employment. Mundkur: Cellworks Group Inc: Current Employment. Christie: Cellworks Group Inc.: Current Employment. Macpherson: Cellworks Group Inc.: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

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UTX mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.3148.3148 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3148-3148

Author(s):

Yasunobu Nagata ◽

Tomas Radivoyevitch ◽

Hideki Makishima ◽

Cassandra M. Hirsch ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

De Novo ◽

Missense Mutations ◽

Loss Of Function ◽

Advisory Committees ◽

Female Ratio ◽

Myeloid Neoplasms ◽

De Novo Aml

Abstract Genetic studies in myeloid neoplasms have revealed that somatic mutations and deletions frequently affect epigenetic regulation via DNA methylation and histone modification. One frequently affected epigenetic complex is polycomb repressive complex 2 (PRC2), which trimethylate Histone3Lysine27 (H3K27) to create a repression mark for the expression of a variety of genes that includes essential homeobox genes. Members of this complex include EZH2, EED and SUZ12, which exert methyltransferase activity, and perhaps indirectly also DNTM3A and ASXL1. The histone demethylase ubiquitously transcribed X (UTX) counters the enzymatic activity of PRC2 by removing di- and trimethyl groups from H3K27. It thus removes repressive marks. We were the first to report UTX mutations in a small portion of CMML and MDS cases. In another cohort, frequent somatic loss-of-function mutations in UTX were found in ALL. The goal of the present study was to understand the functional role of UTX and the consequences of its mutations on H3K27 methylation status, specifically with respect to their relevance to myeloid neoplasia. A total of 1,979 patients with various myeloid neoplasms (n = 1,686) and other hematologic disorders (n = 293) were screened for gene mutations in UTXand other reported driver genes relevant to myeloid neoplasms using whole exome sequencing and/or targeted deep-sequencing. Expression array analyses were performed on 200 samples. In addition, we also used sequencing and expression data from the de novo AML TCGA repository. UTXwas mutated in 55 (2.8%) cases out of 1,979 cases. The mutations were found in 2.5% of MDS, 3.1% de novo AML (including 8.3% CBF AML), 4.8% MDS/MPN, and 1.4% secondary AML (sAML). Of those, 77% were missense mutations and 23% nonsense, splice site and frameshift mutations, without an evident hot spot. The male-to-female ratio was 1.2 (55% vs. 45%). UTX gene expression was significantly higher in females than in males (p<.001). After gender adjustment and dichotomized using a threshold of expression of 2 standard deviations from the mean, low UTX expression levels were identified in 13/183 (7%) individuals. When we focused on clonal burden using variant allele frequencies (VAFs) to investigate clonal architecture within the tumor population, in 37 cases UTX constituted subclonal events and in 18 they were dominant. We then examined the molecular context of UTX lesions. Average mutation burden in patients with UTX mutations was higher than in WT UTX carriers (4 vs. 1.5, p<.001). UTX mutations co-occurred with other driver genetic mutations such as ASXL1, ZRSR2, CUX1, NF1. Surprisingly, EZH2 mutations are also enriched in UTX mutated cases although they function in the opposite direction of H3K27 trimethylation. Focusing on dominant clone and subclonal events in cases with these two mutations, EZH2mutations were enriched in cases with dominant UTX clone. This suggests that they play important roles in the cancer's pathogenesis. To clarify the role of UTX in the maintenance of leukemia, genomic knockouts of UTX were developed in human leukemic cell lines using the CRISPR-Cas9 system. RNA sequencing revealed that knockout cell enrichment for developmental regulators such as Hox genes. In addition, we made knockdowns of human CD34+ cells using short hairpin RNAs against UTX. The cells showed enhanced colony formation and increased replating efficiency consistent with retained clonogenicity. The truncating pattern of UTX mutations in myeloid neoplasms suggests that they are loss-of-function hits. Missense mutations thus need to be confirmed. Functional analysis in vitro shows that low expression of UTX may have functionally equivalent consequences. If so, an additional 7% of patients may have low UTX expression, and may thus phenocopy patients with UTX mutations. Combined, a total of ~10% out of myeloid neoplasm patients may harbor UTX deficiency. Epigenetic modifying drugs related to H3K27 such as inhibitors of EZH2and histone deacetylases are in development. It is controversial to which patients they should be applied. Our findings could be key to a deeper understanding of epigenetic alterations, drug function, and response. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Mukherjee:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. 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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Genotype-Phenotype correlation in Dravet Syndrome with SCN1A mutation increase efficiency of molecular diagnosis

Journal of Epilepsy and Clinical Neurophysiology ◽

10.1590/s1676-26492012000200009 ◽

2012 ◽

Vol 18 (2) ◽

pp. 60-62

Author(s):

MC Gonsales ◽

P Preto ◽

MA Montenegro ◽

MM Guerreiro ◽

I Lopes-Cendes

Keyword(s):

Protein Function ◽

Copy Number ◽

Mutation Screening ◽

Copy Number Variants ◽

Febrile Seizures ◽

Copy Number Variations ◽

Dravet Syndrome ◽

Missense Mutations ◽

The Impact ◽

Doose Syndrome

OBJECTIVES: The purpose of this study was to advance the knowledge on the clinical use of SCN1A testing for severe epilepsies within the spectrum of generalized epilepsy with febrile seizures plus by performing genetic screening in patients with Dravet and Doose syndromes and establishing genotype-phenotype correlations. METHODS: Mutation screening in SCN1A was performed in 15 patients with Dravet syndrome and 13 with Doose syndrome. Eight prediction algorithms were used to analyze the impact of the mutations in putative protein function. Furthermore, all SCN1A mutations previously published were compiled and analyzed. In addition, Multiplex Ligation-Dependent Probe Amplification (MLPA) technique was used to detect possible copy number variations within SCN1A. RESULTS: Twelve mutations were identified in patients with Dravet syndrome, while patients with Doose syndrome showed no mutations. Our results show that the most common type of mutation found is missense, and that they are mostly located in the pore region and the N- and C-terminal of the protein. No copy number variants in SCN1A were identified in our cohort. CONCLUSIONS: SCN1A testing is clinically useful for patients with Dravet syndrome, but not for those with Doose syndrome, since both syndromes do not seem to share the same genetic basis. Our results indicate that indeed missense mutations can cause severe phenotypes depending on its location and the type of amino-acid substitution. Moreover, our strategy for predicting deleterious effect of mutations using multiple computation algorithms was efficient for most of the mutations identified.

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