BCOR Mutations Research Articles

Germline Variants of RUNX-1 in Myeloid Malignancy

Mutation of the master transcription factor RUNX1 (NM_001754) has a well characterized role in the pathogenesis of myeloid neoplasms. RUNX1 is located at 21q22 and is composed of a c-terminal transactivation domain (TAD) (269-480) and an n-terminal RUNT domain (amino acid 76-209) that binds DNA and mediates heterodimerization with CBF-β. If the RUNT domain is disrupted a RUNX1 mutant will behave in a dominant negative fashion. Mutations leaving the RUNT domain intact result in haploinsufficiency compounded by CBF-β binding site competition with the wild-type allele. Germline (GL) RUNX1 mutations cause the autosomal dominant predisposition syndrome familial platelet disorder with propensity to myeloid malignancy (FPDMM). In FPDMM, RUNX1 mutations, including RUNT domain missense mutations or nonsense mutations throughout the gene, confer mild chronic platelet disorders and a lifetime risk of MDS/AML. As with other GL predisposition syndromes a secondary event is considered necessary for progression, typically an additional mutation in the wild type RUNX1 allele. Onset of MDS/AML occurs at a median age of 33 years but the latency can be variable and ease of detection in younger cases likely distorts summary statistics. Inherited mutations in adults may be difficult to distinguish and have not been systematically explored. We hypothesize that a fraction of patients with otherwise typical RUNX1-positive MDS or related disorders are in fact carriers of a RUNX1 GL mutation.DNA obtained from a cohort of 1451 patients with myeloid neoplasia was analyzed using a multiamplicon deep next-generation sequencing (NGS) panel including all ORFs of RUNX1. A total of 124 patients (8.5%) were found to carry 117 unique RUNX1 mutations. Of these, 59 hits were missense and 58 nonsense; and in 94 (76%) of cases the RUNT domain was disrupted. Eighteen (15.3% RUNX1 positive patients) were previously described in FPDMM. We applied various bio analytic criteria to designate somatic status in 57 patients. The remaining 64 RUNX1 mutations were further investigated, when possible, by paired capillary sequencing of CD3- mononuclear DNA and in-vitro expanded CD3+ T-Cell DNA. We found 5/31 (16%) of these cases carried mutation in both myeloid and lymphoid lineages and were designated GL. This included 2 post-RUNT truncations (p.Y281*, p.S410*) and two RUNT domain missense mutations (p.R80C, p.S141L) all of which are expected to produce a dominant negative phenotype. A 5th GL mutation of uncertain significance in the TAD (p.M310I) was also discovered. Excluding this TAD missense mutation, each of these have been described as somatic factors in myeloid disease. The truncation at amino acid 410 is further downstream than all reported FPDMM variants to date. Chart review revealed an anamnestic presence of thrombocytopenia in 80% of proposed FPD patients. Suggestive family history was found in both truncated cases, with hematologic malignancy presenting in a 1st degree relative before 30 years of age. Median age at diagnosis of MDS/AML in GL cases was 46 years (range: 18-68 years) compared to 65 years in the cases designated somatic (range: 37-87 years). Two cases (p.S410* and p.M310I) had cytogenetic abnormalities on the 21st chromosome (trisomy 21 and t(8;21) respectively) and had no other molecular abnormalities detected by our NGS panel. A mutation in GPR98 of unknown significance was found with p.Y281*. Lastly the p.S141L mutant had cooperating mutations in chromatin modifiers ASXL1 and BCOR, and in RNA splicing gene LUC7L2. This study of an exemplary GL leukemia gene suggests that systematic searches for known and potential GL predisposition genes in otherwise typical adult cohorts may reveal a GL role in the evolution of myeloid neoplasms. Future work to determine genetic predisposition to leukemia in adults is essential. DisclosuresSekeres: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. 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.

Clono-Specific Evaluation of Minimal Residual Disease in Acute Myeloid Leukemia

Background:The genetic landscape of adult acute myeloid leukemias (AML) has been recently unravelled. This makes achievable the determination of a comprehensive profile of driver lesions for virtually all patients at diagnosis. Recent studies using multi-target minimal residual disease (MRD) strategies with around 1% sensitivity indicate that the clearance of all molecular events after chemotherapy is associated with better survival. To improve the clono-specificity and the sensitivity of this approach, after a precise determination of AML clonal composition, we combined cytogenetic, FISH, and high sensitivity deep sequencing technologies to monitor the MRD in a series of 69 patients.Methods:Forty-five consecutive patients reflecting the genetic diversity of AML were prospectively included and 24 patients were retrospectively studied. All patients received an anthracycline + cytarabine based regimen. The clonal architecture was established at diagnosis based on NGS-targeted resequencing (122 gene panel) and cytogenetic data. Lesions were next investigated in complete remission (CR). Based on the initial clonal composition, targeted resequencing panels were designed to improve the sensitivity by the use of unique molecular barcodes (Haloplex High Sensitivity, or HS-NGS assay). Cytogenetic events were evaluated by FISH.Results:In the 69 patients, a median of 4 genetic or chromosomal events were identified per patient (range 0-10). One patient had no evaluable target allowing MRD evaluation in 68/69 patients.To determine the threshold of detection of the HS NGS assay, we analyzed the frequency of mutant reads in multiple samples expected to be wild type for 31 given SNVs and 2 indels. A consensus threshold of detection was set at a variant allele frequency (VAF) of 0.2% for all lesions.In CR samples, early initiating events frequently persisted after treatment, especially mutations in DNMT3A, TET2, ASXL1, EZH2, IDH1, TP53, SRSF2, and U2AF1. Mutations in FLT3, NRAS, KIT, NPM1, CEBPA, WT1, IDH2 and BCOR were the most frequently cleared events.Seven patients did not reach CR after one course, and two had no available material after one course. In the 59 remaining patients, we tested whether the global response level of all targets was associated with prognosis. We used the median VAF of the first events of all clonal architectures to separate good responder from poor responders (i.e. VAF = 1.66%). At 2 years, there was a trend to lower leukemia free survival (LFS) probability in poor responders (31.7+/-9.9% vs 51.7+/-9.8%, p=0.08) with no translation in overall survival (OS).We next investigated if the persistence of two or more detectable markers was associated with prognosis. The 58 patients with more than one evaluable event were consequently separated in two groups: patients with 0 or 1 marker above the detection threshold after treatment (n=31), and patients with 2 or more detectable lesions (n=27). At 2 years, DFS was 64.9+/-9.3 % in patients with 0 or 1 detectable marker vs 19.8+/-8.7% in patients with 2 or more detectable markers (p=0.001). OS probability was higher in patients with 0 or 1 detectable marker 84+/-6.6% vs 57.1+/-10.5% (p=0.023). When focusing on the 40 patients with intermediate cytogenetics, persistence of 2 or more markers was associated with lower LFS (57+/-11.8% vs 19.4+/-10.5 p=0.0048) and with a trend to lower OS (85+/-8% vs 61+/-11.9% p=0.07). Similar results were observed when restricting the analyses to the 42 prospectively included patients (At 2 years: LFS 73+/-10% vs 24+/-10%, p=0.0026 and OS 90.2+/-6.6% vs 62.8+/-11.5%, p=0.036).In 50 patients with 3 or more identified events, the persistence of 3 or more markers after one course was associated with a very high risk of relapse (DFS 23.5+/-10.3 % vs 75.8 +/-7.5% at one year, p<0.0001; median DFS at 7 months in the non-responder group and not reached after two years in the responder group), and lower OS probability (84.8+/-6.2 vs 45.2+/-13.5% at 2 years, p=0.026)ConclusionOur study shows the high prognostic value of a personalized multi-target clono-specific MRD evaluation that can be used in nearly all AML patients. Detection of two or more events in more than 0.4% cells after one course is associated with lower survival, in particular in intermediate cytogenetic patients. Larger studies are needed to confirm the results and to evaluate if this strategy could be useful to guide treatment decisions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alterations of BRCA1 and PALB2 Define a Novel Class of Complex-Karyotype AML with a Very Bad Prognosis

BRCA1 is one of the most important gene associated with familial breast and ovarian cancer susceptibility and is involved in the DNA damage repair and cell cycle arrest. Alterations in BRCA1 and their consequences are well characterized in breast and ovarian tumors, while little is known about its role in Acute Myeloid Leukemia (AML).We aimed to investigate the frequency and the interplay of BRCA1 alterations and patterns of somatic mutations (SNVs) and copy number variations (CNVs) of in AML patients.We genotyped 118 AML samples at either diagnosis or relapse by Single Nucleotide Polymorphism (SNP) array (SNP6.0 and Cytoscan HD, Affymetrix) to detect CNVs. In addition, we performed Whole-Exome Sequencing (WES, 100 bp paired-end, Illumina) of 56 genotyped AML patients to detect SNVs and small indels (MuTect and Varscan 2.0). Differences in survival were assessed using Kaplan-Meier survival analysis and Long-Rank test, or Breslow when indicated.We detected BRCA1 loss in 14 out of 118 patients (12%), ranging from 1,6 Kb to the loss of the entire chromosome 17, with none of the BRCA1 loss patients having a normal karyotype. Moreover, BRCA1 losses significantly co-occurred with PALB2 and RAD50 losses. Notably, the PALB2 loss (chromosome 16) mostly involved exon 12 and was detected also in patients without the BRCA1 loss, with a frequency of 11 out of 118 cases (9%), suggesting a pivotal and previously undescribed role in AML pathogenesis. BRCA1 loss patients were enriched for copy number alterations in genes involved in the regulation of mitotic recombination, DNA repair and positive regulation of kinase activity compared with BRCA1 wild-type cases (p <0.01). To investigate which genes and biological functions cooperates with the pathways affected by structural alterations, we integrated the analysis with WES data on 56 out of 118 AML patients. We focused on the genes involved in the DNA damage repair (DDR) and we identified somatic SNVs in BRCA1, TP53 and in the Fanconi Anemia genes BRCA2 and FANCE. Analysis of co-occurrence and mutual exclusivity revealed that BRCA1 loss and DDR-related alterations (BRCA1, PALB2, BRCA2, and FANCE) were significantly associated with TP53 alterations (p=0.0001 and p=0.0003, respectively). In addition, alterations in the DDR genes were mutually exclusive with mutations in KRAS, IDH1/2, BCOR, BCORL1, NPM1 and myeloid transcription factors as RUNX1 and CEBPA. Notably, patients without alteration in the DDR genes were enriched for mutations in DNMT3A and FLT3.Finally, BRCA1 and PALB2 alterations defined group of patients with poor overall survival (OS). In particular, patients with alterations of either BRCA1 or PALB2 had a worst prognosis compared to patients without alterations in the two genes (p=0.009 and p=0.001, respectively). Patients harboring PALB2 loss had a poorer prognosis compared with patients with complex karyotype (p=0.021, Breslow test). In addition, double BRCA1 and PALB2 loss, single BRAC1 or PALB2 hits and lack of alterations in BRCA1 and PALB2 defined three different classes of risks in our AML cohort (p<0.001).In summary, we define for the first time a novel subgroup of AML patients characterized by alterations targeting the DDR pathway, and in particular the BRCA1 and PALB2 genes. Our data suggest that a signature of genes involved in the DDR and cell cycle regulation synergize and led to the uncontrolled proliferation, independently of DNA damage accumulation. Our results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type.Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. [Display omitted] DisclosuresGuadagnuolo:CellPly S.r.l.: Employment. Martinelli:Novartis: Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.

The Mutational Patterns Associated with Cytogenetic Subsets of De Novo Acute Myeloid Leukemia (AML): A Study of 1603 Adult Patients (Pts)

The classification of AML is based on the morphology of leukemic blasts, their immunophenotypes, and the presence of specific chromosomal abnormalities and selected gene mutations. It is widely acknowledged that some gene mutations are enriched in certain cytogenetic subsets, in which they may serve as therapeutic targets. We created a comprehensive oncoprint of mutations associated with centrally reviewed cytogenetic findings in 1603 adults (1080 aged <60 years, and 523 aged ≥60 years) with de novo AML (excluding acute promyelocytic leukemia) who were treated on CALGB/ALLIANCE protocols. The pts were classified into 5 cytogenetic subgroups: (1) cytogenetically normal (CN); (2) with complex karyotype (CK); (3) with t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22), i.e., core-binding factor (CBF)-AML; (4) with balanced rearrangements other than t(8;21) and inv(16)/t(16;16); (5) with unbalanced chromosomal abnormalities in non-complex karyotype.Using a targeted next-generation sequencing panel, we analyzed the mutational status of 80 cancer- and leukemia-associated genes. The mutated genes were assigned to 9 previously described by the Cancer Genome Atlas Research Network (N Engl J Med. 2013;368:2059) functional groups: chromatin remodeling, cohesin complex, kinases, methylation-related, NPM1, RAS pathway, spliceosome, transcription factors, and tumor suppressors. The mutational oncoprint revealed major differences in gene mutation frequencies among the 5 major cytogenetic groups.CN-AML pts had a broad mutational spectrum, involving all functional groups. Mutations in methylation-related genes (61% of pts), NPM1 (57%), and kinases (46%) dominated the mutation pattern.TP53 mutations were most common in pts with CK (38%). The second most common mutation, TET2, was detected only half as often (17%). The mutation patterns associated with "typical" (containing abnormalities resulting in loss of chromosome material from 5q, 7q and/or 17p), and "atypical" (without the aforementioned abnormalities) CKs differed. TP53 mutations were present in 52% of pts with typical but only in 5% of pts with atypical CKs (P<0.001). Conversely, the latter pts harbored mutations in another tumor suppressor gene, PHF6, more often (15% vs. 2%, P=0.03). Furthermore, atypical CKs had a broader spectrum of recurrent mutations, with 6 mutations occurring in ≥10% of pts (IDH2, NPM1, PHF6, TET2 and ZRSR2 mutations and FLT3-TKD), compared with only 2 genes, TP53 and TET2, mutated in ≥10% of pts with typical CKs, suggesting major biological differences between typical and atypical CKs.CBF-AML pts with had few detectable mutations, with a median of 1 (range, 0-5) versus 3 mutations (range, 0-9) detected in the remaining AML pts (P<0.001).In AML with balanced rearrangements, AML pts with MLL-rearrangements often had mutations in RAS pathway genes, whereas they rarely harbored mutations in other functional groups compared with pts with non-MLL-rearranged balanced translocations [MLL-rearranged vs. non-MLL-rearranged: chromatin remodeling, 9% vs. 26%; methylation-related, 12% vs. 23%; spliceosome, 12% vs. 24%]. Some cytogenetic subgroups with particular recurrent chromosome abnormalities presented with specific molecular features, supporting claims that they represent separate AML entities. For instance, 51% of pts with t(6;11)(q27;q23) harbored KRAS mutations, compared with only 3% of all remaining pts. Among t(9;22)(q34;q11.2)-positive pts, 47% had mutations in RUNX1,and 27% mutations in ZRSR2. Pts with inv(3)(q21q26.2/t(3;3)(q21q26.2) had frequent mutations in SF3B1 and BCOR (both 38%).Pts with gains or losses of chromosome material in non-complex karyotypes (unbalanced chromosomal abnormalities) were characterized by high mutation frequencies in methylation-related (55%) and spliceosome genes (34%). Strikingly, spliceosome mutations were enriched in pts with chromosome gains, with almost half of these pts harboring one or more mutations. Except for pts with sole +11, who most often harbored U2AF1 mutations (43%), SRSF2 was the most frequently mutated spliceosome gene in pts with unbalanced chromosomal abnormalities.Our comprehensive depiction of frequencies of single gene mutations and functional groups in recurrent cytogenetic subsets may be useful in guiding mutation testing in AML pts, and result in more focused application of targeted therapy of AML. DisclosuresWang:Immunogen: Research Funding; Incyte: Speakers Bureau. Stone:Pfizer: Consultancy; Sunesis Pharmaceuticals: Consultancy; Novartis: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Agios: Consultancy; Juno Therapeutics: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; ONO: Consultancy; Celator: Consultancy; Amgen: Consultancy; Jansen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Roche: Consultancy.

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

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. DisclosuresCarraway: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.

Clinical and Biological Implications of CUX1 Mutations in Myeloid Neoplasms

Recurrent somatic mutations of CUX1 are described in myeloid neoplasms. CUX1 is located at chromosome 7q22.1; -7/del(7q) involving CUX1 locus are common abnormalities in myelodysplastic syndromes (MDS). Mutations and loss of heterozygosity involving CUX1 have been also described in breast, lung and uterine cancers. Preliminary functional studies, lack of a mutational hotspot and coincidental deletions suggest loss of function/hypomorphic consequences of these molecular defects. CUX1 (p200), contains 4 evolutionarily conserved DNA-binding domains, including 3 CUT repeats and a CUT homeodomain. Functionally, CUX1 regulates many genes involved in DNA replication and chromosome segregation. Cell-based assays have established a role for CUX1 in the control of cell-cycle progression, cell motility, and invasion .The objective of this study is to assess the molecular context and clinical significance of CUX1 mutations and deletions in myeloid neoplasms.We analyzed a subset of 1478 patients [24% lower-risk MDS, 17% higher-risk MDS, 22% primary (p)AML, 14% secondary AML, 14% MDS/myeloproliferative neoplasms (MPN) and 9% MPN] for the presence of CUX1 mutations and deletions. No CUX1 mutations were found in core binding factor AML. We correlated the presence of these lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and associated somatic mutations. Copy number variation and their boundaries were analyzed by Single Nucleotide Polymorphism (SNP) arrays and mutations by multiamplicon deep sequencing utilizing a panel targeting 60 most commonly mutated genes in myeloid neoplasms.In total cohort 4 % of patients had CUX1 mutations and 6% had locus deletions (affecting ch 7q commonly deleted region: 7q22.1) including 90% of del (7q) cases. Expression of CUX1 is significantly lower in AML with -7/del(7q) compared to AML with normal cytogenetics (p<.00001) and also in MDS with -7/del(7q) compared tohealthy controls (p=.004). Additionally, decreased expression of CUX1 was found in 15% of MDS and 8% of AML patients without -7/del(7q) or related mutations. Cases with lower expression had worse OS compared to patients with higher expression (p=.002). In terms of configuration, most mutations were heterozygous, 5% of mutations were hemizygous and 4% were homozygous (due to UPD). Among 75 somatic CUX1mutations; 72% were missense, 20% where frame shift and 8% where non sense.CUX1 mutations were associated with either lower-risk MDS (p=.0001) and pAML (p=.04) while deletions involving the CUX1 locus were significantly related to higher-risk MDS (p=.05). Heterozygous CUX1 mutations were more commonly associated with normal cytogenetics (p=.01). Patients with -7/del(7q) frequently represented del(5q) (p=.04) and thrombocytopenia (p=.001). The OS of patients with CUX1 mutations was shorter (p=.04) as was that of patients with CUX1/deletions (p=.02) when compared to wild type.We subsequently studied the molecular background of CUX1 alterations. CUX1 mutations (vs. wild type) were associated with TET2 (31% vs. 14%, p=.006), ASXL1 (29% vs. 9%, p=.0005), BCOR (28% vs. 8%, p=.0004), and cohesion mutations (26%, vs. 5%, p=.0005), while NPM1 mutations showed the reverse relationship (1% vs. 7%, p=.03). RAS and CUX1 mutations were mutually exclusive (0% vs. 6%, p=.03).When we analyzed clonal hierarchy in the context of CUX1 mutations; dominant CUX1 mutations (24%; mean VAF=49%); were accomplished by ASXL1 (21%) and SRSF2 (14%) mutations which were the most common secondary events in this context. Phenotypically, dominant CUX1 mutations were associated with MDS/MPN (42%) and MDS (33%). 14% of CUX1 mutant cases did not harbor any other alterations and were not associated with a discernable phenotype. Secondary CUX1 lesions (62%; mean VAF=22%) were found in the context of dominant TET2 mutations (16%). The pathomorphologic context of secondary CUX1 mutation did not differ from that of primary lesions. AML seemed to be underrepresented (p=.006) and MPN overrepresented (p=.019) among dominant CUX1 mutant cases.In conclusion, CUX1 lesions including locus deletions with haploinsuffciency, mutations and a fraction of cases with decreased CUX1 expression can be encountered in MDS and related neoplasms, chiefly AML. CUX1 dysfunction is associated with poor survival likely due to its distinct molecular background. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

The Number of RUNX1 Mutations and the Presence of One Intact RUNX1 Allele Influence Cytogenetic Abnormalities, Additional Molecular Mutations and Prognosis in RUNX1 Mutated AML

Background: According to the revised WHO classification of 2016, AML with mutated RUNX1 constitutes a new provisional entity. We previously reported that the subgroup of RUNX1-mutated AML with RUNX1 wild-type (WT) loss is associated with a distinct pattern of cytogenetic and molecular genetic abnormalities and with an adverse prognosis. However, the impact of multiple RUNX1 mutations is unclear yet.Aims: Evaluation of associated genetic alterations and prognosis in AML with >1 RUNX1 mutation as compared to those with (1) RUNX1 WT loss and (2) one RUNX1 mutation.Patient cohorts and methods: The total cohort comprised 467 AML cases with RUNX1 mutations (mut) (296 male, 171 female). Median age was 72 years (range: 18-91 years). All patients were investigated using chromosome banding analysis (CBA) and amplicon sequencing of RUNX1. The cohort was split into the subgroups with RUNX1 WT loss (UPD or RUNX1 deletion detected by genomic arrays) (n=50), cases with >1 RUNX1mut (n = 94) and cases with 1 RUNX1mut and conservation of the RUNX1 WT allele (n = 323). Of these, 50, 55 and 58 cases, respectively, were selected for further mutation analyses of ASXL1, BCOR, CBL, CEBPA, DNMT3A, ETV6, EZH2, FLT3-ITD, FLT3-TKD, GATA2, IDH1, IDH2, KIT, KRAS, MLL-PTD, NPM, NRAS, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and WT1. Variants of unknown significance were excluded from statistical analysis.Results: In the total cohort of 467 cases, CBA revealed a normal karyotype (NK) in 53% of patients, 18% harbored trisomies, 3% showed a complex karyotype (>3 abnormalities), 26% other aberrations. The proportion of cases with trisomies was largest in cases with RUNX1 WT loss (26%), followed by >1 RUNX1mut (19%) and 1 RUNX1mut (16%). In more detail, in the total cohort, 56% of cases with trisomies harbored +8, in 30% +13 was found. A similar pattern was observed for 1 RUNX1mut, whereas in cases with WT loss +13 was the most abundant trisomy (+8: 66% in 1 RUNX1mut vs. 31% in WT loss, p=0.022; +13: 15% vs. 62%, p<0.001). Cases with >1 RUNX1mut showed an intermediate distribution (+8: 44%, +13: 50%).Missense mutations were the most abundant mutation type (53%) inWT loss cases, followed by frameshift mutations (28%). By contrast, in cases with 1 RUNX1mut, frameshift mutations were found more frequent (45%, p=0.016), whereas missense mutations were detected at a frequency of 31% (p=0.006). In cases with >1 RUNX1mut, both were observed at similar frequencies (missense: 36%, frameshift: 38%).Mutation analyses of 163 selected cases revealed SRSF2 (39%), ASXL1 (36%), DNMT3A (19%), BCOR (18%), IDH2 (17%), SF3B1 (17%) and TET2 (17%) mutations as most frequently mutated genes in the total cohort. Cases with RUNX1 WT loss showed a higher frequency of ASXL1mut compared to the other cases (50% vs. 29%, p=0.013), while U2AF1mut were absent from this group (0% vs. 15%, p=0.019). Differences between cases with and without RUNX1 WT loss were also detected for DNMT3A, TET2, SF3B1 (more abundant in WT loss) and IDH2, WT1 (less abundant in WT loss), although these were not statistically significant. For many genes, the group of cases with >1 RUNX1mut showed an intermediate abundance pattern, or mutation frequencies similar to cases with 1 RUNX1mut were observed. Mutations in spliceosome genes (SF3B1, SRSF2, U2AF1, ZRSR2) were very abundant in all subgroups but less frequent (although not statistically significant) in cases with 1 RUNX1mut.Median overall survival (OS) in the total cohort was 14 months. WT loss (OS: 5 months) and >1 RUNX1mut (14 months) showed an adverse impact on prognosis compared to 1 RUNX1mut (22 months; p=0.002 and p=0.048, respectively). Mutations in ASXL1 and KRAS showed a negative impact on OS in the total cohort (10 vs. 18 months, p=0.028; 1 vs. 15 months, p<0.001), whereas DNMT3A mutations negatively affected OS in WT loss only (in WT loss: 1 vs. 7 months, p=0.005).Conclusion: 1) RUNX1 mutated AML cases with WT loss show a high frequency of +13, RUNX1 missense mutations and accompanying ASXL1 mutations. 2) They clearly separate from cases with 1 RUNX1mut, which depict a predominance of +8, RUNX1 frameshift mutations and mutations in IDH2 and WT1. 3) Patients with >1 RUNX1mut show a pattern intermediate between the former two subgroups. 4) Loss of RUNX1 WT and > 1 RUNX1mut showed an adverse impact on OS. Thus, not only the presence and number of RUNX1mut but also the conservation of an intact RUNX1 allele is biologically and clinically relevant. DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Driver Mutations Affecting Natural Killer/T Cell Lymphoma

Natural killer/T cell lymphoma (NKTCL) is an aggressive subtype of non-Hodgkin lymphoma that is rare overall but has a higher prevalence in Chinese and Hispanic populations. Recent sequencing efforts have improved the understanding of the disease and revealed a number of genes that may drive the pathogenesis of NKTCL. These efforts were largely limited by the number of cases studied. Based on previously reported whole exome sequencing data in NKTCL and other lymphoid malignancies and on the frequency and potential biological significance of the mutant, we designed a 334-gene panel and sequenced 105 NKTCLs. We characterized single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations (CNAs) that may have pathogenic roles.Tumor DNA was isolated from 74 Chinese and 31 Hispanic NKTCL patients. For 7 of these, matched normal DNA was extracted from peripheral blood. The 334 genes in the custom panel were captured using the Agilent SureSelect platform, and the exons of these genes were sequenced on an Illumina HiSeq 2500 instrument. For cases without matched normal samples, we discriminated between somatic and germline variants with an in-house machine learning algorithm which is based on information of public variant databases, variant annotations, and sequencing statistics, and followed by manual inspection. CNAs were determined based on the untargeted sequences using an R package, CopywriteR.We identified a total of 479 SNVs and 113 indels in 155 genes in 102 out of 105 patients. Mutations in STAT3 and JAK3 were identified in 30% and 7% of the cases, respectively. The hotspot mutations Y640F, S614R, and G618R in the SH2 domain of STAT3 and A573V of the JH2 domain of JAK3 were observed in most of these cases. 46% of the cases harbored one or more mutations in epigenetic regulators ARID1A, EP300, MLL2, MLL3, and TET2. Transcription co-repressors BCOR and NCOR2 were mutated in 25 of the cases. 13% of the cases contained mutations in HLA-A or CIITA, most of which were inactivating, suggesting possible defects in immune surveillance. DDX3X and TP53 mutations were detected in 19% and 10% of the cases, respectively. TP53 mutations were mutually exclusive with mutations in DDX3X and, except for one case, with STAT3 also. MGA mutations, which were present in 12% of the cases, were usually concurrent with STAT3 mutations. The mutational profiles of Chinese and Hispanic cases were similar, and only CD58 mutations were significantly more common in Hispanic patients (p=0.03). Comparison between NKTCL with localized and systemic presentation demonstrated that CCR7 was mutated more frequently in systemic cases (p=0.02). Prognostic analysis identified EP300 mutations as a potential marker for poorer survival (p=0.01). Frequent copy loss on chromosome 6q was observed and PRDM1, which is present on 6q, was mutated in 10% of the cases.In summary, we identified the profile of mutated genes in a large series of NKTCL. Most of the genes have been previously reported to be mutated in NKTCL, but the frequencies for some of the mutants were quite different. Interestingly, the mutational profiles of NKTCL in Chinese and Hispanic patients were very similar despite the geographic differences. Mutations leading to abnormal activation of the JAK-STAT3 pathway and abnormal chromatin modification were highly prevalent. BCOR, DDX3X and TP53 mutations were also frequent. Variants in CCR7 and EP300 were potential markers for systemic disease and poor prognosis, respectively. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comprehensive mutation profiling of mucinous gastric carcinoma

Mucinous gastric carcinoma (MGC) is a unique subtype of gastric cancer with a poor survival outcome. Comprehensive molecular profiles and putative therapeutic targets of MGC remain undetermined. We subjected 16 tumour-normal tissue pairs to whole-exome sequencing (WES) and an expanded set of 52 tumour-normal tissue pairs to subsequent targeted sequencing. The latter focused on 114 genes identified by WES. Twenty-two histologically differentiated MGCs (D-MGCs) and 46 undifferentiated MGCs (U-MGCs) were analysed. Chromatin modifier genes, including ARID1A (21%), MLL2 (19%), MLL3 (15%), and KDM6A (7%), were frequently mutated (47%) in MGC. We also identified mutations in potential therapeutic target genes, including MTOR (9%), BRCA2 (9%), BRCA1 (7%), and ERBB3 (6%). RHOA mutation was detected only in 4% of U-MGCs and in no D-MGCs. MYH9 was recurrently (13%) mutated in MGC, with all these being of the U-MGC subtype (p = 0.023). Three U-MGCs harboured MYH9 nonsense mutations. MYH9 knockdown enhanced cell migration and induced intracytoplasmic mucin and cellular elongation. BCOR mutation was associated with improved survival. In U-MGCs, the MLH1 expression status and combined mutation status (TP53/BCL11B or TP53/MLL2) were prognostic factors. A comparative analysis of driver genes revealed that the mutation profile of D-MGC was similar to that of intestinal-type gastric cancer, whereas U-MGC was a distinct entity, harbouring a different mutational profile to intestinal- and diffuse-type gastric cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract LB-180: The genetic landscape of Wilms tumor

Abstract Introduction: The National Cancer Institute's Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative seeks to characterize the genomes of high-risk pediatric tumors to identify therapeutic targets. The High-Risk Renal Tumor TARGET initiative includes the analysis of pre-therapy favorable histology Wilms Tumors (FHWT) that relapsed and tumors with diffuse anaplasia (unfavorable histology; DAWT). These two tumor subsets have survival rates of approximately 50% and 60%, respectively. Experimental procedures: Genomic sequencing (whole genome [WGS] or exome [WXS]), global copy number analysis, and global gene expression analysis were performed on a discovery set of 117 (78 FHWT, 39 DAWT) pre-therapy high-risk WTs treated on National Wilms Tumor Study-5 (NWTS-5). To determine the frequency of recurrent variants, targeted sequencing (Illumina HiSeq2500) was performed on a validation set of pre-therapy tumor DNA from a case-cohort of all FHWT treated on NWTS-5 (531 FHWT) and all available 118 DAWT treated on NWTS-5 (these groups include tumors from the discovery set). Results: WGS and WXS revealed an average of 21.74 ± 22.6 high-quality variants per DAWT (range, 3-131) and 13.8 ± 10.9 per FHWT (range 2-58). Genes previously reported to be recurrently mutated in WT were mutated at the following frequencies in the validation set: WTX (6%), CTNNB1 (15%), WT1 (7.5%), DROSHA (11%), DGCR8 (4.5%), XPO5 (2%), SIX1/2 (7%), and MLLT1 (3%). In addition, mutations were identified in three genes that impact the NMYC pathway, which is known to be involved in renal development. These include MYCN P44L/H (4%), MAX R60Q (2%), and novel mutations in NONO (2%); these mutations were mutually exclusive. Novel mutations in BCOR, a transcriptional corepressor that regulates both gene expression during development and chromatin modification, were found in 3% of validation set tumors. Analysis of global gene expression revealed significant up-regulation of genes associated with kidney development, extracellular matrix organization, and epithelial tube development in BCOR-mutant tumors compared with precursor lesions (5 hyperplastic perilobar nephrogenic rests). TP53 mutations were identified in 48% of DAWTs and 1% of FHWTs. The above data do not include copy number changes, which were recurrently detected in WT1, WTX, NMYC, and TP53. Conclusions: Through the TARGET initiative, we have identified several novel, potential driver mutations that occur in WT and have not been reported in other pediatric tumors. The majority of these genes are known to function in processes critical to early development and/or specifically in renal development. Many of these mutations are accompanied by Wnt activating mutations or 11p15 biallelic expression. However, approximately 50% of WTs lack clear driver mutations. Future studies will need to focus on elucidating epigenetic alterations in these tumors as well as genetic changes outside of protein-coding regions. Citation Format: Samantha L. Gadd, Amy L. Walz, Ariadne HAG Ooms, Vicki Huff, Daniela S. Gerhard, Malcolm A. Smith, Jaime M. Guidry Auvil, Leandro Hermida, Tanja Davidsen, Patee Gesuwan, Daoud Meerzaman, Yussanne Ma, Marco A. Marra, Jeffrey S. Dome, Charles G. Mullighan, David A. Wheeler, Oliver A. Hampton, Julie M. Gastier-Foster, Nicole Ross, Elizabeth J. Perlman. The genetic landscape of Wilms tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-180.

Abstract 113: Novel fusion transcripts identified by RNAseq cooperate with somatic mutations in the pathogenesis of acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is a highly heterogeneous disease and somatic mutations and fusion genes have a crucial diagnostic, prognostic and therapeutic role in AML. However, the leukemogenic potential of the fusions, their cooperation with somatic mutations and their prognostic role are still unknown. The aim of the study was to identify novel rare gene fusions having a causative role in leukemogenesis and to identify cooperative somatic mutations as potential targets for personalized therapies in AML cases with rare and poorly described chromosomal translocations. We performed RNAseq on bone marrow samples from 5 AML patients (#59810, #20, #84, #21 and #32). The presence of gene fusions was assessed with deFuse and Chimerascan. Putative fusion genes were prioritized using Pegasus and Oncofuse to select biologically relevant fusions. WES was performed on samples #59810, #20 and #21 and variants were detected using the software Mutect and GATK. The CBFβ-MYH11 chimera was identified in sample #84, with inv(16), thus confirming the reliability of our analysis. Sample #59810 carried the fusion transcript ZEB2-BCL11B (Driver Score, DS = 0.7), an in-frame fusion and a rare event in AML associated with t(2;14). The fusion transcript showed 3 splicing isoforms and ZEB2 and BCL11B transcripts were upregulated in patient's blasts, compared with 53 AML samples with no chromosomal aberrations in the 14q32 region. The WT1-CNOT2 chimera was also detected, which is a novel out-of-frame fusion (DS = 0.008) related to t(11;12) translocation. WES analysis showed SNVs in TET2 and BCOR genes. In sample #20 we identified two different fusion transcripts: CPD-PXT1 (DS = 0.07), which was the reciprocal fusion product of t(6;17) translocation, and SAV1-GYPB, which was cryptic at cytogenetic analysis (DS = 0.8). Interestingly, the latter fusion involved a tumour suppressor gene. Moreover, mutations in BCOR and NRAS were detected by WES. RNAseq on sample #21 identified a novel fusion event between chromosomes 19 and 7, involving the genes OAZ and MAFK (DS = 0.9). The 3’ partner gene is a transcription factor, a functional class recurrently altered in AML. This patient showed SNVs in CBL and SMC1A genes. Finally, no chimeras were confirmed in sample #32 having a t(12;18) translocation. Our data suggest that fusion events are frequent in AML and a number of them cannot be detected by current cytogenetic analyses. Gene fusions cooperate with somatic mutations in the genomic landscape driving AML pathogenesis and its heterogeneity. Moreover, the results indicate that different approaches, including WES, RNAseq bioinformatic and statistic tools need to be integrated in order to identify potential targets for personalized therapies. Acknowledgements: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Anna Ferrari, Giulia Paciello, Elisa Zago, Carmen Baldazzi, Viviana Guadagnuolo, Cristina Papayannidis, Valentina Robustelli, Enrica Imbrogno, Nicoletta Testoni, Massimo Delledonne, Ilaria Iacobucci, Tiziana Clelia Storlazzi, Elisa Ficarra, Pier-Luigi Lollini, Giovanni Martinelli. Novel fusion transcripts identified by RNAseq cooperate with somatic mutations in the pathogenesis of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 113.

Chronologic analysis of clonal evolution in acquired aplastic anemia and sMDS

Acquired aplastic anemia (AA) is a prototype of idiopathic bone marrow failure, which is caused by immune-mediated destruction of hematopoietic progenitors but is also characterized by frequent evolution to clonal myeloid disorders, such as myelodysplastic syndromes or acute myeloid leukemia. However, the chronological behavior of the clonality and its link to myelodysplastic syndrome or acute myeloid leukemia has not been fully explored. To define the clonality and its chronological behavior in AA, we performed targeted sequencing (N=439) in cases with AA. Somatic mutations were detected in 1/3 of our cases. Mutations were most frequently found in DNMT3A, followed by BCOR, PIGA and ASXL1. The prevalence of mutations increased with age. The clone sizes in DNMT3A and ASXL1 were prone to increase, whereas those of BCOR and PIGA were more likely to decrease or remain stable. Mutations in PIGA, BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and more favorable survival. On the other hand, other mutations were associated with worse outcomes. The chronological dynamics of clonality showed marked variability and were not necessarily associated with prognosis.

Mutually exclusive BCOR internal tandem duplications and YWHAE-NUTM2 fusions in clear cell sarcoma of kidney: not the full story.

Internal tandem duplication within the BCOR gene sequence that encodes the PUFD domain, important in the formation of the non-canonical or variant polycomb repressor complex 1 (v-PRC1), was very recently described in 100% of 20 clear cell sarcomas of kidney (CCSKs). None of those 20 cases bore the YWHAE-NUTM2 transcript, previously described by us in CCSK, and which constitutes the only other recurrent genetic aberration observed in CCSK, prompting consideration that these mutations might be mutually exclusive in CCSK. We analysed a cohort of 159 CCSKs and can now not only confirm that there is indeed mutual exclusivity of these BCOR and YWHAE mutations, but also show that a substantial proportion (in this series 11.8%) of CCSKs bear neither mutation when tested by these assays, raising the possibility of distinct aetiologies for subsets of CCSK. Clinical differences observed between the subsets support this notion. As CCSK may show poor chemo-responsiveness, and current treatment protocols mandate the use of doxorubicin with its associated side-effects, advances in understanding the disease biology with a view to more targeted and personalized treatment is a pressing need.

Sporadic and Familial Congenital Cataracts: Mutational Spectrum and New Diagnoses Using Next-Generation Sequencing.

ABSTRACTCongenital cataracts are a significant cause of lifelong visual loss. They may be isolated or associated with microcornea, microphthalmia, anterior segment dysgenesis (ASD) and glaucoma, and there can be syndromic associations. Genetic diagnosis is challenging due to marked genetic heterogeneity. In this study, next‐generation sequencing (NGS) of 32 cataract‐associated genes was undertaken in 46 apparently nonsyndromic congenital cataract probands, around half sporadic and half familial cases. We identified pathogenic variants in 70% of cases, and over 68% of these were novel. In almost two‐thirds (20/33) of these cases, this resulted in new information about the diagnosis and/or inheritance pattern. This included identification of: new syndromic diagnoses due to NHS or BCOR mutations; complex ocular phenotypes due to PAX6 mutations; de novo autosomal‐dominant or X‐linked mutations in sporadic cases; and mutations in two separate cataract genes in one family. Variants were found in the crystallin and gap junction genes, including the first report of severe microphthalmia and sclerocornea associated with a novel GJA8 mutation. Mutations were also found in rarely reported genes including MAF, VIM, MIP, and BFSP1. Targeted NGS in presumed nonsyndromic congenital cataract patients provided significant diagnostic information in both familial and sporadic cases.

Spatial genomic heterogeneity in diffuse intrinsic pontine and midline high-grade glioma: implications for diagnostic biopsy and targeted therapeutics.

IntroductionDiffuse intrinsic pontine glioma (DIPG) and midline high-grade glioma (mHGG) are lethal childhood brain tumors. Spatial genomic heterogeneity has been well-described in adult HGG but has not been comprehensively characterized in pediatric HGG. We performed whole exome sequencing on 38-matched primary, contiguous, and metastatic tumor sites from eight children with DIPG (n = 7) or mHGG (n = 1) collected using a unique MRI-guided autopsy protocol. Validation was performed using Sanger sequencing, Droplet Digital polymerase-chain reaction, immunohistochemistry, and fluorescent in-situ hybridization.ResultsMedian age at diagnosis was 6.1 years (range: 2.9–23.3 years). Median overall survival was 13.2 months (range: 11.2–32.2 months). Contiguous tumor infiltration and distant metastases were observed in seven and six patients, respectively, including leptomeningeal dissemination in three DIPGs. Histopathological heterogeneity was evident in seven patients, including intra-pontine heterogeneity in two DIPGs, ranging from World Health Organization grade II to IV astrocytoma. We found conservation of heterozygous K27M mutations in H3F3A (n = 4) or HIST1H3B (n = 3) across all primary, contiguous, and metastatic tumor sites in all DIPGs. ACVR1 (n = 2), PIK3CA (n = 2), FGFR1 (n = 2), and MET (n = 1) were also intra-tumorally conserved. ACVR1 was co-mutated with HIST1H3B (n = 2). In contrast, PDGFRA amplification and mutation were spatially heterogeneous, as were mutations in BCOR (n = 1), ATRX (n = 2), and MYC (n = 1). TP53 aberrations (n = 3 patients) varied by type and location between primary and metastatic tumors sites but were intra-tumorally conserved. ConclusionSpatial conservation of prognostically-relevant and therapeutically-targetable somatic mutations in DIPG and mHGG contrasts the significant heterogeneity of driver mutations seen in adult HGG and supports uniform implementation of diagnostic biopsy in DIPG and mHGG to classify molecular risk groups and guide therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0269-0) contains supplementary material, which is available to authorized users.

Tracking the Somatic Mutations in Azacitidine-Treated MDS Patients Documents Clonal Development and AZA Responsiveness

Abstract Introduction and hypothesis: Somatic gene mutations develop in ~78% Myelodysplastic syndrome (MDS) patients. MDS progresses into an unstable phase characterized by an accumulation of myeloblasts that is indicated for the DNA demethylation therapy with Azacitidine (AZA). To understand whether AZA is capable to eliminate tumor cells and whether a mutation pattern responds to AZA we herein tracked mutations in the bone marrow (BM) during AZA treatment. Two scenarios were postulated: 1) AZA treatment will/ will not eliminate clones characterized by specific mutations and this will relate to the clinical outcome, 2) during clinical progression on the AZA treatment either the new mutations will develop or the original mutations will be detected. Patients: 40 int-2/high-risk MDS patients (176 samples, median age 70, 22F/18M) indicated for AZA (75mg/m2, 5+2+2) were sequenced. The MDS subtypes included RAEB1, RAEB2, and MDS/AML. Half of the patients progressed from the 5q-syndrome. Libraries were prepared using TruSight DNA amplicon kit. Set of 54 myeloid genes (associated with MDS or AML) were sequenced by Illumina platform HiSeq 2500 with depth &gt;100 per mutation, mutation should be heterozygous, non-synonymous, exonic with frequency &gt;10%. As controls: 4 normal BM samples and 2 cord bloods (also applied for data filtering) and MOLM-13 cell line carrying FLT3-ITD mutation were used. Additional controls included CD3+ T cells isolated from the patient BM samples. Results: 70% of patients were informative for at least one somatic mutation with high impact on the amino acid sequence. Mutations in TET2 (in 13%) were overall the most frequent before AZA was started (followed by mutations in BCOR, RUNX1, STAG2, and NOTCH genes). In 36% of informative patients, the mutation pattern developed on AZA. Interestingly, the most frequent mutations after AZA were in ASXL1 in 10% (followed by mutations in TET2, BCOR, and CUX1 genes). While mutations in ASXL1 and NOTCH genes developped only in the non-5q derived patients, the mutations in RUNX1 developped only in the 5q-derived higher-risk MDS patients. Average number of mutations before AZA and on AZA was 2 per patient. Complete elimination of the mutation pattern was noted in 57% of informative patients during the first 8 months of AZA treatment and this was associated with the therapy responsiveness (PR or CR). In contrast in 7% of informative patients the mutation pattern remained the same and this was associated with the stable disease (leading to progression). Upon progression on the AZA treatment we have observed appearance of the new mutations in 73% of informative patients. 20% of patients progressing after 14 cycles of AZA were non-informative which suggests that these patients may carry mutations in genes not included within the tested set. We have assessed overall survival (OS) according to the mutation status in the following subgroups: non-informative patients, informative patients with mutations that were eliminated by AZA, and those retaining or gaining the mutations upon AZA. The group that developed or retained mutations during first 4-8 cycles of AZA displayed significantly lower OS (p=0.004, median OS:11 months) compared to either non-informative patients (26 months) or those where AZA completely eliminated the mutation pattern (28 months). Conclusions: Tracking the mutations in MDS patients during AZA therapy provides opportunity to detect clonal development in 2/3 of the MDS patients and relate these data to the clinical outcome. Moreover, progression on AZA therapy is usually associated with the development of a new mutation pattern and this coincides with the significantly lower OS. Grants: GACR P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/1. Disclosures Stopka: Celgene: Research Funding.

Prognostic Impact of Molecular Mutations in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS) on Allogeneic Hematopoietic Cell Transplant (HCT) Outcomes: Adverse Impact of TET2 Mutations

▪AML and MDS are heterogeneous myeloid neoplasms with variable biologic and clinical outcomes. Although allogeneic HCT is the only potentially curative therapy for high risk AML and MDS, survival after transplant remains poor, and identifying who benefits is challenging.We hypothesized that next-generation sequencing (NGS) mutational analyses can predict outcome in MDS and AML patients undergoing allogeneic HCT. We performed multi-amplicon targeted pre-HCT NGS using a somatic panel of the 60 most commonly mutated genes in myeloid neoplasias as previously determined by whole exome sequencing, on 123 patients with AML (N=64, 52%) and MDS (N=59, 48%) who subsequently underwent HCT. Median age at transplant was 53 years (range, 20-73). 21 (17%) patients had complex karyotype, 10 (8%) with monosomy 7, 48 (39%) normal, and 48 (39%) with other or unknown cytogenetic abnormalities. 45 (37%) patients were in a complete remission (CR) prior to transplant, while 78 (63%) were in less than a CR; with CR as defined by International Working Group criteria for MDS, or <5% blasts for AML. The majority of patients received myeloablative conditioning (N=83, 68%), and 40 (33%) received a reduced-intensity preparative regimen. Donor source was matched sibling (N=52, 42%), matched unrelated (N=56, 46%), cord-blood (N=12, 10%), and haplo-identical (N=3, 2%). Median follow up was 35 months (range 5-178). Mutations were analyzed individually and by molecular pathway. 88 (72%) patients had at least one mutation, most frequently in STAG2 (10.2%), TET2 (9.8%), ASXL1 (8.1%), and RUNX1 (8.1%). TP53 mutations were more common in MDS patients compared to AML (10% versus 1.6%, P=0.05). NRAS (P=0.019) and TP53 (P=0.022)mutations were more commonly associated with complex karyotype. Mutations in BCOR (P=0.048) and TP53 (P=0.047)were associated with less than CR, while TET2 (P=0.03)mutations were associated with CR prior to HCT. In univariable analyses, the presence of complex karyotype was associated with shorter overall (OS) and relapse-free survival (RFS) (hazard ratio [HR] 2.4; P=0.002 and HR 3.1; P<0.001). Mutations in TET2 (HR 2.1; P=0.042) and EZH2 (HR 2.3; P=0.048), or presence of any mutation in the histone modification pathway (ASXL1, EZH2, KDM6A, SUZ12); (HR 1.7; P=0.039) was associated with poor OS. The presence of any mutation in the DEAD box RNA-helicase family genes (DHX29, DDX54, DDX41) was associated with poor RFS (HR 3.1; P=0.009). Nothing except complex karyotype was specifically associated with higher relapse. Unlike in previous reports, TP53 mutations were not found to be significantly associated with poor OS or RFS, though these cases (N=7) were limited. In multivariable analyses, adjusting for clinical variables, complex karyotype remained significantly associated with poor OS (HR 2.7; P<0.001) and RFS (HR 3.9; P<0.001). TET2 also remained independently associated with poor OS (HR 2.4; P=0.022). Presence of any of the DNA methylation mutations (TET2, DNMT3A, IDH1, IDH2) was associated with poor RFS (HR 1.7; P=0.05). 3-year OS was 23% in patients with a complex karyotype versus 48% in patients without (P=0.002); and 14% in patients with a TET2 mutation and 46% without (P=0.042) (Figure 1).Molecular abnormalities are important variables in determining outcome after allogeneic HCT. We demonstrate that TET2 mutations in AML and MDS predict for poor survival after HCT. Ongoing serial mutational analyses in an extended cohort of patients will enhance our understanding of the role of NGS in informing care decisions for patients undergoing allogeneic HCT for AML and MDS. [Display omitted] DisclosuresMajhail:Gamida Cell Ltd.: Consultancy; Anthem Inc.: Consultancy. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

Strengthening the Evidence of Relevant Somatic Mutations of Individual Patients By Pairing Sequencing Deduced Allelic Burden, Leukemic Blast Percent and Cluster Analysis

IntroductionExome sequencing can detect somatic mutations at an unprecedented scale. However, high false-positive rates due to multiple technical contributors affecting signal-to-noise ratio is an unsolved inherent problem. Furthermore, the current literature does not consistently assess or report the allelic burden of detected and scrutinized mutations, potentially leading to focus on irrelevant mutations. We hypothesized that this issue can be addressed by a structured usage of allele frequencies in combination with detected mutations. Employing improved techniques to assess the molecular signature shortly after diagnosis could also result in improved risk stratification. Here, we present proof-of-principle that identification of relevant mutations, and extent of clonality, can be accomplished by pairing hypothetical allelic burden deduced from sequencing reads to leukemic burden in individual samples.Methods7 samples were used for whole exome sequencing derived from bone marrow aspirated from two different patients. Of these, 4 were leukemia samples (diagnostic and two relapse samples of one T-cell/myeloid mixed-phenotype leukemia and a diagnostic from AML, M0), reflecting different clinical situations, and 3 samples served as controls (keratinocytes, and fibroblasts in the first, remission in the second) to include different clinical situations. Raw data processing, mutations analysis and downstream analysis of sequencing data was performed as described earlier (Hansen & al., MethodsX 2015, Hansen & al., Br J Haematol. 2015), following GATK Best Practice workflow and MuTect default parameters. Cluster analysis was utilized to mathematically divide somatic SNVs into read frequency clusters on the basis of squared Euclidean distance, thus enabling the retrieval of mean allelic burden and expected allele frequencies juxtaposed with flow cytometry-derived leukemic blast percent. The initial condition of the classification model was default set to accommodate separation of background (cluster 1: noise or low read), heterozygous mutations (cluster 2), and an outlier bin for possible homozygous mutations (cluster 3).Results and discussionIn order to reference the leukemic burden of a given sample we employed data from immunophenotyping as a surrogate marker for malignancy. Without filtering of detected mutations by exome sequencing the partly stochastic nature of the signal is evident (fig. 1A). An exponential-like continuum of non-rejected mutations is found when these are sorted according to frequencies (black points), making it difficult to deduce any clonal nature of the malignancy, and to evaluate the validity of possibly relevant mutations found. Using a minimum depth of coverage threshold of 30 the signal-to-noise ratio is increased (fig. 1B), and, subsequently, two clones were resolved at diagnosis (fig. 1C) by cluster analysis. This resolution decreases at lower tumor burden showing expected sensitivity towards lower allelic reads (fig. 1D), although driver mutations of the persistent clone could be detected. At second relapse only one distinct sAML phenotyoe clone and, now homozygous, CDKN2 AR80* could be detected (fig. 1E) with the mutations of secondary clone present at diagnosis (cluster 2) lost due to selective pressure from treatment, i.e. FLT3D835Y. The observations are backed by another diagnostic sample from a patient with AML with NRAS and BCOR mutations (fig. 1F), also with a distinct clone. Here, too much emphasis could easily be given to NRASG12D if the frequency had not been assessed.ConclusionWe have addressed the pertinent question regarding false positive observations arising from deep sequencing or emphasizing mutations found in the low allele frequency fractions. From this dataset we have, despite the low number of samples, accomplished to suggest a formalized approach for single sample mutational analysis. As a consequence, we can now show that malignant clones with high tumor burden can be resolved semi-spatially by sequencing, generally applicable to a wide range of clinical settings. We conclude that this approach is amenable in single patient situations. While further studies are needed to ultimately test the applicability of this approach in the clinical settings, the perspectives to this observation become evident as sequencing depth and cost continue to develop in an inversely correlated manner. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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. DisclosuresSekeres: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.

The Complexity of Interpreting Genomic Data in Patients with Primary and Secondary Acute Myeloid Leukemia (AML)

BackgroundAcute myeloid leukemia (AML) is a complex, heterogeneous neoplasm characterized by the accumulation of complex genetic alterations that are responsible for the initiation and progression of the disease. Secondary AML (sAML) represents a progression from antecedent hematologic disorders such as myelodysplastic syndromes (MDS) or myeloprolifrative neoplasms (MPN). Certain acquired mutations have been reported to be specific for sAML when compared to primary AML (pAML), but many limitations exist when cytogenetic grouping or other parameters are taken into account. In addition, some mutations have been shown to impact survival in some studies, but not others.MethodsWe performed targeted deep sequencing on samples from bone marrow and peripheral blood of pts diagnosed with sAML and pAML and treated at our institution between 1/2003-1/2013. Additional data on pAML was added from The Cancer Genome Atlas (TCGA). A panel of 62 gene mutations described as frequently recurrent mutations in myeloid malignancies were assessed. Cytogenetic grouping was defined by CALGB/Alliance criteria. Differences were compared using Fisher's exact test and the Mann-Whitney U test for categorical and continuous variables, respectively. Overall survival (OS) was calculated from the time of diagnosis to last follow up or death.Results:A total of 496 pts included: 273 with pAML and 223 with sAML. Comparing pAML to sAML, pts were younger (median age 59 vs. 68 years, p<.001) and had a higher WBC at diagnosis (13.5 vs. 3.9 X 109/L, p<.001), respectively. Cytogenetic analysis showed significant differences: 58% of pAML pts had normal karyotype (NK) compared to 37% of sAML (p=.002), whereas 24% and 26% of sAML had intermediate risk (other than NK) and complex karyotype (> 3 abnormalities) compared to 11% and 16% for pAML (p< .001, .009), respectively. Mutations in ASXL1 (p<.001), JAK2 (P =.014), CBL (P =.05), BCOR (P =.02), STAG2 (p =.003), SF3B1 (P =.04), SRSF2 (P =.001 ), and U2AF1 (P =.03) were highly specific for the sAML phenotype, whereas mutations in NPM1 (p<.001 ), FLT3 (p< .001), DNMT3A (p<.001), and IDH2 (P =.02) were more specific for pAML. When the analysis was restricted to pts with NK cytogenetics, only ASXL1 (p<.001) remained specific for sAML and DNMT3A (p<.001) for pAML.Further, when the analysis was restricted to pts with unfavorable risk cytogenetics, only ASXL1 (p=.01) remained specific for sAML. No other mutations were specific for pAML.We then evaluated whether the mutations that were specific to each AML phenotype had an impact on OS. We observed different mutations that impacted OS in each phenotype: DNMT3A (HR 1.81, 95% CI 1.28-2.57, p<.001), TP53 (HR 3.1, 95% 1.74-5.53, p< .001), and SUZ12 (HR 3.18, 95% CI 1.01-10, p=.05) led to worse OS in pAML, whereas mutations in EZH2 (HR 2.12, 95% CI 1.07-4.21, p =.03), PRPF8 (HR 2.32, 95% CI 1.20-4.46, p=.01), and TP53 ( HR 2.92, 95% CI 1.69-5.04, p<.001) lead to worse OS in sAML. Different mutations had a different impact on OS when cytogenetic analysis was taken into account. Mutations in FLT3 (HR 2.15, 95% CI 1.37- 3.35, p<.001) and DNMT3A (HR 2.41, 95% CI 1.57-3.70, p<.001) led to worse OS in NK pAML, whereas none of the mutations impacted OS in NK sAML. Further, in pAML with unfavorable cytogenetics, BCOR (HR 2.41, 95% CI 1.57-3.70, p<.001) and TP53 (HR 2.41, 95% CI 1.57-3.70, p<.001) had led to worse OS, whereas BOCR (HR 2.95, 95% CI 1.03-8.50, p<.001), SF3B1 (HR .19, 95% CI .05-.82, p<.001), SUZ12 (HR .12, 95% CI .01-.99, p<.001),and TP53 (HR 1.9, 95% CI 1.09-3.46, p<.001) only impacted OS in sAML.ConclusionClear genomic variations exist between sAML and pAML. Although some of these genomic changes are more specific to each phenotype in general, this specificity and the impact on OS differed for each cytogenetic subgroup, highlighting the complexity of interpreting genomic information in pts with AML and the need to incorporate both cytogenetic and molecular data in prognosis-driven treatment decisions. DisclosuresSekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; 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.