PHF6 Mutations Research Articles

Clinical characteristics and prognostic study of adult acute myeloid leukemia patients with ASXL1 mutations

ABSTRACT Objectives: A total of 156 adult acute myeloid leukemia (AML) patients were enrolled in this study to explore the clinical characteristics and prognostic impact of ASXL1 mutations. Methods: Clinical characteristics, prognostic impact and the association between ASXL1 mutations and some other mutations were analyzed. Results: We found ASXL1 mutations were most frequently found in M5 subtype and intermediate risk karyotype and were correlated with TET2, DNMT3A and PHF6 mutations. A total of 145 patients were included in prognostic analysis; results showed ASXL1 mutations had no impact on OS and DFS. In normal karyotype-AML (CN-AML) and older (≥60 years) AML, ASXL1 mutations showed adverse impact on OS (P = 0.022; p = 0.019, respectively) and showed adverse prognostic tendency on DFS (p = 0.173; p = 0.108, respectively). ASXL1 mutations were also independent unfavourable prognostic factors for OS on CN-AML and older (≥60 years) AML patients and unfavourable factors for DFS on older (≥60 years) AML in multivariate analysis. Results also indicated that though ASXL1 mutations were associated with TET2, DNMT3A and PHF6 mutations, when coinciding with ASXL1 mutations, the prognosis of AML was not significantly impacted. Discussion: The reliability of our results need to be further confirmed by prospective randomized controlled studies covering a large numbers of AML patients. Conclusion: The results showed ASXL1 mutations may act as a poor prognostic index especially in elder AML and CN-AML patients.

Recurrent Gene Mutations in Pediatric Patients with AML By Targeted Sequencing ―the Jccg Study, JPLSG AML-05―

Introduction Treatment outcomes of pediatric acute myeloid leukemia (AML) have improved via stratification therapy. However, relapse and mortality are still observed in 40% and 30% patients, respectively. Recently, comprehensive analyses using next-generation sequencing have revealed novel genetic alterations in adult AML patients, but insufficient analyses have been performed in pediatric AML patients. Further studies are warranted to evaluate the relationship between a more detailed genetic background and prognosis in pediatric AML patients. Methods We performed targeted sequencing using a 343-gene custom panel and next-generation sequencer in 302 pediatric patients with de novo AML who participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group from 2006 to 2010. One hundred and twenty patients with core binding factor (CBF)-AML, 52 with KMT2A rearrangements, 56 with normal karyotype (NK), 18 with complex karyotype (CK), and 57 with other karyotypes except for patients with acute megakaryocytic leukemia were enrolled. Correlations among gene mutations, other cytogenetic alterations, and clinical characteristics were investigated. Results and Discussion We found the following 6 recurrent gene mutations, which have not been frequently reported in pediatric AML: KMT2C (n = 13, 4.3%), PHF6 (n = 6, 2.0%), MGA (n = 13, 4.3%), TET2 (n = 11, 3.6%), JAK3 (n = 12, 4.0%), and GATA2 (n = 11, 3.6%). KMT2C mutations have been reported in adult and pediatric patients with CBF-AML and in a few patients with other types of AML; however, their clinical significance has not been revealed. Six of 13 patients with KMT2C mutations were found in CBF-AML, whereas the other 7 patients were detected in non CBF-AML (1 patient in KMT2A-rearrangement, 1 in CK, and 5 in other karyotypes). Loss of function mutations in KMT2C have also been reported to be associated with chemotherapy resistance in pediatric AML patients. Although no significant differences were found, 4 of 13 patients with KMT2C mutations did not achieve complete remission (p = .072), and 3 patients relapsed in <12 months. Significant differences were observed in event-free survival (EFS) between patients with and without KMT2C mutations (3-year EFS 36.9% vs. 56.8%; p = .019). In adult AML, PHF6 mutations have been detected in 3% of patients and have been reported as poor prognostic factors. In 6 patients with PHF6 mutations, 3 patients had a normal karyotype, and 2 patients had a complex karyotype. No PHF6 mutations were detected in AML patients with CBF or KMT2A rearrangements. PHF6 mutations were significantly frequently detected in AML patients with myelodysplasia-related changes (4/6, p = .008). Although 5 of 6 patients with PHF6 mutations archived complete remission, finally, 5 patients relapsed or died. There were significant differences in EFS between patients with or without PHF6 mutations (3-year EFS 16.7% vs. 58.2%; p = .041). MGA mutations have been reported thus far in a few AML patients. MGA is a negative regulator of MYC signaling; thus, loss of functions of MGA is considered to contribute to tumorigenesis. However, the clinical significance of MGA mutations has not been reported. MGA mutations were significantly frequently detected in AML patients with FAB M2 (7/13, p = .020). In our cohort, 2 of 3 AML patients with DEK-NUP214 had MGA mutations (p = .004). We did not find correlations between MGA mutations and prognosis (3-year OS 83.3% vs. 75.5%; p= .488, 3-year EFS 66.7% vs. 56.7%; p = .507) TET2 mutations have been found in 8%-19% of adult AML cases and are considered poor prognostic factors. In previous reports, TET2 mutations were also detected in 3.8% of pediatric AML patients, but because of the small number of patients, the association between TET2 mutations and prognosis has not been revealed. TET2 mutations were significantly frequently detected in older AML patients (median age 13.95 vs. 9.0 years; p = .022) and in AML patients with NK (5/11, p = .035). Contrary to previous reports on adult AML, TET2 mutations were not associated with prognosis (3-year OS 77.9% vs. 75.9%; p= .688, 3-year EFS 54.5% vs. 57.4%; p = .907). This suggested that TET2 mutations have a different clinical significance between adult and pediatric AML patients. In conclusion, mutations in the KMT2C and PHF6 genes were associated with poor prognosis, but TET2 mutations were not. Further analysis should be needed about TET2 mutations. Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

Clinical and Genetic Features of Constitutional Partial Trisomy 8 Mosaicism (CT8M) Patients with Cytopenia

Background Constitutional partial trisomy 8 mosaicism (CT8M) is a congenital chromosomal abnormality with an estimated occurrence rate as one out of 25,000-50,000 pregnancies. CT8M has a wide variability in physical manifestation ranging from apparently normal to severe disablement. However, diagnosis of CT8M in adult without physical abnormality is difficult . Acquired trisomy 8, which is restricted to the malignant cells, is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and clinical implication of carrying isolated trisomy 8 is considered as intermediate cytogenetic risk in MDS. However, isolated trisomy 8 without morphological dysplastic features is not definitive evidence for MDS. 15 to 20% of trisomy 8 in MDS are supposed to be derived from CT8M. We therefore diagnosed CT8M patients among patients with cytopenia and analyzed clinical and genetic features to uncover the association with MDS. Methods . Clinical features including cytogenetic analysis were analyzed regularly. Genomic DNA was extracted from whole PB cells or BM mononuclear cells. We performed targeted next-generation sequencing to identify mutations in 68 driver genes of myeloid neoplasms using AmpliSeq for Illumina Myeloid Panel and On-Demand Panel on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. The study was approved by the institutional review board and patients gave written informed consent for the study. Results We identified nine CT8M patients with cytopenia.They comprised 3 males and 6 females at a median age of 56 years (range 24-84 years) (Table). All the patients carried no physical abnormality nor conspicuous phenotypic features. Four patients (Patient #3, #4, #6 and #7) did not show apparent morphological dysplasia at the initial BM examination, and they were not diagnosed as MDS. Their cytopenia has not been exacerbated until now without any treatment, and the duration of stable cytopenia was from 2 to 12 years in these patients. By contrast, five patients with CT8M were diagnosed as MDS . Two patients (#5 and #8) were diagnosed as MDS-single lineage dysplasia (SLD), and their cytopenia has not become worse without any treatment for about 4 years. Other three patients diagnosed as MDS-multilineage dysplasia (MLD) showed various clinical courses. Patient #1 was treated with azacitidine and maintains complete hematological improvement after 34 courses of the treatment. Patient #2 was treated with erythropoietin stimulating agent and azacytidine but developed to AML 3 years after initial diagnosis but leukemic blasts has del(20), not +8. Patient #9 developed advanced pancytopenia in 3 months from initial diagnosis and received red blood cell transfusion regularly. Gene mutations were detected in five out of nine patients with CT8M. In three patients, gene mutations were detected at high (20 to 50%) variant allele frequency (VAF). Patient #2 who was analyzed at the AML phase had gene mutations of SRSF2, SF3B1, STAG2 and NOTCH1. BM sample from patient #9 showed ASXL1 mutation and two TET2 mutations. Patient #4 who did not show apparent myelodysplasia had a high VAF ASXL1 mutation, indicating clonal idiopathic cytopenias of undetermined significance. Two patients had low (2 to 5%) VAF mutations; patient #1 was analyzed after 34 courses of azacitidine had a TET2 mutation; patient #5 with MDS-SLD had a WT1 mutation and two PHF6 mutations. Four patients (#3, #6, #7 and #8) did not have any mutations. The clinical and genetic features showed that CT8M with cytopenia without MDS-related mutations were under 56 years old and did not develop to MDS or stayed at MDS-SLD. Patients with low VAF mutations were also stable. By contrast, patients with advanced diseases gained multiple MDS-related gene mutations with high VAF. One patient without dysplasia had a high VAF ASXL1 mutation. All the patients with gene mutations were age of 56 to 84 years. Conclusion Our results indicated that isolated trisomy 8 may cause cytopenia, but the cytopenia is not exacerbated without MDS-related driver gene mutations. CT8M patients with cytopenia might get gene mutations gradually with age, which leads to MDS or AML. Disclosures No relevant conflicts of interest to declare.

Comprehensive Genomic Profiling of Adult T-Cell Lymphoblastic Lymphoma in Chinese Patients

Background: T-cell lymphoblastic lymphoma (T-LBL) is a rare, aggressive neoplasm of precursor T cells that occurs mostly in adolescents and young adults. Extensive genomic analyses of T-LBL have discovered genes encoding adhesion molecules and extracellular matrix proteins in samples from T-LBL patients, with most studies being performed on patients in the Western hemisphere. We sought to define the mutational profile of T-LBL in Chinese patients. Methods: We collected 15 adult T-LBL biopsies from one center in China with confirmed pathologic diagnosis and treatment information. Median cohort age at diagnosis was 26.0 years. The male/female ratio was 13/2. DNA was extracted from formalin-fixed paraffin embedded samples, and targeted deep next generation sequencing (NGS) analyses were performed. Results: Mutation features were revealed in this Chinese T-LBL analysis. NOTCH1 mutations were found in 67% (10/15) of T-LBL patients (pts) with the highest mutation frequency. PHF6 mutations were seen in 53% (8/15) of cases. TP53mutations were seen in 53% (5/15) of T-LBL cases. JAK1 and JAK3 mutations were seen in 27% (4/15)of T-LBL cases respectively. Mutations at histone modification genesincluded SUZ12 (20%, 3/15), EZH2(13%, 2/15), and EP300 (7%,1/15). Other mutations included PTEN(27% 4/15), DNM2 (27% 4/15). Discussion: The aim of our study was to reveal the genomic landscape of Chinese T-LBL. The cohort collection was unbiased, the patients' features were in concordance with previous reports that the disease of T-LBL is mostly found in the young patients and shows male predominance. The most predominant somatic mutation wasNOTCH1, which is also usually seen in T-ALL. Mutations at histone modification genes were also seen in our study. NGS studies have identified epigenetic modifiers mutations as a hallmark of T-LBL, highlighting an attractive therapeutic target in this disease. In summary, this effort has identified novel mutation features in Chinese T-LBLs that can potentially lead to more effective treatments in Chinese population. Disclosures Li: Guangdong Province Hospital: Employment.

Hotspot U2AF1 Mutations Determine Missplicing Selectivity: Novel Mechanisms Altering Splicing Factors

Mutations in splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) are identified in over 50% of patients diagnosed with myelodysplastic syndrome (MDS). U2AF1 is a U2 auxiliary factor that forms a heterodimer with U2AF2 for the recognition of the 3' splice site (SS) and results in the subsequent recruitment of U2snRNPs during pre mRNA splicing. U2AF1 mutations are present in 11% of MDS and its presence is correlated with an increased risk of progression to AML. Non-canonical mutations are rarely seen in U2AF1 but two highly conserved hotspots (S34, Q157) are frequently seen and result in distinct downstream effects. We performed NGS analysis of a large cohort of patients with myeloid neoplasms (MN; n=3,328) and identified 238 patients with U2AF1 mutations (7%). We analyzed the somatic mutational profile, clonal hierarchy, and splicing profile of patients with U2AF1S34 (n=99), U2AF1Q157 (n=119), and others (n=20; M1/*, A26T/V, R35Q, R118C, E124K, F150L, E152G, C154S, R156H, M172L). The mutational spectrum of U2AF1S34 and U2AF1Q157 was equally dismal but pretty distinct. U2AF1 S34 cases were mostly co-mutated for DNMT3A (5%), TET2 (4%), RUNX1 (2%), ASXL1, CBL, ETV6, KRAS, NRAS (1.3%), STAG2 (1%), CUX1, and TP53 (<1%) while U2AF1Q157 had higher numbers of mutations in ASXL1 (10%), SETBP1, TET2 (3%), NRAS (2%), DNMT3A, PHF6 (2%), JAK2 (2%), CBL, EZH2, TP53 (2%), IDH2, RUNX1, STAG2 (2%), KRAS (1%), and IDH1 (<1%). Mutational rank showed: U2AF1S34 was ancestral in 38% of the cases followed by secondary DNMT3A and NRAS (5%, both). Ancestral U2AF1Q157 was found in 35%, with ASXL1 (19%) being the most common secondary hit. Subclonal U2AF1S34 (44%) was most commonly preceded by DNMT3A (21%), while secondary U2AF1Q157 (40%) had ASXL1 (28%) as the most common first hit. U2AF1S34 and U2AF1Q157 were co-dominant to a miscellanea of mutations. U2AF1S34 cases had a shorter OS than U2AF1Q157 cases (n=82 vs. n=101; 20 vs. 25 mo.; P=.002). Ancestral U2AF1S34 or U2AF1Q157 defined a dismal prognosis compared to secondary U2AF1S34 or U2AF1Q157 (OS: n=63 vs. n=86; 29 vs. 37 mo.; P=.03). To investigate the effects of both mutations on splicing patterns, we analyzed RNA-Seq profiles, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events that were classified as skipping exon (SE), retained intron (RI), and 3' or 5' alternative SS (A3SS, A5SS) (Hershberger, ASH 2019). Overall, 675 AS events in 430 genes were scored significant (pFDR<.05) when the splicing inclusion/exclusion difference was ±10%. U2AF1S34 and U2AF1Q157 caused an equal fraction of SE (79% U2AF1S34; 72% U2AF1Q157). Only 4% of the genes were commonly misspliced by both mutations, while the rest of the genes were uniquely spliced according to each mutation. Some exemplary genes misspliced by both mutations were DDX3X (an RNA helicase) showing a consistent SE at exon 5 and an RI 3-5a and CCNG1 (cell growth regulation) which was enriched for RI 7-7. Among others, U2AF1S34 uniquely affected the splicing of TET2 (SE e3); cell cycle regulators, CDC37L1 (SE), CCNC (SE e8), and HDAC3 (SE e6). We then investigated whether U2AF1 mutations might affect the splicing of other RNA splicing components. This mechanism would lead to the loss of regulation of the spliceosome complex. U2AF1S34 produces selective RIs in SRSF2 (4-4a) and A5SS in SRSF6 (7a and 7). Tumor suppressors and proto-oncogenes were also found to be misspliced by U2AF1S34 including PTEN (RI 3-Ua), CTNNB1 (RI 15-19; 3'SS (19 and 19a), and CCNL1 (RI 4-U). Major regulators of splicing factor activity are phosphatases like PP1R12A and PPP1R12B, which showed an RI 8-7 and an A3SS, respectively. Among genes exclusively misspliced by U2AF1Q157, we identified DEAD-Box helicases [DDX17 (RI), DDX59 (SE e8), DHX29 (RI)], ALAS family members (ALAS1, SE e6; ALAS2, SE e5) and UTX (KDM6A; SE e16). U2AF1Q157 affected the missplicing of DYRK1A (SE e7), a kinase known to phosphorylate SF3B1 at T434 site. In sum, our study suggests that while concurrent mutations in splicing factors lead to lethality, the presence of mutations (as the case of U2AF1S34 and U2AF1Q157) and consequent missplicing of other splicing factors are permissive events in MN and might represent novel mechanisms of disease pathogenesis. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Novel Molecular Pathogenesis and Therapeutic Target in Acute Erythroid Leukemia

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia (AML) characterized by erythroid predominance and dysplasia, which is morphologically classified into two subtypes: pure erythroid and myeloid/erythroid leukemias (PEL and MEL). Clinically, AEL exhibits aggressive and rapid clinical course, which is considered to be linked to frequent TP53 mutations and complex karyotypes found in this subtype. Although multiple sequencing studies were conducted to explain such phenotypic variety and to search promising therapeutic targets, neither the diagnostic discrimination of PEL and MEL subtypes nor the establishment of standard therapeutic strategy has fully been successful. To clarify genetic characteristics and to identify novel therapeutic targets, we comprehensively characterized the mutation and copy number alteration (CNA) profiles and assessed differential impacts of the frequent driver genetic abnormalities on disease phenotypes and clinical outcomes. We initially analyzed 105 cases with AEL (PEL; n=10, MEL; n=69, and unspecified; n=26). Among these, 9 cases were serially analyzed at 2-6 time points. All samples were obtained according to protocols approved by the ethics board of each participating institution. As control, 907 cases with non-erythroid AML (NEL) distinguished by the low fraction (<50%) of erythroid cells in bone marrow were enrolled. Publicly available information on clinical and genetic findings was collected from previous publications, including The Cancer Genome Atlas and BeatAML datasets. In total, 1,012 cases with AML were enrolled in this study. The genetic profile in PEL and MEL were also compared with that of NEL. Finally, we recognized biological significance of the molecular defects by functional analysis of xenograft model, and showed novel therapeutic possibilities targeting pathogenic pathways. Most frequently observed were mutations in TP53 (39%), STAG2 (20%), and NPM1 (15%), and KMT2A-partial tandem duplication (PTD) (18%) in AEL. On the basis of mutational profiles, consensus clustering divided AEL into 4 subgroups. Group A (n=41, 39%) was defined by TP53 mutations into which all PELs were categorized. In CNA analysis, amplification in 9p, 19p, and 21q were significantly more enriched in PEL than NEL. Intriguingly, amplification of 9p, 19p, and 21q lesions commonly included JAK2, EPOR, and ERG loci, respectively. These genes were affected by amplification or mutations more frequently in AEL than NEL (Figure 1). Group B (n=19, 18%) was enriched for mutations in STAG2, which was significantly more frequent in MEL than in PEL (P<0.05). Compared to STAG2-mutated cases in NEL, those in MEL showed significantly higher frequency of KMT2A-PTD. Group C (n=16, 15%) was defined by NPM1 mutations frequently accompanied by PTPN11 mutations (50% in Group C). This combination was also distinct in MEL and was significantly more frequent than in NPM1-mutated NEL cohort (P<0.05). In Group D (n=29, 28%) without any TP53, STAG2, or NPM1 mutations (triple-negative), RUNX1, ASXL1, BCOR, PHF6, and U2AF1 mutations were enriched compared to triple-negative NEL. In addition, novel loss-of-function USP9X mutations were exclusively identified in Group D AEL (P<0.05, compared to triple-negative NEL), which has been implicated in erythroid leukemogenesis through the TGF beta pathway. Serial sample sequencing analysis revealed that JAK2 mutation positive myeloproliferative neoplasms evolved into PEL. Clinically, patients in Group A showed shorter overall survival time compared to those in any other group. To further search for a novel therapeutic target to the most severe type of AEL, JAK2 inhibitor was administered to xenograft models (from 2 cases) having JAK2 and EPOR amplifications and TP53 mutations. Engrafted cells showed in vitro activation of STAT5 and proliferative capacity enhanced with erythropoietin. In vivo analysis showed that inhibition of JAK/STAT pathway significantly suppressed cell growth and prolonged overall survival. In summary, our findings suggest that AEL is classified into 4 major subgroups having unique genetic and clinical features. PEL and MEL are genetically distinct subtypes, which was also highlighted by comparison to NEL. Frequent involvement of EPOR/JAK/STAT pathway suggests therapeutic indication of JAK inhibition for AEL especially in the most aggressive type of AEL cases with TP53 mutations. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Miyazaki:Kyowa-Kirin: Honoraria; Dainippon-Sumitomo: Honoraria; Nippon-Shinyaku: Honoraria; Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria. Takaori-Kondo:Pfizer: Honoraria; Chugai: Research Funding; Janssen: Honoraria; Kyowa Kirin: Research Funding; Takeda: Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Usuki:Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Heuser:Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Ogawa:Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

Using next generation sequencing technology to analyze gene mutations in patients with acute myeloid leukemia and the impact on prognosis

Objective: To analyze the distribution of gene mutations in newly diagnosed acute myeloid leukemia (AML) patients, based on next generation sequencing technology (NGS) and to evaluate their value in AML risk stratification. Methods: The study analyzed 453 newly diagnosed AML(excluded acute promyelocytic leukemia, APL) patients from seven hospitals in Shanghai, from January 1st 2014 to December 31th 2017. RNA and DNA were extracted from pretreatment bone marrow mononuclear cells and targeted sequencing of AML genes were performed. The data of different groups was compared. Results: A total of 453 newly diagnosed AML patients were enrolled in the study, including 247 males and 206 females with a median age of 49.5 (range,11-85) years. A total of 540 mutations/fusion genes were detected in 289 patients, 29.1% (132/259) of whom with two or more mutations/fusion genes. In all patients, NPM1 was the most common mutation(12.8%), followed by ETO and TET2 mutation (11.92% and 11.04%, respectively) . And WT1 over-expression accounted for 10.6%. Patients over the age of 50 were with a higher frequency of mutations associated with epigenetic modification, 11.93% for ASXL1, 13.99% for DMNT3A, 6.58% for IDH1/IDH2, and 13.17% for TET2. The frequency of DMNT3A mutations was three times higher than that of patients under 50 years of age (P=0.017). In this study, a relatively low proportion of genetic mutations was observed in low-risk karyotype group. In the medium-risk karyotype group, the relatively high mutation frequencies were observed in NPM1, TET2, FLT3-ITD, DNMT3A, ASXL1, and CEBPA genes. In the poor-risk karyotype group, the mutation frequencies of ASXL1, TET2, DNMT3A and PHF6 genes were more than 10%, especially ASXL1 and PHF6 mutation frequencies were significantly higher than other molecular risk stratification groups (P<0.05). Of the 254 patients (56%) with normal karyotype AML (NK-AML), 56 patients were detected to have gene mutations about epigenetic modification. The median OS of this group was worse than that of patients without related mutations, while the median LFS had no significant difference. In patients with NK-AML older than 50 years, the OS and LFS of patients with epigenetic modification related gene mutations was 12 months and 10 months, versus 18 months and 12 months of patients without mutations. Conclusions: The gene mutations frequencies in AML patients with different age and molecular risk stratification groups are different. Epigenetics gene mutation frequencies, such as DNMT3A, ASXL1, IDH1/IDH2 and TET2,are higher in patients older than 50 years. A shorter OS can be observed in older patients(>50 years) with epigenetics gene mutation.

Clinical, immunophenotypic, and genomic findings of acute undifferentiated leukemia and comparison to acute myeloid leukemia with minimal differentiation: a study from the bone marrow pathology group

Acute undifferentiated leukemia is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited and it is uncertain if acute undifferentiated leukemia is biologically distinct from acute myeloid leukemia with minimal differentiation, which also shows limited myeloid marker expression and has been reported to have a poor prognosis. We identified 92 cases initially diagnosed as acute undifferentiated leukemia or acute myeloid leukemia with minimal differentiation from pathology databases of nine academic institutions with available diagnostic flow cytometric data, cytogenetic findings, mutational and clinical data. Outcome analysis was performed using Kaplan Meier test for the 53 patients who received induction chemotherapy. Based on cytogenetic abnormalities (N = 30) or history of myelodysplastic syndrome (N = 2), 32 cases were re-classified as acute myeloid leukemia with myelodysplasia related changes. The remaining 24 acute undifferentiated leukemia patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the remaining 30 acute myeloid leukemia with minimal differentiation patients. Compared to acute myeloid leukemia with minimal differentiation, acute undifferentiated leukemia cases were characterized by more frequent mutations in PHF6 (5/15 vs 0/19, p = 0.016) and more frequent expression of TdT on blasts (p = 0.003) while acute myeloid leukemia with minimal differentiation cases had more frequent CD123 expression (p = 0.042). Outcome data showed no difference in overall survival, relapse free survival, or rates of complete remission between acute undifferentiated leukemia and acute myeloid leukemia with minimal differentiation groups (p > 0.05). Acute myeloid leukemia with myelodysplasia-related changes patients showed shorter survival when censoring for bone marrow transplant as compared to acute undifferentiated leukemia (p = 0.03) and acute myeloid leukemia with minimal differentiation (p = 0.002). In this largest series to date, the acute undifferentiated leukemia group shows distinct characteristics from acute myeloid leukemia with minimal differentiation, including more frequent PHF6 mutations and expression of TdT.

Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

AbstractPlant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

Clinical and biological features of PTPN2-deleted adult and pediatric T-cell acute lymphoblastic leukemia

AbstractProtein tyrosine phosphatase nonreceptor type 2 (PTPN2) is a phosphatase known to be a tumor suppressor gene in T-cell acute lymphoblastic leukemia (T-ALL). Because the full clinicobiologic characteristics of PTPN2 loss remain poorly reported, we aimed to provide a comprehensive analysis of PTPN2 deletions within a cohort of 430 patients, including 216 adults and 214 children treated according to the GRAALL03/05 (#NCT00222027 and #NCT00327678) and the FRALLE2000 protocols, respectively. We used multiplex ligation-dependent probe amplification to identify an 8% incidence of PTPN2 deletion, which was comparable in adult (9%) and pediatric (6%) populations. PTPN2 deletions were significantly associated with an αβ lineage and TLX1 deregulation. Analysis of the mutational genotype of adult T-ALL revealed a positive correlation between PTPN2 deletions and gain-of-function alterations in the IL7R/JAK-STAT signaling pathway as well as PHF6 and WT1 mutations. Of note, PTPN2 and PTEN (phosphatase and tensin homolog) deletions were mutually exclusive. Regarding treatment response, PTPN2-deleted T-ALLs were associated with a higher glucocorticoid response and a trend for improved survival in children, but not in adults, with a 5-year cumulative incidence of relapse of 8% for PTPN2-deleted pediatric cases vs 26% (P = .177).

Abstract LB-009: Biomarkers correlating with overall survival (OS) and response to glasdegib and intensive or nonintensive chemotherapy in patients with acute myeloid leukemia (AML)

Abstract Background: The Smoothened inhibitor glasdegib (GLAS) was recently approved in combination with low-dose cytarabine (LDAC) in adults with AML when intensive chemotherapy is not an option. GLAS in combination with intensive chemotherapy has also been well tolerated and shown clinical activity, and is undergoing further evaluation. This analysis aimed to identify molecular drivers that predict overall response (OR) and OS with GLAS. Materials and Methods: We included AML patients receiving LDAC alone or with GLAS 100 mg QD (nonintensive arm) or GLAS 100 mg QD &amp; 7+3 (intensive arm) from Phases 1b and 2 of a multicenter study (NCT01546038). We assessed correlation of OS/OR with mRNA expression of 19 genes, expression levels of 38 cytokines, and mutations in 109 genes. For correlation analyses, a cut-off &amp;lt; or ≥ the median was used for gene expression and cytokine levels and mutated or not for gene mutations. Results: Within the nonintensive arm (LDAC + GLAS, n=68; LDAC, n=30), improved OS with GLAS + LDAC correlated with lower FOXM1 and MSI2, and higher BCL2 and CCND2 expression. For cytokines, lower levels of 6CKINE, BAFF, ICAM-1, MIP-1α, MIP-1β and MMP-3 correlated with improved OS. The low number of responders in the LDAC arm (n=2) prevented correlation analysis of mRNA or cytokines; however, within the LDAC + GLAS arm, OR correlated with high PTCH1 levels, and high EOTAXIN but low BAFF levels. In the intensive arm (GLAS &amp; 7+3, n=59), higher PTCH1 expression correlated with improved OS (median OS 10.8 vs 39.5 months) but no significant correlations were found with OR; no cytokines correlated with OS or with OR. For gene mutation, in the nonintensive arm (LDAC + GLAS, n=65; LDAC alone, n=25), OS differed with PLEKHH1 and STAG2 mutation status, but no genes correlated with OR. In the intensive arm (n=55), mutations in FLT3, TP53, CEP170, PHF6, and ANKRD26 correlated with OS. Patients in this arm with FLT3 mutations (including ITD) responded better vs wild type FLT3 (median OS 13.4 months vs unreached for FLT3 mutant). We also examined mRNA expression and cytokine levels at various times after starting treatment: statistically significant changes included modulation of SMO, BDNF, ITAC, and IL-23 levels in the nonintensive arm, and SMO, MYCN, CDKN1A, and a number of cytokines including IL-8, TNF-α and IL-5 in the intensive arm. Conclusions: In this analysis, expression levels of a select number of genes and circulating cytokines implicated in AML appear to correlate with OS and OR. The improved response with FLT3 mutations and high PTCH1 expression levels in the intensive arm, along with the analysis appearing to indicate modulation of select genes (eg SMO) and cytokines in both arms while on treatment, deserves further investigation. These preliminary findings will need to be verified in larger randomized trials, which are on-going. Citation Format: Akil Merchant, Catriona Jamieson, Michael Heuser, Geoffrey Chan, Panpan Wang, Keith A. Ching, Jillian Johnson, Thomas O’Brien, Jorge E. Cortes. Biomarkers correlating with overall survival (OS) and response to glasdegib and intensive or nonintensive chemotherapy in patients with acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-009.

Genetic variants and clinical significance of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL), the most common childhood malignancy, is characterized by molecular aberrations. Recently, genetic profiling has been fully investigated on ALL; however, the interaction between its genetic alterations and clinical features is still unclear. Therefore, we investigated the effects of genetic variants on ALL phenotypes and clinical outcomes. Targeted exome sequencing technology was used to detect molecular profiling of 140 Chinese pediatric patients with ALL. Correlation of genetic features and clinical outcomes was analyzed. T-cell ALL (T-ALL) patients had higher initial white blood cell (WBC) count (34.8×109/L), higher incidence of mediastinal mass (26.9%), more relapse (23.1%), and enriched NOTCH1 (23.1%), FBXW7 (23.1%) and PHF6 (11.5%) mutations. Among the 18 recurrently mutated genes, SETD2 and TP53 mutations occurred more in female patients (P=0.041), NOTCH1 and SETD2 mutants were with higher initial WBC counts (≥50×109/L) (P=0.047 and P=0.041), JAK1 mutants were with higher minimal residual disease (MRD) level both on day 19 and day 46 (day 19 MRD ≥1%, P=0.039; day 46 MRD ≥0.01%, P=0.031) after induction chemotherapy. Multivariate analysis revealed that initial WBC counts (≥50×109/L), MLLr, and TP53 mutations were independent risk factors for 3-year relapse free survival (RFS) in ALL. Furthermore, TP53 mutations, age (<1 year or ≥10 years), and MLLr were independently associated with adverse outcome in B-cell ALL (B-ALL). MLLr and TP53 mutations are powerful predictors for adverse outcome in pediatric B-ALL and ALL. Genetic profiling can contribute to the improvement of prognostication and management in ALL patients.

PF544 THE DIFFERENT MUTATIONAL LANDSCAPES OF A PANEL GENES BY NEXT GENERATION SEQUENCING IN 967 CASES OF MDS AND AML

Background: Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are myeloid disease with dysplasia characterized by multiple gene mutations in most cases. With advances in Next-generation sequencing techniques, the molecular landscapes in MDS or AML have been reported with further understanding the disease pathogenesis. As well known, MDS may end up in evolving into AML in most cases, especially the subtypes of MDS with excessive blasts. The driver mutation in MDS developing to AML is still open to study. The comparison of mutational landscapes between MDS and AML will provide evidence to better understand the underlying molecular mechanisms of the myeloid dysplasia evolution. Aims: To analyze the different mutational status of 22 genes commonly observed in MDS and AML. Methods: The bone marrow samples were collected from patients with AML (749 cases) and MDS (218 cases) with written consent from June 2017 to January 2019. Next generation sequencing of a panel of 22 genes was used to evaluate the mutations in MDS/AML patients. Results: 719/ 967(74.4%) cases with MDS/AML were detected at least one gene mutation. There were 274 gene mutations in 142/ 218 (65.1%) cases with MDS and 1224 gene mutations in 577/749(77.0%) cases with AML. Both the median mutations and the frequency of gene mutation between MDS and AML were significantly difference (p < 0.05). Compared to MDS, the mutation rate of CEBPA (11.10% vs 1.40%, p < 0.01), NRAS (14.00% vs 6.40%, p < 0.01), KIT (7.30% vs 0.00%,p < 0.01), NPM1 (17.80% vs 5.00%, p < 0.01), FLT3-ITD (15.40% vs 0.90%, p < 0.01), DNMT3A (10.90% vs 4.10%, p < 0.01), IDH2 (9.5% vs 2.8%, p < 0.01), and IDH1 (6.00% vs 1.40%, p < 0.01) genes were significantly higher in AML. While the mutation rate of ASXL1 (5.30% vs 14.7%, p < 0.01), U2AF1 (5.90% vs 22.00%, p < 0.01), RUNX1 (6.30% vs 10.60%, p < 0.05), SRSF2 (2.30% vs 6.00%, p < 0.05), EZH2 (0.50% vs 3.20%, p < 0.01), SETBP1 (0.70% vs 5.00%, p < 0.01), SF3B1 (2.50% vs 7.80%, p < 0.01), and CBL (0.80% vs 3.20%, p < 0.05) genes were significantly higher in MDS. There were no significant difference of the incidence in TET2, FLT3-TKD, JAK2, ETV6, TP53, PHF6 and ZRSR2 gene mutations. Considering the gene mutation increasing with age, the cases were divided into the young (age < 60 years old) and the old (age≥60 years old). In the young cases, the gene mutation profile was similar to the cohort with incidences of CEBPA, NRAS, KIT, NPM1, FLT3-ITD, DNMT3A, IDH1, plus TET2 gene mutations were significantly higher in AML (p < 0.05). The incidences of ASXL1, U2AF1, TP53, EZH2, SETBP1, and CBL gene mutations were significantly higher in MDS (p < 0.05), and the incidences of FLT3-TKD, JAK2, RUNX1, IDH2, ETV6, PHF6, SRSF2, SF3B1 and ZRSR2 gene mutations were not significantly different between AML and MDS cases (p>0.05). The old cohort showed a different gene mutation profile as compared to the young cohort with the incidences of CEBPA, NPM1, FLT3-ITD, and IDH2 gene mutations were significantly higher in AML (p < 0.01), and the incidences of U2AF1 and SF3B1 gene mutations were significantly higher in MDS. There were no significant differences of all the other gene mutations between AML and MDS cases in the old cohort. Summary/Conclusion: There are typical gene mutation patterns in MDS and AML cases. Some genes mutate in MDS and AML cases without differences and others mutate mainly in AML or MDS. The roles and significances of these gene mutation profiles warranted further investigations to understand the pathogenesis underlying MDS/AML.

Mixed-phenotypic acute leukemia with SET-NUP214 fusion gene positive and extramedullary infiltration: report of one case and review of literature

Objective To investigate the clinical and molecular biological characteristics of mixed-phenotypic acute leukemia (MPAL) with SET-NUP214 fusion gene positive and extramedullary infiltration. Methods The clinical characteristics, diagnosis and treatment of one MPAL patient with SET-NUP214 and extramedullary infiltration who was admitted to Hebei Yanda Ludaopei Hospital in November 2017 were analyzed, and the literature was reviewed. Results The patient was diagnosed as MPAL with extramedullary infiltration. Gene detection found SET exon7-NUP214 exon17 fusion positive accompanied with PHF6, SRSF2 and NRAS mutations. After intensive chemotherapy, the patient achieved complete remission, and then received hematopoietic stem cell transplantation (HSCT), followed by early extramedullary relapse after transplantation, and achieved secondary remission after consolidation chemotherapy. Conclusions MPAL with SET-NUP214 fusion gene positive and extramedullary infiltration has a poor prognosis, and it is easy to relapse. Currently, HSCT is the best available treatment strategy for such patients. Key words: Acute leukemia; Mixed-phenotype; Extramedullary infiltration; del(9)(q34); SET-NUP214

Phf6 Loss Enhances HSC Self-Renewal Driving Tumor Initiation and Leukemia Stem Cell Activity in T-ALL.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. SIGNIFICANCE: Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL.This article is highlighted in the In This Issue feature, p. 305.

Complex karyotype in de novo acute myeloid leukemia: typical and atypical subtypes differ molecularly and clinically.

Complex karyotype (CK) with ≥3 abnormalities is detected in 10-12% of patients with acute myeloid leukemia (AML) and associated with poor prognosis. The most common unbalanced abnormalities found in CK result in loss of material from the 5q, 7q and/or 17p chromosome arms. The presence of 5q, 7q and/or 17p abnormalities denotes typical CK and their absence denotes atypical CK. Since molecular features of CK-AML are not well-characterized, we investigated mutational status of 81 leukemia/cancer-associated genes in 160 clinically well-characterized patients. They included 136 patients with ≥3 exclusively unbalanced chromosome abnormalities, 96 of whom had a typical CK and 40 atypical CK, and 24 patients with ≥1 balanced abnormality in addition to ≥2 unbalanced ones. Patients with atypical CK-AML differed from those with typical CK-AML: they carried TP53 mutations less often (P<0.001) and more often PHF6 (P=0.008), FLT3-TKD (P=0.02), MED12 (P=0.02) and NPM1 (P=0.02) mutations. They were younger (P=0.007), had higher WBC (P=0.001) and percentages of marrow (P<0.001) and blood (P=0.006) blasts, higher complete remission rates (P=0.02) and longer overall survival (P<0.001), thus indicating that atypical and typical CK-AMLs constitute distinct disease subtypes. We also identified smaller patient subsets within both typical and atypical CK-AML that differed molecularly and clinically.

PHF6 and DNMT3A mutations are enriched in distinct subgroups of mixed phenotype acute leukemia with T-lineage differentiation

AbstractThe genetic aberrations that drive mixed phenotype acute leukemia (MPAL) remain largely unknown, with the exception of a small subset of MPALs harboring BCR-ABL1 and MLL translocations. We performed clinicopathologic and genetic evaluation of 52 presumptive MPAL cases at Memorial Sloan Kettering Cancer Center. Only 29 out of 52 (56%) cases were confirmed to be bona fide MPAL according to the 2016 World Heath Organization classification. We identified PHF6 and DNMT3A mutations as the most common recurrent mutations in MPAL, each occurring in 6 out of 26 (23%) cases. These mutations are mutually exclusive of each other and BCR-ABL1/MLL translocations. PHF6- and DNMT3A-mutated MPAL showed marked predilection for T-lineage differentiation (5/6 PHF6 mutated, 6/6 DNMT3A mutated). PHF6-mutated MPAL occurred in a younger patient cohort compared with DNMT3A-mutated cases (median age, 27 years vs 61 years, P < .01). All 3 MPAL cases with both T- and B-lineage differentiation harbored PHF6 mutations. MPAL with T-lineage differentiation was associated with nodal or extramedullary involvement (9/15 [60%] vs 0, P = .001) and a higher relapse incidence (78% vs 22%, P = .017) compared with those without T-lineage differentiation. Sequencing studies on flow-cytometry–sorted populations demonstrated that PHF6 mutations are present in all blast compartments regardless of lineage differentiation with high variant allele frequency, implicating PHF6 as an early mutation in MPAL pathogenesis. In conclusion, PHF6 and DNMT3A mutations are the most common somatic alterations identified in MPAL and appear to define 2 distinct subgroups of MPAL with T-lineage differentiation with inferior outcomes.

PHF6 Mutations Are Mutually Exclusive to TP53 Mutations, and Define a Distinct Subgroup of Secondary Acute Myeloid Leukemia Associated with a Primitive Stem/Progenitor Immunophenotype, Absent Complex Karyotype and Relatively Better Outcomes

IntroductionSecondary acute myeloid leukemia (s-AML) includes AML with myelodysplasia-related changes (AML-MRC) transformed from an antecedent diagnosis of myelodysplastic syndrome (MDS) or from myeloproliferative and myelodysplastic overlap syndrome (MPN/MDS), as well as therapy-related AML (t-AML) arising in patients with prior exposure to leukemogenic therapies. S-AML has a dismal prognosis. Although the genomic profiling of AML has been well studied, few studies have focused on the molecular aberrations of s-AML. A recent study has demonstrated shared genomic aberrations by AML-MRC and t-AML including mutations in TP53, SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2. Another recent study has shown that AML patients with mutations in TP53 and chromatin-splicesosome confer poor prognosis and likely represent s-AML. As such, further characterization of the genomic landscape of s-AML is needed. Recurrent somatic PHF6 mutations are rare in de novo AML (~3%), but the frequency and characteristics of PHF6 mutated s-AML are largely unknown.Patients and MethodsAML-MRC and t-AML patients were searched from the patient database at the MSKCC between 1/2014 and 12/2016. All the patients were reviewed and the diagnosis was confirmed. Only patients with genomic sequencing studies (a targeted NGS panel comprising 30 genes with mutations enriched in AML) performed at diagnosis were included.Results152 patients were identified. There were 7 t-AML patients with favorable cytogenetic abnormalities that likely represent a distinct biological subgroup; therefore these cases were excluded from further analysis. AML-MRC (N=75) and t-AML (N=70) patients had similar clinical characteristics including ages, gender distribution, cytogenetic risk, treatment and survival (median survival: 8 months in t-AML and 10 months in AML-MRC, p=0.4); The mutational profiles of AML-MRC and t-AML cohorts were also comparable with recurrent TP53, PHF6, RAS, and RUNX1 mutations, albeit with different frequencies (Figure 1; Red represents t-AML and blue AML-MRC). Therefore we combined AML-MRC and t-AML cohorts for further analysis.43 (29.7%) patients had TP53 mutations, 17 (11.7%) patients had PHF6 mutations, and 85 (58.6%) patients had neither mutation (non-TP53/non-PHF6). TP53 and PHF6 mutations were mutually exclusive. TP53 mutations were nearly exclusively associated with adverse cytogenetic abnormalities including alterations involving chromosomes (chr) 5p and/or 7q (41/43, 95.3%) with the majority being complex karyotype (30/43, 70%). By contrast, only 18% PHF6 mutated s-AML patients had chr 5p/7q abnormalities and none of them had complex karyotype. Among the patients with non-TP53/non-PHF6 mutations, 30% had chr 5p/7q abnormalities and 6% had complex karyotype. Although secondary AML has very poor prognosis, PHF6 mutated patients had better overall survival than the other two groups (Log rank test, p=0.0095; median survival: PHF6 vs TP53 vs. non-TP53/non-PHF6: 24 vs. 7 vs. 9 months). Immunophenotypically, the blasts from PHF6 mutated patients showed a high frequency of loss of CD38 (44% vs 17%, p=0.04) and CD33 expression (69% vs 28%, p=0.004) compared to PHF6 unmutated cases, indicative of a less mature, stem-cell derived phenotypeConclusionPHF6 mutations define a unique subset of s-AML that is associated with a more primitive stem/progenitor immunophenotype, absent complex karyotype and relatively better outcomes. PHF6 mutations are mutually exclusive to TP53 mutations. Studies are needed to further define the genomic classification of s-AML patients in an unbiased way and to elucidate the role of PHF6 mutations in s-AML. [Display omitted] DisclosuresGoldberg:AROG: Research Funding; Abbvie: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Tallman:Orsenix: Other: Advisory board; AbbVie: Research Funding; Cellerant: Research Funding; BioSight: Other: Advisory board; AROG: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Levine:Loxo: Consultancy, Equity Ownership; Gilead: Honoraria; Roche: Consultancy, Research Funding; Epizyme: Patents & Royalties; Imago: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Janssen: Consultancy, Honoraria; Isoplexis: Equity Ownership; Prelude: Research Funding.

Clinical, Immunophenotypic and Genomic Findings of Acute Undifferentiated Leukemia and Comparison to AML with Minimal Differentiation: A Study from the Bone Marrow Pathology Group

Introduction: Acute undifferentiated leukemia (AUL) is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited: the largest study to date reported 16 AUL cases but did not use the current WHO classification and included limited genetic data on 5 cases (Ann Hematol 2013 92:747-758). Moreover, it is uncertain if AUL is biologically distinct from acute myeloid leukemia with minimal differentiation (AML MD), which also shows limited myeloid marker expression and has been reported to have a poor prognosis.Methods: A total of 95 cases (36 AUL cases 59 AML MD) were identified from pathology databases of eight academic institutions with available diagnostic flow cytometric data, cytogenetic findings, and clinical data by searching for diagnoses of “AUL” or “AML MD”. Diagnosis of AUL required absence of any lineage-defining markers including MPO, CD19 and CD3. Using the WHO classification, diagnosis of AML with MD required expression of at least 2 myeloid markers (CD117, CD13 or CD33), absence of myeloid maturation (CD15) or monocytic markers (CD64, CD11b, lysozyme or non-specific esterase). Next generation sequencing with extensive mutational panel data was available in 78 cases. Outcome analysis for overall survival (OS) and relapse-free survival (RFS) and were performed using Kaplan Meier and log rank test for patients who received induction chemotherapy.Results: Based on cytogenetic abnormalities (N=27) or history of MDS (N=2), according to the 2016 WHO Classification, 28 cases (6 AUL and 22 AML MD) were re-classified as AML with myelodysplasia related changes (AML MRC). The remaining 30 AUL patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the 37 AML MD patients (all p > 0.05). Comparison of immunophenotype in the two groups showed that AUL blasts had more frequent expression of TdT (p=0.0003) and lacked myeloid markers (CD117, CD13 or CD33 p<0.0001), while AML MD cases had more frequent CD123 expression (p=0.0421). No differences in B (CD19, CD20, CD10, CD22, CD79a) or T lymphoid antigen (CD2, CD3, CD4, CD5, CD7, CD8) or monocyte marker (CD11b, CD64) co-expression on blasts (p>0.05) were seen between these two groups. The frequency of abnormal karyotype was similar between AUL and AML MD (16/30 [53%] vs 15/37 [41%], respectively). The most common mutations identified in AUL were PHF6 (7/18), SRSF2 (7/18), RUNX1 (7/23), ASXL1 (6/23) and BCOR (5/16). Compared to AML MD, AUL cases were characterized by frequent mutations in PHF6 (7/18 vs 1/23, p=0.013) and SRSF2 (7/18 vs 2/22 p=0.028). Limiting AUL cases to only those with 1 myeloid marker or less also showed similar findings with more frequent mutations in PHF6 (7/16 vs 1/25, p=0.0031), SRSF2 (6/15 vs 3/26 p= 0.018) and trend towards higher BCOR frequency (5/15 vs 2/26, p=0.078) in AUL patients as compared to AML MD. RUNX1 mutation was seen in 7/23 AUL and 8/29 AML MD (p>0.05). 19/30 AUL patients received induction chemotherapy (AML-type regimen in 18 cases and an ALL-type regimen in 1 case) and 15/30 achieved complete remission. In 10 AUL patients who relapsed, 9 showed identical immunophenotype and one case showed expression of CD13 and CD33. Outcome data in the subset of patients who received induction showed no difference in OS, RFS, or rates of complete remission between AUL and AML MD groups (p>0.05). The 28 AML MRC cases that were originally classified as AUL or AML MD presented with lower WBC (p=0.026), more frequent abnormal karyotype (27/28) specifically complex karyotype (20/28 p=0.002), frequent TP53 mutations (p=0.0002) when compared to the AUL group. AML MRC patients showed worse overall OS (p=0.029) as compared with AUL patients and a trend toward worse outcome as compared with AML MD (p=0.068).Conclusions: In this largest series to date, AUL group shows distinct characteristics from AML MD, including more frequent PHF6 and SRSF2 mutations and expression of TdT. However, clinical outcome is similar between the two groups in patients treated with induction chemotherapy. Cases reclassified as AML-MRC had shorter survival compared to de novo AUL and trend towards worse outcome when compared to AML MD patients. These results suggest a genetic rationale for the separation of AUL as a distinct entity from AML MD and also support the WHO classification of cases with history of prior MDS and/or MDS-type karyotype findings as AML-MRC. DisclosuresGarcia:Celgene: Consultancy.

Comprehensive Sequencing Identifies High Frequency of Copy Number Changes in Korean Patients with Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a genetically complex and heterogeneous disease with a wide range of genetic variations thus identified. As genomic profiling and sequencing studies have revealed genetic basis of ALL, a number of genetic variations are gaining clinical significance in connection with risk stratification and targeted therapy. With the need for comprehensive high-throughput analysis, next-generation sequencing (NGS) has become a good candidate for clinical testing. We have designed a comprehensive NGS assay to detect somatic mutations, translocations, and copy number changes and have evaluated its clinical utility in patients with ALL. The panel reliably detected single nucleotide variations (SNV) and copy number analysis was exceptionally helpful in identifying geneic and chromosomal deletions and duplications.A total of 101 samples of 96 patients were tested. Those patients consist of 40 adults (median age 38, range 21-76) and 56 children (median age 6.5, range 0-17), and diagnoses include 76 B-ALLs and 20 T-ALLs. The most-frequently mutated gene was TP53 (7/96, 7.3%). Ras pathway were also frequently observed (21/96, 29%) in B-ALL and were associated with hyperdiploidy (P< 0.001). Mutations in PHF6, PTEN, FBXW7, JAK3 and WT1 were detected in patients with T-ALL, and all the variants were SNVs. PHF6 mutations were most common among T-ALL patients (7/20, 35%), followed by mutations in FBXW7 (5/20, 25%). Deletions outnumbered duplications, and the most frequently deleted was CDKN2A, followed by transcription factor genes including, IKZF1, PAX5, EBF1, and ETV6. IKZF1 deletion was more frequent in adults (P<0.005), and associated with BCR-ABL1 translocation (P<0.001). PAX5 deletion was also associated with BCR-ABL1 (P<0.001) and also observed in translocations representing Philadelphia-like ALL.Fifty patients were with chromosomal abnormality detected by NGS, and some of them showed normal cytogenetics with a conventional method. Hyperdiploidy and interstitial deletion or duplication can be more easily detected; 11 cases were reclassified as B-ALL with hyperdiploidy, and numerical abnormalities and other copy number changes were identified in seven and 20 patients, respectively. Among 31 gene fusions identified by RT-PCR or NGS RNA fusion panel, only 17 (54.8%) were detected by DNA-based NGS testing.Our NGS gene panel and bioinformatics tools showed excellent performance confirming the clinical utility in identification of important variants in patients with ALL. SNVs and CNVs could be identified simultaneously in a single assay, which could be an alternative or supplement for several conventional tests and simplify the testing processes. We found unexpectedly high proportion of CNVs in ALL, suggesting the need for intensive bioinformatics analysis for CNVs and careful interpretation. DisclosuresNo relevant conflicts of interest to declare.