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Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3′-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.

Leukemic relapse is frequently accompanied by progressively aggressive clinical course. To understand the molecular mechanism of leukemic relapse, MLL/AF9-transformed mouse leukemia cells were serially transplanted in C57BL/6 mice (N = 96) by mimicking repeated recurrences, where mutations were monitored by exome sequencing (N = 42). The onset of leukemia was progressively promoted with advanced transplants, during which increasing numbers of somatic mutations were acquired (P

Leukemia accepted article preview online, 01 August 2016. doi:10.1038/leu.2016.212.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

POEMS syndrome is a rare paraneoplastic disease associated with monoclonal plasma cells; however, the pathogenic importance of plasma cells remains unclear. We performed comprehensive genetic analyses of plasma cells in 20 patients with POEMS syndrome. Whole exome sequencing was performed in 11 cases and found a total of 308 somatic mutations in 285 genes. Targeted sequencing was performed in all 20 cases and identified 20 mutations in 7 recurrently mutated genes, namely KLHL6, LTB, EHD1, EML4, HEPHL1, HIPK1, and PCDH10. None of the driver gene mutations frequently found in multiple myeloma (MM) such as NRAS, KRAS, BRAF, and TP53 was detected. Copy number analysis showed chromosomal abnormalities shared with monoclonal gammopathy of undetermined significance (MGUS), suggesting a partial overlap in the early development of MGUS and POEMS syndrome. RNA sequencing revealed a transcription profile specific to POEMS syndrome when compared with normal plasma cells, MGUS and MM. Unexpectedly, disease-specific VEGFA expression was not increased in POEMS syndrome. Our study illustrates that the genetic and transcriptional profiles of plasma cells in POEMS syndrome are distinct from MM and MGUS, indicating unique function of clonal plasma cells in its pathogenesis.

Clonal hematopoiesis of indeterminate potential (CHIP) occurs in an age-related manner and associates with an increased risk of hematologic cancer, atherosclerotic disease, and shorter overall survival. Little is known about the cell of origin, repartition patterns of clonal mutations within the hematopoietic differentiation tree, and its dynamics under evolutionary pressure. Using targeted sequencing, CHIP was identified in 121 out of 437 elderly individuals (27.7%). Variant allele frequencies (VAFs) of 91 mutations were studied in six peripheral blood cell fractions. VAFs were significantly higher in monocytes, granulocytes, and NK-cells compared to B- or T cells. In all cases with available bone marrow material, mutations could be identified in Lin−CD34+CD38− HSCs with subsequent expansion to myeloid primed progenitors. In 22 patients with solid cancer receiving (radio-)chemotherapy, longitudinal study of 32 mutations at 121 time points identified relative VAF changes of at least 50% in 13/32 mutations. VAFs of DNMT3A, were stable in 12/13 cases (P

Background Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. Methods A total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). Results After excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4. Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P A total of 14 genes, together with age, gender, white blood cell counts, hemoglobin, platelet counts, cytogenetic score in IPSS-R, were finally selected for the Cox regression in a proportional hazard model and based on the linear predictor of the regression model, we constructed a prognostic model (novel molecular model), in which patients were classified into 4 risk groups showing significantly different OS (“low”, “intermediate”, “high”, and “very high risk”) with 3-year survival of 95.2%, 69.3%, 32.8%, and 5.3%, respectively (P Conclusions Large-scale genetic and molecular profiling by cytogenetics, NGS and array-CGH not only provided novel insights into the pathogenesis and clonal evolution of MDS, but also helped to develop a powerful prognostic model based on gene mutations and other clinical variables that could be used for risk prediction. Molecular profiling of multiple target genes in MDS is feasible and provides an invaluable tool for improved diagnosis, biologic subclassification and especially prognostication for patients with MDS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract 558 In addition to chromosomal and epigenetic abnormalities, somatic mutations constitute key pathogenic lesions in myeloid neoplasms. Individual somatic mutations or various combinations may be both valuable prognostic markers and targets for new rational therapies. Among them, RAS family genes are ubiquitous oncogenes associated with various cancers. Recurrent canonical mutations in the nucleotide binding domains in NRAS and KRAS result in constitutively activated proteins. In myeloid neoplasms, RAS mutations convey a poor prognosis and are often found in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and, rarely, myeloproliferative neoplasms (MPN). We applied whole exome sequencing to paired germline vs. leukemia samples in 65 cases of MDS, 36 MDS/MPN and 32 sAML. We focused our study on the RAS protein superfamily of small GTPases and identified mutations in 3% and 6% of KRAS and NRAS, respectively. Most significantly, we identified somatic recurrent mutations in the F82 residue of Ras-like without CAAX1 (RIT1) gene in 2 patients with chronic myelomonocytic leukemia (CMML) and secondary AML (sAML), respectively. We confirmed the somatic nature of both mutations in sorted CD3+ cells from each patient (pt). RIT1 gene encodes a member of Ras-related GTPases, involved in the p38 MAPK and AKT signaling pathway that mediates cellular survival in response to stress. RIT1 gene amplification has been found in 26% of hepatocellular carcinoma. However, neither amplification nor mutations of this gene have been reported in myeloid malignancies. We thus focused this line of experimentation on this somatic mutation. To establish clinical associations we further studied a cohort of 322 patients with various myeloid malignancies by Sanger sequencing and detected somatic RIT1 mutations in an additional 6 (2%) cases. All mutations were located in exon 5, in the 81 and 82 residues, which encode the switch II domain of this protein, an effector region very close to the GTP-binding site G3, and which is highly conserved among species. Among the 8 mutant cases, 5 (63%) pts had CMML, resulting in a higher frequency of mutations in this subcohort of pts (5 out of 57 CMML, 9%). The other 3 mutations were found in one primary (p)AML (M5b subtype) (1 out of 58 pAML, 2%) and two high-grade MDS, one refractory anemia with excess blasts (RAEB)-2 and one sAML(RAEB-T in the FAB-classification) (2 out of 80, 2.5%). RIT1 mutations were heterozygous in all cases except for one case with trisomy 1 and duplication of the mutant allele. In the cases of WES, we estimated an allelic frequency of ∼35%, consistent with the presence of a heterozygous mutation in ∼70% of sample cells. Because of the large size of the clone and serial samples showing RIT1 mutation since the time of initial diagnosis, it is likely that RIT1 may be of ancestral origin. As RAS-family gene amplifications have been described in cancer, we also studied the presence of amplifications of the RIT1 locus (1q22) by SNP-A. We found 10 cases characterized by a gain involving the RIT1 region (1q21.1-q44): 4 (40%) cases had a diagnosis of CMML, 4 (40%) had myelofibrosis, whereas the remaining patients had MDS (one RAEB-1 and a RA). Quantitative RT-PCR showed RIT1 overexpression in mutants and in patients with 1q amplification (median normalized relative ratio 0,51 and 0,40, respectively) compared to patients with wild type RIT1 and no amplification in 1q (median normalized relative ratio 0,15; P=.039). We theorized that activating RIT1 mutations may constitute a suitable therapeutic target. Because AKT inhibitors can block AKT phosphorylation and therefore reverse the antiapoptotic effect of mutant RIT1, we tested whether AKT inhibitor V (Triciribine) can selectively abrogate the growth of primary cells with RIT1 mutation. In in vitro suspension cultures, a 65% of reduction proliferation was observed with significant effects even at 0.1μM concentrations. In sum, somatic recurrent RIT1 mutations are novel lesions involved in the molecular pathogenesis of myeloid cancers, presumably early in the development of the disease. Moreover, amplifications of RIT1 also lead to overexpression of this Ras-like GTP-ase. Specifically, these abnormalities appear to be more frequent in patients with CMML, but also can be found in other types of MDS. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

In the treatment of childhood acute lymphoblastic leukemia (ALL), excess shortening of maintenance therapy resulted in high relapse rate, as shown by our previous trial, TCCSG L92-13, in which maintenance therapy was terminated at 1 year from initiation of treatment. In this study, we aimed to confirm the long-term outcome of L92-13, and to identify who can or cannot be cured by shorter duration of maintenance therapy. To obtain sentinel cytogenetics information that had been missed before, we performed genetic analysis with genomic microarray and target intron-capture sequencing from diagnostic bone marrow smear. Disease-free survival (DFS) at 10 years from the end of therapy was 66.0±2.8%. Females (n=138) had better DFS (74.6±3.7%) than males (n=142, 57.5±4.2%, P=0.002). Patients with TCF3-PBX1 (n=11) and ETV6-RUNX1 (n=16) had excellent DFS (90.9±8.7% and 93.8±6.1%, respectively), whereas high hyperdiploidy (n=23) was the most unfavorable subgroup, with 56.6±10.3% of DFS. Short duration of therapy can cure more than half of pediatric ALL, especially females, TCF3-PBX1 and ETV6-RUNX1. Our retrospective observations suggest a gender/karyotype inhomogeneity on the impact of brief therapy.

Array CGH identifies copy number changes in 11% of 520 MDS patients with normal karyotype and uncovers prognostically relevant deletions

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are heterogeneous groups of chronic myeloid neoplasms characterized by clonal hematopoiesis, varying degrees of cytopenia or myeloproliferative features with evidence of myelodysplasia and a propensity to acute myeloid leukemia (AML).1 In recent years, a number of novel gene mutations, involving TET2, ASXL1, DNMT3A, EZH2, IDH1/2, and c-CBL, have been identified in adult cases of chronic myeloid neoplasms, which have contributed to our understanding of disease pathogenesis.2, 3, 4, 5, 6, 7 However, these mutations are rare in pediatric cases, with the exception of germline or somatic c-CBL mutations found in 10–15% of chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML),8 highlighting the distinct pathogenesis of adult and pediatric neoplasms.9 Recently, we reported high frequencies of mutations, involving the RNA splicing machinery, that are largely specific to myeloid neoplasms, showing evidence of myeloid dysplasia in adult.10 Affecting a total of eight components of the RNA splicing machinery (U2AF35, U2AF65, SF3A1, SF3B1, SRSF2, ZRSR2, SF1 and PRPF40B) commonly involved in the 3′ splice-site (3′SS) recognition, these pathway mutations are now implicated in the pathogenesis of myelodysplasia.10 To investigate the role of the splicing-pathway mutations in the pathogenesis of pediatric myeloid malignancies, we have examined 165 pediatric cases with AML, MDS, chronic myeloid leukemia (CML) and JMML for mutations in the four major splicing factors, U2AF35, ZRSR2, SRSF2, and SF3B1, commonly mutated in adult cases. Bone marrow or peripheral blood tumor specimens were obtained from 165 pediatric patients with various myeloid malignancies, including de novo AML (n=93), MDS (n=28), CML (n=17) and JMML (n=27), and the genomic DNA (gDNA) was subjected to mutation analysis (Supplementary Table 1). The status of the RAS pathway mutations for the current JMML series has been reported previously (Supplementary Table 2).11, 12 Nineteen leukemia cell lines derived from AML (YNH-1, ML-1, KASUMI-3, KG-1, HL60, inv-3, SN-1, NB4 and HEL), acute monocytic leukemia (THP-1, SCC-3, J-111, CTS, P31/FUJ, MOLM-13, IMS/MI and KOCL-48) and acute megakaryoblastic leukemia (CMS and CMY) were also analyzed for mutations. Peripheral blood gDNA from 60 healthy adult volunteers was used as controls. Informed consent was obtained from the patients and/or their parents and from the healthy volunteers. We previously showed that for U2AF35, SRSF2 and SF3B1, most of the mutations in adult cases were observed in exons 2 and 7, exon 1, and exons 14 and 15, respectively.10 Therefore, we confirmed mutation screening to these ‘hot-spot' exons. In contrast, all the coding exons were examined for ZRSR2, because no mutational hot spots have been detected. Briefly, the relevant exons were amplified using PCR and mutations were examined by Sanger sequencing, as previously described.10 The Fisher's exact test was used to evaluate the statistical significance of frequencies of mutations for U2AF35, SF3B1, ZRSR2 or SRSF2 in adult cases and pediatric cases. This study was approved by the Ethics Committee of the University of Tokyo (Approval number 948-7). No mutations were identified in the 28 cases with pediatric MDS, which included 13 cases with refractory anemia with excess blasts, 5 with refractory cytopenia of childhood, 2 with Down syndrome-related MDS, 2 with Fanconi anemia-related MDS, 2 with secondary MDS and 4 with unclassified MDS. Similarly, no mutations were detected in 93 cases with de novo AML or in 17 with CML, as well as 19 leukemia-derived cell lines. Our previous study in adult patients showed the frequency of mutations in U2AF35, SF3B1, ZRSR2 or SRSF2 to be 60/155 cases with MDS without increased ring sideroblasts and 8/151 de novo AML patients, emphasizing the rarity of these mutations in pediatric MDS (P

SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1(+/-) mice had a significantly reduced number of hematopoietic stem cells (CD34(-)cKit(+)ScaI(+)Lin(-) cells or CD34(-)KSL cells) compared with Sf3b1(+/+) mice, but hematopoiesis was grossly normal in Sf3b1(+/-) mice. When transplanted competitively with Sf3b1(+/+) bone marrow cells, Sf3b1(+/-) stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1(+/-) mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.