ALBERT

Description: Background Intensive efforts of genome sequencing studies during the past decade identified 〉100 driver genes recurrently mutated in one or more subtypes of myeloid neoplasms, which collectively account for the pathogenesis of 〉90% of the cases. However, approximately 10% of the cases have no alterations in known drivers and their pathogenesis is still unclear. A possible explanation might be the presence of alterations in non-coding regions that are not detected by conventional exome/panel sequencing; mutations and complex structural variations (SVs) affecting these regions have been shown to deregulate expression of relevant genes in a variety of solid cancers. Unfortunately, however, no large studies have ever been performed, in which a large cohort of myeloid malignancies were analyzed using whole genome sequencing (WGS) in an attempt to identify a full spectrum of non-coding alterations, even though its efficacy have been demonstrated in many solid cancers. In this study, we performed WGS in a large cohort of pan-myeloid cancers, in which both coding and non-coding lesions were comprehensively analyzed. Patients and methods A total of 338 cases of myeloid malignancies, including 212 with MDS, 70 with AML, 17 with MDS/MPN, 23 with t-AML/MDS, and 16 with MPN were analyzed with WGS, of which 173 were also analyzed by transcriptome sequencing. Tumor samples were obtained from patients' bone marrow (N=269) or peripheral blood (N=69), while normal controls were derived from buccal smear (N=263) or peripheral T cells (N=75). Sequencing of target panel of 86 genes were performed for all samples. Sequencing data were processed using in-house pipelines, which were optimized for detection of complex structural variations (SVs) and abnormalities in non-coding sequences. Results WGS identified a median of 586,612 single nucleotide variants (SNVs) and 124,863 short indels per genome. NMF-based decomposition of the variants disclosed three major mutational signatures, which were characterized by age-related C〉T transitions at CpG sites (Sig. A), C〉T transitions at CpT sites (Sig. B), and T〉C transitions at ApTpN context (Sig. C). Among these, Sig. C showed a prominent strand bias and corresponds to COSMIC signature 16, which has recently been implicated in alcohol drinking. Significant clustering of SNVs and short indels were interrogated across the genome divided into different window sizes (1Kbp, 10Kbp, 100Kbp) or confining the targets to coding exons and known regulatory regions, such as promoters, enhancers/super enhances, and DNase I hypersensitive sites. Recapitulating previous findings, SNVs in the coding exons were significantly enriched in known drivers, including TP53, TET2, ASXL1, DNMT3A, SF3B1, RUNX1, EZH2, and STAG2. We detected significant enrichment of SNVs in CpG islands, and promoters/enhancers. We also detected a total of 8,242 SVs with a median of 15 SVs/sample, which is more prevalent than expected from conventional karyotype analysis. Focal clusters of complex rearrangements compatible with chromothripsis were found in 8 cases, of which 7 carried biallelic TP53 alterations. NMF-based signature analysis of SVs revealed that large (〉1Mb) deletions, inversions, and tandem duplications and translocations are clustered together and were strongly associated with TP53 mutations, while smaller deletions and tandem duplications, but not inversions, constitute another cluster. As expected, FLT3-ITD (N=15) and MLL-PTD (N=12) were among the most frequent SVs. Unexpectedly, in addition to known SVs associated with t(8;21) (RUNX1-RUNX1T1) (N=6) and t(3;21) (RUNX1-MECOM) (n=1) as well as non-synonymous SNVs within the coding exons (N=30), we detected frequent non-coding alterations affecting RUNX1, including SVs (N=15) and SNVs around splicing acceptor sites (N=5), suggesting that RUNX1 was affected by multiple mechanism, where as many as 38% of RUNX1 lesions were explained by non-coding alterations. Other recurrent targets of non-coding lesions included ASXL1, NF1, and ETV6. Conclusions WGS was successfully used to reveal a comprehensive registry of genetic alterations in pan-myeloid cancers. Non-coding alterations affecting known driver genes were more common than expected, suggesting the importance of detecting non-coding abnormalities in diagnostic sequencing. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Usuki:Mochida Pharmaceutical: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Sanofi K.K.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Daiichi Sankyo: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Limited.: Research Funding; Shire Japan: Research Funding; Janssen Pharmaceutical K.K: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Pfizer Japan: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Chiba:Bristol Myers Squibb, Astellas Pharma, Kyowa Hakko Kirin: Research Funding. Miyawaki:Otsuka Pharmaceutical Co., Ltd.: Consultancy; Novartis Pharma KK: Consultancy; Astellas Pharma Inc.: Consultancy.

Description: Background TET2 is known as an enzyme which converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Loss-of-function mutations in TET2 are frequent in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Normal counterpart of AITL is thought to be follicular helper T cells (Tfh). PTCL-NOS is likely to consist of heterogeneous groups. Some of the PTCL-NOS cases also have features of Tfh. In the last annual meeting, we reported that aged homozygous Tet2 gene-trap mice (Tet2gt/gt), which showed 80% reduction in the Tet2 mRNA level in various hematopoietic cells, developed T-cell lymphoma. To further investigate the mechanism of T-lymphomagenesis, we analyzed methylome, hydroxymethylome, and transcriptome in the lymphoma cells. Material and Method Tet2 gt mice have a trapping vector inserted into the second intron of the Tet2 locus. In all the analyses, we used CD4+ T cells prepared from lymphoma cells developed in these mice, as well as CD4+ T cells prepared from spleen of wild-type mice as a control. To investigate genome-wide methylation and hydroxymethylation statuses, we performed MeDIP and hMeDIP sequencing, and bisulfite sequencing. To examine comprehensive gene expression, we performed microarray-based analysis, followed by Gene Set Enrichment Analysis (GSEA). Result After observation over a year (median, 67 weeks), 5 out of 7 Tet2gt/gt mice developed T-cell lymphomas with Tfh-like immunostaining pattern. No differences were found in the average levels of 5mC between lymphoma and control CD4+ cells throughout the regions around transcription start sites (TSS) +/- 5 kb. In contrast, when the same regions were analyzed for 5hmC levels, those in the lymphoma cells were significantly lower at the regions around TSS +/- 1 kb. When focused on regions having high 5mC contents (MACS score〉5.0), lymphoma cells demonstrated a significant enrichment at regions around TSS +/- 1 kb, intragenic regions, and CpG islands (p=0.013, 0.006, 0.022 respectively). On the other hand, 5hmC was significantly decreased at regions around TSS +/- 1 kb in lymphoma cells than control cells (p=0.018). In a set of genes whose expression was higher in lymphoma cells than control cells, 5hmC levels were significantly lower in lymphoma cells. GSEA analysis revealed upregulation of Tfh-associated genes such as Bcl6 and cMaf (FDR q value=0.0004), key transcription factors for Tfh differentiation, in lymphoma cells compared with control cells. The expression of upregulated Tfh-associated genes was validated by real-time PCR. We focused on the epigenetic change of Bcl6 because it is among the most important transcription factors for Tfh development. It was reported that hypermethylation at intron 1 of Bcl6 upregulated its transcriptional activity in B cell lymphomas. Bisulfite sequencing revealed that the CpG sites in the intron 1 of Bcl6 were massively methylated/hydroxymathylated in lymphoma cells, whereas those in control cells were mostly at an unmodified status. MeDIP sequencing indicated that intron 1 of Bcl6 had more methylated status in lymphoma cells than control cells. We also found that CpG sites in the same region were densely methylated/hydroxymathylated in EL4, mouse T-cell lymphoma cell line, and that the decitabine treatment converted them into unmodified CpG along with the decrease in the Bcl6 expression levels. Discussion and Conclusion Tet2 gt/gt mice developed T-cell lymphoma with both Tfh-like immunohistological character and gene expression pattern. We found distinct changes in methylome, hydroxymethylome, and transcriptome. We also found a tight linkage between the increased methylation of intron 1 of Bcl6 and increased expression of its mRNA level in lymphoma cells developed in Tet2 knockdown mice. The same scenario is indicated in a T-cell lymphoma cell line. These observations imply that, in normal CD4+ T cells, reduced Tet2 function might increase the methylation status of the CpG sites in intron 1 of Bcl6, which may result in upregulation of Bcl6 expression and deviated Tfh generation. These processes might be an initiating event for the development of T-cell lymphoma with the Tfh features. Because of the long latency before lymphoma development in Tet2gt/gtmice, it is likely that additional hits are necessary. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1299 Background: Loss-of-function mutations in TET2 are frequent in human myeloid and lymphoid malignancies. Especially, TET2 mutations were found in 30–47% of angioimmunoblastic T-cell lymphoma (AITL) and 10–38% of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). The tumor origin of AITL is thought to be follicular helper T cells (Tfh). PTCL, NOS is a group of heterogenous T-cell-derived lymphomas, whereas some cases of PTCL, NOS also appear to be derived from Tfh. Several recent papers reported that Tet2 knockout mice demonstrated premalignant status of myeloid lineages, implicating that TET2 mutations have a driver's role in myeloid malignancies; however, there are no clues to how impaired Tet2 function provokes T-cell lymphomas. To address this issue, we analyzed Tet2 gene trap (Tet2gt) mice. Materials and methods: Tet2gt mice (Transgenic Res 17:599, 2008) have a trapping vector inserted into the second intron of Tet2 locus. Lineage-, Sca1+, and c-kit+ (LSK) cells were sorted from Tet2gt E15.5 fetal liver (FL) cells and transplanted into lethally irradiated Ly5.1 wild-type (WT) mice. Bone marrow, spleen, and tumors when developed were analyzed in the recipient mice as well as adult Tet2gtmice by flow cytometory and immunohistochemical staining. Result: Adult Tet2gt mice were obtained under the following ratio; Tet2+/+:Tet2+/gt:Tet2gt/gt=48:52:25. TET2 mRNA expression level in Tet2gt/gt Lin- FL cells was reduced to 20% of that in WT Lin- FL cells, while the 5-hydroxymethyl cytosine level was reduced to around a half of that in WT cells. At 40 weeks of age, the proportion of CD4+/PD1+/Cxcr5+ cells, immunophenotypically similar to Tfh cells, significantly increased in Tet2gt/gt mice compared to WT mice (P=0.022). Two out of 8 Tet2gt/gt mice developed marked splenomegaly (more than 300 mg) at 40 weeks, and 4 out of 6 developed marked splenomegaly with swollen lymph nodes and multi-nodular tumors in liver and lungs at 60 weeks or older (median, 67 weeks). Histological examination of the enlarged spleen and swollen lymph nodes in these 4 mice demonstrated completely destroyed follicular structures. The spleen, lymph nodes, and nodular tumors showed infiltration of morphologically abnormal lymphocytes, which were proven to be CD4+/PD1+ T cells. These cells were also weakly positive for Cxcr5. CD4+ cells purified from the tumors revealed restricted rearrangement patterns of T-cell receptor genes, implying T-cell lymphoma. There were no increased vascular structures representing AITL. CD4+/PD1+ cells purified from the tumors expressed Bcl6 and cMaf, two key transcription factors of Tfh, at significantly higher levels compared to CD4+/PD1+ cells purified from spleen of WT mice. Meanwhile, one of 6 mice transplanted with Tet2gt/gt LSK cells also developed lymphoma with similar pattern of CD4+/PD1+/CXCR5 expression at 40 weeks after transplantation. Discussion: Our observations indicate that reduced expression of TET2 may cause skewed differentiation into Tfh, which eventually develop tumors that pathologically recapitulate PTCL, NOS in human, while the tumor-comprising cells show characteristics of Tfh. The long latency period required for tumor development indicates secondary genetic hits in addition to the downregulation of TET2. These secondary events might determine the specificity of the hematologic malignancies, particularly AITL and PCTL, NOS with propensity for AITL. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Sarcopenia, the loss of muscle mass, has been recognized as a prognostic factor for cancer patients. For example, low body mass index (BMI) was reported to be a risk of poor overall survival (OS) among allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. However, low BMI was not associated with high non-relapse mortality (NRM) rate, and BMI may not directly reflect the physical condition. (Bone Marrow Transplant. 2014;49:1505-12). To evaluate the clinical impact of the muscle volume on the prognosis of allo-HSCT recipients, other biomarkers that directly reflect muscle mass may be warranted. Urinary creatinine excretion (UCE) has been reported to estimate muscle mass and have prognostic value for kidney transplant patients (Transplantation. 2008;86:391-8.). There is no report to evaluate clinical impact of UCE on the prognosis of allo-HSCT recipients. Therefore, we retrospectively analyzed the association between pre-transplant UCE and the transplant outcomes. Methods: We included 173 adult patients with acute myeloid leukemia (AML) in complete remission (CR) who underwent first allo-HSCT from 2006 to 2017 at our institute and measured UCE before allo-HSCT. Concerned the possibility of urine storage failure, two patients with low total daily urine volume (

Description: [Backgrounds] Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of peripheral T-cell lymphoma (PTCL), characterized by generalized lymphadenopathy and autoimmune-like manifestations. Regarding genetic lesions of AITL, frequent mutations in TET2, IDH2, DNMT3A and RHOA have been identified. In some PTCL cases, TET2 and DNMT3A mutations were identified in cell populations beyond the CD4+ T-lymphocytes, in which the tumor cells are contained, suggesting that TET2 and DNMT3A mutations occurred earlier than the commitment to CD4+ T lymphocytes. [Objective] We performed this study to identify the cell-type-specific mutations and further explore mutational profiles in AITL and AITL-related cancer. [Methods] The dataset of targeted sequencing was analyzed for 76 genes in 79 PTCL samples. Mutational origin was analyzed by cell sorting and laser microdissection. [Results] Targeted sequencing identified 168 mutations in 33 genes. Recurrent mutations, in addition to the already known frequent mutations in RHOA/TET2/IDH2/DNMT3A, were found in ODZ1 [4/79 (5%)], Notch1, NAV2, and MTERFD3 [3/79 (4%) for each], MLL2, TET3, FAT2, and LAMA2 [2/79 (3%) for each]. TET2/DMNT3A mutations showed statistically higher allelic burden than the newly identified mutations, suggesting precedence of TET2/DNMT3A mutations. Cell sorting and laser microdissection, followed by amplicon sequencing, revealed that TET2/DNMT3A mutations were identified in both tumor cell-enriched and –depleted populations while RHOA and IDH2 mutations were confined to tumor cell-enriched populations. Most of the newly identified mutations were similarly classified into the above-mentioned two types. It is noteworthy that we found some mutations only in T-cell lymphoma cell-depleted CD20-positive population but not in the tumor-cell-enriched PD-1-positive population. [Conclusion and discussion] Differentiation stages that mutational events arise are likely to be multiple in AITL and AITL-related lymphoma. Moreover, in AITL, Epstein-Bar virus-infected B cells often grow in an oligoclonal manner, sometimes resulting in monoclonal proliferation with fully malignant features. Detection of B-cell specific mutations might suggest premalignant status of B cells in these cases. Disclosures No relevant conflicts of interest to declare.

Description: Backgrounds Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of T-cell lymphoma, characterized by generalized lymphadenopathy and frequent autoimmune-like manifestations. The diagnosis of AITL is sometimes challenging for hematopathologists, because the tumor cell content is generally low and relatively large reactive lymphocytes are confused as tumor cells. Possibly because of the low tumor cell frequency, clonal rearrangement of T-cell receptor gene is undetectable in 30% of the cases. We identified recurrent mutations in RHOA at c.G50T, predicting to generate p.G17V in 70% of AITL and PTCL-NOS harboring AITL features, which implies diagnostic properties for AITL (M S-Y and SC, unpublished). Purpose To establish a novel cost-effective method to diagnose AITL, we performed allele-specific realtime PCR to detect RHOA G17V mutation. Methods Genomic DNA was extracted from 119 AITL and PTCL-NOS samples, which include 47 periodate-lysine-paraformaldehyde (PLP)-fixed, 12 formalin-fixed-paraffin- embedded (FFPE) and 60 frozen tissues. Forty-one out of 60 genomic DNA samples, purified from frozen tissue, were amplified by RepliG kit (QIAGEN). Allele-specific primers for RHOA G17V mutant and wild-type sequences were designed by Wangkumhang's algorithm. The [mut] and [WT] values were individually measured by realtime PCR using each primer set, and the [mut]/([mut]+[WT]) values were calculated. Mutant allele frequencies were determined by amplicon-based deep sequencing using MiSeq. Results The [mut] values were distributed from 1.5×10-7 to 7.6×10-2, and the [WT] values were from 7.9×10-5 to 1.3×10-2. The [mut]/[mut]+[WT] values were distributed from 1.9×10-4 to 8.5×10-1. We set a cut-off value to determine existence of mutation as 2% for MiSeq, and 1.3×10-2 for the [mut]/([mut]+[WT]) value. Then we compared these two methods to detect RHOA G17V mutation. Forty-three cases were positive for the RHOA mutation in this study cohort by MiSeq, including 32 AITL and 11 PTCL-NOS cases. The [mut]/([mut]+[WT]) values of DNA from FFPE samples tend to be lower than those from other samples, and 4 out of 12 FFPE samples determined as mutation-positive by MiSeq were not detected by our allele-specific realtime PCR. We therefore excluded the FFPE samples and analyzed the data of 107 DNA samples, purified from PLP-fixed and frozen tissues. Rank correlation coefficient was 0.753. Sensitivity was 97.4%, and specificity was 97.1%. Positive concordance rate was 94.9%, and negative concordance rate was 98.6%. Conclusions We established a method to detect RHOA G17V hotspot mutation for AITL. It is expected to be highly accurate and cost effective. Disclosures: No relevant conflicts of interest to declare.

Description: Background Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of peripheral T-cell lymphoma (PTCL) characterized by generalized lymphadenopathy, hyperglobulinemia, and autoimmune-like manifestations. Frequent mutations in TET2, IDH2, and DNMT3A have been described in AITL, which are commonly found in myeloid malignancies. However, the molecular pathogenesis specific to AITL is still unknown. Methods To clarify the molecular pathogenesis of AITL, we performed comprehensive gene-mutation analysis. Somatic mutations in 3 AITL and 3 PTCL-NOS specimens were explored using whole-exome sequencing (WES). Targeted resequencing for genes identified by WES was also performed in a cohort of 157 patients with AITL/PTCL-NOS. Results We identified a novel recurrent mutation in RHOA (c.G50T/p.G17V) in 3 AITL and one PTCL-NOS samples by WES. Validation in an extended cohort revealed an extremely high frequency of the identical G17V RHOA mutation in AITL (50/72 [69.4%]), together with mutations in TET2 (39/47 [83.0%]), IDH2 (14/47 [29.8%]), and DNMT3A(12/47 [25.5%]). The G17V RHOA mutation was also found in PTCL-NOS samples at a lower frequency (14/85 [16.5%]), especially in those harboring AITL features (PTCL-NOS with AITL features vs PTCL-NOS w/o AITL features: 13/21 [61.9%] vs 0/38 [0%]). Remarkably, mutations in RHOA, TET2, and IDH2 showed striking correlations. All RHOA-mutated samples were accompanied by TET2 mutations. IDH2 mutations were confined to the samples having simultaneous mutations of RHOA and TET2. Mutations in DNMT3A largely overlapped to TET2 mutations, but its correlation with RHOA or IDH2 mutations was much less clear. TET2 mutations showed a consistently higher allelic burden than RHOA mutations. Gene-mutation analysis of tumor cells and infiltrated cells demonstrated that the G17V RHOA mutation specifically existed in tumor cells, but not in non-tumor cells, while TET2 mutations were identified both in tumor and non-tumor cells. RHOA encodes a small GTPase, which operates as a molecular switch that regulates a wide variety of biological processes through cycling between an active (GTP-bound) and an inactive (GDP-bound) state. We demonstrated that the G17V RHOA mutant did not bind GTP and also inhibited GTP-binding of the wild-type RHOA protein. Accordingly, unlike wild-type RHOA, the G17V mutant was not able to activate transcription from the serum response factor-responsive element (SRF-RE). Conclusions Our data suggests that combination of preceding mutations in TET2 and subsequent tumor-specific G17V RHOA mutation determines distinct disease properties of AITL. Disclosures: No relevant conflicts of interest to declare.

Description: Hematopoietic stem cell transplantation (HSCT) recipients have an increased risk of acute kidney injury (AKI) or chronic kidney disease (CKD). However, serum creatinine level may underestimate the prevalence of these renal complications because of decreased lean body mass or concurrent liver disease, which was frequently observed in a HSCT setting. Cystatin C measurement may be more sensitive for detecting impaired kidney function. We retrospectively reviewed the medical records of 70 HSCT (54 allogeneic and 16 autologous) recipients who had at least one chance to monitor serum cystatin C level during last 2 years in our institution, and evaluated cystatin C as a possible new marker which can predict subsequent renal dysfunction. The occurrence of AKI was defined by the RIFLE classification and CKD staging was based on KDOQI criteria. Of 70 transplant recipients, 20 patients developed AKI after median 50.5 days (range 0–283 days) after HSCT, while worsening CKD stage was observed in 21 patients during observational periods. Cystatin C level was not influenced by pretransplant disease status (P=0.153) or autologous transplant (P=0.311), but significantly elevated after allogeneic transplantation (P