ALBERT

Description: Background: Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease, characterized by expansion of undifferentiated myeloid precursor cells. The outcome for AML has improved through optimal treatment protocols, new drugs, and better supportive care; however, relapse remains common and patients with relapsed AML have poor prognosis. Recent genome-wide analyses revealed several recurrently mutated genes in AML, however, few of these driver mutations have been developed as therapeutic targets to date. In AML, t(8;21) and MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities; however, knowledge of the genetic landscape is limited. Patients and Methods: The AML-05 study is a Japanese nationwide multi-institutional study of children (age 〈 18 years old) with de novo AML, conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). The trial was registered with the UMIN Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm; number UMIN000000511) and conducted in accordance with the principles set down in the Declaration of Helsinki, and approved by the Ethics Committees of all participating institutions. All patients, or their parents / guardians, provided written informed consent. For whole-exome sequencing (WES), whole-exome capture was accomplished by liquid phase hybridization of sonicated genomic DNA using a bait cRNA library (SureSelect Human ALL Exon V5 or V5 Inc RNA 5 kit), following the manufacturer's protocol. Massively parallel sequencing of the captured targets was performed using a HiSeq 2000/2500 (Illumina) with the paired-end 126-133 bp read option. For targeted sequencing, target enrichment was performed using a SureSelect custom kit (Agilent) designed to capture all coding exons of the 338 genes. Similarly, CCND1, CCND2, and CCND3 were also captured and sequenced in t(8;21) AML samples. For cell cycle analysis, cell lines were treated with DMSO, palbociclib (500 nM), or abemaciclib (500 nM) for 24 h. Then, cells were stained with propidium iodide and analyzed using a FACS Canto II flow cytometer (BD Biosciences). Results and Discussion: First, we analyzed paired AML tumor and germline samples from nine pediatric MLL-rearranged AML patients by WES. In total, 52 mutations (mean, 5.8 mutations/patient) were identified, including known mutational targets in AML, such as FLT3, BRAF, SETD2, BCORL1, and WT1. Moreover, novel CCND3 mutation was detected in one patient. Next, we analyzed 56 samples from patients with pediatric MLL-rearranged AML enrolled in the JPLSG AML-05 study, using targeted sequencing. We selected 338 genes, among which were previously reported and putative driver genes, including CCND3, for targeted sequencing. We identified eight mutations in CCND3 in five pediatric MLL-rearranged AML patients (8.9%). All mutations were clustered in the PEST domain. Four of the eight mutations were R271fs, which is a known hot-spot mutation in lymphoid malignancies. Mutations of the other D-type cyclins (CCND1, CCND2) have been reported in t(8;21) AML (Leukemia, 2017); therefore, we also searched for mutations in CCND1, CCND2, and CCND3 by targeted sequencing of samples from pediatric t(8;21) AML patients (n=105). CCND1 (n=3, 2.9%) and CCND2 (n=8, 7.6%) mutations were detected; however, no mutations of CCND3 were detected. By contrast, there were no mutations of CCND1 or CCND2 in MLL-rearranged AML (n=56), suggesting that mutations of D-type cyclins exhibit a subtype-specific pattern in AML. A recent study demonstrated that genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4/6 inhibitor (Cancer Cell, 2017), therefore, we also examined the effects of CDK4/6 inhibitors (abemaciclib and palbociclib) on two t(8;21) AML cell lines (Kasumi-1 and SKNO-1) and five MLL-rearrangement AML cell lines (ML-2, MV4-11, MOLM-13, THP-1, and NOMO-1) were analyzed. All cell lines described above exhibited impaired proliferation after treatment with CDK4/6 inhibitors. Furthermore, treatment of these cell lines with CDK4/6 inhibitors resulted in detection of lower frequencies of S/G2/M phase cells by flow cytometry, suggesting that cells were arrested in G1 phase via CDK4/6 inhibition. These data provide further insights into the genetic basis of, and potential therapeutic strategies in t(8;21) and MLL-rearranged acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

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: Abstract 1591 Introduction The use of fluorodeoxyglucose-positron emission tomography (PET) to aid the prognosis of patients with diffuse large B-cell lymphoma (DLBCL) after treatment with rituximab-containing combination chemotherapy was investigated. Several studies report that less than 20 % of patients who are PET-negative and more than 85 % of PET-positive patients will experience lymphoma recurrence. Since therapy for DLBCL recurrence remains a major problem, the ability to accurately predict the risk of relapse in PET-negative DLBCL patients after chemotherapy would be extremely beneficial. In this study we focused on levels of serum soluble interleukin-2 receptor (sIL-2R) since several reports indicate that sIL-2R at the time of diagnosis is closely linked to the prognoses of DLBCL patients. We measured sIL-2R levels in DLBCL patients shortly after R-CHOP therapy and investigated whether there was any correlation with patient prognoses. Patients and Methods This was a retrospective pooled analysis of DLBCL patients treated with R-CHOP in our institute. Patients were included if they were under the age of 80, had an International Prognostic Index (IPI) score of more than two, and had received R-CHOP with curative intent. Patients who received salvage chemotherapies due to insufficient responses to R-CHOP were also included. We excluded patients with low IPI scores as their sIL-2R levels at the time of diagnosis were usually in the normal range. Patients were also excluded if R-CHOP could not be continued for reasons other than disease progression. sIL-2R and PET analyses were performed when patients first visited the institute after completing 6–8 cycles of R-CHOP. Patients were divided into three groups: Group A consisted of patients who had completed R-CHOP, were PET-negative and whose sIL-2R levels had returned to normal (=600mg/dl); Group C consisted of patients who had switched to salvage therapies as well as those who remained PET-positive after completing R-CHOP. Overall survival (OS) was assessed using the Kaplan-Meier method and the log-rank test was used for comparisons within each group. A multivariate Cox regression analysis was used to adjust for IPI scores. Results From January, 2006 to January, 2012, a total of 178 DLBCL patients aged less than 80 years were referred to our institution, and 70 of these patients were included in this analysis. The median age of patients was 68 years (range 37–79 years), 96 % had advanced stage DLBCL, and 77 % had elevated lactate dehydrogenase levels. 30% of patients had ECOG performance status (PS) less than 2. The median sIL-2R level at the time of diagnosis was 2676 mg/dl (range 612–48000 mg/dl). The numbers of patients assigned to groups A, B, and C were 28 (40 %), 18 (26 %), and 24 (34 %), respectively. A Fisher's exact test showed no significant differences in IPI scores between the three groups (p=0.453). A Mann-Whitney test showed no significant differences in sIL-2R levels at the time of diagnosis between the three groups (A; median 2660 mg/dl, range 612–19000 mg/dl, B; 2905 mg/dl, 970–37100 mg/dl, C; 4347 mg/dl, 734–48000 mg/dl). Patients in groups A and B received 6–8 cycles of R-CHOP (median 7 cycles). Patients in group C received 1–4 salvage treatments (median 2 treatments) before or after completion of R-CHOP (median 6 cycles, range 2–8 cycles). After a median follow-up time of 36 months (range 5–60 months), 24 deaths had occurred (including 20 due to lymphoma), and the 3 year OS rate for the entire cohort was 65.5 % [95 % confidence interval (CI), 51.7–76.2 %]. The 3 year unadjusted OS rate of patients in groups A, B, and C was 95.2% [95%CI, 70.7–99.3%], 60.7% [95%CI, 31.3–80.6%] and 30.6% [95%CI, 12.5–51.0%], respectively (log-rank, p

Description: Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Description: Abstract 1577 Introduction: Except for minor subtypes of disease with prolonged clinical courses, peripheral T cell lymphoma (PTCL) is clinically aggressive and associated with poor survival. Although the International Prognostic Index (IPI) and the PTCL prognostic index (PIT) are used for prognostic stratification, their predictive utility is in need of improvement. PTCL can be subdivided into two types, nodal and extra-nodal, the latter of which is composed of diseases with characteristic clinical presentations. The nodal group includes PTCL not otherwise specified (NOS), angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), and adult T cell leukemia (ATL). While ATL is a distinct disease caused by HTLV-I, the remaining three diseases have several characteristics in common, and their differential diagnosis is sometimes difficult. Patients and Methods: Patients who were newly diagnosed with biopsy-proven, HTLV-I-negative nodal PTCL and referred to our institution between May 1994 and February 2012 were retrospectively analyzed. Patients treated with regimens not intended to induce remission, as well as those with insufficient clinical data, were excluded. This study was Institutional Review Board-approved and complied with the Declaration of Helsinki. The diagnoses were primarily based on histopathology, and in select instances, molecular and immunological analysis was used to support the diagnosis. The overall survival rate (OS) and progression-free survival rate (PFS) were calculated using the Kaplan-Meier method and significance was determined by log-rank test. Univariate and multivariate analyses were performed with the Cox proportional hazards regression model. Results: A total of 77 patients, including 50 PTCL-NOS, 17 AITL, and 10 ALCL patients, 5 of whom were ALK-positive, were identified. The median follow-up time for survivors was 49 months. The median age was 65 years (range, 23–83), and there was a male predominance (male/female ratio: 1.95). 61 had advanced stage (stage III/IV), 20 had more than 1 site of extranodal involvement, and 20 had documented bone marrow involvement. 35 had ECOG performance status of greater than 1. Laboratory data showed elevated serum LDH level in 55. The IPI score was greater than 2 in 47, and the PIT score was greater than 1 in 50. All but one were treated with anthracycline-containing combination chemotherapies. In addition, 16 received high-dose chemotherapy with autologous stem cell rescue. The 5 year OS for the entire population was 42%, and histological diagnosis did not significantly affect OS (the OS for PTCL-NOS, ALCL, and AITL was 35%, 67%, and 47%, respectively). To explore prognostic factors further, univariate analysis was performed using various pretreatment characteristics. Variables significantly associated with poor survival were advanced stage, extranodal involvement 〉 1 site, bone marrow involvement, anemia, monocyte ≥800 /μL, soluble interleukin-2 receptor 〉 3,000 U/mL, serum IgG ≥1700 mg/dL, and serum IgA ≥410 mg/dL. The IPI classification was highly correlated with prognosis in this cohort: the relative risk of death was 2.78, 3.99, and 5.61 times higher for pts with IPI of LI, HI, and H, respectively, when compared with IPI L pts (log-rank test; P = 0.0213). By contrast, the PIT classification did not have prognostic value. Then, by using variables associated with poor survival by univariate analysis and not included into IPI, prognostic variables independent of IPI were identified on multivariate analysis. Monocytosis and elevated serum IgA levels were significantly associated with poor survival independently of IPI. The dichotomized monocyte and IgA were combined to generate an IgA/monocytosis prognostic score and pts were stratified into three risk groups: low (IgA 〈 410 mg/dL and monocyte 〈 800 /μL), int. (IgA ≥410 or monocyte ≥800), and high-risk (IgA ≥410 and monocyte ≥800) populations. The relative risk of death was 8.56 and 2.83 times higher for pts in the high and int. risk groups, respectively, when compared to the low-risk population (P 〈 0.0001). Clearly, the new prognostic score was able to stratify pts by risk in a manner comparable to the IPI. Conclusions: Monocytosis and high IgA levels were a novel prognostic factor independent of IPI in the limited number of HTLV-I-negative nodal PTCL patients included in this retrospective study. Further analysis is warranted in a greater number of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Germline mutations of DEAD-box helicase 41 (DDX41) have recently been implicated in adult-onset myeloid neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). However, with their prevalence being largely based on the studies from Caucasian populations, the prevalence of DDX41 pathogenic variants in other ethnicities and their risks for myeloid neoplasms have not been fully investigated. Moreover, the clinicopathological features of DDX41-mutated myeloid neoplasms and their genetic profiles have not been elucidated either. To address these issues, we investigated germline DDX41 variants among a large cohort of Japanese patients (n=1,609) with sporadic myeloid neoplasms including MDS (n=1,102), myelodysplastic/myeloproliferative neoplasms (n=14), myeloproliferative neoplasms (MPN) (n=31), secondary AML derived from these myeloid neoplasms (sAML) (n=47), and de novo AML (n=410), as well as Japanese healthy controls (n=17,186) using targeted deep sequencing, whole exome sequencing, and/or whole genome sequencing, though which pathogenic germline DDX41 variants and their clinical, pathological, and genetic features were investigated. Risk of detected germline variant for myeloid neoplasms was evaluated by comparing their frequencies between patients and healthy individuals. The germline origin of candidate risk alleles was confirmed using buccal mucosa samples. We identified 4 germline variants that were significantly enriched in the patient cohort (n=58, 3.6 %) compared to healthy controls (n=42, 0.2%) (OR=15; 95%CI: 10.2-22.8), including two truncating variants, p.A500fs (OR=14; 95%CI: 8.7-24.4), p.E7X (OR=12.6; 95%CI: 2.1-86), and two missense alleles, p.Y259C (OR=15; 95%CI: 4.1-60), and p.S363del (OR=11; 95%CI: 3.4-35). Four additional truncating and splice-site variants, Y279X (n=1), R124fs (n=1), c.571+2T〉G (n=2), and c.298+1G〉T (n=1), were also considered as risk alleles, although a small number of each variant precluded an accurate estimation of enrichment in the patient cohort. Overall, as many as 63 (3.9%) patients harbored one of these variants, where each variant allele was invariably heterozygous. Of note, none of these 8 risk variants have been reported in the Caucasian population. To further evaluate the pathogenic role of these DDX41 germline variants, clinical features and somatic genetic events were investigated. The median age at diagnosis did not significantly differ between patients with and without DDX41 variants (60 and 56 years old in variant carrier and in non-carrier, respectively), suggesting that DDX41-mediated myeloid leukemogenesis shows an age-dependence similar to that in other sporadic cases. Compared with DDX41-unmutated cases, DDX41-mutated cases showed a higher male predominance (52/11 and 987/559, respectively; OR=2.7; 95%CI: 1.4-5.1) and were more likely to have an initial diagnosis of MDS rather than de novo AML (55/8 and 1,094/402, respectively; OR=2.5; 95%CI: 1.2-5.4). Patients with germline DDX41 mutations had a median of 1 (0-5) somatic mutations. Somatic DDX41 mutations were found in 43 (2.6%) cases, a majority of which harbored a germline DDX41 risk variants (32/63 (52%) and 11/1,549 (0.7%), respectively; OR=143; 95%CI: 67-311). Other targets of somatic mutations in risk allele-positive cases included ASXL1 (n=9), DNMT3A (n=6), TP53 (n=5), and JAK2 (n=4), for which no significant correlation was observed between risk allele-positive and negative cases. Abnormal cytogenetics and copy number abnormalities were detected in 30 (48%) of the patients with DDX41 risk alleles, none of which were significantly in the risk allele-positive cases. In conclusion, we identified DDX41 germline risk variants among the Japanese population. Germline DDX41 variants were seen in a substantial fraction of Japanese patients with sporadic myeloid neoplasms. Found in the general Japanese population at very low frequencies, these risk alleles account for the largest germline risk for myeloid neoplasms. Somatic DDX41 mutations were common with a prominent mutational hotspot, almost exclusive found in patients with DDX41 risk alleles. Given the high prevalence of DDX41 germline variants and the late onset of associated MDS and sAML, their detection may help better manage patients and carriers who carried DDX41 risk alleles, even when no family history is known. Figure. Figure. Disclosures Ishiyama: Alexion Pharmaceuticals, Inc.: Honoraria. Chiba:Bristol Myers Squibb, Astellas Pharma, Kyowa Hakko Kirin: Research Funding. Asou:Chugai Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; SRL Inc.: Consultancy; Yakult Honsha Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau. Naoe:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding. Usuki:Otsuka Pharmaceutical Co., Ltd.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sanofi K.K.: Research Funding; Shire Japan: Research Funding; SymBio Pharmaceuticals Limited.: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Boehringer-Ingelheim Japan: Research Funding; Pfizer Japan: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K: Research Funding; Novartis: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Miyawaki:Novartis Pharma KK: Consultancy; Otsuka Pharmaceutical Co., Ltd.: Consultancy; Astellas Pharma Inc.: Consultancy.

Description: MDS is a heterogeneous group of myeloid neoplasms caused by genetic and epigenetic alterations. During the past decade, the major driver mutations in MDS have been fully investigated. However, the role of epigenetic alterations, particularly those of DNA methylation, has less intensively been studied, even though abnormal DNA methylation has long been implicated in the pathogenesis of MDS. In this study, we analyzed DNA methylation status of bone marrow mononuclear cells from 320 cases with MDS-SLD (n = 7), MDS-RS (n = 63), MDS-MLD (n = 51), MDS-EB (n = 186), MDS-U (n = 1), and MDS with isolated del(5q) (n = 12), using Illumina 450K methylation array. Mutations in major driver genes (51 genes) and abnormal genomic copy numbers were also interrogated using targeted-capture sequencing. Using unsupervised consensus clustering, we identified 3 subgroups showing unique DNA methylation profiles. Subsequently, we assessed differentially methylated positions (DMPs) associated with each subgroup. Differentially hypermethylated positions (hyper-DMPs) were significantly more enriched in Group 3 (n = 82) (P 〈 0.001), while differentially hypomethylated positions (hypo-DMPs) were more prominent in Group 1 (n = 125). Group 1 was significantly enriched for SF3B1 (46%) mutations (q 〈 0.01), while Group 2 (n = 131) was characterized by the enrichment of ASXL1 (38%), RUNX1 (30%), TP53 (26%), STAG2 (15%), and SETBP1 (6.7%) mutations (q 〈 0.01). In contrast, Group 3 (n = 64) was significantly enriched for TET2 (67%) and IDH1/2 (12% and 15%, respectively) mutations (q 〈 0.01), suggesting a strong association between DNA methylation and gene mutations. To further elucidate mutation-specific DNA methylation patterns, supervised analysis was performed for each mutation. As expected from their enrichment in Group 3 (q 〈 0.01), TET2 and IDH1/2 mutations were significantly associated with hyper-DMPs (P 〈 0.001) involving 1891 and 8330 promotor sites, respectively. Conspicuously, among these hypermethylated promoter sites, 〉1616 were commonly hypermethylated, strongly supporting the common impact of TET2 and IDH1/2 mutations on deregulated DNA methylation. To clarify prognostic impact of abnormal DNA methylation, we first interrogated the correlation between unique methylation subgroups and revised IPSS. Patients with very low or low risk were significantly dominant (74%) in Group 1 (q 〈 0.01), and very high or high risk cases were significantly enriched (68%) in Group 2 (q 〈 0.01). In accordance with this finding, patients in Group 3 showed significantly shorter overall survival (OS) compared to Group 1 (HR: 1.94, 95%CI: 1.11-3.4, P 〈 0.05) and OS was even worse in Group 2 patients (vs. Group 1: HR: 5.18, 95%CI: 3.21-8.36, P 〈 0.001). Strong correlations between epigenetic and genetic profiles were further interrogated using a Bayesian statistical model; on the basis of DNA methylation and gene mutations, the original 3 clusters were re-classified into 5 discrete clusters, clusters A, B, C, D, and E (n = 124, 17, 74, 46, and 59, respectively); patients in Group 1 and 3 largely clustered into Cluster A and E, respectively, while Group 2 was further subclassified into clusters B, C, and D. Clusters B and D were characterized by a conspicuos enrichment of DNMT3A (88%) and TP53 (69%) mutations (q 〈 0.001), while Cluster C was characterized by higher frequency of ASXL1 (71%), RUNX1 (54%), STAG2 (27%), and EZH2 (21%) mutations (q 〈 0.001). In contrast to significant associations between epigenetic regulators and unique methylation clusters, splice factor mutations tended to be clustered into multiple clusters, depending on type of co-occurring mutations. For example, combined SF3B1 and TET2 mutations (n = 20) were enriched in Cluster A, where highly associated with MDS-RS, while patients with SF3B1 and RUNX1 mutations (n = 9) were more grouped in Cluster C, mostly showing MDS-EB phenotype (89%). Similarly SRSF2 mutations with RUNX1 and/or ASXL1 mutations (n = 36) were enriched in Cluster C, largely associated with MDS-EB phenotype (80%), while those with TET2 or IDH1/2 (n = 39) were mainly grouped into Cluster C, many of which showed MDS-EB phenotype (74%). These findings highlight differential roles of mutated epigenetic regulators and splicing factors in abnormal DNA methylation. In conclusion, we elucidated the collaborative impact of DNA methylation profiles and mutation status on heterogeneous pathogenesis and prognosis in MDS. Figure. Figure. Disclosures Nadarajah: MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: 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.

Description: Background Recent reports have highlighted an adverse impact of TP53 mutations on the prognosis of patients with myeloid malignancies. TP53 mutational analysis is useful in classifying these patients with respect to treatment strategies. High Resolution Melt (HRM) analysis is based on differences in the patterns of melting curves obtained when the targeted double strand DNA (dsDNA) dissociates during heating. HRM analysis is more cost- and time-effective than next-generation sequencing (NGS). In this study, we used HRM analysis to evaluate the impact of TP53 mutations on the clinical features and prognosis of patients at our institute with myeloid malignancies. Methods In this retrospective case study, HRM analysis was performed on 23 patients with MDS and 131 with AML, from October 2011 to January 2018. We targeted ten genes recurrently mutated in myeloid malignancies (TP53, NPM1, FLT3-ITD, IDH1, IDH2, NRAS, KRAS, WT1, DNMT3A, CEBPA) using HRM analysis for screening. For TP53 mutation detection, the entire coding region of TP53 gene from exons 1 to 11 was analyzed using the HRM method. Positive samples were validated using Sanger sequencing. We excluded frequently reported single nucleotide polymorphisms (P47S and R72P), silent mutations, and mutations which were not reported in the Catalogue of Somatic Mutations in Cancer database. NGS of TP53 mutations was done in 18 cases to validate mutations found by HRM analysis. Other non-TP53 gene mutations were similarly analyzed. The primary clinical endpoint of this study was overall survival (OS), measured from the time of diagnosis till time of death due to any cause, or till the last follow-up. The secondary clinical endpoint was relapse-free survival (RFS), measured from time of complete remission to relapse, or the last follow-up. The OS and RFS were compared using the log-rank test. Results We evaluated gene mutations in 131 AML and 23 cases of MDS using HRM analysis. Costs for one HRM analysis were $25 (USD, excluding labor) and required approximately 7 hours processing time per sample. If the screen was positive, add-on cost was an additional $20 with 2 hours of processing time. In total, we identified 28 TP53 somatic mutations in 27 cases from this cohort using HRM analysis (17 newly-diagnosed AML, 4 refractory/relapsed AML, and 6 MDS). Out of the total 154 cases, 18 were also analyzed by NGS; TP53 mutations were detected in 4 cases (median variant allele frequencies = 0.42) and no TP53 mutations were detected in the remaining 14 cases, consistent with the results of HRM analysis. Among the 28 somatic TP53 mutations, 26 were missense, 1 frameshift, and 1 affecting splice sites. Most missense mutations detected (22/26) were localized to the DNA-binding domain of the gene. Cases with TP53 mutations frequently had a complex karyotype (P=0.001), but rarely had NPM1 mutations (P=0.039). In comparison with patients with myeloid malignancies but no TP53 mutations, patients with TP53 mutations had lower counts of white blood cells (3,000 ·μL-1 vs 12,700 ·μL-1 ; P=0.006), lower hemoglobin (7.9 g·dL-1 vs 9.3 g·dL-1 ; P=0.048), and lower platelet counts (2.7×104 ·μL-1 vs 5.7×104 ·μL-1 ; P=0.024). TP53 mutations were associated with shorter OS (median OS, 260 days vs 687 days; P=0.02). In newly-diagnosed AML patients, TP53 mutations were associated with lower counts of both white blood cells (3,200 ·μL-1 vs 26,550 ·μL-1; P=0.006) and lower platelet counts (2.0×104 ·μL-1 vs 6.2×104 ·μL-1; P=0.008) in the peripheral blood. Analysis of bone marrow aspirate revealed that more cases with TP53 mutations had a low myeloid:erythroid ratio than those with TP53 wildtype (53.8% vs 12.7%; P=0.02), suggesting that cases with TP53 mutations have a higher proportion of erythroid precursors, such as in acute erythroid leukemia or erythroid/myeloid leukemia (M6) , as per the FrenchAmerican-British (FAB) classification. Among the patients treated with intensive therapy (either conventional chemotherapy or allogeneic stem cell transplantation), cases with TP53 mutations had shorter overall survival (median OS, 265 days vs not reached; P= 0.04) and shorter relapse-free survival (median RFS, 188 days vs not reached; P=0.02). Conclusion Patients with TP53 mutations have a much poorer prognosis than those without, and exhibit refractoriness to conventional therapies. HRM analysis is a cost- and time-effective method to detect TP53 mutations. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) has an indolent clinical course, with overall patient survival at 5 years after diagnosis of more than 85%. Although the cause of death in a significant proportion of MALT lymphoma patients is not the lymphoma itself, some patients experience early relapse or refractory disease. However, the risk factors capable of predicting aggressive disease in MALT lymphoma have not been clearly defined. Patients and Methods This is a retrospective pooled analysis of pathologically confirmed extranodal MALT lymphoma patients treated at our institute. Patients with nodal or splenic marginal zone lymphoma were excluded. The primary endpoint was progression-free survival (PFS), which was assessed using the Kaplan-Meier method. The log-rank test and multivariate Cox regression analysis were used to assess the prognostic value of each clinical variable. Results From January 2000 to June 2013, 52 extranodal MALT lymphoma patients (26 females and 26 males) were referred to our institution. The median age of the patients was 68 years (range, 43–89 years). Thirty-three (63%) patients had limited stage disease (stage I/II) and 48 (92%) patients had an ECOG performance status of less than 2. The cumulative numbers of patients who were diagnosed with orbit, thyroid, salivary gland, stomach, lung, and intestine disease were 7 (13%), 5 (10%), 9 (17%), 16 (31%), 16 (31%), and 4 (8%), respectively. Fifteen (29%) patients had a prior history of autoimmune disease, and a serum electrophoresis study detected M-protein in 9 (17%) patients (5 patients had IgG, 3 patients had IgM, and 1 patient had IgA). There was no significant correlation between the presence of M-protein and prior history of autoimmune disease (Fisher's exact test, p=0.46). Of the 32 patients who underwent cytogenetic studies, 9 (28%) had cytogenetic aberrations involving MALT1. Histological transformation to diffuse large B-cell lymphoma (DLBCL) was confirmed in 3 patients. After a median follow-up time of 53 months (range, 1–142 months), 16 patients had relapsed (MALT lymphoma relapse or transformation to DLBCL), and 1 patient had died due to lymphoma. The 4-year PFS and overall survival rate were 76.3% (95% confidence interval (CI), 60.0–86.7%) and 98.0% (95% CI, 86.6–99.7%), respectively. Among the clinical variables measured at diagnosis, univariate analysis found that advanced stage, three or more extranodal sites, serum soluble IL-2 receptor concentration higher than 700 IU/L, lymphocyte count higher than 1x10^9/l, hemoglobin concentration lower than 12.5 g/dl, and the presence of M-protein adversely affected PFS (p