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Introduction B-acute lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. Early response to induction chemotherapy is one of the important prognostic factors in B-ALL. However, the analytic sensitivity for flow cytometry (FC) is only 10 -4. The feasibility of using next-generation sequencing (NGS) of immunoglobulin for the determination of minimal residual disease (MRD) in B-ALL has been demonstrated. This study aimed to investigate the performance of NGS techniques measuring immunoglobulin heavy chain (IgH)-variable, diversity, and joining (V[D]J) clonal rearrangements compared with FC in detecting MRD for children with B-ALL and to predict the clinical outcome of B-ALL patients. Methods Newly diagnosed younger than 18 years old B-ALL patients who received the treatment strategy of South China children's leukemia Group (SCCLG)-ALL 2016 were recruited. DNA extracted from bone marrow cells at all available time points for each patient was submitted to Simcere diagnostics for sequencing using Illumina NovaSeq platform. We performed IgH V(D)J NGS and FCM on the bone marrow serially obtained at diagnosis (D0), 15 days at induction therapy (D15), 33 days at induction therapy (D33) and then at the end of induction therapy (EOI). We defined MRD positive (MRD +) by IgH V(D)J NGS and FCM as more than 1 blast cell among 10 4 and 10 6 bone marrow cells, respectively. The sensitivity of MRD detection by IgH V(D)J NGS and FCM, and the association of MRD status with clinicopathological characteristics were investigated. Statistical analysis was performed through SPSS Statistics 22. Enumeration data and correlation between MRD data and clinicopathological characteristics were compared by Chi-square test or Fisher's exact test. This trial was registered at www.clinicaltrials.gov as # NCT04977895. Results As of July 27, 2021, 22 patients (median age, 4.5 years; range, 3.0-7.3) were enrolled in the study. Three patients (13.6%) had a t (9;22) translocation consistent with Philadelphia chromosome positive disease. According to risk stratifications, 8 (36.4%), 8 (36.4%), and 2 (9.1%) patients were classified as low risk (LR), intermediate risk (IR), and high risk (HR) groups, respectively. The remaining 4 patients are still under treatment and have not been classified. We identified leukemic IgH clones in 100% of the diagnostic samples and 68.2% (15/22) of the patients were polyclonal. In 11 patients whose samples of all the four timepoints (D0, D15, D33, EOI) have been tested in parallel by FCM and IgH V(D)J NGS, the frequencies of patients with MRD + were 30.4% vs. 90.9% at D15 (P＜0.05) by FCM and IgH V(D)J NGS. IgH V(D)J NGS MRD monitoring could identify MRD + patients with frequency of 45.5% and 18.2% among patients achieved MRD negativity by FCM at D33 (P＜0.05) and EOI (P = 0.46). With an MRD detection limit of 10 -6, 90.9% (10/11), 36.4% (4/11) and 18.2% (2/11) patients were MRD negative by FCM but positive by the NGS test at D15, D33 and EOI, respectively. This suggested that the sensitivity of IgH V(D)J NGS was significantly higher than that of FCM. Correlation of the measured MRD between the two methods in the entire cohort (r = 0.7934, P < 0.0001) as well as in the concordant cases (r = 0.5558, P = 0.0032) was very high. There was a high discordant rate with NGS identifying more patients MRD + at this threshold. Furthermore, NGS MRD was positive but the FCM MRD was negative in 13 samples (P < 0.0001). In addition, positive MRD status of D33 by NGS was significantly associated with the age of B-ALL patients, patients under 6 years more frequently harbored detectable MRD compared with those ≥ 6 years old (87.5% vs. 11.1%, P < 0.01). There was no patient relapsed after a medium follow-up of 10.5 months. Conclusions We demonstrated the higher sensitivity of IgH-V(D)J NGS in MRD detection of B-ALL, which implies that NGS MRD monitoring could be helpful for more accurate risk stratifications and more precise treatment according to risk stratifications. Further study with a larger sample size and a longer follow-up period is need. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Next-generation sequencing (NGS) based on liquid biopsy has been an emerging technology to identify tumor-specific genetic aberrations in adult lymphoma. However, there were few studies on the genomic profiling of plasma circulating tumor-derived DNA (ctDNA) in pediatric mature B-NHL. Methods Paraffin-embedded tissue (FFPE) and plasma samples from the newly diagnosed patients were collected and sequenced by 475 genes panel before, during and post of treatment. Clinical stage system, risk stratification and treatment of pediatric mature B-NHL followed a the modified BFM-95 protocol. Results A total of 53 pediatric mature B-NHLs were enrolled，including 35 Burkitt lymphoma, 18 diffused large B cell lymphoma/high-grade B cell lymphoma (DLBCL/HGBCL). We collected 38 tissue and 124 plasma samples for somatic mutation testing. The number of somatic mutations and the TOP 5 genes detected in the 38 tissues and 31 baseline plasma samples, were 416 vs 496, and MYC(71%), DDX3X(45%)，ID3 (42%), TP53(40%), SMARCA4(29%) (Fig1. A)vs MYC(52%), DDX3X(45%)，ID3(42%), TP53(36%), GNA13(23%) (Fig1. B), respectively. The median allele frequency of mutations in plasma was 3%(ranged from 0.2% to 96.6%) and MYC, DDX3X, ID3, TP53, SMARCA4,ARID1A shows higher max somatic allele frequency (MSAF), indicated that was the early events in tumor genesis. The sensitivity of plasma ctDNA to detecting tissue mutations was 63.4% in the 19 matched samples (11 samples from BL and 8 samples from DLBCL/HGBCL) and the sensitivity in BL and DLBCL/HGBCL were 64.1% and 62%, respectively. All genomic alteration types, including single nucleotide variants (SNVs), indels, and gene fusions were detected in similar proportions in each sample type and the gene mutation rate of every gene detected in paired tissue and ctDNA samples. Among the 37 mature B-NHL patients, 6 patients were collected plasma samples after resection of tumor, of which 4 patients was not detected the somatic gene mutations in plasma (Fig. 2). The abundance of ctDNA in patients with stage IV (N=10) was significantly higher than that of stage I-II (N=6) (P=0.0002) and stage III patients(N=15)(P With a median follow-up of 182 Days, 33 patients have completed anti-tumor treatment, 27 patients completed post-treatment PET-CT, and 20 patients have done ctDNA testing synchronously. PET-CT showed tumor residue in 4 patients, of which 2 patients showed no tumor residue in pathology and ctDNA, 1 patient showed tumor residue in pathology but not in ctDNA and with tumor progression 6 months after treatment, 1 patient unable to take biopsy showed no tumor residue in ctDNA and was no tumor reccourence with regular follow-up. At the last follow-up, 1 patient was disease progression, and all of the 53 patients survived. Conclusion Plasma ctDNA testing by NGS was practicable in pediatric mature B-NHL. The abundance of ctDNA is significantly related to tumor burden. CtDNA testing may be more sensitive than PET-CT for residual disease assessment. Nevertheless, sample size expansion is required to verify such conclusions. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) has become a standard consolidation treatment for patients with invasive lymphoma. The combination of cyclophosphamide, carmustine, and etoposide (CBV) is a commonly used conditioning regimen prior to auto-HSCT for patients with invasive lymphoma. Many modifications of the CBV regimen, including the addition of idarubicin, have been employed, with varying results. We retrospectively analyzed the clinical characteristics and treatment outcomes of 36 patients with invasive B-cell non-Hodgkin lymphoma treated with a conditioning regimen prior to auto-HSCT between January 2015 and June 2018. For our analysis, the patients were divided into two groups: one group received the classic CBV conditioning regimen and the other group received idarubicin at a dose of 8 mg/m2 for 3 days in addition to the CBV regimen. Overall survival (OS), median progression-free survival (PFS), adverse reactions, and hematopoietic reconstitution time were compared between the two groups. OS and PFS were analyzed using the Kaplan-Meier method, and the log-rank test was used for comparison between groups. Cox regression was used for multivariable analysis of other clinical factors. The median follow-up time was 29 months. Among the 36 patients in the cohort, 19 were male and 17 were female, with a male-to-female ratio of 1.12 to 1. The median age of onset was 39.5 years; 18 patients were older than 40 years and 18 were younger than 40 years. Twenty-six patients (72%) had an international prognostic index score of 0-2 and 10 patients (28%) had a score of 3-5. According to Ann Arbor staging criteria, 9 patients (25%) had stage I-II disease and 27 patients (75%) had stage III-IV disease. A total of 21 patients achieved complete remission and 15 patients achieved partial remission before transplantation. There were no significant differences between the groups in terms of neutrophil counts (P = 0.795) or platelet reconstitution time (P = 0.551). Also, there was no difference in adverse reactions between the two groups, suggesting that the addition of idarubicin to the conditioning regimen did not aggravate adverse reactions. OS and PFS were significantly longer in the idarubicin group than among patients who did not receive idarubicin. In the multivariable analysis, the use of idarubicin and complete remission state before auto-HSCT were associated with improved prognosis. These results indicate that the use of idarubicin with the CBV conditioning regimen prior to auto-HSCT is a safe and effective choice for patients with invasive B-cell non-Hodgkin lymphoma. Keywords: B-cell non-Hodgkin lymphoma; conditioning regimen; autologous hematopoietic stem cell transplantation; idarubicin Disclosures No relevant conflicts of interest to declare.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. With current chemotherapies and transplantation therapy, there are still 25-50% T-ALL patients that suffer from relapse and have a poor outcome. MicroRNAs (miRNAs or miRs) are endogenous small non-coding RNAs (containing about 22 nucleotides in length). miRs function at posttranscriptional level as negative regulators of gene expression and exert their regulatory function through binding to target mRNAs and silencing gene expression. To better understand the pathogenesis and develop the new therapeutic targets of T-ALL, we have developed a Pten tumor suppressor knockout T-ALL mouse model and profiled miRs from the mouse Pten deficient T-ALL. miR-26b was one of the miRs that were found down-regulated in the mouse Pten deficient T-ALL. Recent studies showed that the aberrant expression of miR-26b is implicated in several types of cancer. The expression level of miR-26b and its role of in T-ALL, however, are unknown. We investigated if the expression level of miR-26b is aberrant in T-ALL and the effect of potentially altered expression on the growth of human T-ALL cells. Methods: We conducted miR array profiling to identify differentially expressed miRs in the mouse Pten deficient T-ALLs compared with preneoplastic thymocyte controls. We validated expression levels of several miRs, including miR-26b, that are differentially expressed in mouse and human T-ALL cells using quantitative RT-PCR. We also overexpressed miR-26b using a lentivirus based vector in human T-ALL cell lines to assess its effect on cell growth and apoptosis. Results: Employing miR array profiling, we identified a subset of miRs that exhibited marked altered expression in the mouse Pten deficient T-ALL cells. Quantitative RT-PCR validated that the expression level of miR-26b in the mouse Pten deficient T-ALL cells was markedly lower in comparison to that of preneoplastic thymocytes. To determine if miR-26b expression level is also altered in human T-ALL, we performed quantitative RT-PCR on a panel of human T-ALL cell lines. Indeed, the expression level of miR-26b is significantly lower in the human T-ALL cell lines when compared with that of normal thymocytes. To functionally assess if miR-26b plays a role in the cell growth of human T-ALL cells, we expressed exogenous miR-26b in a panel of human T-ALL cell lines. We demonstrated that the expression of exogenous miR-26b significantly reduced the proliferation and promoted apoptosis of several human T-ALL cell lines. Conclusions: Our results demonstrated that miR-26b is down-regulated in T-ALL and the expression of exogenous miR-26b elicits deceased cell proliferation and increased apoptosis of human T-ALL. These results suggest that miR-26b may function as a tumor suppressor in the development of T-ALL and further characterization of the target and regulation of miR-26b may have therapeutic implications. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies sustained by a small population of leukemic stem cells (LSCs) that can resist treatment and act as barriers to cure. Previously, we observed that Hes1 and p21 expression was down-regulated in AML cell lines compared to that of normal bone marrow mononuclear cells. However, the activation status of Hes1-p21 pathway and its regulation in LSCs as well as normal hematopoietic stem cells (HSCs) in AML has not been elucidated. In this study, the Hes1-p21 pathway in LSCs and leukemic progenitors (LPs) was studied in adult CD34+ AML with normal karyotype and no genetic mutations and the upstream miRNA regulators were screened. Our results showed that the level of either Hes1 or p21 was lower in LSCs or LPs than that of HSCs whereas the level of miR-9 was higher in LSCs or LPs than HSCs. An inverse correlation was observed in the expression of Hes1 and miR-9. Furthermore, we validated miR-9 as one of the regulators of Hes1 by reporter gene analysis. Knockdown of miR-9 by lentivirus infection suppressed the proliferation of AML cells by the induction of G0 arrest and apoptosis in vitro. Moreover, knockdown of miR-9 resulted in decreased circulating leukemic cell counts in peripheral blood and bone marrow, attenuated splenomegaly, and prolonged survival in a xenotransplant mouse model. Our results indicate that the miR-9-Hes1-p21 pathway plays an important role in supporting AML cell growth and survival, and that miR-9 has a potential to be a therapeutic target for suppressing AML. Disclosures No relevant conflicts of interest to declare.

MYC protein expression has been identified to be associated with inferior overall survival (OS) and progression-free survival (PFS) when coexpressed with BCL-2 protein in patients with diffuse large B cell lymphoma (DLBCL). But the concurrent expression of MYC and BCL-2 proteins in primary gastrointestinal (PGI)-DLBCL has not been clearly understood. Here, we investigated whether this coexpression has prognostic significance in PGI-DLBCL patients and explored its associations with patients’ clinical parameters. We enrolled 60 PGI-DLBCL patients and 30 age- and sex-matched healthy controls. Expression levels of MYC and BCL-2 were detected from both protein and mRNA levels by immunohistochemistry and real-time RT-PCR. Positive expression levels of MYC and BCL-2 proteins were detected in 35% and 45% of patients, respectively. MYC+/BCL-2+ protein was present in 30% of patients. MYC and BCL-2 protein were correlated with high MYC and BCL-2 mRNA expression, respectively (both p Disclosures No relevant conflicts of interest to declare.

Notch signal pathway is an important mediator of growth and survival in several cancer types, while Notch pathway genes function as oncogenes or tumor suppressors in different cancers. Although aberrant Notch activation contributes to leukemogenesis in T cells, the role of Notch pathway in acute myeloid leukemia (AML) remains unclear. To address this problem, we investigated the expression levels of its downstream effector Hes1 and p21 in primary AML cells and cell lines by realtime PCR and western, and found that both of them was weak in these cells. Induced activation of Hes1 consisently led to AML growth arrest and apoptosis, which was associated with enhanced p21 expression. Besides, overexpression of Hes1 inhibited growth of AML cells in vivo. In conclusion, we reported that Hes1 mediated growth arrest and apoptosis of human AML cells, and demonstrated a novel tumor suppressor role for Hes1 in AML. Disclosures No relevant conflicts of interest to declare.

Abstract 4856 Background: The aim of this study was to evaluate the clinical characteristics, prognostic factors and survival outcomes of patients with gastric diffuse large B-cell lymphoma (DLBCL). Patients and methods: 162 patients with gastric DLBCL were evaluated retrospectively. Comparisons were made between patients of gastric DLBCL with component of mucosa-associated lymphoid tissue lymphoma (DLBCL/MALT) and patients of gastric DLBCL without detectable MALT component (de novo DLBCL). Results: Results according to the distribution of sex, age, stage, performance status, and other clinical characteristics were similar between de novo DLBCL group and DLBCL/MALT group (p>0.05). The ratio of patients with the germinal center B-cell-like (GCB) subtype to non-GCB subtype did not differ significantly between the two groups (1:1.1 versus 1:1.6, p=0.319). However, the proportion of patients with the stage-modified international prognostic index (m-IPI) ≥2 was higher in DLBCL/MALT groups (18%) than taht in de novo DLBCL groups (34%) (p=0.026). In addition, the H. pylori infection rate was 75% in DLBCL/MALT versus 38% in de novo DLBCL (p Conclusion: Gastric DLBCL is a heterogeneous disease that included de novo DLBCL and DLBCL/MALT lymphoma. Compared with the former, the latter has a higher H. pylori infection rate. And what's more, the proportion of patients with m-IPI≥2 is higher in DLBCL/MALT groups. De novo DLBCL was associated with higher 5-year PFS and OS estimates. Non-surgical treatment should be a primary consideration for gastric DLBCL. Immunophenotype classification and m-IPI were the most reliable factors for OS, and advanced stage was for PFS. Disclosures: No relevant conflicts of interest to declare.

Abstract 5094 A dynamic interaction occurs between the lymphoma cell and its microenvironment, with each profoundly influencing the behavior of the other. Here, we demonstrate that adhesion of mantle cell lymphoma (MCL) and other non-Hodgkin lymphoma cells to lymph node stromal cells, follicular dendritic cells (FDCs), confers drug resistance, enhances lymphoma cell clonogenicity, and is associated with induction of histone deacetylase 6 (HDAC6). Furthermore, stroma down-regulated miR-548m contributes to HDAC6 up-regulation, and HDAC6 is a key determinant for FDC-mediated lymphoma cell survival and colony formation. We further showed that stroma-mediated drug resistance and clonogenic growth are reversed by enforced expression of miR-548m and inhibition of HDAC6. The HDAC6-selective inhibitor tubastatin significantly enhances cell death, abolishes cell adhesion-mediated drug resistance, and suppresses clonogenicity and lymphoma growth suppression ex vivo and in vivo. Together, these data suggest that the lymphoma–stroma interaction in lymph node microenvironment directly impacts the biology of lymphoma through epigenetic regulation of miRNA and HDAC, with HDAC6 as a potential therapeutic target to overcome drug resistance in MCL and other B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Abstract 4716 Arsenic trioxide (As2O3) is an ancient drug which was used in traditional Chinese medicine. It has attracted worldwide interests because of its antitumor activity when used in acute promyelocytic leukemia (APL). What's more, the demethylation and antiapoptosis function can also be seen in many papers. The methylation status of SH2 containing phosphatase-1 (SHP-1) promoter is common in malignant hematological diseases, such as MDS. Inducing gene demethylation may be a mechanism of As2O3 in treating hematologic cancers. Whether As2O3 has the effects on demethylation of SHP-1 and on the proliferation and apoptosis of cutaneous T cell lymphoma cell lines, and what is the concrete mechanism? Our study aimed to solve these problems. Methods: The activity of As2O3 was analyzed in two cutaneous T cell lymphoma cell lines, including HuT 78 and HuT 102, and cells derived from cutaneous T cell lymphoma patients. All of the cells were treated with As2O3 of different concentration (2.5μmol/L, 5μmol/L, 7.5μmol/L) and different time (24h, 48h, 72h). The methylation status of SHP-1 promoter and the changes of methylation status after the treatment of As2O3 in the cells was detected by methylation specific polymerase chain reaction(MSP). Then the expression level of SHP-1 mRNA was analyzed by Real-time polymerase chain reaction (Real-time PCR). Meanwhile the proliferative inhibition rate was determined with MTT, and the apoptosis induced by As2O3 was detected by flow cytometry with Annexin V/PI staining. DAPI staining was also used to observe the morphological changes during the apoptosis procedure. Then we explore the possible mechanism of As2O3 in this malignancy. First, protein levels of SHP-1, STAT3, phospho-STAT3 were analyzed using Western blotting. In addition, Real-time PCR was also used to analyze the Bcl2, Bcl-xL, survivin, c-myc, Mcl-1, cyclin-D1 and VEGF mRNA expression level. Results: The MSP results indicated that the SHP-1 promoter in these cells was completely methylated and there was no unmethylation strap. The methylation straps of SHP-1 promoter treated with 5.0μmol/L As2O3 were weakened along with the prolonging of treatment time, meanwhile the unmethylation straps were enhanced. The expression levels of SHP-1 mRNA increased along with the increase of As2O3 concentration and treatment time(P Conclusion: The SHP-1 promoter in cutaneous T cell lymphoma cells is completely methylated resulting in the transcriptional silencing of SHP-1. As2O3 can induce the re-expression of SHP-1 mRNA and protein in cutaneous T cell lymphoma cells via the demethylation effect. What's more, As2O3 can inhibit proliferation and induce apoptosis of these cells. It is reported that, SHP-1 is the negative regulator of STAT3. Our results demonstrated that the demethylation leading to re-expression of SHP1 resulted in down-regulation of phosphorylation of STAT3, so did the target genes of STAT3, including Bcl2, Bcl-xL, survivin, Mcl-1, etc. That's the probable mechanism in this study. In conclusion, As2O3 may be beneficial for advanced-stage MF/SS. Disclosures: No relevant conflicts of interest to declare.

Objiective To explore feasibility and efficacy of unrelated donor peripheral blood stem cell transplantation (UD-PBSCT) in treatment of hematologic malignancies. Methods Ten patients with hematologic malignancies undewent high resolution DNA based typing HLA-matched or 1 locus mismatched UD-PBSCT. Busulfan, cyclophosphamide, Ara-C, MeCCNU and antithymocyte globulin (ATG) were used for preparative regimen in all cases. All patients received mycophenolate mofetile, cyclosporin A and short-term methotrexate with CD25 antibody as the graft-versus-host disease (GVHD) prophylaxis. Results The results showed that rapid engraftment was observed in all cases who presented full donor chimerism at 28 days post transplantation by STR-PCR. The median time of neutrophil recovery >0.5×109/L, platelet recovery >20×109/L was observed at 13, 17.5days respectively post transplantation. The incidence of acute GVHD was 3/10 cases (1case with grade I was recovery by himself, 1case with grade III was cured, 1case with grade VI was died). Conclusion It is concluded that above-mentioned conditioning and GVHD prophylaxis are effective approaches for unrelated donor peripheral blood stem cell transplantation in treatment of hematopoietic malignancies.

Mesenchymal stem cells (MSC) have been shown to elicit immunosuppressive effect on allogeneic lymphocyte response. However, MSC are heterogeneous and data on the inhibitory abilities of different MSC subsets are lacking. The Stro-1 antigen potentially defines a more pure and more primitive MSC subset We compared the suppressive property of expanded Stro-1+ and Stro-1− MSC when add to mixed lymphocyte reactions (MLR) or to mitogen response assays. Results showed that T lymphocyte proliferation was significantly more inhibited by expanded Stro-1+ (11.0%~63.7% of maximal response) than by expanded stro-1− MSC (35.5%~106% of maximal response) (p These findings suggest that in clinics a Stro-1+ pre-selection of MSC might produce a more effective immunosuppression especially for the prevention and the treatment of graft versus host disease.