ALBERT

Description: Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and 〈 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (〈 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

Description: There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with WT ETV6 with potentially dominate negative effects. Whole transcriptome and whole genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both AML and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by ATAC-seq profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Description: Traditional mouse models for hematopoietic stem cell transplant (HSCT) require whole body irradiation to ablate the hematopoietic cells in recipients, with the defect of disturbing non-hematopoietic cells and introducing potential tumorgenesis in a nonautonomous manner. Here, we use a novel approach to produce mice that whole hematopoietic-specific ablation can be conditionally achieved without global body irradiation. Briefly, a Diphtheria toxin receptor (DTR)-GFP reporter element was targeted into the ROSA26 locus to produce DTR-GFP reporter mice, with a loxp-stop-loxp cassette. Then the DTR-GFP reporter mice were crossed to Vav-Cre mice to produce double transgenic mice (DTR-GFP mice). We injected DT to ablate the bone marrow cells from DTR-GFP mice, and transplant WT bone marrow cells into them. Our data showed that all hematopoietic cells including hematopoietic stem cells, Myeloid, lymphoid lineages are GFP positive in DTR-GFP mice. A single dose of DT can kill all the hematopoietic cells from DTR-GFP mice. One month later, WT bone marrow cells were successfully engrafted into the DTR-GFP recipients without irradiation. We are using this model to re-evaluate some leukemia models that irradiated bone marrow niches might be involved in the tumorigenesis.Thus, we establish a de novel HSCT approach without irradiated myeloablation, which will benefit studies of hematopoiesis, leukemogenesis, hematopoietic stem cell niche, as well as other types of tissue transplants that need ablation of recipient hematopoietic system or immune system. Disclosures: Peng: Biocytogen: Employment, Membership on an entity’s Board of Directors or advisory committees. Shen:Biocytogen: Employment, Membership on an entity’s Board of Directors or advisory committees.

Description: Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Ocular adnexal mucosa-associated lymphoid tissue lymphoma (OAML) has an indolent disease course and a generally good response to radiotherapy; hence, limited-stage disease is commonly treated with radiotherapy. However, after localized radiotherapy for limited-stage OAML, a relapse rate of up to 16% was reported and the main relapse sites observed in previous series were in nonirradiated areas. The relapse rate was higher if the disease involved bilateral conjunctivae or tissue beyond the conjunctiva. We designed a phase II study in which patients with newly diagnosed and limited-stage OAML with bilateral or beyond-conjunctival involvement were treated with a regimen of rituximab in combination with cyclophosphamide, vincristine, and prednisolone (R-CVP), which has been widely used for advanced-stage indolent CD20+ B cell lymphoma and in a previous trial has demonstrated efficacy in advanced-stage MALT lymphoma. Patients and methods: Thirty-three patients with Ann Arbor stage I OAML with the adverse factors were enrolled. Patients received six cycles of R-CVP followed by two cycles of rituximab therapy. The enrolment criteria for disease status were Ann Arbor stage IE OAML with bilateral or beyond-conjunctival involvement; bT1 or T〉1 based on the tumor-node-metastasis (TNM) staging system for ocular adnexal lymphoma proposed by the American Joint Committee on Cancer. Patients were required to be ©ø18 years old, with Eastern Cooperative Oncology Group (ECOG) performance scores of 0-2, with no prior chemotherapy or radiation therapy. Exclusions were made for disease confined to unilateral conjunctiva (T1N0M0) or Ann Arbor stage III-IV disease. The primary end point of the study was response rate, defined as the CR and partial response (PR) rates, based on the revised response criteria for malignant lymphoma. The secondary end point was PFS and overall survival. Results: The median age of the patients was 49 years (range, 19-74 years). The anatomic location of disease was the conjunctiva in 12 patients (36.4%), the orbit in 13 (39.4%), the eyelid in five (15.2%), and the lacrimal duct or gland in three (9.1%). Fourteen patients (42.4%) had bilateral disease at presentation. All study patients (100%) responded to the treatment. After the treatment, 28 patients (84.8%) were in CR and five patients (15.2%) in PR. With the median follow up of 26.9 months (range, 7.4-41.0 months), the estimated cumulative CR rate was 93.9% at 3 years (Figure 1). The cumulative CR rate was significantly lower in the patients with beyond-conjunctival disease (T〉1) than in the patients with bilateral conjunctiva-confined disease (bT1) (91.3% vs. 100%; P = 0.022). The cumulative CR rate was lower for patients aged ©ø49 than in patients younger than 49 years (90.4% vs. 100%; P = 0.034). Multivariate analysis indicated that in our study patients beyond-conjunctival involvement (T〉1) was an independent predictor of cumulative CR rate (hazard ratio [HR] 0.424, P = 0.044). Among the study patients, one patient, who had bT1N0M0 disease, relapsed at the same location in the eye at 21.7 months after initiation of treatment. The estimated PFS at 3 years was 95.5 ¡¾ 4.4%. No death was observed and OS was 100%. No patients experienced ophthalmic complications (Figure2). Conclusions: The R-CVP regimen was efficient for disease control up to 3 years because all study patients responded, cumulative CR rate was 93.9% and the PFS was 95.9% at 3 years. The common relapse sites in patients who received radiotherapy were in nonirradiated areas, suggesting that the systemic antitumor coverage of this chemoimmunotherapy may contribute to lowering the risk of distant relapse in our patients. We conclude that the R-CVP regimen can be effective alternative treatment which avoids radiation hazards and efficiently achieves CR in patients with limited-stage OAML, even those with adverse factors of radiotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Background:B-cell-activating factor (BAFF) is a member of the TNF family that critical for maintenance of B-cell development and homeostasis. BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) are three BAFF receptors. It has been reported that BAFF is expressed by neutrophils, monocytes, dentritic cells and macrophages and modulates the proliferation, survival and drug resistance of multiple myeloma (MM) cells. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by direct interaction with MM cells. We hypothesized that BAFF/BAFF receptors play a role in macrophage-induced bortezomib resistance in myeloma. Methods: First, the expression levels of BAFF and its three receptors in primary MM cells, MM cell lines and peripheral blood monocyte(PBMC)-induced macrophages were detected by semiquantitative real time-polymerase chain reaction (qPCR),Western blot and flow-cytometry. Also the concentration of BAFF in the supernatants of MM patients' bone marrow, MM cell lines and macrophages were determined by ELISA. Second, Primary MM cells and MM cell lines were cocultured with macrophages for the indicated time (usually 4-6h and 24h), for some experiments, we added bortezomib to the coculture system. Cell viability and apoptosis of MM cells were verified by Cell Counting Kit-8(CCK8) after treated with recombinant human (rh) BAFF, BAFF neutralizing antibody and BAFF siRNA. The interactions between BAFF and its receptors are unveiled by flow-cytometry. Then, cell survival signaling activations that may confer MM drug resistance were examined by Western blot. Results: Two receptors of BAFF, TACI and BCMA were highly expressed in various MM cell lines. The expressions of BAFF in PBMC-induced macrophages were heterogeneous. Functional studies showed that rhBAFF promoted RPMI8226 and ARP1 myeloma cells growth (P

Description: Background ：Multiple myeloma (MM) is an malignant disease of clonal plasma cell hyperplasia, which incidence is increasing in recent years. Chemotherapy is the main method to treat MM, but because of drug resistance, MM is still an incrurable disease. High-mobility group box 1 protein (HMGB1) is a chromatin associated nuclear protein, which participated in DNA damage repair , rearrangement, and it also as an extracellular damage associated molecular pattern molecule(DAMP) involved in inflammation, neoplastic progression, angiogenesis, invasion and metastasiss and drug resistance of kinds of tumor cells. There’s no research about the role of HMGB1 in multiple myeloma before. Methods: First, We detected HMGB1 mRNA and protein expression level in MM cell lines and primary MM cells by semiquantitative real time-polymerase chain (qRT-PCR)and western blot respectively. The concentration of HMGB1 in the culture supernatants of MM cell lines and primary MM cells was detected by ELISA. Second, we constructed Lentivirus vector with HMGB1 shRNA, infected the lentivirus to MM cells to knockdown HMGB1 expression. Then Chemo-drugs (ADM, quercetin and bortezomib) were added to MM cells, flow cytometry was used for the detection of cell apoptosis. Results:MM cell line and primary MM cell all express HMGB1 with high level. The concentration of HMGB1 in the MM cell supernatants was significantly elevated after chemotherapy treatment compared with untreated cells(p

Description: Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and have very lethal rate. Chemotherapy is the main method to treat AML, but the complete remission rate is still not very optimal. With the development of genetic and molecular biology technologies, more and more molecular biomarkers are found, some of them are useful for us to evaluate the prognosis and can help us to tailor the treatment plan for different patients. TET2, a member of the ten-eleven-translocation(TET) family genes which can modify DNA by catalyzing the conversion of 5mehtyl-cytosine(5-mC) to 5-hydroxymethyl-cytosine(5-hmC), is often inactivated through loss-of-function mutation and deletion in myeloid malignancies. Recent clinical research reported that the lower the expression of TET2 in MDS and AML patients, the better the response to decitabine (DAC, a demethylation agent) will be. However, the mechanism of the phenomenon is still unknown. Our investigation is trying to uncover the mechanism how TET2 protein levels are negatively related with AML sensitivity to decitabine. Methods: We detected TET2 mRNA expression level in acute leukemia cell lines, bone marrow AML specimens and peripheral blood mononuclear cells from healthy donors by semiquantitative real time polymerase chain reaction (qRT-PCR). Western blot is also applied to detect TET2 protein expression. In order to access TET2 methylation status, we used the methylation-specific PCR. And we also checked the mutant status of TET2 in U937 and KG-1 cell line. CCK8 and flow cytometry are used to detect cell proliferation rate, cell apoptosis, and cell cycle profile. Also, we developed TET2 knock-down and overexpression lentivirus to transfect AML cell lines to explore the mechanism why TET2 expression level is related to the response of DAC. Last, gene array is used to compare gene expression level changes between TET2 knock-down cell lines (or TET2 overexpression cell lines) and the control cell lines. Results: The AML cell lines (KG-1, U937, Kasumi, HL-60, THP-1) and AML patients specimens express lower TET2 than that of PBMC from the healthy donor (P

Description: Natural-killer/T cell lymphoma (NKTCL) is a malignant proliferation of CD56+/cytoCD3+ lymphocytes and constitutes a heterogeneous group of aggressive lymphoma prevalent in Asian and South American populations. NKTCL represents a distinct clinicopathologic entity of non-Hodgkin’s lymphoma, characterized by male predominance, strong association with Epstein-Barr virus (EBV) infection, prominent tissue necrosis and aggressive clinical course. However, molecular pathogenesis of NKTCL remains largely elusive. Here we identified somatic mutations by whole-exome sequencing in 25 NKTCL patients and extended validation through targeted sequencing in an additional 80 cases. Functional experiments including RNA unwinding test, colony forming assay, cell proliferation assay and gene expression profiling were also performed. Overall, 50.5% of NKTCL patients displayed somatic mutations of RNA helicase family, tumor suppressors (TP53 and MGA), and/or epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). Recurrent mutations were most frequently discovered in RNA helicase gene DDX3X (21/105 cases, 20.0%). Mutations of DDX3X were seldom overlapped with those of TP53. Functionally, DDX3X mutants exhibited reduced RNA unwinding activity and enhanced cell proliferation. Similar stimulatory effect on cell proliferation was observed in cells transfected with specific siRNA targeting DDX3X. Gene expression profiling revealed an association of DDX3X mutations with activation of NF-kB and MAPK pathways. The clinical follow-up data showed that DDX3X-mutated patients presented a poor prognosis. Our work suggests the heterogeneity of gene mutational spectrum of NKTCL and provides a potential therapeutic target for relevant cases. Disclosures No relevant conflicts of interest to declare.

Description: J Li, L Bao, ZJ Xia and KY Ding contributed equally to this study. Background: Based on the promising results shown in the phase 3 trial (TOURMALINE-MM1, NCT01564537) and the China Continuation Study of MM1, the oral proteasome inhibitor (PI) ixazomib (ixa) was approved in China in April of 2018, in combination with lenalidomide (len) and dexamethasone (dex) (IRd), for patients (pts) with relapsed/refractory multiple myeloma (RRMM). Data on the efficacy and safety of ixa-based therapy in Chinese pts with MM in real-life practice is rather limited. A large national, multi-center, real-world study involving 14 centers from different areas of China was performed to investigate the current status of ixa usage in China and to evaluate the efficacy and safety in routine clinical practice. A total of 246 ixa-treated MM pts was enrolled, with 163 (66.3%) RRMM, 60 (24.4%) newly diagnosed MM and 23 (9.4%) pts received ixa as maintenance. Herein, we reported the data of RRMM in this study. Methods: Medical records, including demographics, disease characteristics, treatment regimen and duration, response rate, adverse events (AEs) and survival, of ixa-treated (at least one cycle completed with response evaluation result) RRMM pts were analyzed. Results: A total of 149 evaluable pts (out of 163 RRMM pts) treated from April 2018, to July 2019 were included in analysis. Baseline features and prior treatment are summarized in Table 1. Patients were categorized into MM1 trial-eligible/-ineligible groups according to the inclusion and exclusion criteria of MM1 study. Median age was 62 years (range 33 - 87) with 52 (34.9%) ≥65 years. Most pts (75.2%) had ISS stage II-III disease. High-risk cytogenetic abnormalities (including del 17p, t (4;14), and/or t (14;16)) were detected in 19 patients (21.1%, among 90 patients with FISH results). Fifty-two (34.9%) pts had a ECOG PS ≥2. Overall, ixa-based regimens were used as the 2nd/3rd/4th/≥5th-line therapy in 29.7%, 33.1%, 16.2% and 17.4% of the pts, respectively. Prior treatment included bortezomib (91.9%), len (52.0%) and thalidomide (58.8%). More than half pts (54.7%) were refractory to previous bortezomib treatment, and 32.2% pts were len-refractory. MM-1 trial-ineligible pts had more advanced ISS stage, higher ECOG PS, more severe anemia, more lines of prior therapy and more refractory diseases. Treatment, outcome and survival were listed in Table 2. Ixa-based regimens included IRd in 70 (47.0%) patients, ixa-dex (Id) in 31 (20.8%) patients and Id plus chemotherapeutics/other agents (44, 29.5%; including cyclophosphamide in 14 pts, thalidomide in 12 pts, adriamycin in 6 pts, melphalan in 5 pts and daratumumab in 3 pts) in 20 (33.3%). (Table 2). One patient received stem cell transplantation (SCT) during follow-up. The best confirmed ORR (≥PR) for all 149 patients was 53.7% (80/149), including 28.2% of patients with ≥VGPR and 7.4% with a CR, with a median time to response of 41.5 days. Surprisingly, ixa-based regimens demonstrated efficacy in pts with PI/len refractory diseases, with an ORR and ≥VGPR rate of 44.4% and 19.9% for PI-refractory pts, and an ORR and ≥VGPR rate of 30.6% and 12.2% for len-refractory pts. Pts eligible for MM1 study shown comparable ORR (76.7%) with that reported in MM1 (ORR 78%). No significant difference in response between different ixa-based regimens was observed. The median PFS of the whole cohort, pts with standard/high cytogenetic risks, pts refractory to bortezomib/len and pts eligible/ineligible for MM1 trial was 8.2, 8.2, 6.8, 6.7, 5.9months, not reached and 6.6months respectively. The median overall survival (OS) of the whole cohort and every subgroup was not reached. Adverse events (AEs) of grade 3/4, reported in 40 (27.2%) patients, included 10.1% thrombocytopenia, 5.4% anemia, 3.4% diarrhea and 6.0% pneumonia. Only 3 (2.01%) pts had a grade 3/4 peripheral neuropathy during follow-up. Discussion and conclusion: Our results show that ixa-based therapy demonstrated good efficacy with limited toxicity for pts with RRMM in real-life clinical practice. Moreover, in pts with PIs- or len- refractory diseases, ixa-based therapy still showed acceptable effectiveness (ORR: 44.4% and 30.6%; mPFS: 6.7 months and 5.9 months). Although 70.5% pts in our real-life cohort were ineligible for MM1 trial, the efficacy and safety profile is similar to that reported in MM1 China Continuation Study. Ixa-based therapy is a reasonable choice for Chinese RRMM pts. Disclosures No relevant conflicts of interest to declare.