ALBERT

Description: BACKGROUND: This study was to evaluate the efficacy and safety of chidamide monotherapy, which is a new histone deacetylase inhibitor (HDAci) of the benzamide class, in relapsed or refractory Angioimmunoblastic T cell lymphoma (AITL), to investigate its genomic expression signatures as well as the mechanisms of chidamide's anti-lymphoma effect and its resistance. METHODS: Two cases with relapsed or refractory AITL were treated with chidamide. We performed a repeated biopsy on relapsed lymphomas and did whole genome next-generation sequencing (NGS) testing on drug -resistant tumor samples. RESULTS: The first patient is a 54-year-old man presented with stage Ⅳ AITL. He underwent autologous stem cell transplantation in his first complete remission (CR) but relapsed 7 months later. In view of resistance to multiple lines of chemotherapy and poor performance status, chidamide was adaministered orally at a standard dose of 30mg twice a week. Pulmonary lesions regressed quickly and a second CR was achieved. Adverse events included grade 2 cytopenias, diarrhea, and reversible QT interval prolongation. The disease free survival was 6 months and AITL relapsed again 4 months ago. He responded to low-dose chidamide combined with lenalidomide and dexamethasone and remains well. Formalin-fixed, paraffin-embeded tumor tissues collected at first relapse were tackled for whole-genome sequencing. The other patient is a 62-year-old woman who had stage Ⅲ AITL. Disease progressed after two cycles of combined chemotherapy, thereafter a standard dose of chidamide monotherapy was initiated. With well tolerability, an unconfirmed CR was achieved and lasted for nearly 3 months. She is still alive but remains refractory to various salvage therapeutics, including chemotherapies, arsenic trioxide, thalidomide, and pralatrexate. A rebiopsy was perferomed and the histopathological findings confirmed the relapse of AITL, associated with Epstein-barr virus infection. Fresh tumor tissues were sent for whole-genome sequencing. In both cases, NGS testing identified mutations of RHOA gene, epigenetic regulators TET2, IDH1, and DNMT3A, as well as CD28. CONCLUSIONS: Chidamide, a low nanomolar inhibitor of HDAC1, 2, 3, and 10, was approved in China for the management of relapsed and/or refractory peripheral T cell lymphoma. It's reported that patients with AITL tended to have higher response rates and more durable responses to chidamide treatment. Our report showed single-agent chidamide is a reasonable approach to treat the formidable disease but seemed difficult to achieve a sustained remission. Various genetic, epigenetic, and immune alterations involve in the pathogensis of AITL, which provide targets for chidamide therapy. High-throughput sequencing approach is very helpful to clarify the mechanisms. Disclosures No relevant conflicts of interest to declare.

Description: Objective The purposes of this study is to investigate the expression of Pim-1 and Pim-2 on macrophages in multiple myeloma (MM) patients; investigate the relationship between co-expression of Pim-1, Pim2 in macrophages and early treatment response and prognosis; investigate the expression of PD-1 and PD-L1 in multiple myeloma; and analyze correlation between the expression of PD-1 and PD-L1 and early treatment response and prognosis, providing preliminary therapeutic evidence for the novel treatment in multiple myeloma. Methods Clinical data and bone marrow biopsy sample of108 patients were selected with newly diagnosed multiple myeloma at West China Hospital of Sichuan University from 2009 to 2014 were collected. Patients that were included were followed up until May 2017. Opal multi-labeling immunohistochemistry of bone marrow was performed, and macrophages were labeled with anti-CD68 antibody in order to detect co-expression of P-im1, P-im2 and macrophages. They were divided into high-expression and low-expression groups according to the degree of their co-expression. Meanwhile detect the expression of PD-1, PD-L1 in MM and they were divided into positive and negative groups. The relationship between the different expression levels of Pim-1 and Pim-2 in macrophages、PD-1、PD-L1 and the early treatment response and prognosis of multiple myeloma were analyzed. The Kaplan-Meier method was used to analyze the influence on disease progression and overall survival in MM patients. The Cox proportional hazards model was used as multivariate analysis used to explore independent risk factors affecting the prognosis of MM patients. Results 1.The median PFS value in high CD68+Pim-1 co-expression group is significantly lower than that of the low expression group(14.0 months vs 24.2 months, Logrank, P=0.0314); the Cox proportional hazards model reveals that the risk of disease progression in high expression of CD68+Pim-1 group is significantly higher than that of the low expression group(risk ratio, 2.22; P=0.04 )Patients with high CD68+Pim-1 expression showed the median survival time is significantly lower than that of the low infiltration group (12.7months vs 37.9months, Logrank, P=0.005); the Cox proportional hazards model reveals that the risk of disease progression in high expression of CD68+Pim-1 group is significantly higher than that of the lower expression group(risk ratio, 4.21; P=0.001 )As for the bone marrow high CD68+Pim-1 expression group ,the median survival time is significantly lower than that of the low infiltration group (18.3 months vs 49.5months, Logrank, P=0.0044); the Cox proportional hazards model reveals that the mortality riskin high expression of CD68+Pim-1 group is significantly higher than that of the lower expression group(risk ratio, 3.64; P=0.01 ).Patients with PD-1 low expression group in bone marrow showed greater response (Complete response/Partial response), while the PD-1 high expression group showed lower response (P=0.013). Conclusion The expression extent of Pim-1 and pim-2 with macrophages in bone marrow are associated with clinical prognosis. The expression extent of pim-2 in MM tumor-associated macrophages display a strong negative correlation to PFS values and the overall survival. The expression extent of pim-1 in MM tumor-associated macrophages display a strong negative correlation to overall survival. Pim-1 and pim-2 are prognostic factors of multiple myeloma. There is a strong negative correlation between expression of PD-1 of bone marrow and the early treatment response. Since the expression degree of PD-1 showed different prognosis in solid tumors and hematological cancer. The evaluation requires combined with other biological indicators Disclosures No relevant conflicts of interest to declare.

Description: Background and Objective: There are 3 members in Pim family, Pim-1, Pim-2 and Pim-3. They are serine/threonine kinase coding proto-oncogene. It is reported that Pim-1 plays a role in solid tumor, leukemia and polycythemia vera, et al. Pim-3, as a newly cloned oncogene has discovered to functioning in hepatic carcinoma, pancreatic cancer, et al. We are trying to find out the roles of Pim1 in acute myeloid leukemia. Methods: 1 Evaluate the expressions of Pim family genes in acute myeloid leukemia patients and analysis the profile of Pim expressions with clinical characteristics. 2 Up-regulating the expression of Pim1 in AML cell lines by transient transfection or long-term infection through GFP-expressing plasmids and lenti-virus system and analyze the proliferation, apoptosis and chemotaxis features of the transfected AML cell lines. Results: 1 Our investigation showed that Pim-1 is up-regulated in around 15% of acute myeloid leukemia (AML) patients. 2 As shown in growth curves and apoptosis assays, over expression of Pim-1induces growth up-regulation and anti-apoptosis. We hypothesized that phenomenon could be originated from the enhanced expression of c-myc, cyclin D1 and Bad phosphorylation shown in western blotting analysis. 3 Our chemotaxis assay and bone marrow stromal cell co-culture model demonstrated that overexpression of Pim-1can enhance the AML cells chemotactic movement toward SDF-1¦Á, with calcium influx increment and phosphorylation of CXCR4. 4 Flow cytometry analysis and confocal immunofluorescence observation demonstrated the up-regulated CXCR4 expression and internalization induced by SDF-1¦Á. 5 We also checked the angiogenesis and adhesion molecule during the process but did not show any contribution. Conclusion: Pim-1, beyond as an oncogene, can promote growth or anti-apoptosis of AML cells, can interact with microenviroment through SDF-1¦Á-CXCR4 axis. The interplay between AML cells and microenviroment mediated by Pim-1 can make sense in AML target therapy and make a good adjunctive for transplantation conditioning regimen. Figure 1. Expression of Pim1 in AML patients and control Figure 1. Expression of Pim1 in AML patients and control Disclosures No relevant conflicts of interest to declare.

Description: Objective: The purpose of this study is to investigate the relationship between the infiltration of tumor associated macrophages (TAMs) and the disease risk stratification and survival of patients with myelodysplastic syndrome (MDS) and to explore the role of TAMs in the clinical prognosis. Methods: We retrospectively collected and analyzed 115 patients initially diagnosed wih myelodysplastic syndromes from January, 2010 to July, 2017 in West China Hospital of Sichuan University. Both bone marrow biopsy specimens and clinical data of the patients enrolled were collected. All patients were assessed prognosis by international prognostic scoring system (IPSS). We quantified the involvement of macrophage (MΦ), alternatively activated macrophage (M2 MΦ) and classic activated macrophage (M1 MΦ) in bone marrow specimen by staining with anti-CD68 monoclonal antibody, anti-CD163 monoclonal antibody, and anti-iNOS monoclonal antibody respectively. Log-rank test was used to evaluate the difference of overall survival (OS) among different subgroups. Logistic regression was used to evaluate the effect of clinical parameters on patients' OS. Cox proportional-hazards models were used to estimate the independent risk factors influencing the prognosis of patients. Results: 1 In the high risk group of MDS, the composition of CD163+MΦ was higher than that of the low risk group(p 〈 0.001). In the low risk group of MDS, the composition of iNOS+ MΦ was higher than that of the group with high risk(p 〈 0.001). 2 The median survival time and 3-year survival rate in the high CD163+MΦinfiltration group were significantly lower than those in the low infiltration group (13 months vs 43 months, 27% vs 64%, Log-rank test, P

Description: 【Introduction 】There is ongoing unmet need for effective therapies in Mayo 2004 stage II-III amyloid light-chain (AL) amyloidosis patients, who undergo early death due to cardiac dysfunction. Lately, in vitro studies demonstrated that doxycycline could induce disruption of fibril formation in transgenic mouse model of AL amyloidosis. Matched case-control study of standard chemotherapy with or without doxycycline confirmed higher hematological response, cardiac responses and superior survival with doxycycline in AL patients. However, the possible advantage of doxycycline on lower early mortality and better long-term survival has not been evaluated in a randomized controlled clinical trial. We designed a randomized unblinded controlled study to investigate the efficacy and safety of co-administration of oral doxycycline with bortezomib-cyclophosphamide-dexamethasone (BCD) regimen in treatment-naïve AL amyloidosis patients with Mayo 2004 stage II-III disease. 【Methods 】The randomized unblinded controlled study took place in 12 hospitals in China. Eligible participants were adults with a confirmed diagnosis of AL amyloidosis, whose Mayo 2004 stage were II or III. Enrolled patients were randomly allocated to receive either doxycycline combined with BCD or BCD alone as initial treatment. We chose stratified blocked randomization (block size of 4) to ensure Mayo stage II and III were evenly distributed between doxycycline group and control group. For both two groups, patients will receive 1.3mg/m2of subcutaneous bortezomib and 40mg of oral or intravenous dexamethasone on days 1, 8, 15 and 22, and 300mg/m2oral or intravenous cyclophosphamide on days 1, 8 and 15 of a 35-day cycle. This process was repeated for 9 cycles. Doxycycline was given orally 100mg twice daily for the experimental group. The primary endpoint is progression-free survival. Secondary endpoints include overall survival, adverse events, hematological response, organ response and safety of treatment. This trial has been registered with ClinicalTrials.gov (number NCT03401372) and recruitment and follow-up are ongoing. We planned to enroll a total of 140 participants. 【Results 】Between April 21st, 2018 and June 30th, 2019, 111 patients were enrolled and randomly assigned to receive doxycycline plus BCD (n=56) or BCD alone (n=55) (Figure 1). The baseline characteristics were shown in Table 1. The median age was 61 (range, 41-78) years with a male: female ratio of 1.64:1. Mayo 2004 stage II disease was present in 28 patients in the control group and 29 patients in the doxycycline group. Mayo 2004 stage III disease was present in 27 patients in the control group and 27 patients in the doxycycline group. The median cTnI was 0.10 (range, 0-1.92) μg/L, NT-proBNP 3647 (range, 271-20507) pg/mL, and dFLC 205.30 (50.28-791.90) mg/L, with no significant difference between either group. Organ involvement included the heart (100%), kidney (61.3%), liver (14.4%), peripheral nerves (10.8%) and gastrointestinal tract (5.4%). The percentage of hepatic involvement and 24-hour urine protein were higher in the doxycycline group. The median duration of doxycycline was 5.9 months. Only one patient discontinued doxycycline due to toxicity (Grade 2 rash). Till now, the median follow-up time was 6.1 months and no patients were lost to follow-up. Fourteen patients have completed 9 cycles of chemotherapy. In total, 22 patients died and disease progression occurred in 3 patients. Two patients discontinued treatment due to withdrawal of consent and one of them proceeded to autologous stem cell transplantation. Three patients discontinued study drug owing to unacceptable diarrhea and received second-line treatment based on ixazomib or melphalan afterwards. The grade 3/4 adverse effects were developed in 8 patients (1 infection, 1 mucositis and 6 diarrhea) in the control group and 6 patients (1 infection, 1 arrhythmia, 1 kidney dysfunction and 3 diarrhea) in the doxycycline group. 【Conclusions 】Our data suggested that addition of doxycycline to standard bortezomib-based chemotherapy was an tolerable regimen for treating patients with AL amyloidosis. If this protocol could significantly improve survival and organ response needs to be confirmed with further follow-up. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The staging system for extranodal natural killer/T-cell lymphoma, nasal type (ENKTL) remains to be an open issue. The purpose of this retrospective study was to determine the performance of four staging systems for nasal ENKTL: Ann Arbor (AA) stage, limited & extensive stage, stage of Chinese Southwest Oncology Group (CSWOG) and Tumor-Node-Metastasis (TNM) stage. Methods This study was comprised of 233 patients with nasal ENTKL. The AA staging system was based on the Lugano modification of AA stage. The limited & extensive staging system was based on the AA stage, and the presence or absence of local tumor invasiveness: limited disease (AA stage I-II without LTI) and extensive disease (AA stage I-II with LTI or AA stage III-IV). The CSWOG staging system was described as follows: stage I, lesions confined within the upper aerodigestive tract without local invasiveness (paranasal sinuses, bony or skin invasion); stage II, localized disease with local invasiveness; stage III, localized disease with regional lymph node (cervical lymph node) involvement; and the others were stage IV. The TNM staging system was described by previous study (Yan et al. PLoS One, 2015). The 3-year overall survival (OS) rates were estimated using the Kaplan-Meier method, and survival curves were compared by the log-rank test. Multivariate analyses were performed using four separate COX models to assess the prognostic values of AA stage, limited & extensive stage, CSWOG stage and TNM stage, respectively, after adjusted for the effects of other covariates. Results After a median follow-up of 35.5 months, estimated 3-year OS rate was 61.3%. Multivariate analyses showed that limited & extensive stage, gender, performance status, lactate dehydrogenase (LDH), chemotherapy and radiotherapy were predictive of OS. The AA, CSWOG and TNM staging systems were not independent prognostic factors. Based these results, patients were stratified into four groups: low-risk limited disease (without risk factor), high-risk limited disease (with at least one risk factor: male, poor performance status, higher LDH), extensive disease with AA stage I-II and extensive disease with AA stage III-IV, for which the 3-year OS rates were 94.0%, 67.9%, 46.2% and 34.0%, respectively (P

Description: Objective. Hemophagocytic lymphohistiocytosis (HLH), also known as Hemophagocytic Syndrome (HPS), is an increasingly recognized clinical syndrome that is characterized by extreme immune activation. HLH was first described as an inherited immune disorder in pediatrics, but it may also arise in adults as the result of persistent antigen stimulation due to infections, autoimmune disorders or malignancies. Early recognition of HLH and appropriate treatment are critically important. For the pediatric patients, the Histiocyte Society Study Group for HLH has developed the HLH-94 and HLH-2004 treatment protocols, but there is no such guideline or consensus for adult HLH. Although there were increasing amount of clinical studies in adult HLH, the majority of them just described the etiologies and clinical profiles, and failed to analyze the treatment effects on outcomes. Therefore, there is an urgent need for more clinical data focusing on treatment in adult HLH patients, in order to clarify optimal therapeutic regimens. Our study retrospectively analyzed the causes, treatment strategies, and relevant outcomes in 104 adult HLH patients in our institution, and with the goal of identifying more appropriate therapeutic strategies for adult HLH patients. Methods. After the approval of our protocol by local institutional Ethics Committee, the medical records of 104 consecutive patients with adult onset HLH in West China Hospital from June 2008 to February 2016 were reviewed. The diagnosis was re-confirmed according to HLH-04 criteria, and demographic data, clinical profiles, treatments and outcomes were collected and analyzed. The latest follow-up visit occurred on 1st July 2016. The different therapeutic effects on prognosis were discussed based on the endpoints which were defined as short-term (30 days) and long-term (last follow-up date) survival rates. Statistical analysis was performed on SAS 9.4 software, and was involved in Log-rank test in univariate analysis and Cox proportional hazard regression model in multivariate analysis. All p values were two-sided and p

Description: Background: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells. The long term survival of AML is not satisfactory, so new treatment should be explored. Here, we show that chidamide(CH), a histone deacetylase inhibitor, combined with decitabine(DE) induces apoptosis of AML cell lines and primary refractory/relapsed AML cells by up-regulating PERP. This may provide a new option for AML treatment. Methods and results: We first examined the half-inhibitory concentrations (IC50) of chidamide and decitabine against THP-1, MV4-11, HL60 and Kasumi-1 cell lines using MTT (Fig1 A-D). And the drug combination is performed according to the IC50. In the double-drug combination experiment, we used MTT to detect the effect of drugs on the proliferation of the four cell lines (Fig1 E-H), used calcusyn 2.0 software to calculate the synergistic effect (Fig2), flow cytometry to detect apoptosis (Fig3 A-D), and western blot to detect the pro-apoptotic protein (C-CASPASE 3 and C-CASPASE 9) and anti-apoptotic proteins (CASPASE 8, BCL-2 and BCL-XL) (Fig3 E-H). We found that chidamide combined with decitabine synergistically inhibited proliferation of AML cell lines, induced apoptosis, up-regulated pro-apoptotic protein levels and down-regulated anti-apoptotic protein levels. To investigate this combination therapeutic effect in vivo, we selected 5 refractory/relapsed AML patients, extracted primary AML cells, and used ATP chemiluminescence kit for drug sensitivity test. The results confirmed that four of the five patients with AML showed sensitivity to combinations (Fig4). To further explore the mechanism of action of CH combination with DE, we performed transcriptome sequencing (Fig5). Analysis of the sequencing results, the gene PERP, which shows the significant difference in the apoptotic pathway, was further examined. The PERP is a new member of the PMP-22/GAS3 family as an apoptosis-associated target of TP53. RT-QPCR and WB verified the role of PERP in apoptosis in DE and CH combination (Fig6). The results showed that the combination could up-regulate the PERP gene than the single drug. When we explored the role of the PERP gene in AML cell lines, we knocked down the PERP gene by lentivirus and detected cell proliferation after infection. Pretreated AML cell lines by lentivirus-infection (Fig7A-F), then we tested for proliferation (Fig7G-I) (Fig8A-C), apoptosis (Fig8D-E), and pro-apoptotic protein expression (Fig8G-I). The results showed that knocking down the PERP gene promoted the proliferation of AML cell lines and attenuated the sensitivity of AML cell lines to chemotherapeutic drugs. We also compare the mRNA level of PERP between 35 AML patients and 20 normal and found that the PERP mRNA of AML patients was significantly lower than the normal (Fig9). MV4-11 cells were exposed to CH and DE alone or in combination, and proteomic sequencing was performed to examine the effect of the drug on cellular protein. The result indicates to some extent that CH contributes more to the combined effect. And the drug causes changes in multiple pathways in the cell (Fig10). Conclusion: Our experiments revealed that CH combined with DE may have therapeutic effects on AML and, to some extent, reveal the mechanism of dual drug combination. Legends to figures Fig1. 50% inhibitory concentration (IC50) values of chidamide and decitabine alone treated AML cell lines. Fig2. Chidamide acts synergistically in AML with DE. Fig3. Chidamide in combination with decitabine significantly induced apoptosis in AML cell lines. Fig4. The sensitivity of relapsed or refractory AML primary cells to chidamide and decitabine alone or in combination. Fig5. Gene expression analysis showed an obvious difference based on treatment. Fig6. Verify transcriptome sequencing results by real-time QPCR and by western blotting with or without drug treatment. Fig7. The effect of down-regulation of PERP by Lentivirus-mediated RNAi on AML cells proliferation. Fig8. PERP knockdown causes AML cells to develop resistance to combination drugs. Fig9.The level of PERP mRNA in peripheral blood mononuclear cells of AML and normal humans. Fig10. Proteomics sequencing results show the differentially expressed protein and a cluster analysis of the functions or pathways enriched by differentially expressed proteins in GO and KEGG pathways compared to single agents. Disclosures No relevant conflicts of interest to declare.

Description: Background: Ruxolitinib, a potent JAK1/JAK2 inhibitor, has been proved to improved splenomegaly and debilitating myelofibrosis-related symptoms in several clinical trials. However, not much is known about the efficacy of ruxolitinib, especially low-dose ruxolitinib, in real world patients in China. Here we assess the efficacy of ruxolitinib in treatment of patients with myeloproliferative neoplasms associated myelofibrosis (MPN-MF) in real world and to analyze factors affect the treatment efficacy. Methods and Results: From July 2017 to June 2019, data of MPN-MF patients treated with ruxolitinib in West China hospital, Sichuan University, China, were retrospectively collected and analyzed. Logistic regression was used for univariate and multivariate analysis of binary variables. Simple linear regression was used in univariate analysis of continuous variables, and multiple linear regression was used in multivariate analysis of continuous variables. Dose of ruxolitinib were decided according to platelet count and financial condition of patients. Of 72 MPN-MF patients, 1 patient was treated with ruxolitinib in an initial dose of 5mg qd, 37 patients with 5mg bid, 15 patients with 10mg bid, 2 patients with 25mg bid. At week 12, 89.3% of patients achieved reduction from baseline in palpable spleen length, of which 44.6% patients achieved ≥50% reduction (Figure 1) ; 95.6% patients achieved reduction from baseline in Total Symptom Score (TSS), of which 44.6% patients achieved ≥50% reduction(Figure 2). At week 48, all 25 patients with bone marrow biopsies achieved improved or stable bone marrow fibrosis grading from baseline, of which 44.0% improved (Figure 3). Both univariate analysis and multivariate analyses showed higher dose of ruxolitinib (〉5mg bid VS ≤5mg bid and〉10mg bid VS ≤10mg bid) was the major factor associated with better spleen response(P10mg VS ≤10mg) was the only factor associated with better TSS improvement(P

Description: Background: Metaphase cytogenetics (MC), which has an important diagnostic, prognostic and therapeutic roles in myelodysplastic syndrome (MDS), is widely used as cytogenetic analyzing tools. It can present entire cytogenetic information at one time, although with some limitations such as Hypocellularity, fewer mitotic cells, secondary myelofibrosis and technicians' subjectivity. Single nucleotide polymorphism(SNP)array based karyotyping ( SNP-A based karyotyping) is a novel diagnostic tool which can detect copy number variations with a high resolution. More importantly, SNP-based array has a unique advantage in detection of loss of heterozygosity, also referred as to uniparental disomy (UPD), which results from duplication of a paternal (unimaternal) or maternal (unipaternal) chromosomal region and concurrent loss of the other allele. However the technology is still relatively expensive, and balanced structural METHOD: We analyzed SNP-A results from 127 patients diagnosed of MDS or MDS related myeloid malignancies(including 6 MDS/MPN, 11 acute myeloid leukemia from MDS, and 110 MDS). 122 patients of them had both MC and SNP-A results. We compared the frequency and diagnostic sensitivity between the cytogenetic aberration findings by MC and genomic alteration findings by SNP-A. In addition, we investigated the novel or additional lesions detected by SNP-A which had not been found by MC, and find further information about the edges of SNP-A. We drew attention to the missing matters of SNP-A which mentioned in MC reports to integrate the limits of SNP-A. We also used multiple-factor analysis to find out the specific situation which MC are not inferior to SNP-A. RESULTS: There are 199 genomic alteration findings in 127 patients by SNP-A ( including 43 UPDs, 57 gain alterations, 86 loss alterations and 13 complicated alterations). The average length of genomic alterations found by SNP-A is 27795.71Kb, the longest one is GainMosaic(1) (248375kb), the shortest is a UPD found in 17q (41.88Kb). In the 122 patients who had both MC and SNP-A results, SNP-A turns to be more effective than MC in significant chromosomal defects(58.2% vs 36.9%,P