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Objective To assess the contents of circulating cytokines in patients with diffuse large B-cell lymphoma (DLBCL), and to examine their relationship with clinicopathological manifestations and prognosis.Method We recruited 72 DLBCL patients, 11 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) patients, and 56 healthy controls from our hospital between the period of January 2017 and January 2020, and measured 7 serum cytokine contents using Beckman Navios flow cytometry. The cytokine level was compared between DLBCL patients and healthy controls using one way ANOVA. Two-sided Spearman test was employed for relationship evaluation between circulating cytokine levels and clinicopathological characteristics, IPI score, and short-term treatment response within DLBCL patients. The inter-group comparison of cytokine levels employed the Mann Whitney test. The support vector machine (SVM) was utilized for the cytokine evaluation-based prediction of DLBCL patient short-term treatment response. Lastly, survival curves were used to assess correlation between the aforementioned cytokines and overall survival.Result The IL-6, IL-10, and IFN-γcontents were markedly enhanced among DLBCL patients, as opposed to healthy controls (P P = 0.00, r = 0.66). Additionally, the international prognostic index (IPI) risk stratification of DLBCL patients was strongly associated with the circulating IL-6 and IL-10 contents. Enhanced IL-6, IL-10, and TNF-α levels often produced worse short-term treatment efficacies (P P Conclusion The IL-6, IL-10, IL-17, TNF-α, and IFN-γ contents can potentially serve as biological indicators of DLBCL tumor immune status, and a combined application with the IPI score can be a robust indicator for DLBCL patient prognosis. Our findings provide novel ideas for the clinical treatment of DLBCL patients.

Supplementary materials

A, A representative diagram of the transduction efficiency evaluation of CART19s 5 days after lentivirus infection. B, Specific target cell lysis by CART19s. T cells or CART19s were co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratio for overnight co-culture (24 hours). C, IFN-γ secretion measurements in supernatants in T cells or CART19 cells co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratios for 24 hours.

Comparison of CART19 levels in different groups. A, Levels of CART19 cells in PB were shown as assessed by means of qPCR assay at serial time points before and after infusion of CART19 respectively. B, CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion were compared. CAR DNA copies in BM (Patient 2, 6, 11, 13) were higher than in PB. C, Paired measurements of CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion, percentage of leukemia cells after FC chemotherapy shows strong correlations (CAR DNA copies in BM VS in PB, Spearman r=0.956, p

Relapsed leukemia cells analysis by FACS.A, leukemia cells with bright CD19 expression obtained CR with MRD negative after CART19 therapy and relapsed with CD19 + leukemia cells (Patient 1). B, leukemia cells with dim CD19 expression obtained CR with MRD negative after CART19 therapy but relapsed with 2 subsets of CD19 + and - leukemia cells (Patient 2 and 3).

Supplementary Tables including Table 1, Table 2 and Table 3

CRS complication after CART19 therapy. A, the peak serum concentration of IL-6, IFN-γ, IL-10, CRP, D-dimer and ferritin of a representative patient (Patient 15) during CRS at the indicated time-points after CART19 infusion are showed. B, D, Peak serum levels of CRP, ferritin and D-dimer in patients who developed grade 3 CRS (n=6) compared with those without CRS or with 1 or 2 CRS (n=10). Data represented the mean{plus minus}SEM. The Mann-Whitney U test was used for statistical analysis. C, Paired measurements of peak serum IL-6, CRP, ferritin and D-dimer showed strong correlations (IL-6 VS CRP, Spearman r=0.617, p=0.014; IL-6 VS ferritin, Spearman r=0.574, p=0.028; IL-6 VS D-dimer, Spearman r=0.789, p

CART19 identification and quantification

The cumulative incidence of non-relapse mortality are shown.

Patient 13 with extramedullary leukemia relapse compromising testis obtained CR after CART19 infusion. A, Ultrasound showed complete elimination of extramedullary relapsed masses involved in testis (red arrows). Images are from before treatment and 90 days after CART19 infusion. B, Pathology showed complete elimination of extramedullary relapsed masses involved in testis. Images are from before treatment and 30 days after CART19 infusion. Left: 10×10, right: 10×40.

Purpose: Patients with relapsed/refractory acute lymphocytic leukemia (R/R ALL) have a poor prognosis. Chimeric antigen receptor–modified T cells against CD19 (CART19) have displayed anti-leukemia activities. However, data from systemic trials in Chinese patients are limited.Experimental Design: T cells transduced with CD19-directed CAR lentiviral vectors were infused in patients with R/R ALL under fludarabine- and cyclophosphamide-based lymphodepletion. The postinfusion responses, toxicities, expansion, and persistence of CART19s in enrolled patients were observed and monitored.Results: We enrolled 15 patients with R/R ALL. The median transduction efficiency of CART19s was 33%. In vitro cytotoxicity assays were conducted and showed prominent antileukemia activities with CART19s. The patients received CART19s infusion at doses of 1.1 × 106/kg to 9.8 × 106/kg. Twelve patients achieved complete remission 1 month after CART19s infusion. CART19s expanded and persisted in peripheral blood and bone marrow. At 150 days, the overall survival rate and leukemia-free survival rate were 65.5% and 37.8%, respectively. The cumulative incidence of relapse and the nonrelapse mortality rate were 54.5% and 7.7%, respectively. Four patients underwent subsequent haploidentical hematopoietic stem cell transplantation. In this trial, 10 patients experienced cytokine release syndrome (CRS). Grade 3 CRS developed in 40% of patients and was associated with a higher disease burden on day −1 and a higher number of previous relapses.Conclusions: This trial demonstrated potent antileukemia activities of CART19s in Chinese patients with R/R ALL. Disease relapse remained the main obstacle. However, patients with a high risk of relapse after CART19s might benefit from subsequent haploidentical hematopoietic stem cell transplantation. Clin Cancer Res; 23(13); 3297–306. ©2016 AACR.

Despite efforts to abrogate the severe threat to life posed by the profound malignancy of mature natural killer/T-cell lymphoma (NKTCL), therapeutic advances still require further investigation of its inherent regulatory biochemical processes. Next-generation sequencing (NGS) is an increasingly developing gene detection technique, which has been widely used in lymphoma genetic research in recent years. Targeted therapy based on the above studies has also generated a series of advances, making genetic mutation a new research hotspot in lymphoma. Advances in NKTCL-related gene mutations are reviewed in this paper.

Objective Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by inflammatory synovitis. We developed a new disease activity evaluation system using important cytokines to help doctors better evaluate disease activity in patients with RA. Methods Flow cytometry was used to detect the levels of seven cytokines. Then, the results were analyzed using an R language decision tree. Results The levels of six cytokines, namely interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-α, and interferon-γ, were significantly different between the active disease and remission stages. Decision tree analysis of the six cytokines with statistical significance identified two judgment rules for the remission stage and three judgment rules for the active disease stage. Conclusion We proposed the use of the decision tree method to analyze cytokine levels in patients with RA and obtain a more intuitive and objective RA disease activity scoring system. This method revealed the relationships of IL-6 and TNF-α levels with inflammatory characteristics in patients with RA, which can help predict disease activity.

A novel pathogenic PYCR2 variant and corresponding brain images in two patients characterized by spastic paraplegia.

About half of patients with severe acute graft vs host disease (aGVHD) show resistance to treatment with first-line steroids. We enrolled 64 patients with grades III-IV SR-aGVHD after allogeneic hematopoietic stem cell transplantation (allo-SCT), to assess the efficacy and safety of the combination therapy of ruxolitinib and etanercept. The overall response rate was 87.5% (95% CI, 79.7%-95.3%) at day 28 of the combination treatment, from which 73.4% reached complete response (CR). A marked reduction ≥75% in daily corticosteroid dosing was documented in 75.4% of patients at day 28. Delayed time from aGVHD to ruxolitinib (OR = 4.88, 95% CI, 0.98-23.56), stages 3-4 liver aGVHD (OR = 8.57, 95% CI, 0.96-46.59) and gut Enterobacteriaceae colonization (OR = 12.39, 95% CI, 1.71-59.77) were related to incomplete response. Grades 3/4 anemia, leukopenia, or thrombocytopenia and CMV-reactivation were found in 29.7%, 26.6%, 39.1%, and 50.0% of patients, respectively. So, 25 (39.1%) experienced complications of severe infection ≥3 grade, in which pulmonary infections were most frequent (15/64, 23.4%). The 2-year overall survival (OS) after the combination therapy was 61.2%. The 2-year incidence of non-relapse mortality and relapse of the underlying malignancy was 26.7% and 15.7%, respectively. Combined treatment with ruxolitinib and etanercept was very effective and relatively safe for severe aGVHD patients, while the various infection complications deserve more attention. This study was registered at the Chinese Clinical Trial Registry (ChiCTR1900024408).

Purpose: Patients with relapsed/refractory acute lymphocytic leukemia (R/R ALL) have a poor prognosis. Chimeric antigen receptor–modified T cells against CD19 (CART19) have displayed anti-leukemia activities. However, data from systemic trials in Chinese patients are limited. Experimental Design: T cells transduced with CD19-directed CAR lentiviral vectors were infused in patients with R/R ALL under fludarabine- and cyclophosphamide-based lymphodepletion. The postinfusion responses, toxicities, expansion, and persistence of CART19s in enrolled patients were observed and monitored. Results: We enrolled 15 patients with R/R ALL. The median transduction efficiency of CART19s was 33%. In vitro cytotoxicity assays were conducted and showed prominent antileukemia activities with CART19s. The patients received CART19s infusion at doses of 1.1 × 106/kg to 9.8 × 106/kg. Twelve patients achieved complete remission 1 month after CART19s infusion. CART19s expanded and persisted in peripheral blood and bone marrow. At 150 days, the overall survival rate and leukemia-free survival rate were 65.5% and 37.8%, respectively. The cumulative incidence of relapse and the nonrelapse mortality rate were 54.5% and 7.7%, respectively. Four patients underwent subsequent haploidentical hematopoietic stem cell transplantation. In this trial, 10 patients experienced cytokine release syndrome (CRS). Grade 3 CRS developed in 40% of patients and was associated with a higher disease burden on day −1 and a higher number of previous relapses. Conclusions: This trial demonstrated potent antileukemia activities of CART19s in Chinese patients with R/R ALL. Disease relapse remained the main obstacle. However, patients with a high risk of relapse after CART19s might benefit from subsequent haploidentical hematopoietic stem cell transplantation. Clin Cancer Res; 23(13); 3297–306. ©2016 AACR.

// Wanmao Ni 1 , Beili Hu 2 , Cuiping Zheng 3 , Yin Tong 4 , Lei Wang 1 , Qing-qing Li 1 , Xiangmin Tong 1 , Yong Han 1 1 Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Zhejiang Provincial People’s Hospital, Hangzhou, Zhejiang, P. R. China 2 Medical of College, Zhejiang University, Hangzhou, Zhejiang, P. R. China 3 The Center Hospital of Wenzhou, Wenzhou, Zhejiang, P. R. China 4 Department of Hematology, Shanghai General Hospital, Shanghai, P. R. China Correspondence to: Xiangmin Tong, email: tongxiangmin@163.com Yong Han, email: onlune@126.com Keywords: flow cytometry, immunophenotyping, support vector machine, acute myeloid leukemia, minimal residual disease Received: July 04, 2016 Accepted: September 29, 2016 Published: October 04, 2016 ABSTRACT We investigated the ability of support vector machines (SVM) to analyze minimal residual disease (MRD) in flow cytometry data from patients with acute myeloid leukemia (AML) automatically, objectively and standardly. The initial disease data and MRD review data in the form of 159 flow cytometry standard 3.0 files from 36 CD7-positive AML patients in whom MRD was detected more than once were exported. SVM was used for training with setting the initial disease data to 1 as the flag and setting 15 healthy persons to set 0 as the flag. Based on the two training groups, parameters were optimized, and a predictive model was built to analyze MRD data from each patient. The automated analysis results from the SVM model were compared to those obtained through conventional analysis to determine reliability. Automated analysis results based on the model did not differ from and were correlated with results obtained through conventional analysis (correlation coefficient c = 0.986, P > 0.05). Thus the SVM model could potentially be used to analyze flow cytometry-based AML MRD data automatically, objectively, and in a standardized manner.

Prognostic factors for patients with acute promyelocytic leukemia (APL) treated in the context of arsenic trioxide (ATO)-based frontline regimes have not been established clearly. We retrospectively analyzed the clinical features, immunophenotypes, Fms-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD), and outcomes of 184 consecutive newly diagnosed APL patients treated by intravenous ATO-based therapy. The median age was 40 years (14-77 years). The early death rate was 4.9% (9/184 patients). With a median follow-up time of 36 months (9-74 months), the 3-year relapse-free survival (RFS) and overall survival (OS) were 93.3% and 92.2%, respectively. Interestingly, there was no meaningful association between 3-year RFS and initial white blood cell count, FLT3-ITD status, or type of PML-RARA isoforms. In multivariable analysis, the CD56 expression was the only independent risk factor in terms of RFS (hazard ratio, 4.70; P=0.005). These results suggested that ATO-based therapy may ameliorate the unfavorable influence of previously known high-risk features; moreover, CD56 expression remains to be a potentially unfavorable prognostic factor in APL patients.

Background/Aim: As the knowledgebase of acute myeloid leukemia (AML) has grown, classification systems have moved to incorporate these new findings. Methods: We assessed 32,941 patients with AML whose records are contained in the Surveillance, Epidemiology, and End Results (SEER) database. Results: Half of all patients diagnosed between 2001 and 2013 did not have a World Health Organization (WHO) classification. Acute promyelocytic leukemia and acute panmyelosis with myelofibrosis were associated with the longest leukemia-specific survival (110 and 115 months, respectively), and AML with minimal differentiation and acute megakaryoblastic leukemia with the shortest (30 and 28 months, respectively). For patients in the WHO groups AML not otherwise specified (AML-NOS) and AML with recurrent genetic abnormalities (AML-RGA), the risk of death was greater for older patients and less for married patients. Black patients with any type of AML-NOS also had a higher risk of death. Patients whose case of AML did not receive a WHO classification were older and this group had a higher risk of death when compared to patients with a WHO type of AML-NOS. Conclusion: Our findings highlight the divergent outcomes of patients with AML and the importance of using the WHO classification system and demographic factors to gauge their prognosis.

In this study, we confirmed that combining HHT with ACR can result in synergistic cytotoxicity to AML cells in vitro and in vivo. Combining HHT and ACR simultaneously inhibited PI3K/AKT and WNT/β-catenin signaling in AML cells. Significant increases in growth inhibition and apoptosis were induced by an AKT inhibitor when the WNT3A gene of THP-1 cells was silenced. HHT+ACR could synergistically induce the apoptosis of CD34(+)/CD38(-) primary AML cells. These results highlight β-catenin and AKT are promising targets for combination therapy for AML.

To examine the effects of triptolide (TPL) on T-cell leukemia cells and identify their underlying mechanisms.The cytotoxicity of TPL was assessed by MTT assay. Cell apoptosis was determined using annexin V and DAPI staining and analyzed by flow cytometry or fluorescence microscopy. The activation of caspase pathways and the expression of nuclear factor κB (NF-κB) p65 were examined by Western blotting. Differences in microRNA (miRNA) expression in Molt-4 and Jurkat cells before and after TPL treatment were identified using microarrays and real-time RT-PCR, respectively.TPL 20-160 nmol/L treatment potently inhibited cell growth and induced apoptosis in T-cell lymphocytic leukemia cell lines. Molt-4 and Jurkat cells, however, were more sensitive to TPL than L428 and Raji cells. After 24 h of treatment, bortezomib abrogated the growth of Molt-4 and Jurkat cells with an IC(50) of 15.25 and 24.68 nmol/L, respectively. Using Molt-4 cells, we demonstrated that treatment 20-80 nmol/L inhibited the translocation of NF-κB p65 from the cytoplasm to the nucleus and that phosphorylated NF-κB p65 in nuclear extracts was down-regulated in a dose-dependent manner. Similar results were also seen in Jurkat cells but not in L428 cells, as these cells are resistant to TPL and bortezomib (a NF-κB inhibitor). Twenty-three miRNAs were differentially expressed after TPL treatment. Functional analysis revealed that TPL treatment could inhibit expression of miR-16-1* and that transfection of miR-16-1* led to significantly decreased apoptosis induced by TPL.Our in vitro studies suggest that TPL might be an effective therapeutic agent for treatment of T-cell lymphocytic leukemia and that its cytotoxic effects could be associated with inhibition of NF-κB and down-regulation of miR-16-1*.

Background and Aim: Acute graft versus host disease (aGVHD) remains a major problem after allogeneic hematopoietic stem cell transplantation (allo-SCT). Steroids currently represent the gold-standard treatment for aGVHD based on prospective randomized trials, while fewer than half of patients have a lasting complete response. The long-term mortality rate of steroid-refractory (SR) aGVHD remains around 70%. To date, no consensus has been reached regarding the optimal salvage treatment for SR-aGVHD. We performed the first prospective, multi-center clinical trial to assess the efficacy and safety of the combination of ruxolitinib and etanercept as a novel approach to treat grades II-IV SR-aGVHD . Methods: Thirty-one malignant hematologic disease patients with grades II-IV SR-aGVHD after allo-SCT from three centers in East China were enrolled from January 2017 to January 2018. All of them received ruxolitinib and etanercept as salvage-therapy for SR-aGVHD. Ruxolitinib was initiated at a dose of 5-10 mg BID for 2 months, and then tapered gradually for another one month. Etanercept was administrated at 25mg BIW for 2-8 weeks. Results: At day 30 after starting the combination treatment, overall response (complete and partial response: CR+PR) was 90.3% with 74.2% being CR. The incidences of CR per organ were 93.5%, 84.2% and 82.6% for skin, liver, and gut involvement, respectively. The median time from combination therapy to the optimal response was 12 days. The patients who received ruxolitinib within 14 days after aGVHD onset have a higher CR rate that those with delayed ruxolitinib therapy (94.7% vs. 50.0%, p=0.007). And the patients without gut infections have a higher CR rate than infected cohort (90.0% vs. 54.5%,p=0.037). Only time from aGVHD to ruxolitinib (RR=3.17, 95%CI 2.40~5.33, p=0.009) were independent predictors for incomplete response. III-IV Cytopenia and CMV-reactivation were observed during ruxolitinib treatment in 29.0% and 41.9% of patients. A significant decline in the level of IL-6, IFN-y and TNF-α was observed during 2-week treatment. The number of peripheral effector Tregs and Treg/Th17 ratio were significantly increased in responding patients. Compared with the historical cohort of basiliximab and etanercept for SR-aGVHD in our center (n=31), no significant difference was found on the baseline, such as age, gender, underlying disease, disease status, conditioning intensity, as well as SR-aGVHD characteristics. Although the ORR in patients treated with ruxolitinib and etanercept is identical with the historical cohort (90.3% vs. 90.3%), ruxolitinib group achieved rapider remissions in liver aGVHD and gut aGVHD than the historical cohort (median time to remission for liver aGVHD: 21days vs. 28 days, p=0.0049; median time to remission for gut aGVHD: 11 days vs. 17 days, p=0.0026). Among 31 patients in the study cohort, 9 cases (29.0%) were complicated with severe infection >= 2 grade, including military tuberculosis(n=1), CMV-interstitial pneumonia(n=1), carbapenem-resistant enterobacteriaceae (CRE) sepsis(n=2), invasive fungal infection (IFI,n=1), human herpesvirus-6 acute limbic encephalitis (n=1) and bacteria pneumonia (n=3). The 1-year overall survival (OS) and 1-year non-relapse mortality (NRM) were 73.9% and 22.9%, respectively, and the 1-year relapse rate was 8.3% after ruxolitinib. Conclusion: Combined treatment with ruxolitinib and etanercept resulted in a rapid CR to visceral aGVHD and meanwhile reserve graft anti-leukemia (GVL) effect as the relapse rate of primary disease is relatively lower. The various infection complications associated with ruxolitinib merit more attention. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Musashi-2 (MSI2) expression of 116 adult B-cells acute lymphoblastic leukemia (B-ALL) patients was measured by real-time PCR. Kaplan–Meier analysis showed that patients with high MSI2 expression had inferior overall survival (OS) ( P = 0.004), event free survival (EFS) ( P = 0.001) and relapse free survival (RFS) ( P = 0.018) in BCR-ABL-negative B-ALL. Multivariate models revealed that, besides WBC more than 30 × 10 9 /L and IK6 variant of IKZF1, high MSI2 expression was also an independent prognostic factor for adult BCR-ABL-negative B-ALL. Our data suggest that high MSI2 expression could indicate poor prognosis and facilitate risk and treatment stratification in adult BCR-ABL-negative B-ALL.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel candidate of tumor suppressor that can selectively induce apoptosis experimentally in a spectrum of human cancer cells including leukemia cells. However, a recent study suggests that mda-7/IL-24 promotes the survival of chronic lymphocytic leukemia B-cells. In this study, we showed that mda-7/IL-24 was constitutively expressed in leukemia cell lines and primary acute myeloid leukemia samples. Using a conditionally replicating adenovirus expressing mda-7/IL-24 (ZD55-IL-24), we showed that enforced expression of mda-7/IL-24 in leukemia cells induced autophagy, which was triggered by the upregulation of Beclin-1. Immunofluorescence and coimmunoprecipitation studies suggested that mda-7/IL-24 protein interacts with Beclin-1. Class III PI3K/Beclin-1 complex was shown involved in the mda-7/IL-24-induced autophagy. Moreover, autophagy inhibition by phosphatidylinositol 3-kinase inhibitor, wortmannin, resulted in a reduced Beclin-1 expression and autophagosome formation associated with significantly enhanced cell death. Importantly, the combination of ZD55-IL-24 with wortmannin elicited a strongly enhanced antileukemia efficacy in established leukemia xenografts. These results suggest that mda-7/IL-24-induced autophagy in leukemia cells may provide survival advantage and mda-7/IL-24 combined with agents that disrupt autophagy is a promising new strategy for the treatment of leukemia.

Fludarabine, a nucleoside analogue, plays a major role in the treatment of B-cell lymphocytic leukemia, hairy cell leukemia, and indolent lymphomas. There is a controversy about antitumor activity of fludarabine in multiple myeloma (MM). The aim of this study was to evaluate the activity of fludarabine against human myeloma cells both in vivo and in vitro. We demonstrated that myeloma cell line RPMI8226 was efficiently inhibited by fludarabine, concomitantly with decreased phosphorylation of Akt, down-regulation of the inhibitor of apoptosis proteins (IAP) family, including XIAP and survivin, and induction of apoptosis related to activation of caspase cascade. Contrary to dexamethasone, the effect of fludarabine on RPMI8226 cells was independent of interleukin-6. Fludarabine also induced cytotoxicity in dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) cells at 48 h with IC50 of 13.48 microg/mL and 33.79 microg/mL, respectively. In contrast, U266 cells were resistant to fludarabine. Moreover, RPMI8226 myeloma xenograft model was established using severe combined immunodeficient mice. The tumors treated with fludarabine at 40 mg/kg increased less than 5-fold in 25 d comparing with approximately 10-fold in the control tumors, demonstrating the antitumor activity of fludarabine in vivo. These results suggest that fludarabine may be an important therapeutic option for MM patients who are resistant to dexamethasone.

Overexpression of human telomere repeat binding factor 2 (TRF2), which may play an important role in the fate of cancer cells, has been observed in adult T-cell leukemia. Previous reports have shown that the inhibition of TRF2 results in the apoptosis of cancer cells. In this study, we demonstrated that arsenic trioxide (As2O3) induced in vitro growth inhibition and/or apoptosis of human T-cell leukemia cell line Molt-4 in a caspase-independent manner. Telomerase activity was not inhibited, although the level of the reverse transcriptase subunit of the human telomerase gene (hTERT) mRNA expression was down regulated during the early times and then recovered to the level found in untreated controls about 48 hours after treatment with As2O3. Furthermore, a remarkable telomere shortening related to exposure of As2O3 was observed in 50 population doubling. Inc ontrast, the alteration of telomere length did not occur after exposure to higher concentration of As2O3 (10 microM) for 24 hours and 48 hours, respectively, suggesting that the shortening of telomeres induced by As2O3 is dependent of a series of cell division cycles. Chromosomal analysis showed that As2O3 exposure caused chromosomal end-to-end fusion in human T-cell leukemia cells while downregulation of TRF2 was observed. Finally, the inhibition of TRF2 protein expression and the sensitivity to As2O3 in a panel of leukemia cell lines were checked. The data revealed that inhibition of TRF2 rendered leukemia cells more susceptible to As2O3. In conclusion, the downregulation of TRF2 by As2O3 contribute to chromosomal end-to-end fusion, and apoptosis in leukemia cells, suggesting that TRF2 could be an attractive target for new therapies of leukemia.

Although recent data shows that arsenic trioxide (As2O3) is capable of inducing cell death via cell cycle arrest and apoptosis both in acute promyelocytic leukemia (APL) and in non-APL cells, the mechanisms of As2O3-mediated cell death are not fully understood. In this study, we investigated the in vitro effects of As2O3 on cell growth inhibition and cell death in human T-lymphocytic leukemia and myelodysplastic syndrome (MDS) cell lines. As2O3 significantly inhibited the proliferation of Molt-4 and Mutz-1 cells in dose- and time-dependent manner. Autophagic cell death (programmed cell death type II) and apoptosis (programmed cell death type I) were activated together in leukemia cell lines after exposed to As2O3. Numerous large cytoplasmic inclusions and vacuoles were observed in As2O3-treated cells using electron microscope. Furthermore, 3-methyladenine (an autophagy inhibitor) significantly reduced autophagic cell death and sequentially induced apoptosis. Finally, leukemia cells treated with 4 microM As2O3 showed a considerable up-regulation of Beclin-1 (a Bcl-2-interacting protein) expression, which was independent of transcription of mRNA and required protein synthesis. In addition, Molt-4 cells treated with As2O3 exhibited the down-regulation of Bax protein expression, suggesting that Bax may be involved in accumulating of Beclin-1 and triggering autophagic cell death in As2O3-treated leukemia cells. These results may lead to a better understanding of the mechanism of action of As2O3, and provide a suggestion that As2O3 may be of therapeutic value for the treatment of patients with human T-lymphocytic leukemia and myelodysplastic syndrome.

Purpose: To test the effect of Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, on human leukemic cell lines (HL-60).Methods: HL-60 cells were treated with CPT. Cell growth inhibition (%) was quantitated using MTT assay. Apoptosis detection with Annexin V-FITC/propidium iodide staining was followed by flow cytometry. Caspase-3, caspase-8, and caspase-9 colorimetric assay kit was used to determine caspase protease activity. Loss of mitochondrial membrane potential was examined by flow cytometry with JC-1 staining. Bax, PARP, p53, p21 and cytochrome C were determined using Western blot.Results: Morphologic assessment, Annexin V-FITC/propidium iodide staining results and sub-G1 percentage indicate that the cytotoxic effect of CPT was mediated by induction of apoptosis. Furthermore, increased Bax expression, decreased Bcl-2 expression, loss of mitochondria membranepotential (MMP), release of cytochrome C, activation of caspase enzyme, cleavage of PARP and accumulation of p53 and p21 were detected during the apoptotic process. Caspase inhibitor partially abrogated CPT-induced apoptosis.Conclusion: The results show that CPT induced apoptosis of HL-60 cell lines by mitochondria pathway, and suggest that CPT may serve as a potential therapy for leukemia.Keywords: Cryptotanshinone, Salvia miltiorrhiza Bunge, Caspase, Membrane potential, Mitochondrial, Apoptosis, Leukemia, Cytochrome C, Cell cycle

Abstract 3638 Background: CAG regimen (cytarabine, aclarubicin, G-CSF) has been commonly used in China and Japan for the treatment of AML and MDS. This study is to summarize the data and to analyze the efficacy as well as the toxic effects of CAG regimen in acute leukemia (AL) and MDS pts. Methods: The databases of PubMed, Wanfang Data, as well as American Society of Hematology (ASH) annual meeting abstracts were searched for articles published in English, Chinese and Japanese languages between January 1995 and December 2010. Eligible studies were relevant clinical trials on AL and MDS pts treated with CAG regimen. Complete remission (CR) rates and odds ratio (OR) were compared through a meta-analysis using a random-effects or fixed-effects model. Results: 37 trials with a total of 1045 AL and MDS pts were included for analysis. Among the 1045 pts treated with CAG, 819 pts were AML, 215 pts were de novo MDS or transformed AML (MDS/tAML), 6 pts were ALL, and 5 pts were biphenotypic acute leukemia (BAL). The AML CR rate of CAG from 29 studies was 58.0% (95% CI, 53.1%-62.7%). The MDS/t-AML CR rate from 12 studies was 45.7% (95% CI, 39.0%-52.4%). The AML CR rate was significantly better than that of MDS /tAML (Q=8.072, p0.05). The CR rate for the 367 elderly AML pts was 52% (95% CI 51.5%-62.3%). The CR rate was also significantly higher in pts with favorable (64.5%, 95% CI 38.8%-83.9%) and intermediate (69.6%, 95% CI 60.4%-77.5%) cytogenetics than those with unfavorable one (29.5%, 95% CI 19.7%-41.8%) (p Conclusions: CAG regimen induced significantly higher CR rates in AML than in MDS pts. The CR rates of CAG regimen was significantly better than those of other induction regimens in AML pts. This regimen was well tolerated with low cardiotoxicity and early death rate. Disclosures: No relevant conflicts of interest to declare.

Arsenic Trioxide (As2O3) has been used successfully in the treatment of patients with relapsed or refractory acute promyelocytic leukemia (APL) without severe marrow suppression. Currently, the action of As2O3 on many other hematopoietic malignancies is under investigation. Much evidence has shown that caspase-3 plays essential executing role in apoptosis of many leukemia cell lines. But, the exact mechanism of As2O3-induced apoptosis in Molt-4 cell line which is originated from acute lymphoblastic leukemia is not well understood. Here, we investigate the action of As2O3 on Molt-4 cells and involved mechanism. Significant dose- and time-dependent inhibition of cell growth was observed by MTT assay. Following the treatment of As2O3 for 72 h, As2O3 at 4 μM exhibited 50% inhibition of growth in Molt-4 cells. The effect of As2O3 on the cell cycle was determined in Molt-4 cells by FACS analysis. DNA flow cytometric analysis with three independent experiments indicated that As2O3 induced a G1 and a G2-M phase arrest in Molt-4 cells following 6μM of exposure. Similar results were observed in Molt-4 cells following 2μM and 4μM exposure. These results indicated that As2O3 inhibited the cellular proliferation of Molt-4 cells via a G1 and a G2-M phase arrest of the cell cycle. To confirm and evaluate the induction of apoptosis, we performed the staining of cells with annexin V and PI. As with the percentages of sub-G1 group by FACS analysis, the proportion of apoptotic cells was increased in a dose-and -time dependent manner. Taken together, these results indicate that induction of apoptosis can be another mechanism of the antiproliferative effect of As2O3 besides G1 and G2-M phase arrests of the cell cycle in Molt-4 cells. We subsequently studied the activation of initiator caspase-8 and executioner caspase-3 in Molt-4 cells by Western blotting. Molt-4 cells that had undergone apoptosis on culturing with As2O3 displayed the initial activation of caspase-8 with the appearance of the large cleavage fragment of 43 to 41 kd. Despite the higher basal level of procaspase-3 expression in the Molt-4 cells prior to As2O3 treatment, we were unable to detect cleaved, activated caspase-3 following As2O3 treatment. Next, we checked whether inhibition of caspases-3 could abrogate the proapoptotic effects of As2O3. For this purpose the caspase-3 inhibitor, z-DEVD-fmk, was used. The results shown that addition of z-DEVD-fmk did not rescue Molt-4 cells from apoptosis induced by As2O3. These results clearly differ from other observations made with other leukemia cells and might explain, at least in part, that As2O3 induces apoptosis in Molt-4 cells is caspase 8-Dependent and caspase 3-Independent.

The regimen of cytarabine, aclarubicin and G-CSF (CAG) has been widely used in China and Japan for treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We searched literature on CAG between 1995 and 2010 and performed a meta-analysis to determine its overall efficacy using a random-effects or fixed-effects model. Thirty five trials with a total of 1029 AML (n = 814) and MDS (n = 215) patients were included for analysis. The CR rate of AML (57.9%) was significantly higher than that of MDS (45.7%) (p < 0.01). No difference in CR was noted between the new (56.7%) and relapsed/refractory AML (60.1%) (p > 0.05). The CR rate was also significantly higher in patients with favorable (64.5%) and intermediate (69.6%) karyotypes than those with unfavorable one (29.5%) (p < 0.05). Remarkably, the CR rate of CAG was significantly higher than those of non-CAG regimens (odds ratio 2.43). CAG regimen was well tolerated, with cardiotoxicity in 2.3% and early death in 5.2% of the cases. In conclusion, CAG regimen was an effective and safe regimen for the treatment of AML, and may be more effective than non-CAG regimens. Randomized controlled trials are strongly recommended to evaluate its efficacy and safety in comparison with the current standard treatment.