When is the best time and grade to start ruxolitinib in corticosteroid-refractory acute graft-versus-host-disease: A multi-center research.

OBJECTIVE: Ruxolitinib was recently approved to treat corticosteroid-resistant acute graft-versus-host disease (GvHD). However, it is unknown as to whether starting ruxolitinib at a lower versus higher acute GvHD grade or earlier versus later affected outcomes. This study identified the impact of starting acute GvHD grade and start time after declaring corticosteroid resistance and the effect on complete and overall response rates to ruxolitinib therapy. METHODS: Retrospective, observational multi-center study. We divided cohorts into starting ruxolitinib ≤ 7 days (N = 45) versus at > 7 days after declaring corticosteroid resistance (N = 24). RESULTS: In ≤ 7 days cohort complete response (CR) rates at day 28 were 69% (54, 81%) versus 25% (11, 47%; p = .001) in > 7 days cohort, and overall response (OR) rates were 91% (78, 96%) versus 80% (48, 92%; p = .25). CONCLUSIONS: Our data suggest that starting ruxolitinib in ≤ 7 days of declaring corticosteroid failure regardless of G vHD grade improves complete response rate but not OR rates. Starting ruxolitinib at grade I and within 7 days may get a more significant response.

Adoptive immune transfer from donors offers Anti-HBV protection to HBsAb-negative patients after Allo-HSCT.

Adoptive transfer of hepatitis B virus (HBV) immunity may occur following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we investigated the adoptive transfer of HBV immunity in 112 patients without HBV surface antibody (HBsAb) (HBsAb-) at the time of their first allo-HSCT. After allo-HSCT, HBV-DNA（87.5%） and HBsAg（11.1%%）cleared in HBsAg+ patients. All HBsAg- patients acquired HBsAb immediately. Nevertheless, HBsAb titers subsequently declined, and 39/67 (58.2%) patients lost HBsAb during follow-up. The 5-year overall survival (OS) was better in patients who lost HBsAb. Multivariate analysis showed that the independent risk factors for OS were lack of cytomegalovirus (CMV) clearance, acute graft-versus-host disease (aGVHD), and no HBsAb loss. Overall, adoptive immune transfer offers anti-HBV protection to patients without HBsAb, as they acquire HBsAb and clear HBV-DNA and HBsAg, while HBsAb loss after allo-HSCT predicts better survival.

Machine Learning Models for the Diagnosis and Prognosis Prediction of High-Grade B-Cell Lymphoma.

High-grade B-cell lymphoma (HGBL) is a newly introduced category of rare and heterogeneous invasive B-cell lymphoma (BCL), which is diagnosed depending on fluorescence in situ hybridization (FISH), an expensive and laborious analysis. In order to identify HGBL with minimal workup and costs, a total of 187 newly diagnosed BCL patients were enrolled in a cohort study. As a result, the overall survival (OS) and progression-free survival (PFS) of the HGBL group were inferior to those of the non-HGBL group. HGBL (n = 35) was more likely to have a high-grade histomorphology appearance, extranodal involvement, bone marrow involvement, and whole-body maximum standardized uptake (SUVmax). The machine learning classification models indicated that histomorphology appearance, Ann Arbor stage, lactate dehydrogenase (LDH), and International Prognostic Index (IPI) risk group were independent risk factors for diagnosing HGBL. Patients in the high IPI risk group, who are CD10 positive, and who have extranodal involvement, high LDH, high white blood cell (WBC), bone marrow involvement, old age, advanced Ann Arbor stage, and high SUVmax had a higher risk of death within 1 year. In addition, these models prompt the clinical features with which the patients should be recommended to undergo a FISH test. Furthermore, this study supports that first-line treatment with R-CHOP has dismal efficacy in HGBL. A novel induction therapeutic regimen is still urgently needed to ameliorate the poor outcome of HGBL patients.

A practical update on the epidemiology and risk factors for the emergence and mortality of bloodstream infections from real-world data of 3014 hematological malignancy patients receiving chemotherapy.

Background: Bloodstream infection (BSI) is a common and serious complication after patients with hematologic malignancies (HM) receiving chemotherapy. This study examined real-world data seeking to characterize HM BSI and identify risk factors for BSI emergence and mortality. Methods: We retrospectively analyzed the pathogenic epidemiology, antibiotic resistance, and BSI risk factors in a single-center cohort including 3014 consecutive patients with HM receiving chemotherapy between 2013 and 2016. Results of the pathogenic epidemiology were validated via comparison to available reported data. Results: We found that 725 patients (24.1%) had BSIs. Gram-negative (G-) bacteria represented 64.7% of the 744 isolated pathogenic strains, while Gram-positive (G+) bacteria and fungi accounted for 27.7% and 7.7% of the BSIs, respectively. The most common isolates were Klebsiella pneumoniae (19.2%), and 95.1% of the multidrug-resistant strains (MDR) were extended-spectrum beta-lactamase producing strains. G- bacteria were the main microflora responsible for BSI in our cohort of Chinese HM patients compared to studies in developed countries or in neutropenic children with HM or solid tumors. Multivariate analysis revealed that male sex, age ≥ 45 and < 65 yr, hospital length of stay ≥ 9d, neutropenia ≥ 7d before cultures, ≥ 2 antibiotics, and infections (gastrointestinal, perirectal, or urinary tract) independently predicted BSI emergence. Furthermore, age ≥ 65 yr, neutropenia ≥ 7d before blood cultures, no HM remission, lower white blood cell count, ≥ 3 antibiotics, respiratory infections, and Acinetobacter baumannii and Stenotrophomonas maltophilia BSI were independent predictors of 30-day mortality. Conclusions: G- bacteria were the predominant microflora during the study period and antibiotic resistance levels of the pathogens detected were high, especially for MDR strains. The mortality of BSI patients was high in this large cohort. Close attention should be paid to the risk factors identified here to facilitate timely and effective clinical management of such patients.

Mesenchymal stem cells enhance the impact of KIR receptor-ligand mismatching on acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation in patients with acute myeloid leukemia but not in those with acute lymphocytic leukemia.

Killer cell immunoglobulin-like receptor (KIR) receptor-ligand mismatch has been shown to be protective for acute and chronic graft-versus-host disease (aGVHD, cGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute leukemia. Mesenchymal stem cells (MSC) have been considered as one of the most promising prophylaxis for severe GVHD. However, there are no prospective or retrospective studies determining whether they can work synergistically on GVHD. To investigate the potential influence of KIR matching and MSCs, and their synergism on aGVHD and cGVHD after allo-HSCT in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Data from 104 patients with AML and 50 patients with ALL treated with allo-HSCT in the transplantation unit were retrospectively analyzed. KIR genotyping was performed by the PCR-SSO method. The amplicons were quantified on the Luminex 200 flow analyzer and analyzed using the Quick-Type for Lifecodes software to generate KIR data. Cox proportional hazards models were used in multivariate analyses. KIR receptor-ligand matching was associated with an increased risk of grade II-IV aGVHD compared to KIR receptor-ligand mismatching (p < 0.001) in AML patients, but KIR ligand-mismatching had no significant effect on aGVHD or cGVHD in ALL patients. In contrast, MSCs reduced the incidence of grade II-IV aGVHD in both AML and ALL patients (AML: p = 0.006; ALL: p = 0.008) regardless of KIR mismatching. The combination of KIR receptor-ligand mismatch and MSC transplantation significantly suppressed grade II-IV aGVHD occurrence in AML patients (p = 0.039). In the KIR mismatch group, the incidence of aGVHD was 2.8% in patients receiving MSC compared to 14.6% in those who did not (p = 0.047). KIR receptor-ligand mismatch, MSC transplantation and their combined use significantly reduced the risk of aGVHD after allo-HSCT. These data provide a clinically applicable strategy to reduce aGVHD, thus improving allo-HSCT outcome.

Iron overload as a risk factor for poor graft function following allogeneic hematopoietic stem cell transplantation.

Hematological malignancies are increasingly treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, iron overload is a frequent adverse effect of allo-HSCT and is associated with poor prognosis. In the present study, we investigated hematopoiesis in iron-overloaded mice and elucidated the effects of iron overload on the bone marrow (BM) microenvironment. Iron-overloaded BALB/C mice were generated by injecting 20 mg/mL saccharated iron oxide intraperitoneally. Hematoxylin-eosin staining was performed to evaluate the effects of an iron overload in mice. BM cells obtained from C57BL/6 mice were transplanted into irradiated BALB/C mice (whole-body irradiation of 4 Gy, twice with a 4-hours interval) by tail vein injection. Two weeks after allo-HSCT, the hematopoietic reconstitution capacity was evaluated in recipients by colony-forming assays. Histopathological examinations showed brown-stained granular deposits, irregularly arranged lymphocytes in the liver tissues, and blue-stained blocks in the BM collected from mice received injections of high-dose saccharated iron oxide (20 mg/mL). Iron-overloaded mice showed more platelets, higher-hemoglobin (HGB) concentration, fewer granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte colony-forming units (CFU-E), and mixed granulocyte/erythrocyte/monocyte/megakaryocyte colony-forming units (CFU-mix) than healthy mice. Iron-overloaded recipients presented with reduced erythrocytes and HGB concentration in peripheral blood, along with decreased marrow stroma cells, CFU-GM, CFU-E, and CFU-mix relative to healthy recipients. Taken together, our findings demonstrate that iron overload might alter the number of red blood cells after transplantation in mice by destroying the BM microenvironment, thereby affecting the recovery of BM hematopoietic function.

Epidermal growth factor stimulates exosomal microRNA-21 derived from mesenchymal stem cells to ameliorate aGVHD by modulating regulatory T cells.

Regulatory T cells (Tregs), a subset of CD4+ T cells, may exert inhibitory effects on alloimmune responses including acute graft-versus-host disease (aGVHD), and several microRNAs are implicated in the pathophysiological process of GVHD. Therefore, we aimed in the present study to characterize the functional relevance of epidermal growth factor (EGF)-stimulated microRNA-21 (miR-21) in regulating bone marrow-derived mesenchymal stem cells (BMSCs) in a mouse model of aGVHD. We first isolated and cultured BMSCs and Tregs. Then, we examined effects of miR-21 knockdown or overexpression and EGF on cell activities of BMSCs and the expression of PTEN, Foxp3, AKT phosphorylation, and extent of c-jun phosphorylation by gain- and loss-of-function approaches. The results showed that miR-21 promoted the proliferation, invasion, and migration of BMSCs. Furthermore, miR-21 in BMSCs-derived exosomes inhibited PTEN, but enhanced AKT phosphorylation and Foxp3 expression in Tregs. In addition, EGF enhanced c-jun phosphorylation to elevate the miR-21 expression. Furthermore, EGF significantly increased the efficacy of BMSCs in a mouse model of aGVHD, manifesting in reduced IFN-γ expression and lesser organ damage. Moreover, EGF treatment promoted the Foxp3 expression of Tregs in BMSCs-treated aGVHD mice. Taken together, EGF induced the BMSCs-derived exosomal miR-21 expression, which enhanced Foxp3 expression in Tregs, thereby improving the therapeutic effect of BMSCs on aGVHD.

Procalcitonin as a marker of Gram-negative bloodstream infections in hematological patients with febrile neutropenia.

The aim of this study was to explore the predictive value of procalcitonin (PCT) in Gram-negative bloodstream infections (BSIs) in hematological patients with febrile neutropenia. A total of 1466 samples (396 blood culture (BC)-positive, 1052 BC-negative, and 18 contaminated specimens) were included, comprising 268 Gram-negative, 88 Gram-positive, 19 fungal, and 21 polymicrobial BSIs. Median PCT value (0.72 ng/mL; IQR: 0.23-3.87) was significantly higher in Gram-negative than Gram-positive (0.34 ng/mL; IQR: 0.14-2.23; p < .01), or fungal (0.27 ng/mL; IQR: 0.13-0.40; p < .01) BSIs. In mono-microbial BSIs, the best PCT cutoff distinguishing Gram-negative BSIs from all other fever causes was 0.56 ng/ml, with a specificity of 76.8%. PCT levels were significantly higher in BSIs from multidrug-resistant (MDR) Gram-negative strains than from non-MDR (p < .01). This study confirms that elevated PCT may predict Gram-negative BSIs in hematological patients with febrile neutropenia, and demonstrates higher PCT levels in MDR Gram-negative BSIs in these patients.

G-CSF-primed haplo-identical HSCT with intensive immunosuppressive and myelosuppressive treatments does not increase the risk of pre-engraftment bloodstream infection: a multicenter case-control study.

A multicenter retrospective study in 131 patients (44 females/87 males) with hematological disorders who underwent G-CSF-primed/haplo-identical (Haplo-ID) (n = 76) or HLA-identical (HLA-ID) HSCT (n = 55) from February 2013 to February 2016 was conducted to compare the incidence and risk factors for pre-engraftment bloodstream infection (PE-BSI). In the Haplo-ID group, 71/76 patients with high-risk (n = 28) or relapsed/refractory hematological malignancies (n = 43) received FA5-BUCY conditioning (NCT02328950). All received trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis. Blood cultures and catheter tip cultures were obtained to confirm the BSI. PE-BSI was detected in 24/131 HSCT patients (18/76 in Haplo-ID and 6/55 in HLA-ID) after 28 febrile neutropenic episodes. Among 28 isolates for the 24 patients, 21 (75%) were Gneg bacteria, 6 (21.4%) Gpos and 1 (3.6%) fungi. Bacteria sources were central venous line infection (7/29.2%), gastroenteritis (6/25%), lower respiratory tract infection (LRTI; 5/20.8%), perianal skin infection (4/16.7%), and unknown (2/8.3%). The duration of neutropenia (P = 0.046) and previous Gneg bacteremia (P = 0.037) were important risk factors by univariate analysis, while the type of HSCT was not. A trend of TMP-SMX-resistant BSI in both groups may be due to routine antibacterial prophylaxis strategies. Our data show that G-CSF-primed Haplo-ID HSCT did not increase the risk of PE-BSI, even with intensive immunosuppressive treatments.

Bone marrow stromal cells enhance the survival of chronic lymphocytic leukemia cells by regulating HES-1 gene expression and H3K27me3 demethylation.

The majority of patients with chronic lymphocytic leukemia (CLL) are not cured by traditional chemotherapy. One possible explanation for this is that the microenvironment protects CLL cells from both spontaneous- and cytotoxic-mediated apoptosis. The present study was designed to investigate the mechanisms accounting for these effects, since this information is crucial to understanding CLL physiopathology and identifying potential treatment targets. The CLL cell line L1210 and primary CLL cells were cultured under different conditions: With serum, cyclophosphamide (CTX), or with monolayers and conditioned medium (CM) from the stromal cell line HESS-5. Apoptosis, Hes family BHLH transcription factor 1 (HES-1) gene and protein expression, and histone H3K27me3 DNA demethylation were determined. Co-culture of L1210 cells with HESS-5 cells significantly inhibited serum deprivation- and CTX-induced apoptosis of leukemia cells, and resulted in a significant increase in short-term proliferation. Soluble factors in the CM from HESS-5 cells had a negligible effect. The HESS-5 cell-mediated inhibition of apoptosis of CLL cells was associated with increased HES-1 expression and hypomethylation of the H3K27me3 gene in the leukemia cells. These results indicate that stromal cells enhance the survival of CLL cells by regulating the HES-1 gene and protein expression, as well as H3K27me3 DNA demethylation, and suggest that specific interactions between stromal and leukemia cells may enhance the resistance of leukemia cells to chemotherapy.

In vitro effects of reprogramming factors on the expressions of pluripotent genes and CD34 gene in human acute promyelocytic leukemia HL-60 cells.

OBJECTIVE: Our study aims to explore the in vitro effects of reprogramming factors on the expressions of pluripotent genes and CD34 gene in HL-60 cells. METHODS: According to the construction of lentiviral vector LV-OSCK of reprogramming factors (Oct-4, Sox2, Klf4, c-Myc), 293T cells were transfected to detect virus titer. The endogenous pluripotent genes (Oct4, SOX2, c-Myc and Klf4) and CD34 mRNA and protein expressions were detected by AP staining, immunofluorescence staining, qRT-PCR and flow cytometry. RESULTS: Expressions of Oct4, SOX2, c-Myc and Klf4 were 0.220±0.013, 0.186±0.009, 0.287±0.015 and 0.153±0.007. These levels were significantly higher in the experimental group than the control and blank groups. CD34 protein expression in the experimental group was also discovered to be significantly higher than the other two groups. CONCLUSION: The reprogramming factors could increase the expressions of pluripotent genes and CD34 gene in HL-60 cells.

Bone marrow-derived mesenchymal stem cells overexpressing MiR-21 efficiently repair myocardial damage in rats.

OBJECTIVE: We investigated the ability of bone marrow derived mesenchymal stem cells (BMSCs) overexpressing microRNA-21 (miR-21) to repair cardiac damage induced by anthracyclines in rats. METHODS: Sprague-Dawley (SD) rats of 2~3 weeks old were selected to isolate and culture BMSCs. A lentivirus harboring pLVX-miR-21 was generated and transfected into rat BMSCs. The rats were assigned into an untreated negative control group, and groups injected with adriamycin alone or with adriamycin followed by BMSCs, pLVX-BMSCs or pLVX-miR-21-BMSCs (n = 10 each). Proliferation and migration of cells were detected by cholecystokinin-8 (CCK- 8) and transwell. MiR-21 expression, mRNA expressions of B cell lymphoma 2 (Bcl2), BAX (BCL-2-associated X protein) and vascular endothelial growth factor (VEGF) were tested by qRT-PCR. Western blotting was applied to detect protein expressions of Bcl-2, Bax and VEGF. RESULTS: Using CCK- 8 and transwell assays, we found that pLVX-miR-21-BMSCs, which overexpressed miR-21, exhibited greater proliferation and migration than untransfected BMSCs or pLVX-BMSCs. Ultrasonic cardiograms and immunohistochemical analysis demonstrated that among the five groups, the pLVX-miR-21-BMSC group exhibited the most improved heart function and enhanced angiogenesis. Moreover, the pLVX-miR-21-BMSC group showed enhanced expression of Bcl-2, VEGF and Cx43 and reduced expression of Bax, BNP and troponin T. CONCLUSION: These findings suggest miR-21 overexpression enhanced the proliferation, invasiveness and differentiation of BMSCs as well as expression of key factors (Bcl-2, VEGF and Bax) essential for repairing the cardiac damage induced by anthracyclines and restoring heart function.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells.

OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

A 5-day cytoreductive chemotherapy followed by haplo-identical hsct (FA5-BUCY) as a tumor-ablative regimen improved the survival of patients with advanced hematological malignancies.

Haplo-HSCT has been used when HLA-matched siblings are not available. Conditioning regimens aim to reduce tumor burden prior to HSCT and provide sufficient immunoablation. We report the outcome of haplo-HSCT in 63 consecutive patients from 2/2013 to 12/2015 (19 females/44 males) with high-risk or relapsed/refractory hematological malignancies (n=29-AML; 8-sAML; 19-ALL; 5-advanced-MDS; 2-CML-BC). Median age was 20 years (range: 1.1-49). Twenty-one patients achieved remission prior to transplant, while 42 did not. Patients received FA5-BUCY, i.e., 5-day salvage chemotherapy (Fludarabine/Ara-C) and conditioning (Busulfan/Cyclophosphamide). GvHD prophylaxis included ATG, CsA, MMF and short-term MTX. All patients received stem cells from bone marrow and peripheral blood, and achieved successful engraftment, except two who died before. With a median follow-up of 269 days (120-1081), 42/63 patients are still alive and disease-free. Two-year OS and RFS were similar in patients not in remission and in those in complete remission (61.3% vs 56.3%, p=0.88; 58.3% vs 56.3%, p=0.991). Non-relapse mortality and relapse incidence were 22.2% and 11.1%, respectively. Severe acute-GvHD occurred in 4/63 patients. Transplant-related mortality was low at day+100 (17.5%) and for the entire study period (20.6%). Unexpectedly, few patients experienced mild-to-moderate toxicity, and main causes of death were infection and GvHD. BM blast counts, age, and donor-recipient gender-pairs did not affect the outcome. Less chemotherapy cycles prior to HSCT might result in more favorable outcome. Thus, haplo-HSCT with FA5-BUCY appears promising for advanced disease, especially when TBI and amsacrine, used for FLAMSA, are not available and in pediatric patients for whom TBI is not recommended.

Lymph Node Flow Cytometry as a Prompt Recognition of Ultra Early Onset PTLD: A Successful Case of Rituximab Treatment.

Ultra early posttransplantation lymphoproliferative disorder (PTLD) is a rare and fatal complication after hematopoietic stem cell transplantation (HSCT). Here we report, by lymph node (LN) flowcytometry, that we early recognized ultra early PTLD after an HLA-matched sibling allo-HSCT followed by a successful treatment with anti-CD20 antibody (rituximab) in a patient in progress disease for angioimmunoblastic T-cell lymphoma (AITL). The patient was conditioned with a reduced intensity conditioning (RIC) regimen. One week after transplantation, the patient developed high fever, generalized fatigue, high Epstein-Barr virus (EBV) load, and LN enlargement. An LN lymphocyte suspension and peripheral blood flowcytometry was performed to find majority of LN lymphocytes highly expressed CD20. By highly suspicious PTLD, 4 doses of rituximab (375 mg/m(2) qw) were given immediately followed by reducing and withdrawing immunosuppressant reagent. PTLD was later confirmed by pathology. The patient had good response to rituximab, showing absence of fever, reduction in LN size, and no detectable EBV-DNA. Twenty months after HSCT, the patient remains well without evidence of AITL and PTLD. The current report is one of the earliest cases of PTLD after HSCT. Taken together, by LN flowcytometry as a prompt recognition, rituximab can be an effective preemptive therapy for ultra early developed PTLD.

Constitutive activation of NF-κB signaling by NOTCH1 mutations in chronic lymphocytic leukemia.

NOTCH1 mutations occur in approximately 10% of patients with chronic lymphocytic leukemia (CLL). However, the relationship between the genetic aberrations and tumor cell drug resistance or disease progression remains unclear. Frameshift deletions were detected by gene sequencing in the NOTCH1 PEST domain in three naive CLL patients. These mutations were associated with chromosomal abnormalities including trisomy 12 or 13q deletion. Of note, one of the patients developed Richter's transformation during FCR treatment. Immunofluorescent and western blot analyses revealed a markedly higher intracellular domain of NOTCH (ICN) expression in the mutated cells compared with their unmutated counterparts and normal CD19+ B lymphocytes (P<0.01 and P<0.001, respectively). In addition, strong DNA-κB binding activities were observed in the mutant cells by gel shift assays. RT-PCR analysis revealed elevated RelA mRNA expression in the mutant cells, while RelB levels were variable. Reduced levels of RelA and RelB mRNA were observed in unmutated CLL and normal B cells. Compared to unmutated CLL and normal B cells, increased apoptosis occurred in the mutant cells in the presence of GSI (ICN inhibitor) and PDTC (NF-κB inhibitor), particularly under the synergistic effects of the two drugs (P=0.03). Moreover, IKKα and IKKß, the active components in the NF-κB pathway, were markedly inhibited following prolonged treatment with GSI and PDTC. These results suggested that NOTCH1 mutations constitutively activate the NF-κB signaling pathway in CLL, which is likely related to ICN overexpression, indicating NOTCH1 and NF-κB as potential therapeutic targets in the treatment of CLL.

Younger patients with chronic lymphocytic leukemia benefit from rituximab treatment: A single center study in China.

Chronic lymphocytic leukemia (CLL) is characterized by high heterogeneity in clinical features and outcomes in Western countries and China. In this study, the clinical and laboratory data of 210 CLL patients who were admitted to a single center in China between 2002 and 2011 were retrospectively analyzed. CLL patients had a median age of 60.2 years (range, 35-92 years) and CLL occurred more often in elderly female patients than in male patients (female:male, 1.2:1). The overall response rate [ORR, complete remission (CR) + partial remission (PR)] in the entire cohort of patients was 69.5% and the median overall survival (OS) was 67 months (95% CI, 57.88-76.11). In univariate analysis, an age of >60 years, chromosome 17p deletion (17p-) and elevated ß2-MG were associated with a worse OS. Patients with all three poor prognostic factors had a worse outcome than patients with only one or two factors. Patients with 17p- had a significantly lower ORR (P=0.008) and shorter OS (P=0.001) than those without 17p-. Rituximab (R)-based treatment was able to overcome the poor prognosis associated with 17p-. Moreover, the addition of R to fludarabine (F) and cyclophosphamide (C) treatment significantly improved the OS (P=0.012) compared with FC alone in younger patients. However, there were no significant benefits for older patients (P=0.07). This implies that the age of a patient is important in their response to therapy and survival.

A novel nonradioactive CFDA assay to monitor the cellular immune response in myeloid leukemia.

BACKGROUND: Donor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, AML is a highly diverse disease and the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach. OBJECTIVE: To monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system to assess the antileukemia immunity by flow cytometry, correlated with [3H]-thymidine uptake. METHODS: The myeloid blasts derived from five patients with AML relapsed post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML progenitor cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail (50ng/mL of SCF; 25ng/mL of IL-3; 100ng/mL of GM-CSF; 100ng/mL of G-CSF; 2U/mL of EPO; 0.47g/L of transferrin; and 5×10(-5)mmol/L of 2-ME). The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake. The simultaneous use of TO-PRO-dye and calibrate beads allowed not only the cell viability to be known but also allowed quantification of the effector function. RESULTS: Here, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with our nonradioactive, CFDA-based assay provided comparable results with the classic [3H]-thymidine assay with an even lower ratio of effector to target cells. CONCLUSION: Taken together, the novel, nonradioactive, CFDA-based assay was a robust tool to monitor the antileukemic immune response after DLT in myeloid leukemias.

[Clinical study of umbilical cord-derived mesenchymal stem cells for treatment of nineteen patients with steroid-resistant severe acute graft-versus-host disease].

OBJECTIVE: To evaluate the safety and efficacy of umbilical cord-derived mesenchymal stem cells (MSCs) infusion in patients with steroid-resistant severe acute graft-versus-host disease (aGVHD). METHODS: A total of 19 patients with steroid-resistant severe aGVHD received MSCs infusion treatment. The treatment response, transplantation-related mortality, events associated with infusion and relapse rate were analyzed. RESULTS: Two patients with grade II, 5 patients with grade III and 12 patients with grade IV aGVHD received a total of 58 infusions of MSCs. The mean total dose of MSCs was 2.13 (range 0.60 - 7.20)×10(6) cells per kg bodyweight. Seven patients received one infusion, 2 patients received two infusions, and 10 patients received three or more infusions. Eleven patients had a complete response and 4 had a partial response and 4 had no response. No patients had side-effects during or immediately after infusions, and no MSCs related tumorigenesis was detected to date. Eleven patients survived and 8 died, 4 for aGVHD, 1 for infection and 2 for aGVHD with concomitant infection and 1 for underlying leukemia relapse. The cell viability of freshly prepared MSCs is 93% (92% - 95%) by trypan blue staining. The cell viability of programmatically frozen and thawed MSCs is 72% (70% - 74%). CONCLUSION: Infusion of umbilical cord-derived MSCs expanded in vitro is an effective therapy for patients with steroid-resistant severe aGVHD without negative impact on relapse. Freshly prepared MSCs are superior to frozen and thawed cells in terms of cell viability.

[Clinical analysis on 40 patients with chronic lymphocytic leukemia].

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum ß2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum ß2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.