ALBERT

Description: Mantle cell lymphoma (MCL) is one subtype of the B cell non-Hodgkin lymphomas (NHL) and has a distinguash course. Despite all efforts, MCL is nearly incurable that refractory or relapse(r/r) course will occur in almost all patients. Due to the rapid development of chemotherapy resistance, it is difficult to treat r/r MCL. A number of treatment regimens have been evaluated with various degrees of success. Cellular immunotherapy has the potential to lead significant and sustained clinical remissions in lymphoma patients, in which modification of T cells with chimeric antigen receptors (CARs) is a powerful regimen. T cells containing CARs with costimulatory domains show better activity against tumor cells. We conducted a clinical trial testing a "second-generation" CD19-specific CAR with 4-1BB costimulatory domains in patients with r/r MCL. Six patients with r/r MCL were enrolled and all were received T-cell infusions. The age of patients ranged from 52 to 81 years old. Five patients were male and one patient was female. Five patients had stage IV MCL with bone marrow involvement and one patient was stage II. Four patients had received more than 3 lines regimens and two patients had received first-line chemotherapy regimen. Four patients received FC regimen before CAR-T infusion and two patients were not received fludarabine due to age ≥80 years old. Treatment was well tolerated, two patients developed transient infusion reaction. No patients have grade 3-4 Cytokine Release Syndrome (CRS). Three patients have grade 2 CRS and two patients have grade 1 CRS. Objective response rate is 66.67%. One patient achieves complete remission(CR) and three patients have partial remission(PR). The response of the other two patients is stable disease(SD). The patient who has CR response has remained progression-free for 36 months and is still being followed up. Two patients who have PR response have progressed in 3 months and the other one has durable response for 7 months. In the patients who has response, the peak of fever appears at approximately day 10 after CAR-T infusion, moreover interleukin(IL)6,IL2αand interferon(IFN)-γ almost reaches the peak on the day 14. In conclusion, adoptive immunotherapy with CD19-specific T cells was well tolerated and was associated with antitumor activity. Further large-sample and multicenter studies are needed to fully evaluate this strategy for such patients. Disclosures No relevant conflicts of interest to declare.

Description: The myeloproliferative neoplasms (MPNs) are hematologic malignancies with a chronic clinical course and a risk of developing thrombosis and acute leukemia. JAK2 is a member of the Janus family of non-receptor tyrosine kinases, and the somatic activating point mutation of JAK2 (JAK2V617F) has been found in Philadelphia chromosome-negative MPNs in approximately 95% of polycythemia (PV), 60% of essential thrombocytosis (ET), and 50% of JAK2 V617F-positive MPD (PMF) patients. Although several JAK2 inhibitors are in the early stage of clinical trials and have been shown to help to improve symptoms and quality of life in patients, their long-term effectiveness in patients remains to be determined. New therapeutic strategies need to be developed for treating PV more effectively. Because hematopoietic stem cells (HSCs) harbor JAK2V617F in PV patients, PV should be compared to Philadelphia chromosome-positive chronic myeloid leukemia (CML) that is also derived from HSCs and has a myeloproliferative phenotype similar to PV in mice. Therefore, we reasoned that a gene essential for CML development might also play a critical role in PV development. We have shown that the survival and self-renewal of CML-initiating cells require the arachidonate 5-lipoxygenase gene (Alox5) and that Alox5 is essential for CML development. Therefore, in this study we investigated the role of Alox5 in pathogenesis of PV in mice. We showed that JAK2V617F upregulates Alox5 expression both in vitro (Ba/F3 cells) and in vivo (the spleen cells in PV mice). To examine whether Alox5 is required for induction of PV by JAK2V617F, we transduced bone marrow (BM) cells from wild type (WT) or Alox5 homozygous knockout (Alox5-/-) mice with JAK2V617F-GFP, followed by transplantation into lethally-irradiated recipient mice. We found that the average percentage of JAK2V617F-expressing white blood cell counts (GFP+ WBCs) in recipients of JAK2V617F-transduced Alox5-/- donor BM cells was significantly lower than that in recipients of JAK2V617F-transduced WT donor BM cells (P〈 0.001). Total numbers of WBCs, red blood cells (RBCs), hemoglobin (HGB) and hematocrit (HCT) were also significantly lower in the absence of Alox5. With time (at 9 months after induction of PV), the effect of Alox5 deficiency on PV development became more significant. Because JAK2V617F-transformed HSCs is shown to be a PV-initiating cell population, we further tested whether loss of Alox5 affects this cell population in PV mice. We found that the percentage and total number of HSCs (Lin-Sca-1+c-Kit+) in mice receiving JAK2V617F-transduced Alox5-/- BM cells was significantly lower than that in mice receiving JAK2V617F-transduced WT BM cells. These findings suggest that Alox5 could be an effective target gene for PV. We tested this idea by treating PV mice with an Alox5 inhibitor zileuton (300 mg/kg, once a day) or a placebo. Western blot analysis of protein lysates from the spleens of PV mice showed that 5-lipoxygines (5-LO, a protein product of the Alox5 gene) is upregulated by JAK2V617F, and inhibition of Alox5 function by zileuton restored the 5-LO level back to the control level. In addition, the number of WBCs in placebo-treated mice rose significantly with time, whereas the number of WBCs in zileuton-treated mice did not. The inhibition of PV development by zileuton correlated with much smaller infiltrated spleen in zileuton-treated PV mice than in placebo-treated PV mice. Furthermore, we compared the levels of JAK2V617F-expressing cells (GFP+) in PV mice. FACS analysis showed that the percentage of Gr-1+ or Mac+ PV cells in peripheral blood was significantly lower in zileuton-treated PV mice than in placebo-treated PV mice. Consistent with the inhibition of PV cells by zileuton in mice, survival of zileuton-treated PV mice was significantly improved compared to placebo-treated PV mice (Fig. 3I). At 5 months after induction of PV, about 40% placebo-treated PV mice died, but all zileuton-treated mice survived. To understand the underlying mechanisms, we treated JAK2V617F-expressing BaF/3 cells with zileuton, and found that zileuton inhibited activation of AKT and β-catenin by JAK2V617F, and did not affect activation of PI3K, MAPK and JAK2. Together, these results demonstrate that Alox5 represents a critical pathway in JAK2V617F-induced PV and that targeting of Alox5 provides a new strategy for treating PV. Disclosures: No relevant conflicts of interest to declare.

Description: Eperythrozoon ovis, used thought to be a rickettsia but now identified as a mycoplasma, is an erythrocytic agent that causes hemolytic anemia both in animals and human. To demonstrate the epidemiological status of Eperythrozoon ovis infection in Chinese population, 1458 healthy volunteers, 247 patients with hematologic disorders and 106 susceptible people with direct contact to suspected animals were investigated by classical blood smear examination. The positive samples were identified by a specific PCR assay. The microscopic results showed a lot of small organisms attaching on the surface of erythrocytes (Fig 1). The partial 16s rRNA gene of these organisms was amplified using the conserved primers and confirmed as hemoplasma by sequence alignment analysis. Moreover, complement regulatory protein (CR1, CD35), indicating the function of red cell membrane, was tested by flow cytometry with mouse anti-human CD35 and caprine-anti-mouse IgG-FITC reagents. The expression of CR1 might help to elucidate the mechanism why the Eperythrozoon always lead to anemia and icterus in human being (Fig 2). The Eperythrozoon infection rate in healthy was 239/1458, 95/247 in hematologic disease patients and 55/106 in susceptible people, respectively in our results. Results of flow cytometry in peripheral blood sample showed the co-relation between the CD35 expression and the eperythrozoon infection status. The CD35 values increased obviously in the infected cases. In the 4 outbreak cases with FUO (Fever of unknown origin, finally diagnosed as eperythrozoonsis), CD35 reached the peak which might indicate the immuno-defense from the body against the hemotrophic mycoplasma. Gradually, the CD35 value decreased markedly in the long-term infectious cases, which may be explained by anemia and icterus resulting from destroyed membrane of the erythrocyte. Fig. 1: Field emission scanning electron microscope to diagnose the Eperythrozoonsis. The mildly (Fig. 1--1) and severely (Fig. 1–2) infected RBCs. Erythrocyte in picture 1 (Fig. 1--1) had not been deformed but cells in (Fig. 1–2) and (Fig. 1–3) were badly misshapen with some holes on its surface. Transmission electron microscope showed the location of these organisms in erythrocyte depressions (Fig. 1–4) and fibril of these organisms connected with RBC was also observed when these organisms disassociated from erythrocyte (Fig. 1–5). Blood smear stained by Wright-Giemsa mixture demonstrated severe bacteremia (Fig. 1–6). Fig. 1:. Field emission scanning electron microscope to diagnose the Eperythrozoonsis. The mildly (Fig. 1--1) and severely (Fig. 1–2) infected RBCs. Erythrocyte in picture 1 (Fig. 1--1) had not been deformed but cells in (Fig. 1–2) and (Fig. 1–3) were badly misshapen with some holes on its surface. Transmission electron microscope showed the location of these organisms in erythrocyte depressions (Fig. 1–4) and fibril of these organisms connected with RBC was also observed when these organisms disassociated from erythrocyte (Fig. 1–5). Blood smear stained by Wright-Giemsa mixture demonstrated severe bacteremia (Fig. 1–6). Fig. 2: The expression of CD35 antigen on the surface of RBCs was assessed by flow cytometry. Compared to CD35 expression from the controls, no obvious differences were observed from the healthy people(around 4.69) (Fig 2-1). In the positive samples, the CD35 values increase from the negative 4.69 to 10.61 (Fig 2--2). In the outbreak cases, however, a sharp increase of CD35 antigen expression at the RBC external membrane were observed with the values of 28.76(Fig. 2–3 and 2–4), which were significantly higher than the value of the control samples (p

Description: Abstract 4121 The activation of the JAK/STAT pathway caused by the JAK2 gene mutations is an important pathogenetic mechanism of myeloproliferative neoplasm(MPN). Recently, many evidences suggest that there are factors besides the mutations of JAK2 gene participate in the pathogenesis of MPN. Suppressors of cytokine signaling (SOCS) proteins are potent inhibitors of JAK/STAT pathway, therefore we hypothesized that the down regulation of SOCS protein system may be a possible pathogenetic mechanism of MPN through the activation of the JAK/STAT pathway. In order to testify our hypothesis, we investigated mutated points, the expression and methylation status of the SOCS1, SOCS2 and SOCS3 gene in 100 MPN patients(44 polycythemia vera (PV), 38 essential thrombocythemia (ET) and 18 idiopathic myelofibrosis(MF)). We obtained some interesting results: (1) By using DNA sequence analysis, two mutations of SOCS3 were identified with in the coding region in 1 of PV patients and 1 of ET patients (2%), respectively, and both of these 2 patients are with JAK2V617F mutation.(wide type ACG, coding Threonine, alterering to mutant type AAG, coding Lysine). Furthermore, three types of nonsense mutations were identified in SOCS3:Firstly,38 (38%) mutations of SOCS3 were identified with in the coding region in 19 of PV patients,17 of ET patients and 2 of MF respectively, (wide type CCC, coding Proline, alterering to mutant type CCA, coding Proline); Secondly, 44 (44%) mutations of SOCS3 were identified with in the coding region in 21 of PV patients,18 of ET patients and 5 of MF respectively, (wide type GTA, coding Valine, alterering to mutant type GTG, coding Valine); At last, 35 (35%) mutations of SOCS3 were identified with in the coding region in 13 of PV patients,20 of ET patients and 2 of MF respectively, (wide type GAT, coding Aspartic acid, alterering to mutant type GAC, coding Aspartic acid).Five nonsense mutations were found in SOCS2: 2 of PV patients,3 of ET patients, (wide type AAT, coding Asparagine, alterering to mutant type AAC, coding Asparagine). On the contrary, the presence of JAK2V617F mutation did not affect the nonsense mutations of SOCS2 or SOCS3. (2) By using Methylation Specific PCR (MSP), SOCS1 hypermethylation was identified in 27 patients. Hypermethylation of the SOCS2 promoter was identified in 9 of 100 (9%) patients. Hypermethylation of the SOCS3 promoter was identified in 35 of 100 (35%) patients. There was no hypermethylation of the SOCS1, SOCS2 and SOCS3 gene in 173 normal controls. (3) By using semi-quantitative PCR, the RNA expression levels of SOCS1, SOCS2 and SOCS3 were also investigated. We observed hypermethylated patients had lower SOCS1 or SOCS3 mRNA levels than unmethylated MPN samples, also observed that among patients with unmethylated SOCS1 and SOCS3, mRNA expression was higher from patients carrying the JAK2V617F mutation as compared with JAK2 wild type patients. On the contrary, the presence of JAK2V617F mutation did not affect the expression of SOCS1 or SOCS3 mRNA in methylated patients. Moreover, SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F negative myeloproliferative neoplasm. (4) According to SOCS1, SOCS3 methylation was not significantly correlated with survival or other clinical variables. In conclusion, SOCS1 and SOCS3 hypermethylation can activate the JAK/STATsignaling pathway in alternative or together with JAK2 mutations. These alterations might represent a potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Description: Chimeric antigen receptor T (CAR-T) cell therapy has emerged as a novel treatment modality for B-cell malignancies. CD19-specific CAR-T cells induce high rates of initial response among patients with relapsed B-cell acute lymphoblastic leukemia (ALL). However, cytokine release syndrome (CRS) is the most common and severe toxicities of CAR T-cell therapy for ALL, and clinical experience is limited. Here, we describe the clinical presentation and management of 30 patients who presented with CRS following CAR-T cell therapy for relapsed/refractory ALL at our hospital. 12 of the 30 patients (40%) developed grade 1-2 CRS, 14 patients (46.7%) presented with grade 3-4 CRS and 2 patients (6.7%) died of grade 5 CRS. Compared with grade 1-2 CRS, grade 3-4 CRS correlated negatively with overall survival and progression-free survival (P =0.02). We found that higher ferritin levels and percentages of CD19 positive cells in blood lymphocytes cells at time of CAR-T cell infusion were associated with more severe CRS. Grade 3-4 neurotoxicity was frequently present in patients with grade ≧3 CRS. We also observed that the organ disfunctions occurred in sequence after fever onset during the period of CRS. Neurotoxicity, cardiovascular disfunction and cytopenia in some patients manifest as biphasic. Compared to use of tocilizumab for CRS ≧ grade 3, early intervention of tocilizumab for hyperpyrexia duration ≧ 6h alleviates the severity of CRS, and no patients died of severe CRS since this management approach was performed. As use of novel CAR-T cell therapy expands, the data from our clinical experience may help others anticipated the clinical course of organ function and manage CRS in CAR-T therapy. Figure Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4715 From July 2006 to February 2009, total of 146 patients with non-APL AML were admitted in our department. Cytogenetic analyses, detection of Flt3-ITD, NPM1 gene mutation and VEGF and its receptors (Flt-1, KDR) were performed on all patients. Forty-nine patients less than 60 years of old and with normal karyotype were selected for prognostic analyses. Flt3-ITD was positive detected in 15 cases (30.61%), NPM1 mutation in 18 cases (36.73%), VEGF in 46 cases (93.88%), Flt-1 in 41 patients (84.04%) and KDR in 38 cases (77.55%). After one or two courses of induction therapy with IDA regimen (Idarubicin + cytarabine: 3+7) in all 49 patients, total CR rate was 67.43% (33/49). One patient died because of severe invasive fungal infection. Among the remaining 15 non-CR patients, 10 were Flt3-ITD positive and NPM1 negative, and all with higher expression of VEGF and KDR. All the CR patients were treated with consolidation regimen with high dose cytarabine (3g/m2, q 12h iv for 3 consecutive days) for 6 courses. After follow-up at least for 6 months, only 12 patients are alive up to now. In these 12 patients, Flt3-ITD were all negative expressed, 6 patients were NPM1 positive, 2 patients VEGF negative, 3 patients both KDR and Flt-1 negative. There are no patients alive with positive expressed of Flt3-ITD, KDR and negative expressed of NPM1. Therefore, we supposed that negative expression of Flt3-ITD and KDR plus positive expression of NPM1 could be a favorable parameter for outcome prediction in AML patients with normal karyotype. Disclosures: No relevant conflicts of interest to declare.

Description: Activation of Notch signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with Notch ligand Delta1 has enabled a clinically relevant ex vivo expansion of short-term HSPCs. In vitro studies have also revealed a role of low O2 tension in HSPC regulation. A molecular link has been demonstrated in several stem/progenitor cell populations between Notch and hypoxia pathways but their interaction has not been investigated in human HSPCs. G-CSF mobilized human CD34+ cells from 4 healthy subjects were cultured in the presence of cytokines (SCF, FLT3L and TPO) in hypoxia (1.5-2% O2) or normoxia (21% O2) in vessels coated with fibronectin alone or combined with increasing concentrations of the immobilized ligand Delta1 (2.5, 5, 10 and 20 µg/mL). After 21 days in culture, cells were counted and characterized using CFU assays, flow cytometry for lineage (Glycophorin A+, CD13+, CD20+, CD3+ and CD41+ cells) and HSC (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholow) phenotypes, and transplantation in immunodeficient (NSG) mice. In normoxia, the total number of cells increased 118-fold compared to baseline in the absence of Delta1 with limited residual CD34+ cells (1.5 ± 0.7%), extensive differentiation toward the myeloid lineage (96.3 ± 0.3% CD13+ cells) and minimal engraftment potential in NSG mice (0.2 ± 0.2% human CD45+ cells). With increasing concentrations of Delta1 in normoxia, consistent with the hypothesis that Delta1 delays differentiation, the total number of cells increased less (41-, 25-, 11- and 7-fold relative to baseline, respectively) CD34+ cells expanded more (4-, 4-, 3- and 2-fold relative to baseline, respectively), and CFU numbers increased more (8-, 7-, 4- and 3-fold relative to baseline, respectively) than without Delta1. However, phenotypically defined HSCs were undetectable or markedly decreased at the lowest Delta1 concentrations used (2.5 and 5 µg/mL) and their numbers were maintained or only minimally increased at the highest Delta-1 concentrations tested (10 and 20 µg/mL) relative to uncultured CD34+ cells. Accordingly, only cells cultured with 10 and 20 µg/mL Delta1 resulted in levels of engraftment in NSG mice (5.5 ± 5.4% and 5.4 ± 0.9% human CD45+ cells, respectively) comparable to uncultured cells (7.0 ± 0.1% human CD45+ cells). In hypoxia, total cell counts increased less than in normoxia both without (8-fold relative to baseline) and with increasing concentrations of Delta1 (11-, 11-, 9-, 9-fold relative to baseline, respectively) due to diminished myeloid differentiation. Total CD34+ cells decreased 1.7-fold in hypoxia in the absence of Delta1, but expanded modestly in the presence of Delta1 (3-, 3-, 2- and 2-fold, respectively). CFU numbers followed a similar trend. However, in hypoxic cultures with 2.5, 5 and 10 µg/mL Delta1, phenotypically defined HSCs increased 2.5-, 6.6- and 1.3-fold, respectively, compared to uncultured cells. Importantly, hypoxia combined with 2.5, 5 and 10 µg/mL Delta1 concentrations resulted in increased human cell engraftment in NSG mice (21.2 ± 4.4%, 29.3 ± 11% and 11.8 ± 5.4% human CD45+ cells, respectively) compared to uncultured cells (7.0 ± 0.1% human CD45+ cells). When 20 µg/mL Delta1 was used in hypoxia, engraftment potential in NSG mice was decreased (1.1 ± 0.6% human CD45+ cells). We next performed limiting dilution analysis to measure the frequencies of long-term repopulating HSCs (LT-HSCs) within the CD34+ cell compartment at baseline and after 21 days in hypoxic or normoxic cultures supplemented with the optimized concentrations of Delta1 (10 µg/mL in normoxia and 5 µg/mL in hypoxia). LT-HSCs in uncultured CD34+ cells were measured at the expected frequency (1 in 7,706; 95% CI of 3,446 to 17,232). When analyzed at 3 months post-transplantation, a limited (1.5-fold) increase in LT-HSC frequency (1 in 5,090; 95% CI 2.456 to 10,550) was obtained from Delta1 normoxic cultures compared to uncultured cells. In contrast, the frequency of LT-HSCs (1 in 1,586; 95% CI 680 to 3,701) was 4.9-fold higher in hypoxic Delta1 cultures compared to uncultured cells, and 4.2-fold higher than in normoxic Delta1 cultures. Similarly, absolute numbers of LT-HSCs per 100,000 Day 0 equivalent CD34+ cells increased from 13 (baseline) to 216 (normoxia) and 694 (hypoxia). Our data indicate that hypoxia potentiates Notch-induced expansion of human HSPCs and may be of benefit in stem cell transplantation and gene therapy applications. Disclosures Cheruku: Novartis: Research Funding. Larochelle:Novartis: Research Funding.