Culturing adequate CAR-T cells from less peripheral blood to treat B-cell malignancies.

OBJECTIVE: Chimeric antigen receptor-modified T (CAR-T) cells have shown impressive results against relapsed/refractory B cell malignancies. However, the traditional manufacture of CAR-T cells requires leukapheresis to isolate large amounts of peripheral blood T cells, thus making some patients ineligible for the procedure. METHODS: We developed a simple method for CAR-T cell preparation requiring small volumes of peripheral blood. First, CD3+ T cells isolated from 50 mL peripheral blood from patients (B-cell malignancies) were stimulated with immobilized anti-CD3/RetroNectin in 6-well plates and then transduced with CAR-expressing lentiviral vector. After 4 d, the T cells were transferred to culture bags for large-scale CAR-T cell expansion. In vitro and animal experiments were performed to evaluate the activity of the manufactured CAR-T cells. Finally, 29 patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 patients with B-cell lymphoma were treated with the CAR-T cells. RESULTS: The CAR-T cells were expanded to 1-3 × 108 cells in 8-10 d and successfully killed B cell-derived malignant tumor cells in vitro and in vivo. For patients with B-ALL, the complete remission rate was 93% 1 month after CAR-T cell infusion; after 12 months, the overall survival (OS) and leukemia-free survival rates were 69% and 31%, respectively. For patients with lymphoma, the objective response rate (including complete and partial remission) was 78% 2 months after CAR-T cell infusion, and after 12 months, the OS and progression-free survival rates were 71% and 43%, respectively. Cytokine-release syndrome (CRS) occurred in 65.51% and 55.56% of patients with B-ALL and B-cell lymphoma, respectively; severe CRS developed in 20.69% of patients with B-ALL and in no patients with lymphoma. CONCLUSIONS: Our novel method can generate sufficient numbers of CAR-T cells for clinical use from 50-100 mL peripheral blood, thus providing an alternative means of CAR-T cell generation for patients ineligible for leukapheresis.

Safety and efficacy of CAR-T cell targeting BCMA in patients with multiple myeloma coinfected with chronic hepatitis B virus.

BACKGROUND: Reactivation of hepatitis B virus (HBV) infection is a well-recognized complication in patients with chronic or resolved HBV infection undergoing anticancer therapy. There is a risk of HBV reactivation after infusion of chimeric antigen receptor (CAR) T cells for patients with refractory/relapsed (R/R) multiple myeloma (MM). METHODS: We administered B cell maturation antigen (BCMA) CAR-T cell by infusion to nine patients with R/R MM with chronic or resolved HBV infection. Patient serum was analyzed to determine the expression of five components of HBV and the copy number of HBV DNA. HBV reactivation was defined if a patient re-exhibited hepatitis B surface antigen (HBsAg) or HBV DNA regrowth after CAR-T therapy. RESULTS: In one patient who was HBsAg-positive, no HBV reactivation was observed during the follow-up period of 9.8 months after administration of anti-HBV drugs before and after CAR-T therapy. Among eight patients with MM who had resolved HBV infection, two patients administered prophylactic anti-HBV drugs did not exhibit HBV reactivation. Of the six patients who did not use prophylactic antiviral drugs, five did not exhibit HBV reactivation, while one showed recurrence of HBsAg without detection of HBV DNA or damage to liver function. The best objective response rate was 100%, and the progression-free survival (PFS) at 12 months was of 88.89% (median PFS was not observed). CONCLUSIONS: These findings showed that BCMA CAR-T cell therapy could be used in patients with R/R MM with chronic or resolved HBV infection and that antiviral drugs should be administered in these patients during CAR-T cell therapy.

Chromosome t(7;11)(p15;p15) translocation in acute myeloid leukemia coexisting with multilineage dyspoiesis and mutations in NRAS and WT1: A case report and literature review.

The chromosomal translocation t(7;11)(p15;p15) and the resulting nucleoporin 98-homeobox A9 (NUP98-HOXA9) gene fusion is rare but recurrent genetic abnormity in acute myeloid leukemia (AML). The present study describes a case of AML plus maturation (-M2) with multilineage dyspoiesis in a 30-year-old male in whom a 46,XY,t(7;11)(p15;p15) karyotype was detected through chromosome analysis. Subsequent molecular and sequencing analysis demonstrated a NUP98-HOXA9 fusion gene with a type I fusion between NUP98 exon 12 and HOXA9 exon 1b, and mutations in neuroblastoma V-Ras oncogene homolog and Wilms tumor 1. The patient achieved hematological complete remission (CR) following two courses of induction chemotherapy. However, the NUP98-HOXA9 fusion gene remained detectable during the hematological CR period and following intensive consolidation chemotherapy. The disease relapsed 11 months after diagnosis, and the patient became refractory, with complications from an infection causing eventual mortality. The present case and literature review suggest that patients with AML and t(7;11) may have unique biological and clinical characteristics, and a poor prognosis.

Bortezomib combined with lenalidomide as the first-line treatment for the rare synchronous occurrence of multiple myeloma and pulmonary adenocarcinoma: A case report.

BACKGROUND: Simultaneous multiple myeloma (MM) and pulmonary adenocarcinoma is a rare occurrence, and thus, treatment is a challenge. This study reports on 1 such case of MM with concurrent lung cancer, where an accurate diagnosis was made and the patient underwent treatment for both cancers. CASE SUMMARY: A 68-year-old man presented with 2 months of progressive lower back pain. Visualization with magnetic resonance imaging (MRI) revealed multiple collapsed vertebrae from T12 to S3, as well as an altered signal intensity at the T3 vertebra. The patient was diagnosed with MM upon examination. A chest computed tomography (CT) scan revealed a round mass in the left lower lobe of the lungs, and a CT-guided needle biopsy uncovered a moderately differentiated adenocarcinoma. There were no additional notable findings in the left lung using positron emission tomography computed tomography (PET-CT). Therefore, a diagnosis of MM with pulmonary adenocarcinoma was made. Surgery was performed to excise the lung cancer. Bortezomib was used as first-line induction therapy against both tumors and lenalidomide was used for maintenance. The patient went into complete remission. Using this combined chemotherapy, the patient has survived for over 3 years since a diagnosis was made despite relapsing twice after the first year. CONCLUSION: This report clearly delineates the diagnosis and treatment of a rare case of synchronous MM and pulmonary adenocarcinoma, as well as depicts a potentially positive outcome for the patient. It also overviews some diagnostic and therapeutic implications for clinicians.

Omacetaxine mepesuccinate induces apoptosis and cell cycle arrest, promotes cell differentiation, and reduces telomerase activity in diffuse large B­cell lymphoma cells.

Clinical studies have demonstrated that omacetaxine mepesuccinate exerts beneficial effects on acute myelogenous leukemia. It has been suggested that omacetaxine mepesuccinate, used alone or with interferon­α or cytarabine, induces remission in patients with chronic myelogenous leukemia. These effects are possibly mediated by its ability to induce apoptosis of leukemia cells and inhibit the activity of telomerase. To determine whether omacetaxine mepesuccinate is beneficial in diffuse large B­cell lymphoma (DLBCL), two DLBCL cell lines [a germinal center B cell­like subtype (GCB) and an activated B cell­like subtype (ABC)] were treated with omacetaxine mepesuccinate at various concentrations for different durations. The present study indicated that omacetaxine mepesuccinate exerts proapoptotic effects in the two cell types in a dose­ and time­dependent manner. The ABC subtype demonstrated increased sensitivity compared with the GCB subtype. At 40 ng/ml, omacetaxine mepesuccinate exhibited a marked proapoptotic effect on DLBCL cells compared with the other tumor cells investigated. Furthermore, omacetaxine mepesuccinate induced cell cycle arrest at G0/G1 phase, and promoted cell terminal differentiation of pro­B cells. The present study also demonstrated that omacetaxine mepesuccinate exerted its antitumor effect by reducing telomerase activity. In conclusion, the present study demonstrated that omacetaxine mepesuccinate may induce apoptosis and cell cycle arrest, promote cell differentiation, and reduce telomerase activity in DLBCL cells, thus aiding the development of omacetaxine mepesuccinate­based DLBCL therapeutic strategies.

[Clinical features and survival analysis of patients with CD56 expression in de- novo acute myeloid leukemia with t（8;21）].

OBJECTIVE: To explore the clinical features and survival of patients with CD56 expression in de- novo acute myeloid leukemia（AML）with t（8;21). . METHODS: Clinical data of 82 de novo AML with t（8;21）who were newly diagnosed from Jan 2008 to Apr 2014 were analyzed retrospectively, 50 expressed CD56 and 32 not. Clinical characteristics and prognoses were compared between patients expressing and nonexpressing CD56. RESULTS: There were no statistically significant differences in terms of age, gender, white blood cell count（WBC）, percentage of bone marrow blasts, extramedullary infiltration rate, the early mortality or the presence of additional cytogenetic abnormalities between CD56 + and CD56- groups（P＞0.05). The expressions of lymphatic antigens CD19 between CD56 + and CD56- groups showed significant difference （30.0% vs 53.1% , P=0.036). The complete remission and 3-year overall survival（OS）showed no significant differences between CD56+ and CD56-groups, while 3- year disease- free survival（DFS）showed significant differences（25.8% vs 46.9%, P=0.014). Multivariable analysis for DFS identified CD56 positivity as an independent predictor. DFS of who received allogeneic hematopoietic stem cell transplantation（HSCT）was better than those treated with intermediate- dose cytarabine/high dose cytarabine（IDAC）as postremission therapy. CONCLUSION: The expression of CD56 in de-novo AML with t（8;21) appeared to be associated with poorer prognosis.

[Clinical features and survival analysis of patients with CD20 positive adult B-lineage acute lymphoblastic leukemia].

OBJECTIVE: To explore the clinical and survival significance of CD20 positive adult patients with B-lineage acute lymphoblastic leukemia (B-ALL). METHODS: The clinical features and survival of 168 adult patients with B-ALL diagnosed and treated in our department from May 2007 to July 2011 were analyzed retrospectively, 58 expressed CD20 and 110 not. RESULTS: The sex, distribution of age, anemia, thrombocytopenia, infiltration of liver, spleen and lymph nodes, the expression of myeloid lineage marker, incidence of Ph chromosome, complete remission within 4 weeks showed no significant differences in CD20 positive and negative groups (P>0.05); median white blood cell count at diagnosis and the rate of patients with high white blood cell count in CD20 positive group were 19.2×109/L and 37.9% respectively, which were significantly higher than those of 6.93 × 109/L and 20.9% in CD20 negative group (P<0.05); cumulative incidence of relapse between two groups showed significant difference (P<0.05); multivariable analysis for overall survival and progress-free survival identified CD20 positivity as independent predictor. CONCLUSION: The expression of CD20 in adult patients with B-ALL appeared to be associated with high white blood cell count and poor prognosis.

Expression of neuron-specific enolase in multiple myeloma and implications for clinical diagnosis and treatment.

OBJECTIVE: To determine the expression of neuron-specific enolase (NSE) in patients with multiple myeloma (MM) and to evaluate its clinical value as a tumor marker and, an indicator of disease progression and treatment efficacy. METHODS: Using electrochemiluminescence immunoassay (ECLIA), we measured the serum levels of NSE in 47 healthy subjects (control group), 25 patients with small cell lung cancer (lung cancer group), and 52 patients with MM (MM group). For the MM group, serum NSE levels were measured and other disease indicators and related symptoms were monitored before and after chemotherapy. The relationship between NSE expression and other MM-related factors was analyzed. In addition, immunohistochemical staining was performed on bone marrow biopsy specimens from patients with MM. RESULTS: In the control group, serum NSE levels were within the normal range as previously reported, while the lung cancer group and the untreated MM group exhibited NSE levels that were significantly higher relative to the control group (P<0.05). The difference in NSE expression between the lung cancer group and untreated MM group was statistically significant (P<0.05). NSE levels were significantly decreased in MM patients after chemotherapy and were positively correlated with an MM disease index [beta-2 microglobulin (ß2-MG)]. Changes in NSE were not related to the response rate to chemotherapy but rather were correlated with progression-free survival. CONCLUSIONS: Patients with MM may have increased serum NSE levels, and changes in NSE may provide insight into treatment efficacy of chemotherapy and disease progression. Perhaps NSE expression is a viable biomarker for MM and can be a useful reference for the design and adjustment of clinical MM treatment programs.

[Hyperthermia increases the sensitivity of RPMI 8226 cells to adriamycin].

This study was purposed to explore the effect of hyperthermia on sensitivity of multiple myeloma cells RPMI 8226 to adriamycin (ADM) and its mechanism. The working concentration of ADM against RPMI 8226 cells was defined by MTT assay. RPMI 8226 cells were divided into 4 groups: control group, hyperthermia (42°C) group, chemotherapy (ADM) group and combination group (42°C + ADM), the survival rate of RPMI 8226 cells in 4 groups was detected by trypan blue exclusion, the inhibitory effect of hyperthermia on proliferation of RPMI 8226 cells was detected by MTT assay, the cell cycle distribution, apoptosis rate of cells, intracellular ADM concentration and P-gp expression level were measured by flow cytometry. The 1/4 IC(50) of ADM was defined as the working concentration in the experiment. The results indicated that the hyperthermia promoted the entering the cells from in G(0)/G(1) phase into S and G(2)/M phases, the expression of P-gp protein on cells in hyperthermia and combination groups was down-regulated, the intracellular ADM concentration in combination group obviously increased. It is concluded that the hyperthermia combined with ADM obviously enhance the inhibitory effect on proliferation of RPMI 8226 cells. The hyperthermia increases the sensitivity of RPMI 8226 cells to chemotherapy through down-regulating the expression of P-gp protein on cells and increasing the intracellular ADM concentration.

[ABVD chemotherapy scheme at day 1 and 8 for treatment of primary Hodgkin's lymphomas].

OBJECTIVE: To explore the effect of adriamycin, bleomycin, vincristine and dacarbazinum (ABVD) chemotherapy scheme executed at day 1 and day 8 for primary Hodgkin's lymphomas (HL). METHODS: 62 patients with primary HL in stages II - IV treated in our department from October 2005 to October 2006 were divided into group A and B at random with 31 patients in each group. The patients in group A received ABVD chemotherapy scheme executed at day 1 and day 8 for 6 - 8 cycles. The patients in group B received ABVD chemotherapy scheme executed at day 1 and day 15 for 6 - 8 cycles. The patients of the groups received radiotherapy by the same doctor after chemotherapy according to the patients condition and the radiotherapy regimens were not affected by the grouping. RESULTS: The complete remission rate (CR) in group A after chemotherapy was 90.3% (28/31); the one-year and two-year disease free survival (DFS) rates were 87.1% (27/31) and 80.0% (20/25) respectively. The CR rate in group B after chemotherapy was 83.9% (26/31); the one-year and two-year DFS rates were 80.6% (25/31) and 72.0% (18/25) respectively. The discrepancy of CR rates and the one-year and two-year DFS rates between the two groups was not significant (P > 0.05). The incidences of therapeutic side effects such as myocardial ischemia grade III - IV liver function impairment, pulmonary fibrosis and serious marrow inhibition between the two groups were not significant too (P > 0.05). Average chemotherapy period for the patients in group A was 159 days; it was 69 days shorter than that in group B. CONCLUSION: The CR rate, 1-year DFS rate and 2-year DFS rate of ABVD chemotherapy scheme executed at day 1 and 8 are similar to those of ABVD chemotherapy scheme executed at day 1 and 15 for primary HL in stages II - IV. The side-effects of chemotherapy between group A and B are similar too. The chemotherapy period in group A is shortened significantly.

[The efficacy of high-dose cytarabine for patients with t(8;21) AML and with normal karyotype AML].

OBJECTIVE: To compare the efficacy of high-dose cytarabine (HD-Ara-C) based chemotherapy for post-remission treatment in patients with t(8;21) (q22;q22) AML-M2 and those with normal karyotype AML-M2. METHODS: AML-M2 patients were grouped into with (21 cases) or without (23 cases) t(8;21) (q22;q22) karyotype groups. After achieved remission by induction therapy, all patients received four cycles of HD-Ara-C (3 mg/m2 per 12 hours by three-hour infusion day 1 to day 3) with either mitoxantrone (7 mg m(-2) d(-1)) or aclarubicin (30 mg m(-2) d(-1)) or etoposide (70 mg m(-2) d(-1)) for 3d as post-remission treatment. RESULTS: Relapse rate in the t(8;21) and the normal karyotype groups was 29% and 57% respectively (P<0.05); 3 year disease-free survival (DFS) rate was 71% and 43% respectively (P < 0.05). and 3 year over-all survival (OS) rate was 76% and 65% respectively (P >0.05). CONCLUSION: Four cycles of high-dose cytarabine based combination chemotherapy as post-remission treatment improves long-term disease-free survival in patients with t(8;21) (q22;q22) AML-M2.

[Application of all-trans retinoic acid combining chemotherapy and As4S4 in the maintenance treatment of patients with acute promyelocytic leukemia].

OBJECTIVE: To compare the efficacy of all-trans retinoic acid (ATRA) combining chemotherapy and As4S4 with ATRA combining chemotherapy for the maintenance treatment of patients with acute promyelocytic leukemia (APL). METHODS: Sixty patients with APL induced to complete remission by ATRA and consolidated by chemotherapy were randomly divided into two groups. Thirty patients as As4S4 group received ATRA + As4S4 + chemotherapy, and another thirty patients as non-As4S4 group were treated only with ATRA + chemotherapy as maintenance therapy. The therapeutic effects, side effects and PML-RARalpha gene expression were analyzed. RESULTS: The three-year continuous complete remission (CCR) rate was 90.0% for As4S4 group and 61.1% for non-As4S4 group, the difference being statistically significant. Significant difference was also found in the positive rate of PML-RARalpha fusion gene between the two groups. The side effects were mild. CONCLUSION: APL patients in maintenance therapy with ATRA + 6-MP + MTX + As4S4 can obtain a higher CCR.

[Detection and screening for human cytomegalovirus infection in allogeneic hematopoietic stem cell transplantation recipients by different methods].

The aim of study was to explore the better detection method for cytomegalovirus (CMV) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients and to compare the efficiency of fluorogenic quantitative PCR (FQ-PCR), flow cytometry (FCM) and ELISA. The plasma DNA loading and serum level of IgM antibody against CMV in 214 clinical specimens from 19 allo-HSCT patients were detected by real-time FQ-PCR and ELISA respectively, the pp65 antigen in 118 peripheral blood leukocyte samples were measured by FCM. The results showed that the positive rates of pp65 antigen, IgM antibody and DNA load were 30.85% (58/188), 13.08% (28/214) and 35.51% (76/214) respectively, the coincidence between their sequential detection positive rates and clinical diagnosis were 7/8, 7/8 and 3/8 respectively. There was no statistical significant difference between the positive rate of pp65 antigen and of DNA amount (P > 0.05), and they have manifested relationships (P < 0.05). The positive rate of IgM antibody detected by ELISA was obvious lower than that of DNA quantitated by FQ-PCR and pp65 antigen detected by FCM, but the difference between them showed statistical significance (P < 0.05), Smaller relativity was found between IgM antibody detection and the other two methods (P > 0.05). It is concluded that FQ-PCR and FCM are sensitive, rapid, suitable and reliable methods for monitoring recipient reactive CMV infection of allo-HSCT recipients and are worthy to extensively use for guiding antiviral therapy.

Minimally differentiated acute myeloid leukemia with t(12;22)(p13;q11) translocation showing primary multidrug resistance and expressing multiple multidrug-resistant proteins.

Here we report a rare chromosomal translocation, t(12;22)(p13;q11), which was detected in a 53-year-old female patient diagnosed as having minimally differentiated acute myeloid leukemia (AML-M0) according to the French-American-British classification criteria. Chromosome painting analysis with probes for chromosomes 12 and 22 confirmed the result of the conventional cytogenetic analysis. Reverse transcriptase polymerase chain reaction revealed the TEL-MN1 fusion transcript. Interestingly, she presented primary multidrug resistance and did not respond to several kinds of chemotherapy regimens. Moreover, she could not achieve remission after two doses of monotherapy with Mylotarg. Flow cytometry analysis detected high levels of expression of P-glycoprotein, multidrug-resistant-related protein, lung-related protein, and glutathione S-transferase pi in this case at presentation. As far as we know, this is the first report of t(12;22)(p13;q11) translocation involving TEL and MN1 genes in an AML-M0 patient.

[MEKK2 regulates the production of interleukin 2].

OBJECTIVE: To study the role of MEKK2 on the production of IL-2 in Jurkat cells stimulated by PHA/anti-CD28 antibody. METHODS: The MEKK2 and JNK kinase activities were measured in both dominant negative MEKK2 Jurkat (dnMEKK2 Jurkat) cells and parental Jurkat cells. The AP(1) and IL-2 promotor activities were measured by luciferase activity assay. The IL-2 mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After stimulation by PHA/anti-CD28, JNK was activated in parental Jurkat cells but not in dnMEKK2 Jurkat cells. The luciferase report gene activities of AP1 and IL-2 promotors were increased by 4- and 5-folds in parental cells whereas only by 1 fold in dnMEKK2 Jurkat cells. The level of IL-2 mRNA and IL-2 protein were increased in parental Jurkat cells but not in dnMEKK2 Jurkat cells. CONCLUSION: MEKK2 plays an important role on the production of IL-2 in Jurkat cell stimulated with PHA/anti-CD28 antibody. It is a potential drug target for the treatment of GVHD and autoimmune disease.

[Multidrug resistance mechanisms in cell line HL-60/VCR].

BACKGROUND & OBJECTIVE: Drug resistance is a major factor in chemotherapeutic failure of leukemia. Multidrug resistant cell lines are the good models for investigating the mechanisms and reversal of acquired drug resistance. This study was designed to explore the multidrug resistance (MDR) mechanisms in cell line HL-60/VCR. METHODS: Flow cytometry and a panel of antibodies were used to analyze the expression of MDR proteins (P-gp, MRP, LRP, BCRP, GST-pi) and apoptosis-modulating proteins (bcl-2, bcl-x, bax, bad) in MDR cell line HL-60/VCR and drug sensitive cell line HL-60. RESULTS: The expression levels of MDR proteins (P-gp, MRP, BCRP, GST-pi) were (18.62, 1.19, 1.50, 1.32-flod) higher in HL-60/VCR than in HL-60, while the expression of LRP level was similar. The levels of apoptosis-modulating proteins(bcl-2, bcl-x, bad) were (2.48, 1.25, 1.08-fold) higher in HL-60/VCR than in HL-60, while the pro-apoptosis protein bax contrarily decreased in HL-60/VCR. CONCLUSION: Various MDR mechanisms were involved in multi-drug resistance HL-60/VCR cell line, which including increasing expression of drug-resistance protein (P-gp, MRP, BCRP, and GST-pi); the apoptosis-modulating proteins (bcl-2, bcl-x, bax, and bad) might take part in the mechanism of drug resistance.