A nomogram prediction of citrate reaction during mononuclear cell collection in solid tumor patients.

PURPOSE: Citrate reaction is one of the main adverse events in peripheral blood mononuclear cell (MNC) collection. The aim of this study was to elucidate the risk factors for citrate reaction in patients with advanced solid tumor collection and to construct a nomogram to predict the risk. METHODS: One hundred forty-eight patients with advanced solid tumor who underwent peripheral blood MNC collection in our hospital between January 2021 to December 2021 were selected. The general data, creatinine level before collection, Ca2+ concentration before collection, absolute value of monocyte lymphocytes before collection, circulating blood volume, anticoagulant dosage, and blood collection duration were included in Logistic regression analysis to identify the risk factors of citrate reaction. According to the results of the multivariate logistic model, nomogram was established and receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value of the model. RESULTS: Among the 148 solid tumor patients, 35 patients (23.6%) of the 148 patients developed citrate reaction. Multivariate analysis showed that the risk factors for citrate reaction in the process of collection included sex (odds ratio [OR] = 6.718; 95% confidence interval [95% CI]: 2.191-20.594, P = .001), age (OR = 0.957; 95% CI: 0.921-0.996, P = .03), and processed circulating blood volume (OR = 1.001; 95% CI: 1.000-1.002, P = .01). Logistic regression can analyze independent risk factors and establish risk prediction model. The predictive performance of the model is good, and the area under ROC curve is 0.799. CONCLUSIONS: The MNC collection process is safe. The incidence of citrate reaction in the collection of peripheral blood MNCs from patients with advanced solid tumor is related to the age, gender, and processed circulating blood volume of patients. The nomogram can be used to assess a patient's risk of citrate reaction.

Phase I clinical trial of EGFR-specific CAR-T cells generated by the piggyBac transposon system in advanced relapsed/refractory non-small cell lung cancer patients.

PURPOSE: This phase I clinical trial is designed to assess the safety and feasibility of the epidermal growth factor receptor (EGFR) chimeric antigen receptor (CAR) T-cell generated by the piggyBac transposon system in advanced relapsed/refractory non-small cell lung cancer (NSCLC) patients. Compared to viral systems, the piggyBac transposon system is a simpler, more economical, and alternative way to introduce chimeric antigen receptor (CAR) transgenes into T cells. METHODS: This study recruited nine patients with advanced relapsed/refractory EGFR-positive NSCLC for two cycles of the piggyBac-generated EGFR-CAR T cells at dose of 1 × 106 cells/kg or 3 × 106 cells/kg of body weight. The patients were monitored for adverse events, clinical response, and persistence of plasma GFR-CAR T cells. RESULTS: Infusions of piggyBac-generated EGFR-CAR T cells were well tolerated in all nine patients. The most common adverse events were grade 1 to 3 fever and there were no patients who experienced grade 4 adverse events or serious cytokine release syndrome. After treatment, eight of nine patients showed detectable EGFR-CAR T cells in their peripheral blood. One patient showed a partial response and lasted for more than 13 months, while six had stable disease, and two had progressed disease. The progression-free survival of these nine patients was 7.13 months (95% CI 2.71-17.10 months), while the median overall survival was 15.63 months (95% CI 8.82-22.03 months). CONCLUSION: This Phase I clinical trial revealed that the non-viral piggyBac transposon system-engineered EGFR-CAR T-cell therapy is feasible and safe in treatment of EGFR-positive advanced relapsed/refractory NSCLC patients. Future study will assess it in more patients or even possibly with a higher dose. Trial registration number NCT03182816.

Changes and Clinical Significance of Detailed Peripheral Lymphocyte Subsets in Evaluating the Immunity for Cancer Patients.

OBJECTIVE: The evaluation of lymphocyte subsets is widely regarded as an important factor for monitoring tumor progression and response to therapy. This study was designed to establish a comprehensive and detailed assessment of peripheral lymphocyte subsets with a multi-parametric flow cytometry assay for response prediction and prognosis evaluation of cancer patients. METHODS: Peripheral blood samples collected from 40 cancer patients and 23 age- and sex-matched healthy volunteers were tested for 29 lymphocyte subsets by flow cytometry. The univariate analysis was applied to establish the reference interval of healthy samples, and the ratio and proportion of 29 lymphocyte subsets between patient samples and healthy controls were compared to evaluate their clinical significance by Mann-Whitney U-test model. RESULTS: The reference ranges of 29 lymphocyte subsets were established with a normal distribution and no significant differences were observed between genders. Compared with healthy control group, lower proportion and ratio of specific parameters, such as Naïve Th cells (p<0.01), Naïve Tc cells (p<0.01), CM (central memory) Tc cells (p<0.01), Naïve T cells/Memory T cells (p<0.001), Naïve T cells/EM (effector memory) T cells (p<0.001) and Naive Th cells/Memory Th cells (p< 0.001), and higher proportion and ratio of EM Th cells (p<0.001), EM Tc cells (p<0.01), effector Tc cells (p<0.05), EM Th cells/CM Th cells (p<0.01) and EM Tc cells/CM Tc cells (p<0.01), as well as Breg (p<0.001), B cells (p<0.05) and CD16-NK cells (p<0.001) were found in cancer cohorts. CONCLUSION: This study suggests that the changes in certain lymphocyte subsets might be helpful to evaluate the immunity of cancer patients, and holds great potential for clinical application.

Overexpression of miR­21 is involved in acute monocytic leukemia­associated angiogenesis by targeting IL­12.

Angiogenesis is important in pathophysiological processes, including the pathogenesis of acute monocytic leukemia (AML). MicroRNA­21 (miR­21) is overexpressed and exhibits oncogenic activity in cancer. However, the biological mechanism underlying the effect of miR­21 in AML remains to be fully elucidated. In the present study, the expression levels of miR­21 and vascular endothelial growth factor (VEGF) were determined in 26 patients with AML and 28 healthy individuals. The secretion of VEGF was also measured following the transfection of THP­1 cells with miR­21 mimic or inhibitor. The supernatants of the THP­1 cells, which were transfected with miR­21 mimic, inhibitor or small interfering RNA (si)VEGF, respectively, were used to incubate human umbilical vein endothelial cells (HUVECs), following which tube formation of the HUVECs was measured. miR­21 targets were predicted using a biological target prediction website and confirmed using a luciferase assay. The effects of interleukin (IL)­12 were investigated by examining the tube formation of HUVECs and the secretion of VEGF following recombinant human (rh) IL­12 pretreatment. The results revealed that miR­21 and VEGF expression was significantly increased in the peripheral blood monocytes of the patients, compared with the healthy controls. There was negative correlation between the expression of IL­12 and miR­21 in the serum of patients with AML. Furthermore, supernatant VEGF levels from the miR­21 mimic­transfected THP­1 cells were increased, whereas a decreasing trend was observed in the miR­21 inhibitor group. The angiogenic ability of the HUVECs pretreated with supernatant from the THP­1 cells transfected with miR­21 mimic was higher, and was lower in THP­1 cells co­transfected with miR­21 mimic and siVEGF, compared with the miR­21 mimic only group. A luciferase assay demonstrated that IL­12 was the direct target of miR­21, and the level of IL­12 in the supernatant of THP­1 cells transfected with miR­21 mimic was increased. IL­12 pretreatment increased VEGF expression and angiogenic ability in HUVECs. The inactivation of miR­21 or activation of its target gene may be a potential therapeutic strategy in human AML.

Association of VEGF and VDR gene- gene and gene- smoking interaction on risk of multiple myeloma in Chinese Han population.

AIMS: To investigate the association of several single nucleotide polymorphisms (SNPs) within vascular endothelial growth factor (VEGF) and vitamin D receptor (VDR) gene polymorphisms and additional gene- gene and gene- smoking interaction with multiple myeloma (MM) risk in Chinese population. METHODS: Generalized multifactor dimensionality reduction (GMDR) was used to screen the best interaction combination among SNPs and smoking. Logistic regression was performed to investigate association between 6 SNPs within VEGF and VDR gene, additional gene- gene and gene- smoking interaction on MM risk. RESULTS: MM risk is significantly higher in carriers with the rs699947- A allele within VEGF gene than those with CC genotype (CA+ AA versus CC), adjusted OR (95%CI) =1.72 (1.19-2.33), and higher in carriers with rs2228570- T allele within VDR gene than those with CC genotype (CT+ TT versus CC), adjusted OR (95%CI) = 1.68 (1.26-2.17). We also found a significant two-locus model (p=0.0010) involving rs699947 and rs2228570, and a significant two-locus model (p=0.0107) involving rs2228570 andsmoking. Participants with rs699947- CA+AA and rs2228570- CT+TT genotype had the highest MM risk, compared to participants with rs699947- CC and rs2228570- CC genotype, OR (95%CI) = 3.12 (1.82 -4.61). Smokers with rs2228570- CT+TT genotype had the highest MM risk, compared to never- smokers with rs2228570- CC genotype, OR (95%CI) = 3.27 (1.74-4.86). CONCLUSIONS: We found that the A allele of rs699947 within VEGF and T allele of rs2228570 within VDR gene, interaction between rs699947 and rs2228570, rs2228570 andsmoking were all associated with increased MM risk.

Adenovirus co-expressing CD40 ligand and interleukin (IL)-2 contributes to maturation of dendritic cells and production of IL-12.

The aim of the present study was to construct a chimeric adenovirus (Ad)5/F35 co-expressing human CD4O ligand (CD4OL) and interleukin (IL)-2 (Ad5/F35 CD40L-IL-2). The infection efficiency to human monocyte-derived dendritic cells (Mo-DCs), expression of genes, phenotype changes and IL-12 production of Mo-DC by Ad5/F35 CD40L-IL-2 were investigated. CD40L and IL-2 from total RNA extracted from human peripheral blood mononuclear cells (PBMCs) were cloned by reverse transcription-polymerase chain reaction and used to construct Ad5/F35 CD40L-IL-2. The infection efficiency, expression of CD40L, and phenotype changes of Mo-DC infected with Ad5/F35 CD40L-IL-2 were analyzed using flow cytometry. The quantities of IL-2 and IL-12 in the supernatants of Mo-DC following infection of Ad5/F35 CD40L-IL-2 were measured by enzyme-linked immunosorbent assay. The CD40L and IL-2 genes were successfully cloned and the Ad5/F35 CD40L-IL-2 was constructed. Ad5/F35 CD40L-IL-2 efficiently infected Mo-DCs with an infection efficiency of >75%, and the infected Mo-DCs expressed CD40L and secreted IL-2. The expression levels of cluster of differentiation (CD)80, CD86, CD40, and human leukocyte antigen-antigen D related on Mo-DC were moderate; however, CD83 was low prior to infection of Ad5/F35 CD40L-IL-2. Those molecules, particularly CD83, were markedly upregulated 24 h after the infection. Increasing quantities of IL-12 in the supernatants were detected subsequent to infection at different time points in a time-dependent manner. Thus, Ad5/F35 CD40L-IL-2 efficiently infected human Mo-DCs and its products, CD40L and IL-2, were subsequently expressed. In addition, infection with Ad5/F35 CD40L-IL-2 stimulated the maturation of Mo-DC and high levels of IL-12 production.

Association of MDM2 polymorphism with risk and prognosis of leukemia: a meta-analysis.

OBJECTIVE: In this study, we performed an updated meta-analysis by summarizing all available relevant association studies to evaluate whether the murine double minute-2 (MDM2) T309G polymorphism is associated with risk of leukemia and to determine its prognostic effect. MATERIAL AND METHODS: Studies published in PubMed, Embase and the Cochrane Controlled Trial Register were searched till June 2014 using the search terms 'MDM2', 'polymorphism' and 'leukemia'. RESULTS: Eleven studies were included in this meta-analysis, with a total of 2,478 patients accrued. There were 8 studies providing data on single nucleotide polymorphism at position 309 (SNP309) and risk of leukemia and 7 studies providing data on SNP309 and overall survival. Our analysis showed that patients having G/G mutations had a significantly higher risk of developing leukemia (HR 1.90, 95% CI 1.56-2.31, p < 0.00001), while the association between G/T and leukemia was not significant (HR 1.18, 95% CI 0.96-1.45, p = 0.11). In addition, SNP309 was not significantly associated with patient survival (HR 1.29, 95% CI 0.79-2.13, p = 0.31). CONCLUSIONS: Our meta-analysis showed that the MDM2 T309G variation, especially homozygous G/G, might be associated with an increased risk of leukemia. Additional studies are needed to confirm the findings as well as to understand the underlying mechanisms.

Arsenic trioxide-based therapy in relapsed/refractory multiple myeloma patients: a meta-analysis and systematic review.

Multiple myeloma (MM) is a clonal malignancy characterized by the proliferation of malignant plasma cells in the bone marrow and the production of monoclonal immunoglobulin. Although some newly approved drugs (thalidomide, lenalidomide, and bortezomib) demonstrate significant benefit for MM patients with improved survival, all MM patients still relapse. Arsenic trioxide (ATO) is the most active single agent in acute promyelocytic leukemia, the antitumor activity of which is partly dependent on the production of reactive oxygen species. Due to its multifaceted effects observed on MM cell lines and primary myeloma cells, Phase I/II trials have been conducted in heavily pretreated patients with relapsed or refractory MM. Therapy regimens varied dramatically as to the dosage of ATO and monotherapy versus combination therapy with other agents available for the treatment of MM. Although ATO-based combination treatment was well tolerated by most patients, most trials found that ATO has limited effects on MM patients. However, since small numbers of patients were randomized to different treatment arms, trials have not been statistically powered to determine the differences in progression-free survival and overall survival among the experimental arms. Therefore, large Phase III studies of ATO-based randomized controlled trials will be needed to establish whether ATO has any potential beneficial effects in the clinical setting.

Comparative study of the efficacy of allogeneic hematopoietic stem cell transplantation from human leukocyte antigen-haploidentical related and unrelated donors in the treatment of leukemia.

AIMS: To compare the efficacy of hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA)-haploidentical related donors (RD) and unrelated donors (URD) in the treatment of leukemia. METHODS: Ninety-three leukemia patients underwent allogeneic HSCT were divided into two groups: 51 cases of RD-HSCT and 42 cases of URD-HSCT. In the RD-HSCT group, a preconditioning regimen with fludarabine (Flu) + busulfan (Bu) + cytosine arabinoside (Ara-C) was used in 42 patients and total body irradiation (TBI) + Flu + Ara-C was used in the remaining 9. RESULTS: In the URD-HSCT group, a modified preconditioning regimen with Bu + cyclophosphamide was used in 35 patients, while the other 7 patients underwent treatment with TBI + Flu. After transplantation, the occurrence rate of grade II-IV acute graft-versus-host disease (GVHD) was 46.0 and 51.2% in the two groups. Likewise, the rate of chronic GVHD was 46.0 and 63.4%, respectively. No significant differences in the occurrence of acute and chronic GVHD were detected between the two groups. The differences in early-stage infection rate after transplantation, recurrence rate, 3-year survival rate, and disease-free survival rate between the two groups were not significant. CONCLUSION: HLA-haploidentical RD-HSCT with enhanced preconditioning and administration of immunosuppressants showed a clinical efficacy similar to that of URD-HSCT against leukemia, without the risk of increased infection and GVHD.

[Clinical analysis of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation].

The objective of this study was to explore the incidence and therapeutic efficacy of cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical data of 140 patients undergoing allo-HSCT in our department of hematology from 2010-01 to 2012-01 were retrospectively analyzed. The results showed that the incidence of CMV infection was 4.3% (48/140), the time for the first detection of positive CMV-DNA was at day 45 (33 to 68) after allo-HSCT, and the CMV quantitative range was 1.25×10(3) - 5.5×10(6). There were 2 cases of CMV-related interstitial pneumonia and 5 cases of hemorrhagic bladder inflammation. A total of 65 patients suffered from graft versus host disease (GVHD), in which 32 cases (49.2%) were accompanied with CMV infection, CMV-DNA negative in patients treated with ganciclovir, foscarnet sodium anti-CMV was at day 45 (33 to 68) with the effective rate of 100%. 12 patients with CMV infection were accompanied with transient neutropenia and thrombocytopenia. It is concluded that after allo-HSCT the CMV infection occurs frequently. The patients with GVHD have a higher incidence of CMV infection. Ganciclovir and foscarnet sodium are reliable to be used for treatment of CMV infection with fewer adverse reactions.

[The effects of donor peripheral blood stem cell infusion in the prevention of relapse in high risk leukemia after haplotype hematopoietic stem cell transplantation].

OBJECTIVE: To explore the preventative effect of donor peripheral blood stem cell (PBSC) infusion mobilized by granulocyte colony-stimulating factor (G-CSF) for the relapsing patients with leukemia after haplotype hematopoietic stem cell transplantation (HSCT), as well as its therapeutic effect and safety. METHODS: G-CSF was given at 5 - 10 µg×kg(-1)×d(-1) to donor and PBSCs were obtained on day 5 and frozen and allotted for storing. PBSC infusion was given to all the 20 patients on day 90 after HSCT, and the second treatment was given to 4 patients on day 30 after the first infusion. The occurrence of graft-versus-host disease (GVHD), relapse rate of high risk leukemia and long-term survival were evaluate after PBSC infusion. RESULTS: A total of 19 patients had acute GVHD after PBSC infusion for a median of 25 (12 - 60) months, 4 of them were ≥ degree III. The cumulative incidence rate was 22.9%, and all of them accepted PBSC infusion twice. Thirteen patients had assessable chronic GVHD, 10 of them were restricted, and no one died of it. Nine patients died of relapse of leukemia. The remaining 11 patients survived leukemia free, including 4 with chronic myelogenous leukemia, 4 with acute myeloid leukemia (AML), 1 with lymphoma-leukemia and 2 with myelodysplastic syndrome-AML (MDS-AML). Kaplan-Meier analysis showed the disease free survival rate of 2-year was 52.5%. CONCLUSION: The prophylactic donor PBSC infusion mobilizing with G-CSF is effective, safe and feasible for the relapse of leukemia.

[Treatment of severe intestinal acute graft-versus-host disease with CD25 monoclonal antibody in haploidentical hematopoietic stem cell transplantation].

OBJECTIVE: To study the feasibility of CD25 monoclonal antibody (basiliximab) in the treatment of severe III-IV intestinal acute graft-versus-host disease (GVHD) following haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Twenty patients, 13 males and 7 females, who developed III-IV intestinal acute GVHD after haplotypic HSCT between October 2004 and September 2009, were treated with basiliximab (20 mg/d, d1, 4) and prednisolone (1 mg×kg(-1)×d(-1)) from the day of diagnosis. The therapy was repeated in the second week if the intestinal symptoms showed no improvement. The therapeutic effect was analyzed and the adverse reaction and cytomegalovirus (CMV) infection were observed. RESULTS: Ten patients had a complete remission and 5 were in a partial remission. The total effective rate was 75.0%. The clinical symptoms started to lessen in 1-12 days after using basiliximab (average: 7 days). During the 6-64 month follow-up (average: 25 months), 8/10 cases with a complete remission had no acute GVHD relapse, and the other 2 relapsing patients experienced a remission after a re-administration of basiliximab. Nine patients survived with a longest period of 64 months. Four withdrew corticosteroids and the other 5 stayed on a low-dose maintenance corticosteroid regimen. The 2-year disease-free survival was 47.5% by Kaplan-Meier calculation. CONCLUSIONS: Basiliximab is feasible in the treatment of III-IV intestinal acute GVHD after haplotype HSCT. It does not increase the relapse of leukemia or the incidence of infections.

[A preliminary report on treatment of myelodysplastic syndrome with cyclosporin a and androgen].

The study was aimed to explore clinical result of cyclosporin A (CsA) and androgens for treatment of myelodysplastic syndrome (MDS) with refractory anemia. Four cases of MDS-RA were treated with CsA and androgens, while the changes of blood counts, bone marrow and chromosome were observed. The results showed that substantial hematological response was observed in all four patients, that their anemia improved and all transfusion-dependent patients achieved transfusion independence. In conclusion, CsA and Adr therapy was well tolerated. CsA and Adr therapy offer an alternative treatment of MDS with RA. The mechanisms of the benifical effect from this therapy remain the subject of an ongoing study.

[Cloning and Expression of Various Deletants of Gsalpha Gene in Escherichia Coli]

Gsalpha gene mutation has been discovered in some human tumors. In our previous studies, three novel deletants of Gsalpha gene, Gsalpha L-1(500 bp), Gsalpha L-2(300 bp), and Gsalpha L-3(200 bp), and wild type Gsalpha-4(1 200 bp) were found in human leukemia cell lines and detected in leukemic cells from patients with acute leukemia. To investigate the construction, function and biological significance of the deletants, the plasmids of Gsalpha L-1, Gsalpha L-2 and wild Gsalpha-4 were transformed into E. coli DH5, amplified by PCR, and cloned in expression vector pET22b(+), and then transformed into E. coli, respectively. As a result, higher levels of expression of three recombinants were obtained in form of inclusion bodies. The results suggested that these Gsalpha isoforms have an open reading frame of gene and can be expressed in vitro. The data lay a foundation to study the relation of Gsalpha gene to leukemogenesis.