Case Report: CD19 and CD20 monoclonal antibodies with sequential chemotherapy for refractory acute B-lymphocytic leukemia in children.

Objective: This paper observes the efficacy of chemotherapy combined with CD19 and CD20 monoclonal antibodies in clearing minimal residual disease (MRD) and bridging transplantation for refractory acute B-lymphoblastic leukemia (B-ALL) in children and reviews the literature. Methods: A 4-year-old boy diagnosed with B-ALL in our hospital was treated with the SCCLG-ALL-2016 protocol. MRD and gene quantification decreased after induction but remained persistently positive, with poor efficacy. After this patient received three cycles of consolidation chemotherapy combined with blinatumomab and rituximab, MRD and fusion gene quantification became negative, and he received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Results: During the use of monoclonal antibodies, neurotoxicity, CRS, or other side effects did not occur. Before transplantation, MRD became negative, and the bone marrow had been in complete remission since transplantation (13 months). Conclusion: Chemotherapy combined with blinatumomab for refractory B-ALL in children can bring a better remission rate for patients and is a means of bridging transplantation. Nevertheless, sequential CD20 monoclonal antibody therapy is the first report , and no adverse effects were observed in our case. It is well tolerated and can be used as one of the treatments for refractory B-ALL.

[Clinical Features and Prognosis of Multiple Myeloma Patients with Secondary Primary Malignancies].

OBJECTIVE: To explore the clinical characteristics and prognosis of multiple myeloma(MM) patients with secondary primary malignancies. METHODS: The clinical data of newly diagnosed MM patients admitted to the First Affiliated Hospital of Zhengzhou University from January 2011 to December 2019 were retrospectively analyzed. The patients with secondary primary malignancies were retrieved, and their clinical features and prognosis were evaluated. RESULTS: A total of 1 935 patients with newly diagnosed MM were admitted in this period, with a median age of 62 (18-94) years old, of which 1 049 cases were hospitalized twice or more. There were eleven cases with secondary primary malignancies (the incidence rate was 1.05%), including three cases of hematological malignancies (2 cases of acute myelomonocytic leukemia and 1 case of acute promyelocytic leukemia) and eight cases of solid tumors (2 cases of lung adenocarcinoma, and 1 case each of endometrial cancer, esophageal squamous cell carcinoma, primary liver cancer, bladder cancer, cervical squamous cell carcinoma, and meningioma). The median age of onset was 57 years old. The median time between diagnosis of secondary primary malignancies and diagnosis of MM was 39.4 months. There were seven cases with primary or secondary plasma cell leukemia, the incidence rate was 0.67%, and the median age of onset was 52 years old. Compared with the randomized control group, the ß2-microglobulin level in the secondary primary malignancies group was lower (P=0.028), and more patients were in stage I/II of ISS (P=0.029). Among the 11 patients with secondary primary malignancies, one survived, ten died, and the median survival time was 40 months. The median survival time of MM patients after the secondary primary malignancies was only seven months. All seven patients with primary or secondary plasma cell leukemia died, with a median survival time of 14 months. The median overall survival time of MM patients with secondary primary malignancies was longer than that of the patients with plasma cell leukemia (P=0.027). CONCLUSION: The incidence rate of MM with secondary primary malignancies is 1.05%. MM patients with secondary primary malignancies have poor prognosis and short median survival time, but the median survival time is longer than that of patients with plasma cell leukemia.

[Gene Mutation and Overexpression of Newly Diagnosed Multiple Myeloma Patients].

OBJECTIVE: To analyze the characteristics of gene mutation and overexpression in newly diagnosed multiple myeloma (NDMM) patients. METHODS: Bone marrow cells from 208 NDMM patients were collected and analyzed. The gene mutation of 28 genes and overexpression of 6 genes was detected by DNA sequencing. Chromosome structure abnormalities were detected by fluorescence in situ hybridization (FISH). RESULTS: Gene mutations were detected in 61 (29.33%) NDMM patients. Some mutations occurred in 5 or more cases, such as NRAS, PRDM1, FAM46C, MYC, CCND1, LTB, DIS3, KRAS, and CRBN. Overexpression of six genes (CCND1, CCND3, BCL-2, CCND2, FGFR3, and MYC) were detected in 83 (39.9%) patients, and cell cycle regulation gene was the most common. Single nucleotide polymorphisms (SNP) changes were detected in 169 (81.25%) patients, the TP53 P72R gene SNP (70.17%) was the most common. Abnormality in chromosome structure was correlated to gene overexpression. Compared to the patients with normal chromosome structure, patients with 14q32 deletion showed higher proportion of CCND1 overexpression. Similarly, patients with 13q14 deletion showed higher proportion of FGFR3 overexpression, whereas patients with 1q21 amplification showed higher proportion of CCND2, BCL-2 and FGFR3 overexpression. CONCLUSION: There are multiple gene mutations and overexpression in NDMM. However, there is no dominated single mutation or overexpression of genes. The most common gene mutations are those in the RAS/MAPK pathway and the genes of cyclin family CCND are overexpression.

[Clinical Characteristics and Therapeutic Efficacy of Multiple Myeloma Combined with Renal Amyloidosis].

OBJECTIVE: To evaluate the clinical characteristics of multiple myeloma (MM) combined with renal amyloidosis and its curative efficacy and prognosis. METHODS: The clinical data of 22 cases of newly diagnosed multiple myeloma combined with renal amyloidosis treated in our hospital from November 2011 to July 2015 were analyzed retrospectively. RESULTS: According to Intenational Staging System (ISS), among above-menthioned 22 patients the ISS II accounted for 77.2% (17/22), ISS III accounted for 22.8% (5/22). The patients with renal impairment accounted for 36.4% (8/22), with anemia 40.9% (9/22), with serum album < 35 g/L 86.4% (19/22), with urinary protein positive 100% (22/22). The evaluation of the curative efficacy of the 22 cases was as follows: CR 13.6% (3/22); VGPR 4.5% (1/22); PR 22.8% (5/22); SD 45.5% (10/22); PD 13.6% (3/22). Out of 9 patients with effective treatment, 3 cases (3/9, 33.3%) achieved "improved" in renal amyloidosis, 4 cases (4/9, 44.5%) achieved stable in renal amyloidosis, 2 cases (2/9, 2%) achieved "worsened" in renal amyloidosis. Among 17 cases who were followed up, 7 cases died, 10 cases survived, the average duration of follow-up for these cases was 11 (1-37) months, the median overall survival (OS) time was 19 (95% CI 9.2-28.8) months. CONCLUSION: MM with renal amyloidosis is rare, refractory and has a poor prognosis. Whether there is impairment of kidney function or not, renal amyloidosis shall be taken into consideration if the MM patients got massive proteinuria especially nephritic syndrome. Bortezomib may improve the curative efficacy.

Poly(ADP-ribosyl)ation of Apoptosis Antagonizing Transcription Factor Involved in Hydroquinone-Induced DNA Damage Response.

The molecular mechanism of DNA damage induced by hydroquinone (HQ) remains unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor (AATF), PARP-1, and phosphorylated H2AX (γ-H2AX) were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-1-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, γ-H2AX expression increased to 265%. Co-immunoprecipitation (co-IP) assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly(ADP-ribosyl)ation (PARylation) regulated AATF expression. In conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation.

[Aplastic anemia with macrocytic anemia: a study based on long-term follow-up].

OBJECTIVE: To elucidate the clinical features, response rate, prognosis and clonal evolution of aplastic anemia (AA) with macrocytic anemia (mAA). METHODS: The clinical features at initial diagnosis and data in follow up of mAA hospitalized from January 2000 to October 2011 were analyzed retrospectively. RESULTS: (1) Of 153/568 (26.9%) cases of mAA at initial diagnosis, 114(74.5%)were non-severe AA (NSAA), 39(25.5%)severe AA (SAA) and 0 very severe AA (VSAA), while the proportion was 16.2%, 45.2%, and 38.6% in 376 normocytic anemia AA (nAA), and the difference is statistically significant(χ(2) = 181.390; P = 0.000). The median age of mAA was significantly higher than that of nAA \[30(4 - 70)years vs 19 (3 - 68) years, P = 0.001\]. (2) There were no statistical difference in hemoglobin, absolute neutrophil count (ANC), platelet count (PLT), response rate after 6 months treatment and overall survival (OS) between mAA and nAA grouped in SAA and NSAA respectively. In SAA, the reticulocyte count (Ret) of mAA was significantly higher than that of nAA \[23.90(2.99 - 61.00)×10(9)/L vs 13.1(0 - 70.60)×10(9)/L, P = 0.000\] and the proportion of erythroid cells in bone marrow of mAA was also higher \[23.5 (0 - 58) vs 14.5 (0 - 65), P = 0.043\], while they did not differ significantly in NSAA. (3) The proportion of AA with PNH clones or abnormal cytogenetics did not differ significantly in mAA and nAA groups before treatment. The incidences of AA evolved to PNH in mAA and nAA was not statistically significant (7/153 vs 9/376, χ(2) = 1.099, P = 0.294) and so was the incidence of evolution to MDS/AML(3/153 vs 13/376, χ(2) = 0.399, P = 0.528). CONCLUSION: In presented with macrocytic anemia at initial diagnosis of AA, higher proportion of NSAA, elderly age, higher Ret and proportion of erythroid cells are features, but being no statistical difference in the response rate, OS, and proportion of clonal evolution.

[Immunophenotyping characteristics of adult patients with acute lymphoblastic leukemia in different ages].

The purpose of this study was to investigate the immunophenotyping characteristics of adult acute lymphoblastic leukemia (ALL) patients in groups of different ages. Immunophenotyping was performed in 260 ALL patients by flow cytometry using a panel of monoclonal antibodies and CD45/SSC gating. The results indicated that (1) all the 82 cases of T-cell acute lymphoblastic leukemia (T-ALL) expressed CD7 (100%) while the positive rate of CD2 remarkably decreased with aging. The positive rate of CD2 in patients aged 14 to 18 years (adolescents) was 91.67%, which is significantly higher than that in cases aged 19 to 35 years (young adults) and > 35 years (older adults) (65.71% and 43.48% respectively, p < 0.05); the positive rate of CD34 and HLA-DR increased with aging, there was significant difference of the HLA-DR expression between the older adults group (39.13%) and the other two groups (4.17% in adolescents and 11.43% in young adults respectively (p < 0.05). Moreover, there were significant differences of the myeloid antigen (MyAg) and CD13 expression between the older adults and younger adults (p < 0.05). (2) As to adult B-cell acute lymphoblastic leukemia (B-ALL), the positive rates of CD19 and HLA-DR in 178 cases were 100%; the positive rate of CD33 in young adults was significant higher than that in adolescents (p < 0.05), the differences of the other marker expressions failed to reach statistical significance in adult B-ALL patients. It is concluded that the immunophenotypes of adult T-ALL are evidently heterogeneous in different ages, and expression with more aberrant phenotypes indicates poor prognostic significance in patients older than 35 years. There is no significant association of immunophenotypes with ages among different age groups of adult B-ALL.

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

The aim of this study was to explore the clinical features and survival of adult patients with CD20 positive B-lineage acute lymphoblastic leukemia (B-ALL). The clinical manifestations, examination results, therapeutic effect and survival rate of 119 adult B-ALL patients diagnosed and treated in our hospital from May 2004 to January 2008 were analyzed retrospectively. The results showed that among 119 cases, CD20 positive B-ALL accounted for 40 cases (33.61%), CD20 negative B-ALL patents accounted for 79 cases (66.39), the percentage of male patients in CD20 positive and negative groups were 72.50% and 50.63%, the leukocyte counts at diagnosis in these two groups were (27.35+/-30.29)x10(9)/L and (0.11+/-81.72)x10(9)/L, respectively, there were significant differences (p<0.05), whereas the distribution of age, infiltration of liver, spleen, lymph nodes and central nervous system, the hemoglobin and platelet levels, the expression of myeloid lineage marker, the incidence of Ph chromosome, the ratio of hyperdiploid and normal karyotype, the complete remission rate within 4 weeks, induction death rate and relapse rate and so on in CD20 positive and negative groups showed no significant differences (p>0.05). The analysis of Kaplan-Meier curve on survival rate demonstrated that the median overall survival time and 3-year overall survival rate of adult B-ALL patients in CD20 positive and negative groups were 11.0 months and 12.0 months, 28% and 20% respectively, there were no statistical differences (p=0.832). It is concluded that the expression of CD20 in adult B-ALL appears to be associated with sex and leukocyte count, but not associated with other clinical features, which indicates no significant influence on the prognosis of patients.

[Effect of CTL on K562 cell induced by exosomes and in combination with CPG OND].

To investigate the specific anti-leukemia effect of cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) activated by exosomes alone or in combination with CpG ODN in vitro and the feasibility of exosomes as remedial vaccine, the DCs induced from normal volunteer PBMNCs were divided into 7 groups. Three groups of them were added with the exosomes: Kexo (exosomes derived from K562 cells) or DCexo (exosomes derived from DCs induced from K562 cells) or FTexo (exosomes derived from DCs induced from K562 cells and pulsed by freeze-thawing antigen of K562 cells) as experimental groups (Kexo, DCexo and FTexo). The other three groups were added with CPG ODN while added the exosomes (Kexo, DCexo and FTexo), and were used as experimental groups also (Kexo+CpG, DCexo+CpG and FTexo+CpG). The seventh group DCs was added with nothing as blank control. These DCs above mentioned were cultured continuously for 72 hours. The T lymphocytes were co-cultured with DCs for another 72 hours to generate CTL. Then, the killing effects of them on K562 cells were determined by MTT assay. The results showed that all experimental groups pulsed by exosomes displayed stronger killing effect, compared with control group (p<0.05). DCexo and FTexo displayed stronger killing effect too, compared with Kexo (p<0.05). CPG ODN as an adjuvant could enhance the killing effect (p<0.05). It is concluded that the special killing effect on K562 cells can be induced by exosomes, CPG ODN as an adjuvant can enhance the killing effect. Exosome is hopeful as a remedial vaccine to be used for the leukemia therapy.

[Effects of novel human chemokine-like factor superfamily 8 on proliferation and EGFR expression of HL-60 cells].

This study was aimed to investigate the effect of chemokine-like factor superfamily 8 (CKLFSF8) on proliferation and expression of epidermal growth factor receptor (EGFR) of HL-60 cells. Expression of CKLFSF8 mRNA on HL-60 cell line was assayed by RT-PCR; the target gene was transfected into the cells by lipid vector, cell proliferation was determined by MTT assay, while expression of EGFR in HL-60 was determined by immunocytochemical technique. The results indicated that expression of CKLFSF8 existed in HL-60 cells. After transfection, cell proliferation was inhibited (P < 0.05) and the expression of EGFR in HL-60 cells was also discovered to be inhibited (P < 0.05). It is concluded that the proliferation and expression of EGFR in HL-60 cells can be inhibited by transfection of CKLFSF8. The novel chemokine may provide a new approach in the treatment of leukemia.

[The properties of exosomes secreted by human umbilical cord blood dendritic cells and its enhancement of CTL cytotoxicity].

AIM: To investigate the properties of exosomes secreted by dendritic cells(DCs) derived from human umbilical cord blood mononuclear cells(HUBMCs) and its enhancement of CTL cytotoxicity. METHODS: The HUBMCs were separated from human umbilical cord blood(HUCB). The generation of DCs were induced for 12 days in medium containing SCF, IL-4 and GM-CSF. Tumor antigen extracted by repeated freeze-thawing were added to culture medium and cultured successively for 2 days. The DCs were collected and exosomes were separated from culture supernatant through centrifugation at 100,000 g. The exosomes-associated properties were analyzed by FCM, SDS-PAGE, transmission electron microscope(TEM) and MTT colorimetry. RESULTS: After being induced for 12 days with SCF, IL-4 and GM-CSF the number of DCs had a 10-fold increase and displayed typically morphological characteristics of DCs. FCM analysis showed that the DCs loaded with tumor antigen expressed highly MHC-I, MHC-II, CD40, CD80, CD86, CD11c and CD54. Exosomes could be separated from culture supernatant of DCs by ultracentrifugation. Mixed lymphocyte reation(MLR) and cytotoxicity assay showed that exosomes secreted by DCs could effectively stimulate T cell proliferation and enhance their cytotoxicity. CONCLUSION: A large number of DCs are obtained from UBMCs and the exosomes are secreted from them in vitro. The exosomes can present tumor antigen to T cells and enhance the cytotoxicity of CTLs.

[Production of specific CTL induced by exosomes derived from K562 cells].

The aim of this study was to investigate whether exosomes derived from K562 cells and human monocyte-derived dendritic cells (DCs) transfected with total RNA of K562 cells are capable of inducing antigen-specific cytotoxic T lymphocytes (CTL) responses in vitro. DCs were generated from peripheral blood mononuclear cells (PBMNC) of healthy volunteers in the presence of GM-CSF and IL-4, and then were transfected with K562 RNA by using DOTAP lipofection. Exosomes was extracted from the supernatant of DCs and K562 cells. The T cell were activated to be tumor specific CTL after DCs and exosomes were co-cultured with autologous T cells derived from healthy volunteers' PBMNC. The effect of CTL on K562 cells was detected by MTT assay. The results showed that treatment of T cells with exosomes derived from K562 cells or DCs transfected with total RNA of K562 cells could significantly promote their killing ability on K562 cells as compared with untreated T cells (P < 0.05). The killing ability of T cells treated with exosomes on K562 cells was stronger than on HL-60 cells (P < 0.05). It is concluded that the specific CTL immune response to leukemia cells can be induced by exosomes derived from K562 cells.