[Genetic analysis of a case of B-acute lymphoblastic leukaemia with double Philadelphia chromosomes and double derivative chromosome 9s].

OBJECTIVE: To explore the genetic basis for a rare case of acute B-lymphocytic leukemia (B-ALL) with double Philadelphia chromosomes (Ph) and double derivative chromosome 9s [der(9)]. METHODS: A patient with double Ph and double der(9) B-ALL who presented at Shanghai Zhaxin Intergrated Traditional Chinese and Western Medicine Hospital in June 2020 was selected as the subject. Bone marrow morphology, flow cytometry, G-banding karyotyping, fluorescence in situ hybridization (FISH), genetic testing and chromosomal microarray analysis (CMA) were used to analyze bone marrow samples from the patient at various stages. RESULTS: At initial diagnosis, the patient's bone marrow morphology and flow immunotyping have both supported the diagnosis of B-ALL. G-banded karyotyping of the patient indicated double Ph, in addition with hyperdiploid chromosomes involving translocations between chromosomes 9 and 22. BCR-ABL1 fusion gene was positive. Genetic testing at the time of recurrence revealed presence of a heterozyous c.944C>T variant in the kinase region of the ABL1 gene. FISH showed a signal for ABL1-BCR fusion on both chromosome 9s. CMA showed that the mosaicism homozygosity ratio of chromosome 9 was about 40%, and the mosaicism duplication ratio of chromosome 22 was about 43%. CONCLUSION: Since both der(9) homologs were seen in 40% of cells, the possible mechanism for the double der(9) in this patient may be similar to that of double Ph, which might have resulted from non-disjunction during mitosis in the Ph chromosome-positive cell clone.

Involvement of miR-125a in resistance to daunorubicin by inhibiting apoptosis in leukemia cell lines.

In this study, we investigated whether miR-125a participated in the resistance of the leukemia cell lines to the chemotherapeutic agent daunorubicin. Higher expression of miR-125a is correlated with lower treatment response and shorter overall survival in acute leukemia patients. Overexpression of miR-125a induced drug resistance in HL-60, K562, and THP-1cell lines through reducing apoptosis. We also showed that miR-125a mediated daunorubicin resistance in leukemia cell lines through the decrease of GRK2 and Puma which were proved to be direct targets of miR-125a. This study may provide novel therapeutic targets for therapy and improve predictions of therapeutic responses in leukemia to daunorubicin.

Case report: Preventive infusion of donor-derived CD7 chimeric antigen receptor T cells after allogeneic hematopoietic stem cell transplantation.

Chimeric antigen receptor T cells (CAR T) targeting CD7 for T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) showed promising efficacy and safety in some clinical trials. However, most of them were bridged with allogeneic hematopoietic stem cell transplantation (allo-HSCT). We described successful treatment with preventive donor-derived anti-CD7 CAR-T therapy in a case of refractory T lymphoblastic lymphoma following allo-HSCT, who could not receive autologous anti-CD7 CAR-T products due to the low-quality of T lymphocytes. To date, the patient's complete remission has persisted for 20 months after HSCT.

De novo myelodysplastic syndrome in a Rothmund-Thomson Syndrome patient with novel pathogenic RECQL4 variants.

Rothmund-Thomson syndrome (RTS) is a rare autosomal-recessive disorder with clinical features consisting of rash, poikiloderma, sparse hair, short stature, juvenile cataracts, skeletal abnormalities, and cancer predisposition. Genetic studies involving detection of pathogenic RECQL4 variants provide the diagnostic certitude. Osteosarcoma was found in two-thirds RECQL4-mutated RTS patients, while hematological malignancies were rarely reported. The variant diversity of RECQL4 gene has not been fully identified and mutations associated with hematologic malignancies are not well described. In this study, we presented a pedigree of RTS from a Chinese family, among which the proband was diagnosed with de novo myelodysplastic syndrome (MDS). Comprehensive medical examination and chromosome karyotyping were performed on the proband. Whole exome sequencing (WES) was performed on the proband, his sister and his mother. The familial cosegregation of sequence variants derived from WES was conducted by polymerase chain reaction-based Sanger sequencing. Structures of candidate RECQL4 mutants were done by in silico analysis to assess pathogenicity. Three novel RECQL4 germline variants, including c.T274C, c.G3014A, and c.G801C, were identified by WES and validated by Sanger sequencing. Prediction of conformation indicated that the structural stability of human RECQL4 protein was largely affected with these variants. The co-occurring U2AF1 p.S34F and TP53 p.Y220C mutations might contribute to the development of MDS. Our study expands the mutational spectrum of RECQL4 and provides underlying molecular mechanism for the development of MDS in RTS patients.

Administration of granulocyte-macrophage colony-stimulating factor enhanced chimeric antigen receptor T-cell expansion and cellular immunity recovery without inducing cytokine release syndrome.

Background: Neutropenia and cytokine release syndrome (CRS) are two major toxicities of chimeric antigen receptor (CAR)-T cell therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an ideal candidate treatment for neutropenia except for its potential aggravation of CRS. We hypothesized that the optimal timing of supplemental with GM-CSF in a shortage of host immunity and CAR T-cell was chosen as avoidance of CRS. In the study we evaluated the safety and efficacy of GM-CSF intervention post-CAR T-cell therapy while circulating CAR T-cell declined. Materials and methods: Nine patients received GM-CSF therapy who displayed moderate neutropenia with absolute neutrophil counts (ANC) < 1,500 cells/mm3 with concomitant declination of circulating CAR T-cell. Results: The median duration of GM-CSF intervention was 15 days (4-30). CAR T-cell expansion was observed in peripheral blood (PB) of seven patients (7/9). The median baseline and peak CAR T cells count in PB of the seven patients with CAR T-cell expansion were 0.85 × 106/L (0-50.9) and 6.06 × 106/L (1.43-112.55). And the peaks of CAR T-cell levels in PB appeared in day 7 (2-11) following the initiation of GM-CSF administration with increases of 2.84 × 106/L (0.38-61.65). Also, increased white blood cells in PB were observed in all patients. The median onset and duration time of WBC recovery were 9 (1-14) and 17 (3-53) days. Moreover, the increment of WBC, neutrophil, lymphocyte and CD3-CD16 + CD56 + natural killer cell in PB was observed. In addition, no CRS or fatal infection occurred during GM-CSF treatment. Conclusion: This study provides evidence for the clinical feasibility of combining CAR T-cell therapy with the GM-CSF to treat neutropenia patients with concomitant declination of circulating CAR T-cell.

The impact of JAK2V617F mutation on different types of thrombosis risk in patients with essential thrombocythemia: a meta-analysis.

To assess the effect of JAK2V617F on different thrombotic risks in essential thrombocythemia (ET) patients, we identified eligible studies from several databases including Pubmed, Embase, and Cochrane Central Register of Controlled Trials (up to November 2014). Twenty-two studies of 2922 ET patients were included in exploring the relationship between JAK2V617F and the risk of thrombosis. Compared to JAK2V617F-negative ET patients, JAK2V617F-positive ET patients had higher odd risks (ORs) of arterial thrombosis [OR = 2.59 (1.84-3.65)] and venous thrombosis [OR = 2.10 (1.53-2.88)]. The JAK2V617F-positive group was also more prone to increased risk of microcirculatory disturbances [OR = 1.50 (0.97-2.32)]. Moreover, JAK2V617F may indicate increased risk of either arterial [OR = 1.71 (1.22-2.39)] or venous thrombosis [OR = 2.90 (1.54-5.46)] before diagnosis of ET. During follow-up, JAK2V617F might not be related to arterial thrombosis [OR = 1.90 (0.90-2.08)], but rather venous thrombosis [OR = 1.95 (1.08-3.53)]. In conclusion, JAK2V617F increased the risk of arterial and venous thrombosis in ET patients, while understanding its role in microcirculatory disturbances will require further studies.

Spautin-1, a novel autophagy inhibitor, enhances imatinib-induced apoptosis in chronic myeloid leukemia.

Imatinib mesylate (IM), a targeted competitive inhibitor of the BCR-ABL tyrosine kinase, has revolutionized the clinical treatment of chronic myeloid leukemia (CML). However, resistance and intolerance are still a challenge in the treatment of CML. Autophagy has been proposed to play a role in IM resistance. To investigate the anti-leukemic activity of specific and potent autophagy inhibitor-1 (spautin-1) in CML, we detected its synergistic effect with IM in K562 and CML cells. Our results showed that spautin-1 markedly inhibited IM-induced autophagy in CML cells by downregulating Beclin-1. Spautin-1 enhanced IM-induced CML cell apoptosis by reducing the expression of the anti-apoptotic proteins Mcl-1 and Bcl-2. We further demonstrated that the pro-apoptotic activity of spautin-1 was associated with activation of GSK3ß, an important downstream effector of PI3K/AKT. The findings indicate that the autophagy inhibitor spautin-1 enhances IM-induced apoptosis by inactivating PI3K/AKT and activating downstream GSK3ß, leading to downregulation of Mcl-1 and Bcl-2, which represents a promising approach to improve the efficacy of IM in the treatment of patients with CML.

Polyclonal rabbit antithymocyte globulin induces apoptosis and has cytotoxic effects on human leukemic cells.

UNLABELLED: The possibility of antileukemic activity of antithymocyte globulin (ATG) was investigated in 8 human leukemic cell lines and primary leukemic cells from 15 leukemia patients. The study demonstrated that ATG induced apoptosis and reduced proliferation in both cell lines and primary leukemic cells, particularly in lymphatic origin cells, indicating that ATG has broad-spectrum antileukemic activity, especially for cells of lymphatic origin. BACKGROUND: Polyclonal ATGs are currently used to prevent graft-versus-host disease in allogeneic stem cell transplantation patients and to treat patients with severe aplastic anemia. It contains antibodies against antigens expressed on various hematopoietic cells, we hypothesized that it induces cell death not only in healthy cells but also in malignant hematopoietic cells. MATERIALS AND METHODS: In this study, several human leukemic cell lines and primary leukemic cells from 15 patients with leukemia were used to investigate the ability of polyclonal ATGs to induce apoptosis and proliferation. RESULTS: Polyclonal ATGs induced cell apoptosis in primary leukemic cells and in cell lines in a dose-dependent manner, and induced apoptosis in different populations through a variety of targets. Cell proliferation was significantly reduced in the presence of polyclonal ATGs; it arrested cells in the G0-G1 phase by cell cycle analysis. Treatment with polyclonal ATGs plus complement increased cytolysis of the leukemic cells; complement augments polyclonal ATG-induced leukemic cell death. CONCLUSION: These data show that polyclonal ATG has broad-spectrum antileukemic activity, especially for cells of lymphatic origin, as it induced cell death through a variety of targets. This study provides an experimental basis for the application of polyclonal ATGs in allogeneic hematopoietic stem cell transplantation and in patients with lymphatic leukemia.

[Distribution of abnormal cell clone with deletion of chromosome 20q in marrow cell lineages and apoptosis cells in myelodysplastic syndrome].

This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.

[Prognostic factor analysis of 77 old patients with acute myelogenous leukemia].

BACKGROUND & OBJECTIVE: The manifestations of old acute myelogenous (AML) patients have their special biological and clinical characteristics, with lower response rate to therapy and shorter survival time. This study was to investigate the prognostic factors of elderly patients with AML retrospectively. METHODS: 77 patients aged> or =60 years with AML from 1994 to 2005 were admitted to our study and all the possible prognostic factors were analyzed with Kaplan-Meier survival analysis. The significant factors were further analyzed by Cox proportional hazard model analysis. RESULTS: Seventy-two patients were evaluated. The patients aged 60-70 (median survival time was 350 days) had significantly longer survival time than those aged more than 70 (median survival time is 60 days)(P<0.001), which their CR ratios were 71.4% and 29.4% (P=0.001). The patients with performance status 0 or 1 (median survival time was 402 days) had significantly longer survival time than those with performance status 2, 3 or 4 (median survival time was 31 days)(P<0.001), which their CR ratios were 75% and 15% (P<0.001). The patients with primary AML (median survival time was 98 days) had significantly longer survival time than those with secondary AML (median survival time was 32 days)(P=0.007), which their CR ratios were 50% and 0% (P=0.023). The patients treated with sub-standard dosage of anthracycline (median survival time was 293 days) had significantly longer survival time than those treated with reduced dosage of anthracycline (median survival time was 35 days)(P=0.006), which their CR ratios were 63.6% and 33.3% (P=0.02). The patients with bone marrow blast cell ratio< or =50% (median survival time was 98 days ) had significantly longer survival time than those with bone marrow blast cell ratio >50% (median survival time was 55 days)(P=0.006). The patients with favorable karyotype (median survival time was 293 days) had significantly longer survival time than those with unfavorable or normal karyotype (median survival time was 31 days)(P=0.005). The patients without CD34 expression (median survival time was 201 days) had significantly longer survival time than those with CD34 expression(median survival time was 36 days)(P<0.001). The patients with the peripheral blood white blood cell count (PBWBC)>10x10(9)/L (50%) had significantly higher CR ratio than those with PBWBC< or =10x10(9)/L (25%)(P=0.043). The patients received chemotherapy (50%) had significantly higher CR ratio than those received supportive therapy (0%)(P=0.001). In the stepwise COX proportional hazard regression model, all the seven factors related to OS remained independent and significant. CONCLUSIONS: Factors, including age >70, PS 2 to 4, percentage of blasts in bone marrow >50%, secondary AML, unfavorable karyotype, expression of CD34, lower dosage.