Prognostic Significance of LPCAT1 in Adult Acute Myeloid Leukemia Patients with FAB Subtype M2 / 中国实验血液学杂志

OBJECTIVE@#To study the prognostic value of LPCAT1 in acute myeloid leukemia (AML).@*METHODS@#TaqMan-based reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect relative expression of LPCAT1 in 214 newly diagnosed adult AML patients and 24 normal controls. Survival functions were estimated using the Kaplan-Meier method and were compared by the Log-rank test. A Cox proportional hazard regression model was used to identify prognostic factors.@*RESULTS@#The expression level of LPCAT1 in adult AML was 34.37%(1.83%-392.63%), which was significantly lower than 92.81%(2.60%-325.84%) of normal controls (P<0.001). The prognostic significance of LPCAT1 was evaluated in 171 non-acute promyelocytic leukemia patients with complete clinical information and prognostic data. Survival analysis showed that the expression level of LPCAT1 had no significant effect on the prognosis of the whole cohort. However, in AML patients with FAB subtype M2 (AML-M2), the 2-year relapse-free survival (RFS) rate of patients with low LPCAT1 expression was 35.4%(95%CI: 0.107-0.601), which was significantly lower than 79.2%(95%CI: 0.627-0.957) of patients with high LPCAT1 expression (P=0.012). Multivariate analysis showed that low expression of LPCAT1 was an independent risk factor for RFS of AML-M2 patients (HR=0.355, 95%CI: 0.126-0.966, P=0.049).@*CONCLUSION@#In adult AML patients LPCAT1 shows low expression. Low LPCAT1 expression is an independent risk factor for RFS in M2-AML patients.

Clinical Significance of Low Expression of LncRNA CASC15 in Acute Myeloid Leukemia with NPM1 Mutations / 中国实验血液学杂志

AbstractObjective: To identify the expression and methylation patterns of lncRNA CASC15 in bone marrow (BM) samples of acute myeloid leukemia (AML) patients, and further explore its clinical significance.@*METHODS@#Eighty-two de novo AML patients and 18 healthy donors were included in the study. Meanwhile, seven public datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were included to confirm the expression and methylation data of CASC15. Receiver operating characteristic (ROC) curve analysis was applied to determine the discriminative capacity of CASC15 expression to identify AML. The patients were divided into CASC15high group and CASC15low group by X-tile method, and the prognostic value of CASC15 was identified by Kaplan-Meier method and univariate and multivariate Cox regression analysis.@*RESULTS@#The expression level of CASC15 was significantly decreased in BM cells of AML patients compared with healthy donors (P<0.001). ROC curve analysis suggested that CASC15 expression might be a potential biomarker to discriminate AML from controls. The expression of CASC15 was high at the early stage of hematopoiesis, and reached a peak at the stage of multipotent progenitors differentiation, then decreased rapidly, and was at a range of low level fluctuations in the subsequent process. Among FAB subtypes, CASC15 expression in M0 was significantly higher than that in M1-M7. Clinically, CASC15low patients were more likely to have NPM1 mutations than CASC15high patients (P=0.048), while CASC15high patients had a significantly higher frequency of IDH1 and RUNX1 mutations (P=0.021 and 0.014, respectively). Moreover, CASC15low group had a shorter overall survival (OS) in patients with NPM1 mutations. Furthermore, multivariate analysis confirmed that CASC15 expression was a significant independent risk factor for OS in NPM1 mutated AML patients. In addition, CASC15 methylation level in BM samples of AML patients was significantly decreased compared with healthy donors. Patients with CASC15 high methylation had poor OS and disease-free survival.@*CONCLUSION@#The expression of CASC15 is decreased in AML, and low CASC15 expression may predict adverse prognosis in AML patients with NPM1 mutations. Moreover, CASC15 methylation level in AML is significantly decreased, and high CASC15 methylation may predict poor prognosis in AML.

Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells / 中华血液学杂志

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.

Development and functional verification of CAR-T cells targeting CLL-1 / 中华血液学杂志

Objective: To explore the development of a CAR-T cells targeting CLL-1 and verify its function. Methods: The expression levels of CLL-1 targets in cell lines and primary cells were detected by flow cytometry. A CLL-1 CAR vector was constructed, and the corresponding lentivirus was prepared. After infection and activation of T cells, CAR-T cells targeting CLL-1 were produced and their function was verified in vitro and in vivo. Results: CLL-1 was expressed in acute myeloid leukemia (AML) cell lines and primary AML cells. The transduction rate of the prepared CAR T cells was 77.82%. In AML cell lines and AML primary cells, CLL-1-targeting CAR-T cells significantly and specifically killed CLL-1-expressing cells. Compared to untransduced T cells, CAR-T cells killed target cells and secreted inflammatory cytokines, such as interleukin-6 and interferon-γ, at significantly higher levels (P<0.001) . In an in vivo human xenograft mouse model of AML, CLL-1 CAR-T cells also exhibited potent antileukemic activity and induced prolonged mouse survival compared with untransduced T cells [not reached vs 22 days (95%CI 19-24 days) , P=0.002]. Conclusion: CAR-T cells targeting CLL-1 have been successfully produced and have excellent functions.

Prognostic implications and functional enrichment analysis of LTB4R in patients with acute myeloid leukemia / 南方医科大学学报

OBJECTIVE@#To explore the expression patterns, prognostic implications, and biological role of leukotriene B4 receptor (LTB4R) in patients with acute myeloid leukemia (AML).@*METHODS@#We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database for mRNA expression analyses, survival analyses, Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R. The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN. The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis. A protein-protein interaction network was constructed using STRING. GSEA analyses of the differentially expressed genes (DEGs) were performed based on datasets from TCGA-LAML stratified by LTB4R expression level. We also collected peripheral blood mononuclear cells (PBMCs) from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR. We also examined serum LTB4R protein levels in the patients using ELISA.@*RESULTS@#The mRNA expression level of LTB4R was significantly increased in AML patients (4.898±1.220 vs 2.252±0.215, P < 0.001), and an elevated LTB4R expression level was correlated with a poor overall survival (OS) of the patients (P=0.004, HR=1.74). LTB4R was identified as an independent prognostic factor for OS (P=0.019, HR=1.66) and was associated with FAB subtypes, cytogenetic risk, karyotype abnormalities and NPM1 mutations. The co- expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis, including neutrophil inflammation, lymphocyte activation, signal transduction, and metabolism. The DEGs were enriched in differentiation, activation of immune cells, and cytokine signaling. Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA (P=0.044) and protein (P=0.008) in AML patients, and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2 (r= 0.466, P=0.040).@*CONCLUSION@#LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.

IRF2-INPP4B-mediated autophagy suppresses apoptosis in acute myeloid leukemia cells

BACKGROUND: The present study aimed to investigate the underlying role of interferon-regulatory factor 2 (IRF2)-inositol polyphosphate-4-phosphatase, type-II (INPP4B) axis in the regulation of autophagy in acute myeloid leukemia (AML) cells. METHODS: Quantitative real time PCR (QRT-PCR) and western blot were performed to determine the expression levels of IRF2, INPP4B and autophagy-related markers in AML cell lines. Autophagy was assessed by elevated Beclin-1 expression, the conversion of light chain 3 (LC3)-I to LC3-II, downregulated p62 expression and green fluorescent protein (GFP)-LC3 puncta formation. The colony formation and apoptosis assays were performed to determine the effects of IRF2 and INPP4B on the growth of AML cells. RESULTS: IRF2 and INPP4B were highly expressed in AML cell lines, and were positively correlated with autophagy-related proteins. Overexpression of IRF2 or INPP4B stimulated autophagy of AML cells, whereas inhibition of IRF2 or INPP4B resulted in the attenuation of autophagy. More importantly, IRF2 or INPP4B overexpression reversed autophagy inhibitor, 3-methyladenine (3-MA)-induced proliferation-inhibitory and pro-apoptotic effects, while IRF2 or INPP4B silencing overturned the proliferation-promoting and anti-apoptotic effects of autophagy activator rapamycin. CONCLUSION: IRF2-INPP4B signaling axis attenuated apoptosis through induction of autophagy in AML cells.

Comparison of biochemical and immunological profile of pediatric patients with acute myeloid leukemia in relation to healthy individuals / Comparação do perfil bioquímico e imunológico de pacientes pediátricos com leucemia mieloide aguda e de indivíduos saudáveis

ABSTRACT OBJECTIVE: To compare the biochemical and immunological profiles of pediatric patients with acute myeloid leukemia (AML) with healthy children and adolescents. METHODS: This was a cross-sectional study in which 21 therapy-naïve patients with AML were compared with a group of 24 healthy individuals. The following data were analyzed: serum proteins, leucocytes and subgroups, erythrocytes, hematocrit, hemoglobin, platelets, cytokines in peripheral blood mononuclear cells cultures under spontaneous and BCG- or PHA-stimulated conditions, immunoglobulin A, and erythrocytic glutathione. Statistical analysis was performed using SPSS software, considering as significant p-values < 0.05. RESULTS: Serum albumin levels were higher (p < 0.0001) in the control group, as well as all the parameters related to red blood cells (p < 0.0001). For leucocytes and subgroups, no statistical difference was found between the AML and the control groups. For cytokines, the concentrations were significantly higher under spontaneous and BCG-stimulated conditions for TNF-a, IL-6, IL-10, and IFN-? in the control group. Under PHA-stimulated conditions, the concentration was higher (p = 0.002) only for IL-6. No difference was found between the two groups for the other cytokines and for IgA in the saliva. Erythrocytic glutathione was higher (p < 0.0001) in AML patients. CONCLUSIONS: It was possible to characterize the biochemical and immunological profile of pediatric patients with AML, as well as highlight some significant differences in these parameters when comparing with healthy children and adolescents.

RESUMO OBJETIVO: Comparar o perfil bioquímico e imunológico de pacientes pediátricos portadores de leucemia mieloide aguda (LMA) em relação a um grupo de crianças e adolescentes saudáveis. MÉTODOS Estudo transversal, em que foram avaliados 21 pacientes com LMA virgens de terapia e 24 indivíduos saudáveis. Foram analisados: proteínas séricas, leucócitos e subgrupos, eritrócitos, hematócrito, hemoglobina e plaquetas, citocinas em cultura de células mononucleares do sangue periférico sob condição espontânea e estimulada por BCG ou PHA, imunoglobulina A e glutationa eritrocitária. Análise estatística foi feita com o software SPSS considerando p < 0,05. RESULTADOS: Albumina sérica foi superior (p < 0,0001) no grupo de controle, bem como todos os parâmetros relacionados com os glóbulos vermelhos (p < 0,0001). Para os leucócitos e subgrupos não houve diferença estatística entre os pacientes com LMA e o grupo controle. As concentrações foram significativamente mais elevadas sob condições espontânea e estimulada por BCG para as citocinas TNF-a, IL-6, IL-10 e IFN-? no grupo controle. Sob condição estimulada com PHA a concentração foi superior (p = 0,002) apenas para a IL-6. Não houve diferença estatística para as demais citocinas e para IgA salivar entre os dois grupos. Glutationa eritrocitária foi superior (p < 0,0001) nos pacientes LMA. CONCLUSÕES: Diante do exposto, foi possível caracterizar o perfil bioquímico e imunológico de pacientes pediátricos com LMA, bem como evidenciar diferenças significativas em alguns desses parâmetros ao se compararem os indivíduos doentes e o grupo de crianças e adolescentes saudáveis.

Estimation of salivary amylase and total proteins in leukemia patients and its correlation with clinical feature and radiographic finding.

Background: Leukemia is a fatal disease. The oral manifestations of the leukemias occur early in the course of the disease and these oral features can at times act as a diagnostic indicator. Saliva has been used as a diagnostic aid in a number of systemic diseases. Materials and Methods: In our study, samples of unstimulated saliva of 30 leukemia patients who were not on chemotherapy were collected and analyzed for salivary amylase and total protein. The oral manifestations and radiographic changes (OPG) were recorded. The correlation between the oral manifestations and the salivary components (salivary amylase and total protein) was assessed for prognostic significance. Results: In the present study when the mean values of salivary amylase (1280±754 U/ml) and total protein (647.2±320.7 mg%) were compared with that in control subjects. There was a statistically significant difference for amylase levels (P<.05). On intraoral examination the study subjects showed pallor, gingivitis, gingival enlargement, petechiae, and ecchymosis. On the OPG, the radiographic features included generalized rarefaction of bone (20%), thinning of lamina dura (3.4%), generalized alveolar crest bone resorption (30%), thinning of walls of alveolar crypts (6.7%), besides others, e.g., periapical abscess (10%). Conclusions: The saliva of leukemic patients demonstrated obvious changes in composition. A rise in salivary amylase and total protein levels was evident, with the increase in amylase levels being statistically significant.

Granulocytic sarcoma masquerading as Ewing's sarcoma: a diagnostic dilemma.

An eleven-year-old boy presented with a swelling in his left elbow. Radiologically the features were that of an Ewing's sarcoma involving the ulna. Histopathology showed small round cell tumor strongly positive for Monoclonal Imperial Cancer research fund 2 (MIC2) antigen. Similar cells in the bone marrow were involved with MIC2 positivity. The patient developed skin lesions, which on biopsy were found to be chloromas. The initial biopsies were reevaluated with special stains revealing granulocytic sarcomas in acute myeloid leukemia masquerading as Ewing's due to its MIC2 positivity. The possibility of myeloid neoplasms should be considered routinely with known MIC2 positive round cell tumors.

Erythrocyte enzyme abnormalities in leukemias.

Red cell enzymes were assayed in a total of 67 patient including 24 patients with AML (19 relapse, 5 remission), 16 patients with ALL (10 relapse, 6 remission), 22 patients with CML and 5 patients with blastic CML. Diagnosis of leukemia was based on clinical presentation, peripheral blood smear and bone marrow examination (as per FAB classification). PK activity was significantly high in case of CML and blastic CML (p<0.01). Red cell HK was high in all leukemia subtypes. There was no alteration in red cell G6PD. Notably there was no PK deficiency in AML or G6PD deficiency in ALL. Activities of G6PD and PK could be correlated in cases of CML, AML, (p<0.05) and ALL (p<0.01) i.e. when there was increased activity of G6PD, PK activity also tended to be higher. HK activity showed a positive correlation with PK and G6PD activity in cases of CML (p<0.05), however in acute leukemia there was no such correlation. Alteration of enzyme activities among red cells in leukemia occurred only during relapse. At the time of remission there has been no significant alteration in any of the enzyme activities. It would therefore, appear that enzyme alterations seen in leukemia patients is due to abnormal pluripotent stem cell that has given to a leukemia cell. The fact that enzyme alterations have primarily occurred at the time of relapse would further substantiate that abnormalities of red cell enzymes may be the result of a derivation some circulating red cells from the abnormal pluripotent stem cell. With the recovery of normal stem cells function during remission, enzyme abnormalities tend to become normal.

Alteraciones bioquímicas y citoquímicas de hidrolasas ácidas lisosomales en leucemias / Biochemical and citochemical alterations of lisosomal acid hydrolases in leukemias

La actividad enzimática total de ß-galactosidasa (ß-gal), hexosaminidasa (hex) y fosfatasa ácida (Fac) fue determinada bioquímicamente, tanto en suero como en sobrenadantes de homogenatos celulares de sujetos normales y pacientes portadores de leucemias, empleándose sustratos paranitrofenilados específicos. La actividad de ß-gal en leucemias mieloides, tanto agudas (LMA-M1) como crónicas (LMC), sólo mostró un incremento significativo en sobrenadantes de homogenato celulares, respecto a los valores de neutrófilos normales. En leucemias linfoidales agudas (LLA), como crónicas (LLC), su comportamiento no ofreció variaciones. Tanto los sueros como los sobrenadantes de homogenatos celulares de LMA-M1 y LMC mostraron un franco incremento en la actividad de hex, mientras en LLA y LLC esta actividad no mostró variaciones. La actividad sérica de fosfatasa ácida estuvo incrementada en el 86% de las LMC. En los sobrenadantes de homogenatos celulares de LLA y LLC, esta actividad enzimática se mostró significativamente disminuida respecto de los valores de linfocitos normales. En los tres casos de LMA-M4 analizados, fue observado en el contenido celular niveles elevados de ß-gal, hex y Fac (lo que estaría correlacionado con la presencia de monocitos y/o monoblastos normales, con alto contenido de hidrolasas ácidas). Citoquímicamente fue demostrado en médula ósea y sangre periférica de pacientes con LMA-M1 una ligera o nula actividad de hex, en tanto que en LLA la reacción fue localizada asimétricamente en un polo celular o en gránulos citoplasmáticos. Los resultados encontrados demuestran una gran heterogeneidad en el contenido lisosomal de los diferentes tipos de leucemias

Estudio de la vitamina B12 y sus transportadores en la leucemia aguda y síndrome mieloproliferativo / Study of vitamin B12 and its carriers in acute leukemia and myeloproliferative syndrome

Se realizaron determinaciones de la vitamina B12 y sus proteínas transportadoras en un grupo de pacientes con leucemia aguda y síndrome mieloproliferativo y los resultados se compararaon con un grupo control. El grupo con policitemia vera se compara con 5 pacientes que padecen de policitemia secundaria. Se encontró un aumento de la capacidad latente de unión (UBBC) dependiente de la transcobalamina II en los pacientes con leucemia aguda. Igual resultado se encontró en aquéllos con leucemia mieloide crónica y policitemia vera, pero en éstos dicha elevación se debió a un aumento conjunto de los niveles de transcobalamina I y II (ARBC). Los resultados obtenidos en los pacientes con leucemia mieloide crónica, que fueron estudiados evolutivamente, sugieren que la vitamina B12 y sus proteínas transportadoras pueden ser un elemento que se deba considerar para decidir la duración del tratamiento de esta enfermedad