ONCOLOGY LETTERS 18: 6057-6065, 2019 PLCB4 upregulation is associated with unfavorable prognosis in pediatric acute myeloid leukemia SHIWEN WU1, WEI ZHANG2, DONGQIN SHEN3, JIANLE LU2 and LI ZHAO2 Departments of 1Clinical Laboratory, 2Central Laboratory and 3Nursing, The First Hospital of Lanzhou University, Lanzhou University, Lanzhou, Gansu 730000, P.R. China Received May 7, 2019; Accepted August 21, 2019 DOI: 10.3892/ol.2019.10921 Abstract. Phospholipase C (PLC) is a membrane-associated Introduction enzyme that regulates several cellular behaviors including cell motility, growth, transformation and differentiation. PLC Acute myeloid leukemia (AML) is a highly heterogeneous is involved in cancer migration, invasion and drug resistance. disease, characterized by poor differentiation and abnormal However, the expression status and prognostic role of PLCB4 in proliferation of myeloid progenitors (1). AML is the most acute myeloid leukemia (AML) remain unclear. In the present common type of acute leukemia in adults, with an estimated study, the complete clinical and mRNA expression data of annual incidence of >20,000 new cases in the United States in 285 pediatric patients with de novo AML were obtained from 2019 (2). In 2013 it was reported that it was the leading cause the Therapeutically Available Research to Generate Effective of cancer deaths among children and individuals <35 years Treatments database. The association between PLCB4 expres- of age in China (3). Although ongoing advances in the treat- sion and clinical and molecular features was explored. The ment of AML have led to significant improvements in the expression of PLCB4 was significantly higher in patients clinical outcome, the overall survival (OS) rate remains low, with AML who relapsed compared with those with long-term mainly due to high recurrence rate and drug resistance (4). complete remission. Patients with PLCB4 upregulation had The specific mechanisms involved in these processes remain significantly lower overall survival (OS) and event free survival poorly defined. (EFS) rate compared with those with low PLCB4 expression. Phospholipase C (PLC) plays an essential role in cell Multivariate Cox's regression analyses demonstrated that metabolism, as it hydrolyzes membrane-bound phospholipid high PLCB4 expression was an independent risk factor of phosphatidylinositol 4,5-biphosphate (PIP2) into two second adverse OS (P<0.01; HR, 2.081) and EFS (P<0.01; HR, 2.130). messengers, inositol 1,4,5-triphosphate (IP3) and diacylglyc- Following stratification analysis according to transplant status erol (DAG) (5). A growing body of evidence indicates that in cases of first complete remission, the patients with high increased expression of PLC promotes invasion and metastasis expression of PLCB4 had significantly lower OS and EFS rate in several solid tumor types, including gastric carcinoma, in the chemotherapy group, but not the stem cell transplant breast cancer, hepatocellular carcinoma, pancreatic cancer, group. Furthermore, PLCB4‑associated genes were identified esophageal cancer and colorectal cancer (6,7). using Spearman's rank correlation analysis. KEGG pathway Of the PLC isoforms, PLCB1 promotes breast cancer cell analysis revealed that PLCB4 and its associated genes were migration via actin remodeling (8), and additionally regulates mainly involved in three potential pathways, including the cell cycle progression in AML (9-12). PLCB2 is significantly Rap1 signaling pathway. Overall, the findings of the present upregulated in human breast cancer and exhibits oncogenic study suggest that increased PLCB4 expression is associated functions and poor prognostic effects, by promoting cell divi- with poor clinical outcome in pediatric patients with AML, sion, motility and invasion (13,14). and thus may represent a potential prognostic biomarker and PLCB4 encodes the β4 isoform of PLC isoenzymes, therapeutic target for AML. a superfamily that regulates the metabolism of inositol lipids (15). Increased PLCB4 expression is associated with poor OS rate in patients with solid tumors, including mesothe- lioma, melanoma and gastrointestinal tumors (16-18). PLCB4 contributes to solid tumor progression (16,17), however, its Correspondence to: Dr Li Zhao, Department of Central Laboratory, The First Hospital of Lanzhou University, Lanzhou University, function in hematological tumors, particularly AML, has not 1 Donggang West Road, Lanzhou, Gansu 730000, P.R. China been explored. Leukemia stem cells (LSC) are the source of E-mail: [email protected] drug resistance and relapse of AML, however, PLCB4 expres- sion in LSCs remain to be elucidated (19). In the present study, Key words: phospholipase C β4, acute myeloid leukemia, prognosis, the effect of PLCB4 expression and its clinical significance pediatric, recurrence in AML were investigated. PLCB4 expression may serve as a novel diagnostic or therapeutic target in pediatric patients with AML. 6058 WU et al: PLCB4 UPREGULATION AND PROGNOSIS OF PEDIATRIC AML Figure 1. PLCB4 expression in AML. (A) PLCB4 expression in patients with AML of different clinical status (relapse and long-term CR). (B) PLCB4 expres- sion in AML patients of different survival status (alive and deceased) at 5-years follow-up. *P<0.05; ****P<0.0001. AML, acute myeloid leukemia; CR, complete remission; PLCB4, phospholipase C β4. Materials and methods Statistical analysis. Continuous variables were presented as the median with interquartile range, and categorical variables Patients. All patients and corresponding clinical information were presented as frequencies and percentage proportions. were obtained from the Therapeutically Available Research Pearson's χ2 or Fisher's exact tests were performed for cate- to Generate Effective Treatments (TARGET) database gorical variables. Mann-Whitney U tests were performed to (https://ocg.cancer.gov/programs/target/) on 11 November analyze the difference of continuous variables between two 2018. Samples were excluded if clinical information and groups, while Kruskal-Wallis test were used for the comparison RNA expression data were incomplete. A total of 285 pedi- of multiple groups, and a Bonferroni test for the post hoc test. atric patients with de novo AML were enrolled (median OS was defined as the time from diagnosis until death for any age, 10 years; range, 0-23 years), including 147 males and reason, or until the last follow-up. Event-free survival (EFS) 138 females. Of these patients, 36 accepted stem cell trans- was defined as the time from diagnosis to death, relapse, plants (SCTs) in first complete remission (CR1), and the induction failure or last follow-up. Survival analysis was remaining patients accepted chemotherapy. Median follow-up based on the Kaplan-Meier method and the log-rank tests to time of the patients was 1,355 days (range, 2-4,037 days). compare the differences between survival curves. Univariate and multivariate Cox's regression analyses were used to assess Gene expression analyses. The raw counts of mRNA expres- the association between PLCB4 expression and prognosis, as sion profiles (level 3) were downloaded from the TARGET well as other clinical variables. The sensitivity and specificity database and log2 transformed. Patients were divided into of the PLCB4 expression signature was evaluated according high- and low-PLCB4 groups, using the median of PLCB4 to the area under the curve (AUC) of receiver operating char- expression level as the cut-off value. A comparison of clinical acteristic (ROC) curves of 5-year OS and EFS. ROC curves and molecular characteristics was performed between patients were generated using the survival ROC packages based on with high and low PLCB4 expression. R software (version 3.3.3; www.r-project.org/). The SPSS software (version 23.0; IBM Corp.) was applied for statistical Functional and pathway enrichment analysis of analysis. The figures were generated using GraphPad Prism PLCB4‑associated genes in AML. Spearman's rank correla- (version 6.01; GraphPad Software, Inc.) or R software. P<0.05 tion analysis was performed to identify PLCB4-associated was considered to indicate a statistically significant difference. genes using R software (version 3.3.3; www.r-project.org). Correlation coefficient values <-4 or >4 were considered Results to be significantly correlated with PLCB4 expression. Gene Ontology (GO; http://geneontology.org) functional PLCB4 expression in AML. Amongst the 285 patients, analysis (20) and Kyoto Encyclopedia of Genes and Genomes 49 remained in long-term complete remission (CR) until the (KEGG; http://david.ncifcrf.gov) pathway enrichment anal- last follow-up, and 236 experienced relapse. PLCB4 expres- ysis (21,22) of the PLCB4-associated genes were performed sion was significantly upregulated in patients with relapse using the online Database for Annotation, Visualization and (median, 4.858) compared with patients with long-term CR Integrated Discovery database (version 6.8; https://david. (median, 4.087) (P<0.05; Fig. 1A). Significant differences ncifcrf.gov/) (23). P<0.05 was considered as the cut-off crite- were also observed in the classification of survival status rion for significant differences. (P<0.01; Fig. 1B). Median PLCB4 mRNA expression was significantly lower in patients who survived (median, 3.807) PLCB4 expression in leukemia stem cells. The expression compared with those that had died (median, 5.473) at 5-years of PLCB4 in primitive progenitor cells was assessed, based follow‑up. No significant differences in PLCB4