Comprehensive characterization of TGFB1 across hematological malignancies.

TGFB1, which encodes TGF-ß1, a potent cytokine regulating varies cellular processes including immune responses. TGF-ß1 plays context-dependent roles in cancers and is increasingly recognized as a therapeutic target to enhance immunotherapy responses. We comprehensively evaluated expression of TGFB1 and its clinical and biological effects across hematological malignancies. TGFB1 expression was first explored using data from the GTEx, CCLE, and TCGA databases. The expression and clinical significances of TGFB1 in hematological malignancies were analyzed using Hemap and our In Silico curated datasets. We also analyzed the relationship between TGFB1 with immune scores and immune cell infiltrations in Hemap. We further assessed the value of TGFB1 in predicting immunotherapy response using TIDE and real-world immunotherapy datasets. TGFB1 showed a hematologic-tissue-specific expression pattern both across normal tissues and cancer types. TGFB1 expression were broadly dysregulated in blood cancers and generally associated with adverse prognosis. TGFB1 expression were associated with distinct TME properties among different blood cancer types. In addition, TGFB1 expression was found to be a useful marker in predicting immunotherapy responses. Our results suggest that TGFB1 is broadly dysregulated in hematological malignancies. TGFB1 might regulate the immune microenvironment in a cancer-type-specific manner, which could be applied in the development of new targeted drugs for immunotherapy.

Reduced expression of lncRNA DLEU7-AS1 is a novel favorable prognostic factor in acute myeloid leukemia.

The objective of our study was to measure DLEU7-AS1 expression in de novo acute myeloid leukemia (AML) whilst also analyzing its clinical relevance. We used gene expression data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Cancer Cell Line Encyclopedia (CCLE) and Genotype-Tissue Expression project (GTEx) to assess the expression profile of DLEU7-AS1 in pan-cancers, cancer cell lines and normal tissues. Reverse transcription-quantitative PCR was used to measure DLEU7-AS1 expression in bone marrow from 30 normal individuals and 110 patients with de novo AML. DLEU7-AS1 expression was found to be markedly reduced in the AML samples of the TCGA pan-cancer datasets. In our PCR validation, DLEU7-AS1 expression was significantly decreased in the AML samples compared with that in controls (P<0.001). Low DLEU7-AS1 expression (DLEU7-AS1low) correlated positively with lower blood platelet counts (P=0.029). In addition, low DLEU7-AS1 expression was more frequently observed in the intermediate (58%; 44/76) and favorable karyotypes (65%; 15/23) compared with that in the poor karyotype (10%; 1/10; P=0.005). In particular, patients with high expression levels of DLEU7-AS1 (DLEU7-AS1high) showed lower complete remission rates (P=0.002) than patients with DLEU7-AS1low. Survival analysis revealed that patients with DLEU7-AS1low had longer overall survival (OS) than patients with DLEU7-AS1high (P<0.05). Multivariate Cox analysis demonstrated that in patients with non-acute promyelocytic leukemia (non-M3) who were ≤60 years old, DLEU7-AS1 expression was an independent prognostic factor for OS. Furthermore, we found distinct correlations among the expression of DLEU7-AS1, infiltration by immune cells and immune checkpoint genes in AML.

Methylation-independent CRIP1 expression is a potential biomarker affecting prognosis in cytogenetically normal acute myeloid leukemia.

Abnormal expression of CRIP1 has been identified in numerous solid tumors. However, CRIP1 expression and its regulation are little known in acute myeloid leukemia (AML). The purpose of this study was to evaluate the expression and regulation of CRIP1 and the clinical implications of CRIP1 aberration in AML. Real-time quantitative PCR was carried out to detect the level of CRIP1 expression in 138 AML patients and 38 controls. CRIP1 methylation was detected by methylation-specific PCR and bisulfite sequencing PCR. Five public available AML datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were further analyzed. The level of CRIP1 expression was up-regulated in AML patients compared with controls (P = 0.045). CRIP1 high patients had a significantly lower complete remission (CR) rate than CRIP1 low patients (P = 0.020). CRIP1 high group had a shorter overall survival (OS) and leukemia-free survival (LFS) than CRIP1 low group in cytogenetically normal AML (CN-AML) patients (P = 0.007 and 0.012, respectively). Multivariate analysis further confirmed that high CRIP1 expression was an independent risk factor for LFS in CN-AML patients (P = 0.005). However, we found that CRIP1 expression was not associated with the status of its promoter, which was nearly fully unmethylated both in controls and AML patients. Furthermore, our results were validated using the published GEO datasets and TCGA datasets. Our findings suggest that high CRIP1 expression is independently related with unfavorable prognosis in CN-AML.

The M2 macrophage marker CD206: a novel prognostic indicator for acute myeloid leukemia.

Hematological malignancies possess a distinctive immunologic microenvironment compared with solid tumors. Here, using an established computational algorithm (CIBERSORT), we systematically analyzed the overall distribution of 22 tumor-infiltrating leukocyte (TIL) populations in more than 2000 bone marrow (BM) samples from 5 major hematological malignancies and healthy controls. Focusing on significantly altered TILs in acute myeloid leukemia (AML), we found that patients with AML exhibited increased frequencies of M2 macrophages, compared to either healthy controls or the other four malignancies. High infiltration of M2 macrophages was associated with poor outcome in AML. Further analysis revealed that CD206, a M2 marker gene, could faithfully reflect variation in M2 fractions and was more highly expressed in AML than normal controls. High CD206 expression predicted inferior overall survival (OS) and event-free survival (EFS) in two independent AML cohorts. Among 175 patients with intermediate-risk cytogenetics, the survival still differed greatly between low and high CD206 expressers (OS; P < .0001; 3-year rates, 56% v 32%; EFS; P < .001; 3-year rates, 47% v 25%). When analyzed in a meta-analysis, CD206 as a continuous variable showed superior predictive performance than classical prognosticators in AML (BAALC, ERG, EVI1, MN1, and WT1). In summary, M2 macrophages are preferentially enriched in AML. The M2 marker CD206 may serve as a new prognostic marker in AML.

Reduced expression of PDLIM4 gene correlates with good prognosis in acute myeloid leukemia.

This study was aimed to investigate the expression pattern of gene PDLIM4 (PDZ and LIM domain 4) and analyze its clinical correlation with the patients suffered from acute myeloid leukemia (AML). The expression pattern of PDLIM4 in AML was detected by using EvaGreen real-time quantitative PCR (RQ-PCR). The results showed that the PDLIM4 transcript significantly decreased in 94 AML patients, compared with 21 controls (P < 0.01). The decrease of PDLIM4 transcript was found in 42 (45%) AML patients. PDLIM4 low-expression occurred among the subtypes of M1/M2/M3 more frequently than that in M4/M5/M6 (56% vs 20%, P < 0.01). AML patients with PDLIM4 low-expression had an overall survival (OS) higher than that in AML patients without PDLIM4 low-expression (P < 0.05). Analysis with receiver operating characteristic curve (ROC) displayed that PDLIM4 expression possesses the diagnostic value to differentiate AML from controls, with ROC curve area of 0.865 (95% CI: 0.801-0.930). It is concluded that reduced PDLIM4 expression is a common and favorable event for the good prognosis in AML, and can be used as a potential diagnostic biomarker of cancer.

[Expression of SALL4 gene in patients with acute and chronic myeloid leukemia].

The aim of this study was to investigate the expression level of the SALL4 gene and its clinical significance in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Real-time quantitative PCR (RQ-PCR) was performed to detect the expression level of SALL4 mRNA in bone marrow mononuclear cells (BMMNC) from 35 AML, 12 CML patients and 24 iron deficiency anemia patients as controls. The results indicated that the expression level of SALL4 in AML (0%-14%, median 1.43%) was obviously higher than that in controls (0% - 1%, median 0%) (P < 0.001). SALL4 expression was positive in 65.7% (23/35) AML patients. The frequency of SALL4 expression was in M2 (86.7%, 13/15) > M3 (75.0%, 6/8) > M1 (60.0%, 3/5) > M4 (14.3%, 1/7), and the difference among 4 groups was statistically significant (P = 0.008); there was no correlation of the frequency of SALL4 expression with the age, sex, white blood cell WBC count, hemoglobin concentration, platelet count and chromosomal abnormalities of AML patients (P > 0.05). All the 13 CML cases showed positive expression of SALL4 gene (1% - 128%, median 19.39%), which was higher than that in controls (P < 0.001). The analysis of receiver operating characteristic (ROC) curve showed the area under ROC curve (AUC) of AML and CML were 0.983 (95% confidence interval: 0.95 - 1.017) and 0.997 (95% confidence interval: 0.986 - 1.007) respectively. It is concluded that SALL4 expression is a common molecular event and can be considered as a molecular marker for assisting diagnosis of AML and CML.

Overexpression of miR-378 is frequent and may affect treatment outcomes in patients with acute myeloid leukemia.

MicroRNA miR-378 plays important roles in tumorigenesis by enhancing cell survival, reducing apoptosis, promoting tumor growth, angiogenesis and promoting cell migration and invasion. Abnormal expression of miR-378 has been observed in various types of cancers. The aim of this study was to investigate the expression status of miR-378 and its clinical significance in patients with acute myeloid leukemia (AML) using real-time quantitative PCR. miR-378 overexpression was identified in 26 of 84 (31%) AML patients. The patients with miR-378 overexpression had lower hemoglobin level than those without miR-378 overexpression (66 versus 78 g/L, respectively, P=0.010). The frequency of miR-378 overexpression in FAB-M2 subtype was higher than other subtypes (44% versus 20%, P=0.032). Moreover, the frequency of miR-378 overexpression was higher in patients with t(8;21) than in others (64% versus 24%, P=0.012). The status of miR-378 expression was not correlated with the mutations of eight genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, C/EBPA and U2AF1). The difference in relapse-free survival was observed between patients with and without miR-378 overexpression (P=0.049). These findings suggest that miR-378 up-regulation is a common event and might have an adverse impact on prognosis in AML.

[Expression of RAGE-1 gene in acute myeloid leukemia].

The purpose of this study was to detect the expression of RAGE-1 transcript in the bone marrow mononuclear cells (BMMNC) from patients with acute myeloid leukemia (AML) and to investigate the relationship of RAGE-1 expression level with clinical variables. The expression level of RAGE-1 gene in BMMNC from 94 newly diagnosed AML patients was measured using RQ-PCR. The relationship between RAGE-1 expression level and clinical parameters (age, sex, blood cell counts, diagnosis and prognosis) was investigated, and the levels of RAGE-1 expression were compared in patients before and after treatment. The results showed that overexpression of RAGE-1 transcript was found in 28% (26/94) AML patients (1.34 - 16.34, median 3.07). No significant difference was observed in sex, age, blood parameters and FAB subtypes between the groups with and without RAGE-1 overexpression. There was also no significant difference in the frequency of RAGE-1 overexpression among different cytogenetic risk groups and among the patients with different types of karyotypes. The level of RAGE-1 transcript significantly decreased in those patients obtained complete remission after treatment. The overall survival of AML patients with RAGE-1 overexpression was similar as that in those without RAGE-1 overexpression. It is concluded that RAGE-1 overexpression is a common event in AML, but has no impact on the prognosis of patients.

SF3B1 mutation is a rare event in Chinese patients with acute and chronic myeloid leukemia.

OBJECTIVE: Somatic mutations of SF3B1 gene have recently been identified in myelodysplastic syndrome and chronic lymphocytic leukemia. The frequency and clinical relevance of SF3B1 mutations have been rarely studied in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The present study was aimed to analyze the frequency of SF3B1 mutations in AML and CML. DESIGNS AND METHODS: High-resolution melting analysis (HRMA) was established to detect the mutation hotspots (codon E622, H662, K666, and K700) of SF3B1 gene in 275 AML and 81 CML patients. RESULTS: Heterozygous SF3B1 mutations were detected in three AML patients by HRMA. Direct DNA sequencing identified one K666T, one K666N and one K700E mutations. All three AML patients had normal karyotypes. One case also had NPM1 and DNMT3A mutations, one had FLT3 internal tandem duplication and DNMT3A mutations, and the other had NPM1 mutation. No SF3B1 mutations were detected in CML patients. CONCLUSIONS: SF3B1 mutation is a rare molecular event in Chinese AML and CML patients.

Development of a high-resolution melting analysis for the detection of the SF3B1 mutations.

SF3B1, located on chromosome 2q33.1, encodes a core component of RNA-splicing machinery, and its mutation has been described in myelodysplastic syndromes (MDS) characterized with ring sideroblasts (RS). To explore the reliability and sensitivity of the high-resolution melting analysis (HRMA) technique for the identification of the SF3B1 mutations, mutations in 92 patients with MDS were detected in this study. The sensitivity could reach 5%, obviously higher than the 25% of direct DNA sequencing. A low frequency (5.4%) of SF3B1 mutations were identified in patients with MDS, including three cases of K700E, one case of H662Q, and one case of K666M. Further, SF3B1 mutations were more frequently recurrent in the 33% of patients with MDS characterized with RS, whereas in other subtypes of MDS, only 2.3% of patients were detected with SF3B1 mutations (p=0.006). In conclusion, a rapid, reproducible, sensitive, and high-throughput HRMA assay has been established for the scanning of SF3B1 mutations.

Aberrant hypomethylation of SALL4 gene in patients with myelodysplastic syndrome.

The abnormalities of SALL4 gene, which encodes a zinc-finger transcription factor and is essential for developmental events, have been found to be involved in tumorigenesis. In this study, we investigated the methylation status of the CpG island of SALL4 promoter region in myelodysplastic syndrome (MDS) using methylation-specific PCR (MSP). Aberrant hypomethylation of SALL4 gene was found in 21.7% (18/83) of the cases analyzed. A significantly positive correlation was identified between the level of SALL4 transcript and the status of SALL4 hypomethylation (R=0.641, P<0.001). No correlation was found between SALL4 hypomethylation and clinical parameters. However, the frequency of SALL4 hypomethylation significantly increased in higher risk MDS (14% in Low/Int-1 versus 39% in Int-2/High, P=0.031). The association between SALL4 hypomethylation and the mutations in three methylation modifiers (IDH1, IDH2 and DNMT3A) was not observed. Although the estimated 50% survival time of the SALL4-hypomethylated group was shorter than that of SALL4-methylated group (11.0 months vs. 20.0 months), the difference was not statistically significant (P=0.430). These findings suggest that hypomethylation of SALL4 promoter is a common event in MDS.

Development of high-resolution melting analysis for the detection of the MYD88 L265P mutation.

OBJECTIVE: Recurrent L265P mutation of myeloid differentiation primary response gene 88 (MYD88) has been identified in a proportion of diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia. The present study aimed to establish a rapid, sensitive, and reliable method using high-resolution melting analysis (HRMA) to detect MYD88 L265P mutation in DLBCL. DESIGNS AND METHODS: The sensitivity of HRMA in the detection of MYD88 L265P mutation was evaluated. MYD88 L265P mutation was further screened in 120 patients with DLBCL. The results of HRMA were validated by direct DNA sequencing. RESULTS: For the detection of MYD88 L265P mutation, the reproducible maximal sensitivity of HRMA was 5% higher than that obtained by direct DNA sequencing (25%). Heterozygous MYD88 L265P mutations were identified in 11 (9.2%) DLBCL cases, all of which were diagnosed as non-germinal-center B cell (non-GCB) DLBCL. CONCLUSIONS: The HRMA assay is a rapid, sensitive, reliable, and high-throughput method to screen MYD88 L265P mutation and could be used in clinical diagnostic laboratories.

Aberrant hypomethylation of SALL4 gene is associated with intermediate and poor karyotypes in acute myeloid leukemia.

OBJECTIVE: SALL4 gene has been identified to stimulate the expansion of hematopoietic stem cell (HSCs) and enhance the self-renewal of HSCs. Overexpression of SALL4 has been found in several cancers. The present study was aimed to investigate the methylation status of SALL4 promoter region in acute myeloid leukemia (AML). DESIGNS AND METHODS: The methylation status of SALL4 promoter was analyzed in 84 patients with AML using methylation-specific PCR (MSP) and its clinical significance was evaluated. RESULTS: Aberrant hypomethylation of SALL4 gene, which was correlated with SALL4 expression, was found in 17.8% (15/84) cases. The patients with SALL4 hypomethylation had significantly older age and higher WBCs than those without SALL4 hypomethylation. The incidence of SALL4 hypomethylation was higher in M1 subtype than in M2 and other subtypes (50%, 26% and 6%, respectively, P=0.001). SALL4 hypomethylation was associated with cytogenetically intermediate and poor groups. Although survival time of the SALL4-hypomethylated AML was shorter than that of SALL4-methylated group (4 months vs 9 months), the difference was not statistically significant (P=0.356). CONCLUSIONS: Hypomethylation of SALL4 promoter is a common event and is associated with the intermediate and poor karyotypes in AML.

U2AF1 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome.

Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.

IDH1 and IDH2 mutation analysis in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome.

The somatic mutations of isocitrate dehydrogenase genes (IDH1 and IDH2) have been identified in a proportion of hematologic malignancies. We examined IDH1 R132 and IDH2 R140/R172 mutations by high resolution melting analysis and direct sequencing in Chinese patients with different myeloid malignancies including 198 acute myeloid leukemia (AML), 82 myelodysplastic syndrome (MDS), 85 chronic myeloid leukemia, and 57 myeloproliferative neoplasms. IDH1 and IDH2 mutations were found in four (2.0%) and ten (5.0%) AML and in two (2.4%) and three (3.6%) MDS cases, but not in other patients. IDH1 and IDH2 mutations were heterozygous and mutually exclusive. IDH1/2 mutations were significantly more frequently observed in cytogenetically normal AML or MDS compared to those without mutations. There was no difference in overall survival of both AML and MDS patients with or without IDH1/2 mutations (P = 0.177 and 0.407, respectively). In conclusion, IDH1/2 mutations are recurrent but rare molecular aberrations in Chinese AML and MDS.

Recurrent DNMT3A R882 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome.

Somatic mutations of DNMT3A gene have recently been reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We examined the entire coding sequences of DNMT3A gene by high-resolution melting analysis and sequencing in Chinese patients with myeloid malignancies. R882 mutations were found in 12/182 AML and in 4/51 MDS, but not in either 79 chronic myeloid leukemia (CML), or 57 myeloproliferative neoplasms (MPNs), or 4 chronic monomyelocytic leukemia. No other DNMT3A mutations were detected in all patients. R882 mutations were associated with old age and more frequently present in monoblastic leukemia (M4 and M5, 7/52) compared to other subtypes (5/130). Furthermore, 14/16 (86.6%) R882 mutations were observed in patients with normal karyotypes. The overall survival of mutated MDS patients was shorter than those without mutation (median 9 and 25 months, respectively). We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS, and may be an adverse prognostic event in MDS.

Gene expression of helicase antigen in patients with acute and chronic myeloid leukemia.

The aim of this study was to investigate the expression status of the helicase antigen (HAGE) transcript and its clinical significance in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The expression of HAGE cDNA in bone marrow mononuclear cells from AML and CML patients was detected by using real-time quantitative PCR. The results indicated that overexpression of HAGE transcript (117.12% - 9842.70%, median 434.96%) was detected in 14.8% (11/74) AML patients. AML patients with HAGE cDNA expression were significantly older than those HAGE-negative patients (median 67 and 45 years, respectively, p = 0.001). HAGE cDNA expression was more frequently present among the patients with acute monoblastic leukemia (M(4) and M(5), 7 of 20, 35.0%), compared to the patients with acute non-monoblastic leukemia (M(1), M(2), M(3) and M(6), 4 of 54, 7.4%) (p = 0.007). 28.6% (8/28) cases with normal karyotypes showed HAGE cDNA overexpression, significantly higher than 7.5% (3 of 40) in those with chromosomal abnormalities (p = 0.041). Overexpression of HAGE transcript was found in 9 (34.6%) CML cases and more frequently observed at accelerated phase and blast crisis (4/4, 100%) than that at chronic phase (5/22, 22.7%) (p = 0.008). It is concluded that HAGE cDNA expression is relevant to specific subtypes of AML and to the progression of CML.

Aberrant methylation of CCAAT/enhancer binding protein zeta promoter in acute myeloid leukemia.

The aberrant changes of tumor suppressor genes (TSGs) are now recognized as an important mechanism contributing to the development of acute myeloid leukemia (AML). CCAAT/enhancer binding protein zeta (C/EBPζ), a candidate TSG, has been found to be involved in cancers including AML. We detected the methylation status of C/EBPζ promoter in 133 patients with AML using the methylation-specific polymerase chain reaction (MS-PCR) and examined C/EBPζ transcript in 32 patients using real-time quantitative PCR. The abnormal methylation of C/EBPζ gene promoter was found in 62 (46.6%) AML cases. No correlation was found between C/EBPζ promoter hypermethylation and the age, sex, WBC counts, platelet counts and FAB subtypes of AML patients (P>0.05). The trend that the frequency of C/EBPζ methylation increased as karyotype became more adverse was observed (R=0.167, P=0.075). There was a significant correlation between C/EBPζ expression and C/EBPζ methylation in AML patients (R=0.606, P=0.002). Our data suggest that the aberrant methylation of C/EBPζ promoter may be involved in AML.