Hua-Yuan Zhu

Downregulated Dicer expression predicts poor prognosis in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Alterations in microRNAs (miRNAs) expression have been proposed to play a role in CLL pathogenesis. Dicer and Drosha are the main regulators of miRNA biogenesis, and deregulation of their expression has been indicated as a possible cause of miRNA alterations observed in various cancers. To investigate the role of Dicer and Drosha in CLL, we assessed the expression of Dicer and Drosha and their correlation with other prognostic factors, including Binet stages, immunoglobulin heavy chain variable gene (IGHV) mutation status, TP53 mutation status, ZAP‐70 protein and CD38 expression level in 165 CLL patients by using real‐time polymerase chain reaction methods. Patients with unmutated IGHV genes had significantly lower expression of Dicer than patients with IGHV mutations. The lower expression level of Dicer was also significantly associated with higher level of CD38 and ZAP‐70, and more aggressive Binet stage. We also analyzed Dicer expression in different cytogenetic subgroups. Lower Dicer level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22.3) in contrast to higher level in good risk cytogenetics (deletion in 13q14 as the sole abnormality). Furthermore, the lower expression of Dicer in CLL shows a strong association with shorter overall survival (OS) (P = 0.0046) as well as with reduced treatment free survival (TFS) (P = 0.0006). By contrast, no differences in the expression of Drosha among these groups of patients were observed. Our data suggest that Dicer expression may play an important role in the progression and prognosis of CLL. (Cancer Sci 2012; 103: 875–881)

The prognostic significance of TP53 mutations in Chinese patients with chronic lymphocytic leukemia is independent of del(17p13)

The poor prognosis of chronic lymphocytic leukemia (CLL) patients with del(17p13) is well established. Several studies have shown that cases with TP53 mutations and TP53 mutations without del(17p13) may be adverse prognostic factors. We studied 173 well-characterized CLL patients by direct sequencing to detect TP53 mutations (exons 2–11). TP53 mutations were detected in 14.5% (25 of 173) of samples. Most patients with del(17p13) had TP53 mutations (72.2%). Mutations in the absence of del(17p13) were found in 8.3% in our cohort, which were higher than other countries. Compared with cases without TP53 alterations, TP53 mutations and deletions were both associated with advanced stages and unmutated immunoglobulin heavy-chain variable region status. Survival analysis showed that the occurrence of TP53 mutations and del(17p13) were associated with shorter overall survival (OS), treatment-free survival (TFS), and resistance to chemotherapy. TP53 mutations were the variables strongly associated with OS and TFS by multivariate Cox regression analysis. Moreover, we also found that cases with TP53 mutations in the absence of del(17p13) had a similar clinical and biological course and similar poor short OS as cases carrying del(17p13) in Chinese patients with CLL.

Aberrant microRNA expression in Chinese patients with chronic lymphocytic leukemia

MicroRNAs (miRNAs) are a class of small endogenous RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. Many reports have indicated that miRNAs play a critical role in malignancies, and regulations in the progression of leukemia. However, the miRNAs expression level in Chinese patients with chronic lymphocytic leukemia (CLL), and its prognostic value remain elusive. We identified various degrees of down-regulation of miR-15a, miR-16-1, miR-29b, miR-181a and miR-181b in CLL mononuclear cells. Moreover, we have identified miR-29b and miR-181a/b expression significantly correlated with IGHV mutational status. Transcript levels of predicted target genes BCL-2 and TCL-1 were also determined, and the expression levels were significantly upregulated in CLL patients compared with normal controls (P<0.001). Higher expression of TCL-1 was significantly correlated with aggressive disease features. In addition, an inverse correlation was observed in the CLL samples we examined between miRNAs (miR-16-1, miR-181a, miR-181b) and BCL-2 level; furthermore, an inverse correlation was also observed between miRNAs (miR-16-1, miR-181a, miR-181b) and TCL-1, which suggest that these miRNAs may implicate in negatively regulating target mRNA at transcriptional level. These different miRNAs may play an important role in the pathogenesis of CLL and might be applied for the assessment of prognosis in patients with CLL.

Enhancing the action of rituximab by adding fresh frozen plasma for the treatment of fludarabine refractory chronic lymphocytic leukemia.

Complement deficiencies have been identified in many chronic lymphocytic leukemia (CLL) patients. Supplying fresh frozen plasma (FFP)‐derived complement can enhance complement‐dependent cell lysis by the rituximab. The objective of our study was to evaluate the clinical efficacy and safety of the treatment by adding FFP to rituximab in fludarabine refractory CLL patients. Twenty‐two patients were treated with two units of FFP followed with rituximab, 375 mg/m2, as a single agent, repeated every 1–2 weeks. Patients received a median of four courses of the combined FFP and rituximab treatment (range: 2–6). Sixteen patients (72.7%) responded to treatment and seven (31.8%) achieved a complete remission. Three (13.6%) of which had no evidence of minimal residual disease after treatment. Patients with high expression of ZAP‐70 or CD38, unmutated immunoglobulin heavy chain variable region, mutated p53, or adverse cytogenetic features, achieved response to treatment at rates that appeared similar to those who did not have such characteristics. With a median follow‐up of 12 (4–19) months, the median overall survival and progression free survival have not been achieved. Toxicity was minimal, and the treatment was well tolerated. Our data suggest that the adding FFP to rituximab is an effective nonmyelotoxic regimen for the treatment of fludarabine refractory CLL patients.

MDM2 promoter SNP309 is associated with an increased susceptibility to chronic lymphocytic leukemia and correlates with MDM2 mRNA expression in Chinese patients with CLL

A single nucleotide polymorphism (SNP) at position 309 in the promoter region of MDM2 leading to increased expression of MDM2 and attenuated function of p53 has recently been suggested as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL) although this has been questioned. The MDM2 SNP309 genotypes in 173 CLL patients and 260 healthy controls were detected by the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method, which was confirmed by direct DNA sequencing. Compared with the T/T genotype, the SNP309 G/G genotype instead of T/G heterozygote was associated with a significantly increased risk of CLL (OR = 2.84; 95% CI 1.61–5.03; p < 0.001). Age at onset of CLL was similar irrespective of MDM2 status. MDM2 mRNA expression within CLL of G/G genotype was significantly higher than that in T/G (p = 0.009) and T/T genotypes (p < 0.001). Excluding patients with p53 deletions or mutations enhanced the significance of the findings (G/G vs. T/T, p < 0.001; G/G vs. T/G p = 0.001), which prompted us to study the role of the polymorphism in p53 wild‐type individuals. In the p53 wild‐type groups, survival analysis showed that the patients with MDM2 SNP309 G/G and T/G genotypes both had significantly shorter treatment‐free survival (TFS) than SNP309 T/T genotype. Notably, univariate and multivariate analyses showed that MDM2 SNP309 genotypes were associated with TFS. These data show that MDM2 309G polymorphisms contribute to the risk of developing CLL. The unfavorable MDM2 SNP309 G/G genotype was associated with an increase of MDM2 mRNA expression. MDM2 SNP309 was found to be associated with TFS in p53 wild‐type Chinese CLL populations.

Clinical significance of lymphoid enhancer-binding factor 1 expression in acute myeloid leukemia.

Abstract Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/β-catenin signaling pathway and its dysregulation is associated with a number of malignant diseases such as leukemia. We explored the expression profile of LEF1 in acute myeloid leukemia (AML) and determined its specific prognostic significance in this disease. The LEF1 mRNA level in patients with previously untreated AML was significantly higher than in normal controls. Patients with AML with relatively higher LEF1 expression were more likely to achieve a complete remission (CR) following induction therapy in comparison to those with a lower LEF1 level. Moreover, we provide the first evidence that primary AML samples with AML1–ETO or PML–RARα have a higher LEF1 level compared with those without each fusion gene. High LEF1 expression predicts a significantly better overall survival for patients with intermediate-risk cytogenetics. High LEF1 level was associated with a favorable relapse-free survival in patients with FLT3-ITD wild-type. Finally, a scoring system based on LEF1 level and mutation status of FLT3-ITD or NPM1 is reliable to predict the outcome for AML with intermediate-risk cytogenetics. Our results indicate that LEF1 contributes to the pathophysiology of AML and could serve as a novel predictor of better treatment response. LEF1 level may be incorporated into an improved risk classification system for certain specific subtypes of AML.

Clinical significance of down-regulated cylindromatosis gene in chronic lymphocytic leukemia.

Abstract Loss of cylindromatosis gene (CYLD) expression has been observed in various cancers, including chronic lymphocytic leukemia (CLL). As a deubiquitination enzyme, CYLD regulates the proliferation, development and activation of lymphoid cells. Here we determined the CYLD mRNA expression by quantitative polymerase chain reaction (PCR) in 125 patients with CLL. CYLD was considerably down-regulated in CLL cells compared to normal B cells. Low CYLD expression was associated with unmutated status of the immunoglobulin heavy-chain variable-region (IGHV) gene (p = 0.0018) and CD38 positivity (p = 0.0499). Patients with high CYLD expression showed a trend toward improved overall survival (OS) (10-year OS: CYLD high: 94.74%, CYLD low: 52.71%; p = 0.0534). For patients with mutated IGHV gene, high CYLD was also associated with better OS (10-year OS: CYLD high: 100%, CYLD low: 66.67%; p = 0.0547). In conclusion, low CYLD expression identifies a subgroup of patients with CLL with inferior outcome, indicating the role of CYLD as a tumor suppressor in the pathogenesis of CLL.

Overexpressed BAG3 is a potential therapeutic target in chronic lymphocytic leukemia

Bcl-2-associated athanogene 3 (BAG3), a member of BAG family, is shown to sustain cell survival and underlie resistance to chemotherapy in human neoplastic cells. We aimed to determine the exact role and underlying mechanisms of BAG3 in human chronic lymphocytic leukemia (CLL). One hundred human CLL samples and 20 normal B-cell samples from healthy controls were collected. We measured the BAG3 expression in these cells and explored its relationship with known prognostic factors for CLL. The roles of BAG3 in cell apoptosis and migration were evaluated by small interfering RNA-mediated knockdown of BAG3 in primary CLL cells. We showed that BAG3 expression level was increased in CLL cells compared with normal B cells. Moreover, BAG3 expression was particularly upregulated in CD38 positive, unmutated immunoglobulin heavy-chain patients and those with lymphadenopathy and/or splenomegaly. Importantly, patients with increased BAG3 expression level have poor overall survival in subgroups with positive ZAP-70 or those without any “p53 abnormality”. In addition, knocking down of BAG3 expression resulted in increased apoptotic ratio and decreased migration in primary CLL cells. Our data indicate that BAG3 is a marker of poor prognostic in specific subgroups of CLL patients and may be a potential therapeutic target for this disease.

High LEF1 expression predicts adverse prognosis in chronic lymphocytic leukemia and may be targeted by ethacrynic acid

Aberrant activation of lymphoid enhancer-binding factor-1 (LEF1) has been identified in several cancers, including chronic lymphocytic leukemia (CLL). As a key transcription factor of the Wnt/β-catenin pathway, LEF1 helps to regulate important genes involved in tumor cell death mechanisms. In this study, we determined LEF1 gene expression levels in CLL (n = 197) and monoclonal B-cell lymphocytosis (MBL) (n = 6) patients through real-time RT-PCR. LEF1 was significantly up-regulated in both MBL and CLL patients compared with normal B cells. Treatment-free survival (TFS) time and overall survival (OS) time were much longer in CLL patients with low LEF1 expression than in those with high LEF1 levels. Furthermore, Wnt inhibitor ethacrynic acid (EA) induced both apoptosis and necroptosis in primary CLL cells. EA also enhanced the cytotoxicity of both fludarabine and cyclophosphamide against CLL cells in vitro. Finally, we demonstrated that EA functions by inhibiting the recruitment of LEF1 to DNA promoters and restoring cylindromatosis (CYLD) expression in CLL cells. Our results showed, for the first time, that high LEF1 expression is associated with poor survival for CLL patients. Combined with other chemotherapeutic drugs, EA may be a promising therapeutic agent for CLL.

Low T3 syndrome is a strong prognostic predictor in diffuse large B cell lymphoma.

The aim of this study was to evaluate the prognostic effect of low triiodothyronine (T3) syndrome on patients with diffuse large B cell lymphoma (DLBCL). A hundred and eighty‐eight patients with detailed thyroid hormone levels at diagnosis of DLBCL were enrolled. Low T3 syndrome was defined as a low serum free T3 (FT3) level with low or normal serum free tetraiodothyronine (FT4) and thyroid stimulating hormone levels. Multivariate Cox regression analysis was used to screen prognostic factors associated with progression‐free survival (PFS) and overall survival (OS). Receiver‐operator characteristic curves and the corresponding areas under the curve were calculated to assess the predictive accuracy of International Prognostic Index (IPI) and low T3 syndrome. Twenty‐four patients were diagnosed with low T3 syndrome, which was associated with worse PFS and OS in the rituximab era. It was an independent prognostic factor for PFS and OS, especially for those with IPI 0−2, extranodal sites ≤1 and stage III−IV. Synchronously low FT3 and FT4 had poorer survival outcome compared to only low FT3 and adding criterion of low T3 syndrome improved the prognostic capacity of IPI for predicting PFS and OS in DLBCL. Low T3 syndrome was found to be a strong prognostic predictor in DLBCL.