Dan-Xia Zhu

Serum microRNAs are promising novel biomarkers for diffuse large B cell lymphoma

MicroRNAs (miRNAs) are regulatory RNA molecules that are deregulated in many disease types, including cancer. Recently, miRNAs have shown promise as markers for cancer diagnosis. The aim of this study was to investigate whether serum miRNAs can be used as biomarkers for the detection of diffuse large B cell lymphoma (DLBCL). We measured the levels of miRNAs (miR-15a, miR-16-1, miR-21, miR-29c, miR-34a, miR-155, and miR-223) in serum samples from patients with DLBCL and healthy controls using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We show here that miRNAs are present in human serum in a remarkably stable form. Four of miRNAs (miR-15a, miR-16-1, miR-29c, and miR-155) were significantly elevated in DLBCL serum when compared with normal controls (P < 0.05), while miR-34a was downregulated in DLBCL serum when compared with controls (P < 0.05). Receiver operating characteristic analyses reflects strong discriminating DLBCL from controls, with area under the curves of 0.7722, 0.7002, 0.6672, 0.8538, and 0.7157 for miR-15a, miR-16-1, miR-29c, miR-34a, and miR-155, respectively. At the cut-off value of 0.0006 for miR-15a, the sensitivity was 80% and the specificity was 76%; at the cut-off value of 0.0886 for miR-16-1, the sensitivity was 94% and the specificity was 51%; at the cut-off value of 1.395 for miR-34a, the sensitivity was 100% and the specificity was 70%; at the cut-off value of 0.0022 for miR-155, the sensitivity was 83% and the specificity was 65%. In conclusion, these data suggest that serum miRNAs are potentially useful tools as novel noninvasive biomarker for the diagnosis of DLBCL.

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.

Intermediate prognosis of 6q deletion in chronic lymphocytic leukemia

Cytogenetic features have an important role in the definition of distinct disease subsets in CLL. The deletion of 6q is known to occur at a relatively low frequency in CLL, and the detailed analysis of hematologic and clinical features of patients with CLL with 6q deletion is limited. To verify the incidence and prognostic significance of 6q deletion in Chinese patients with CLL, fluorescence in situ hybridization (FISH) was used in 240 patients with CLL. del(6q23) was found in 18 patients (7.5%), and only five patients had deletion in 6q23 as the sole abnormality. Strong correlations between del(6q23) and clinical parameters were not found. A difference in terms of survival in patients with del(6q23) as compared with patients without this anomaly was not able to be demonstrated. However, a significant difference was found when comparing the del(6q23) group with the del(17p13) or del(11q22.3) group (p = 0.023), or isolated del(13q14) group (p = 0.019). Our findings place the del(6q23) cytogenetic subset of CLL in an intermediate prognosis position between patients with del(11q22.3) or del(17p13), and patients with isolated del(13q14). FISH probes to detect deletions of 6q might be useful in clinical practice in the work-up of patients with CLL.

Richter transformation in 16 of 149 Chinese patients with chronic lymphocytic leukemia.

Abstract No confirmed risk factors are known to predict Richter syndrome (RS), and the value of clinical prognosticators conventionally applied to chronic lymphocytic leukemia (CLL) is not firmly established in this setting. The objective of this study was to present evidence for RS in Chinese patients with CLL and risk factors for CLL transformation to Richter syndrome. With a median follow-up of 43 months from CLL diagnosis in 149 Chinese patients, 16 (10.7%) patients progressed to diffuse large B-cell lymphoma (DLBCL). According to correlation analysis, a high level of lactate dehydrogenase (LDH) and CD38 positivity were found to be independent predictors of transformation to RS. Survival analysis showed that presence of RS, advanced Binet stage, high level of LDH, high level of β2-microglobulin, high concentration of thymidine kinase (TK), ZAP-70 and CD38 expression, unmutated immunoglobulin heavy chain variable (IGHV) gene status and del(17p13) were adverse factors in determining overall survival (OS). Only del(17p13) was strongly associated with survival by multivariate Cox regression analysis. Median OS after transformation was 16 months (95% confidence interval, 5.6–26.4 months). The results support that RS is associated with a poor outcome, and a policy of close monitoring and careful biopsy is needed in patients carrying transformation risk factors.

Prognostic significance of serum immunoglobulin paraprotein in patients with chronic lymphocytic leukemia.

The aim of this study was to explore the clinical and other associated laboratory features of chronic lymphocytic leukemia (CLL) patients with immunoglobulin (Ig) paraproteinemia. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) were performed to measure serum Ig paraprotein. The correlations between serum Ig paraprotein and other prognostic factors were analyzed. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. In 133 Chinese CLL patients, 27 (20.3%) patients occurred Ig paraproteinemia at diagnosis. According to the correlation analysis, advanced Binet stage (r=0.314, P<0.001), direct antiglobulin test (DAT)-positive (r=0.366, P<0.001), high level of serum β2-microglobulin (β2-MG) (r=0.296, P=0.001) and thymidine kinase (TK) 1 (r=0.227, P=0.037), unmutated immunoglobulin heavy chain variable gene (IGHV) status (r=0.284, P=0.002), ZAP-70-positive (r=0.305, P=0.001), CD38-positive (r=0.284, P=0.002), and cytogenetic abnormalities of del(17p13) or del(11q22.3) (r=0.208, P=0.032) emerged as factors significantly related to the occurrence of Ig paraproteinemia. Survival analysis showed that the patients with Ig paraproteinemia had significantly shorter survival times than the patients without serum Ig paraprotein (P=0.013). Binet stage (P=0.028), high levels of lactate dehydrogenase (LDH) (P=0.004), IgG paraproteinemia (P=0.048), IgM paraproteinemia (P=0.001), ZAP-70-positive (P=0.003), DAT-positive (P=0.013), unmutated IGHV status (P=0.009), and del(17p13) (P=0.001) were the adverse factors in determining overall survival (OS). Del(17p13) (P=0.006), ZAP-70 (P=0.030), and IgM paraproteinemia (P=0.040) were the variables strongly associated with OS by multivariate Cox regression analysis. It was showed that serum Ig paraprotein might be applied for the assessment of prognosis in patients with CLL.

TP53 mutation is not an independent prognostic factor in patients with mantle cell lymphoma at advanced stage

Mantle cell lymphoma (MCL) is incurable in most patients. Several molecular markers have been identified as possible prognostic factors in MCL, including TP53 mutations. Direct sequencing was used to detect 32 cases with leukemic presentation of MCL form exons 2–11 in order to explore the prognostic significance of TP53 mutations in Chinese patients. Within the MCL cohort, 6(18.8%) patients harbored TP53 mutations. TP53 mutations in the absence of del(17p13) were found in 5.0% of MCL cases (1 of 20) compared with 83.3% of MCL cases (5 of 6) with del(17p13). Compared with patients without TP53 mutations, TP53 mutations were associated with risk factors including age, higher serum lactate dehydrogenase, lymphocytosis, high-risk (HR) international prognostic index, HR mantle cell lymphoma international prognostic index, complex karyotype, and higher occurrence of TP53 deletions. In contrast, it is found that TP53 mutations were correlated with mutated immunoglobulin heavy-chain variable region status and CD38 negative. TP53 mutations were the significant factors in predicting survival in univariate analysis, but unfortunately they were not the unique variable associated with overall survival by multivariate Cox regression analysis. TP53 mutation was insufficiently an independent prognostic factor in patients with MCL at advanced stage.