Dong-Mei Wang

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.

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.

The expression of SOX11, cyclin D1, cyclin D2, and cyclin D3 in B-cell lymphocytic proliferative diseases

SOX11 is mainly correlated with embryo neurogenesis and remodeling of tissues. D cyclins (cyclin D1, cyclin D2, and cyclin D3) work in cell transformation. We assessed the expression of SOX11, cyclin D1, cyclin D2, and cyclin D3 mRNA in 152 patients with B-cell lymphocytic proliferative diseases (B-LPD) using qRT-PCR and we detected SOX11 protein using immunohistochemistry in 15 B-LPD patients, to clarify the clinical significance of the four genes in B-LPD. Data showed the transcriptional levels of SOX11 and cyclin D1 were higher for the mantle cell lymphoma (MCL) samples compared with chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), hairy cell leukemia (HCL), splenic marginal zone lymphoma (SMZL), and healthy collators. The expression levels of cyclin D1 and cyclin D2 were both higher in DLBCL than in SMZL. The expression levels of the four genes were highly related to each other. Three of 4 MCL patients showed nuclear staining for SOX11, while other 11 B-LPD examples were negative. Furthermore, we also found the ZAP70-positive CLL patients had higher SOX11 expression levels than ZAP70-negative CLL patients. It was revealed that MCL patients have higher expression levels of SOX11 and cyclin D1 mRNA, specially expressed nuclear SOX11 protein.

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.

High-dose methylprednisolone can induce remissions in patients with fludarabine-refractory chronic lymphocytic leukaemia.

PURPOSE To evaluate the clinical efficacy and safety of high-dose methylprednisolone (HDMP) in patients with fludarabine-refractory chronic lymphocytic leukaemia (CLL). METHODS Twelve patients who were refractory to fludarabine-based treatment were treated with 2-6 cycles of HDMP (1g/m(2) for 5days). RESULTS Ten patients (83.3%) responded to treatment and three (25.0%) achieved a complete remission (CR). Two (16.7%) of which had no evidence of minimal residual disease (MRD) after treatment. Patients with leukaemia cells that have high expression of ZAP-70 or CD38, unmutated immunoglobulin heavy chain variable region (IGHV), mutated p53 or adverse cytogenetic features achieved response to treatment at rates that appeared similar to those achieved by patients who did not have such disease characteristics. With a median follow-up of 13 (4-30) months, the median overall survival (OS) and the progression-free survival (PFS) have not been achieved. Treatment with HDMP was well tolerated, notably in the patients having poor myeloid reserve and pretreated cytopaenias. CONCLUSIONS HDMP is an effective non-myelotoxic regimen for the treatment of patients with fludarabine-refractory CLL.

Expression level of DEK in chronic lymphocytic leukemia is regulated by fludarabine and Nutlin-3 depending on p53 status

Human oncogene DEK has been shown to be upregulated in a number of neoplasms. The purpose of this study was to investigate DEK expression level in chronic lymphocytic leukemia (CLL), analyze the correlation between DEK expression and CLL prognostic markers, and characterize the role of DEK in the response to either chemotherapeutic drugs or nongenotoxic activators of the p53 pathway. DEK mRNA was evaluated by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR), and primary CLL samples were treated in vitro with either fludarabine or Nutlin-3 to explore the interaction of p53 status and DEK mRNA expression. The median expression levels of DEK mRNA were 6.792 × 10−2 (1.438 × 10−2−3.201 × 10−1) in 65 patients with CLL. A marked increase of DEK mRNA expression was observed in the CLL patients with unmutated immunoglobulin heavy chain variable (IGHV) gene (p = 0.025), CD38-positive (p = 0.047), del(17p13) (p = 0.006). Both fludarabine and Nutlin-3 significantly downregulated DEK in the primary CLL cells which were with normal function of p53, or without deletion or mutation of p53 (p = 0.042, p = 0.038; p = 0.021, p = 0.017; p = 0.037, p = 0.017). However, the downregulation of DEK was not observed in the primary CLL cells which were with dysfunction of p53, or with deletion or mutation of p53 (p = 0.834, p = 0.477; p = 0.111, p = 0.378; p = 0.263, p = 0.378). These data show that DEK might be applied for the assessment of prognosis in patients with CLL, and fludarabine and Nutlin-3 regulate DEK expression depended on p53 status.