Response and acquired resistance to crizotinib in Chinese patients with lung adenocarcinomas harboring MET Exon 14 splicing alternations.

Approximately 10% of lung adenocarcinomas harbor aberrations that are targetable using the approved multitargeted TKI crizotinib. MET exon 14 skipping mutation predicts for response to crizotinib in human lung adenocarcinomas. However, a substantial part of patients still has no sufficient tissue to perform genomic analysis. As a promising noninvasive biomarker and potential surrogate for the entire tumor genome, circulating tumor DNA (ctDNA) has been applied to the detection of driver gene mutations. Here we described the MET exon 14 splicing mutations in cell-free circulating-tumor DNA by next-generation sequencing (NGS) technology. Patient firstly responded to crizotinib therapy within four months, however, three acquired mutation in the MET kinase domain, D1228N/H and Y1230H, were found at the time of disease progression. To our knowledge, this is the first clinical report of three mutations simultaneously arising in a patient with MET exon 14 splicing mutation.

TP53 Pro72 allele potentially increases the poor prognostic significance of TP53 mutation in chronic lymphocytic leukemia.

Previous studies have investigated the associations between TP53 mutations and codon 72 polymorphisms and prognosis in chronic lymphocytic leukemia (CLL). However, the joint effect of TP53 mutations and TP53 codon 72 polymorphisms on CLL prognosis remains uncertain. We used direct sequencing to detect TP53 mutations and codon 72 genotype in 207 patients with CLL. The Pro/Pro genotype was associated with an increased incidence of TP53 mutations and deletion, but had no apparent effect on biological tumor behavior or clinical response. Compared to patients with wild-type p53, patients with TP53 mutations and the Pro72 allele (Arg/Pro + Pro/Pro genotypes) were associated with unmutated immunoglobulin heavy-chain variable region status and chemorefractoriness. Overall survival (OS) in the entire patient group was differed significantly between patients with TP53 mutations and either the Pro72 allele or Arg/Arg homozygotes (P = 0.014). Notably, patients with TP53 mutation and the Pro72 allele experienced a 23.7-fold increase in hazard ratio (95% CI 3.38-165.9; P = 0.001) for OS compared with patients with wild-type p53 and those with the Arg/Arg genotype. The TP53 Pro72 allele potentially increases the prognostic significance of TP53 mutations in CLL.

Risk of hepatitis B virus (HBV) reactivation in non-Hodgkin lymphoma patients receiving rituximab-chemotherapy: a meta-analysis.

BACKGROUND: The addition of Rituximab to standard chemotherapy (C) has been reported to improve the end of treatment outcome in non-Hodgkin lymphoma (NHL) patients. Nevertheless, rituximab has been associated with hepatitis B virus reactivation (HBV-R). OBJECTIVES: The aim of this systematic review and meta-analysis is to research the relationship between rituximab and HBV-R. STUDY DESIGN: We searched the commonly used databases both in English and Chinese from November 1997 to June 30, 2012. Meta-analysis was performed in fixed/random-effects models using Review Manager 5.1 and STATA 10.0. Publication bias was examined through Egger's test and Begg's funnel plot. RESULTS: Nine eligible articles were selected in this review (8 studies in English and 1 studies in Chinese), which included 971 adult patients and met all inclusion and exclusion criteria. Of rituximab-associated HBV-R cases reported through case series (n=387), 304 were HBcAb (+)/HBsAg (-) and 83 HBsAg (+). The pooled effect of rituximab-based therapy on HBV-R significantly increased under fixed-effects model [Relative risk (RR) 2.14, 95%CI 1.42-3.22, P=0.0003]. In subgroup analysis, rituximab-associated HBV-R in isolated HBcAb (+) patients remained high, and the RR was 5.52 (95%CI 2.05-14.85, P=0.0007). The RR of HBV-R in NHL patients with HBsAg (+) treated with R-based therapy when compared with the control population was 1.63 by the random-effects model. CONCLUSIONS: Rituximab therapy may increase the risk of developing HBV-R in NHL patients with HBcAb(+).

High expression of cyclic nucleotide phosphodiesterase 7B mRNA predicts poor prognosis in mantle cell lymphoma.

In order to determine the relationship between expression of cyclic nucleotide phosphodiesterase isoform 7B (PDE7B) and mantle cell lymphoma (MCL) progression, PDE7B mRNA expression was measured by qRT-PCR in 21 untreated MCL patients with bone marrow involvement and 20 healthy donors. The expression level of PDE7B was markedly higher in MCL patients compared with normal controls (P=0.001), and the higher level of PDE7B expression was associated with unfavorable cytogenetic characteristics in MCL. It was showed that higher expression of PDE7B might be a novel unfavorable prognostic indicator in MCL, which possess important clinical significance.

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.

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.

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.

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.

[mRNA levels detected by real-time quantitative RT-PCR in chronic lymphocytic leukemia and their clinical significance].

This study was aimed to investigate the expression level of murine double minute 4 (MDM4) mRNA in chronic lymphocytic leukemia (CLL) and its prognostic value in CLL. By means of ß-actin as internal reference, the real-time quantitative RT-PCR was set up. The expression of MDM4 mRNA in 66 CLL patients was measured by fluorescence dye SYBR Green I. The dispersion of MDM4 expression ratio of groups with different prognostic factors was described by using Mann-Whitney U test. The results showed that the median MDM4 mRNA expression level was 0.037098 (0.088245-0.014875) in CLL patients. The expression level of MDM4 mRNA was significantly higher in patients with P53 gene deletion than that in patients without P53 gene deletion (0.13167 vs 0.030927) (p < 0.001), and also significantly higher in patients with P53 mutation than that in patients without P53 mutation (0.13167 vs 0.03077) (p < 0.001). MDM4 expression was also associated with Binet stages (p = 0.044) and ATM gene deletion (p = 0.046), but was not associated with LDH (p = 0.216), ß(2)-MG (p = 0.314), TK1 (p = 0.300), ZAP-70 (p = 0.559), CD38 (p = 0.513) and IgVH mutation status (p = 0.333). It is concluded that the expression level of MDM4 is significantly higher in patients with P53 deletion or mutation. MDM4 expression is significantly associated with Binet stages and ATM gene deletion. MDM4 may be an important prognostic factor in CLL.

Polymorphisms and haplotypes in multidrug resistance 1 gene are not associated with chronic lymphocytic leukemia susceptibility and prognostic parameters of chronic lymphocytic leukemia in Chinese population.

The human multidrug resistance (MDR1, ABCB1) gene encodes P-glycoprotein (P-gp), which affects the pharmacokinetics of many drugs. Here, we investigated whether common MDR1 single nucleotide polymorphisms (SNPs) (C1236T, C3435T, and G2677T/A) affect predisposition to chronic lymphocytic leukemia (CLL). Genotyping was performed in 150 patients with CLL and 117 controls using polymerase chain reaction (PCR)-based assays. Haplotypes were inferred using the PHASE algorithm. We found comparable allele and genotype frequencies among patients with CLL and controls. Moreover, patient and control groups did not differ regarding MDR1 haplotype distribution (p = 0.97). Furthermore, no correlation was shown between the MDR1 1236, 3435, or 2677 genotypes and Binet stage, unmutated immunoglobulin heavy-chain variable region (IGHV) status, CD38 expression, ZAP-70 expression, and p53 deletion. In conclusion, our results do not support a major influence of MDR1 variants on the risk of CLL, and these genomic polymorphisms are not associated with clinical prognostic factors in Chinese patients with CLL.

[Expression of cyclic nucleotide phosphodiesterase 7B in patients with mantle cell lymphoma and its prognostic significance].

To investigate the expression level of cyclic nucleotide phosphodiesterase (PDE) 7B mRNA and its prognostic value in mantle cell lymphoma (MCL), the real-time quantitative RT-PCR (QPCR) was used to detect pde7b expression levels of bone marrow mononuclear cells from 20 newly diagnosed MCL patients with bone marrow involvement and peripheral blood mononuclear cells from 20 normal persons, the association of pde7b expression levels with prognostic indexes was analyzed by statistical software. The results showed that the median values of pde7b mRNA expression level in 20 MCL patients and normal controls were 8.7 × 10(-4) (4 × 10(-5) - 6.9 × 10(-3)) and 0.5 × 10(-4)(0.18 × 10(-4) - 1.7 × 10(-4)) respectively (p = 0.001). No association was found between pde7b expression and patients' clinical baseline information (gender and age), as well as certain prognostic factors, leukocyte count, lactate dehydrogenase level, CD38 expression and immunoglobulin heavy-chain variable region mutation status, but pde7b mRNA expression was significantly associated with cytogenetic abnormality, ß(2)-microglobulin level and ZAP-70 expression. It is concluded that the pde7b mRNA expression is obviously higher in MCL patients compared with normal controls and significantly correlates with unfavorable cytogenetic characteristics in MCL. The pde7b may be used as a novel prognostic indicator in MCL, and has important clinical significance.

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/m(2), 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.

[Cytogenetics of chronic lymphocytic leukemia stimulated by CpG-oligodeoxynucleotides and IL-2].

This study was to explore the stimulating effect of CpG-oligodeoxynucleotides (CpG-ODN) in combination with interleukin-2 (IL-2) on cytogenetic features of chronic lymphocytic leukemia (CLL) cells. Peripheral blood or bone marrow cells of 115 patients with CLL were cultured for 72 hours with CpG-ODN plus interleukin-2 (IL-2), and routine karyotype analysis was performed with R-banding technique. The metaphase number≥20 was considered as successful stimulation. The results showed that among the 115 CLL patients, successful stimulation rate was 74.8%. The rate of chromosome aberrations was 58.1%. One kind of aberration was detected in 21 cases (24.4%), two kinds of aberration in 6 cases (7.0%), complex aberrant karyotype in 23 cases (26.7%), included highly complex aberrant karyotype in 9 cases (10.5%), respectively. A total of 163 abnormalities of 102 kinds were detected in 86 patients. Number aberrations were 116 (71.2%), and structural abnormalities were 47 (28.8%). The most frequent number aberration was trisomy 12 (14.0%), and structural aberration was 15q+ (5.8%). It is concluded that most of CLL patients have chromosome abnormality, and the number abnormality are more frequent than the structural aberrations. CpG-ODN plus IL-2 can effectively raise the number of cells at metaphase and the detection rate of chromosome aberrations in CLL patients.

[Bag3 gene expression in chronic lymphocytic leukemia and its association with patients' prognosis].

The aim of this study was to investigate bag3 gene expression in chronic lymphocytic leukemia (CLL)patients and its association with clinical prognosis. A total of 46 blood samples from untreated CLL patients were collected, SYBR Green-based real-time PCR was used to detect the bag3 mRNA expression, and its association with prognostic index was analyzed by statistical software. The results showed that the median values of bag3 level detected by real-time PCR in 46 CLL patients and normal controls were 0.021 (0.0007 - 1.124) and 0.0025 (0.0005 - 0.014) respectively, the former was significantly higher than the latter. The bag3 level in drug-resistant group was obviously higher as compared with the drug-responsive group. No association was found between bag3 expression and patient clinical baseline information (gender and age) as well as established prognostic factors (lymphocyte count, disease stage, IgVH mutation status, cytogenetics analysis and CD38, ZAP 70 expression). It is concluded that the bag3 expression in CLL patients is markedly higher than that in normal controls, while the high bag3 level in CML patients is probably related with drug resistance, but is not related with clinically established prognostic factors.

[Detection of puma mRNA levels by real-time quantitative RT-PCR in chronic lymphocytic leukemia and its clinical significance].

This study was aimed to investigate the expression level of puma (p53 up-regulated modulator of apoptosis) mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. The puma mRNA expressions in 100 CLL patients and 11 normal controls were measured by relative quantification RT-PCR with fluorescent dye SYBR Green I, the beta-actin was used as internal reference. The difference of puma expression rate between groups with different prognostic factors was described using the Mann-Whitney U test. The relative quantitative value of puma expression was calculated by means of 2 (-ΔCt). The results indicated that the correlation coefficients of the standard curves in qRT-PCR were ≥ 0.99. The coefficients of variations (CV) within group or between groups were < 5%, and the sensitivity reached 10² copies/microg RNA. The median puma mRNA expression level was 1.038 x 10⁻³ (4.106 x 10⁻4 - 2.806 x 10⁻³) in CLL patients, which was 1.220 x 10⁻³ (7.233 x 10⁻4 - 1.405 x 10⁻³) in normal controls. There was no difference of puma mRNA expression between CLL patients and normal controls (U = 544.5, p = 0.957). Puma expression was significantly correlated with Binet stages (p < 0.001), expression of CD38 (p = 0.002), ZAP-70 protein (p = 0.012), LDH levels (p = 0.009) and beta2-MG (p = 0.046). The puma expression level in patients with earlier Binet stage (Binet stage A) was obviously higher than that in patients with later Binet stage (Binet stage B, C). The puma expression levels in patients with positive expression of CD38 and ZAP-70 protein, elevating levels of LDH and beta2-MG were sharply lower than those in patients without above-mentioned unfavorable factors. The puma expression was also correlated with molecular cytogenetic abnormalities, the puma expression levels in patients with trisomy 12 (p = 0.003) and 14q32 translocation (p = 0.045) detected by FISH were significantly lower than those in patients without above-mentioned molecular cytogenetic abnormalities. It is concluded that the qRT-PCR assay is reliable and sensitive. Puma mRNA expression is significantly correlated with a great deal of prognostic factors, and may be a prognostic marker of 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.