Chun Qiao

miR-17-92 cluster microRNAs confers tumorigenicity in multiple myeloma

miRNAs play important roles in the regulation of cell proliferation, differentiation and apoptosis. The deregulation of miRNAs expression contributes to tumorigenesis by modulating oncogenic and tumor suppressor signaling pathways. Oncogenic transcription factor Myc can control expression of a large set of microRNAs (miRNAs). Previous studies have shown that the expression of miR-17-92 cluster, a polycistron encoding six microRNAs (miRNA), has close relationship with the expression of Myc. In current study, silencing Myc in multiple myeloma (MM)cells induced cell death and growth inhibition, and downregulated expression of miR-17-92 cluster. Overexpression of miR-17 or miR-18 could partly abrogated Myc-knockdown-induced MM cell apoptosis. One of the mechanism of Myc inhibiting MM cell apoptosis is through Myc activates miR-17-92 cluster and subsequently down-modulates proapoptotic protein Bim. Although miR-17-92 cluster are located at 13q31.3, the expression of miR-18, miR-19 and miR-20 (especially miR-19) in patients with del(13q14) was higher than those without del(13q14). Patients with miR-17, miR-20 and miR-92 high-expression had shorter PFS compared to those with miR-17, miR-20 and miR-92 low-expression. These results suggest the Myc-inducible miR-17-92 cluster miRNAs contribute to tumorigenesis and poor prognosis in multiple myeloma.

Distinctive IgVH gene segments usage and mutation status in Chinese patients with chronic lymphocytic leukemia

BACKGROUND AND OBJECTIVES The incidence of chronic lymphocytic leukemia (CLL) in Asian countries is lower than that in the Western ones, where CLL is the most common leukemia. It is a clinically heterogeneous disease, with survival ranging from a few months to decades. The mutation status of the immunoglobulin variable heavy chain (IgVH) gene has significantly improved prediction of the risk for disease progression. We investigated the frequency and mutation status of IgVH gene expression in Chinese patients with CLL. METHODS IgVH gene segments usage and mutation status were investigated by multiplex RT-PCR, and the relationship between IgVH somatic mutation status and the expression of CD38 and ZAP-70 was analyzed in 65 CLL patients. RESULTS Forty-five (69.2%) patients had mutated IgVH, and 20 (30.8%) had unmutated IgVH. The most frequently expressed VH gene family was found to be VH3 (47.7%) followed by VH4 (40%), VH1 (6.2%), VH2 (4.6%) and VH7 (1.5%), with no expression of VH5 or VH6 gene families. VH1-69 and VH3-21 which commonly overused in Western CLL were very low in our cohort. IgVH gene mutation status was significantly associated with the expression of CD38. CONCLUSIONS The frequency of IgVH gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. The expression of them may be simple and reliable surrogates for the identification of IgVH mutations.

Serum thymidine kinase 1 concentration in Chinese patients with chronic lymphocytic leukemia and its correlation with other prognostic factors

Chronic lymphocytic leukemia (CLL) shows a remarkable heterogeneity, with some patients having an almost normal lifespan, others surviving only several months after diagnosis despite intensive therapy. The aim of this study was to investigate the serum thymidine kinase 1 (TK1) concentration in Chinese patients with CLL and its correlation with well-established other prognostic factors. Enhanced chemiluminescent dot blot assay was performed to measure serum TK1 concentration in 80 CLL patients. The concentration of TK1 was significantly increased in patients with Binet C (P = 0.002), higher levels of serum lactate dehydrogenase (LDH) (P = 0.012) and β2-microglobulin (β2-MG) (P = 0.025), unmutated IGHV status (P < 0.001), or higher expression levels of ZAP-70 (P = 0.014) and CD38 (P = 0.018) groups compared to the patients with Binet A, lower levels of serum LDH and β2-MG, mutated IGHV status, or lower expression levels of ZAP-70 and CD38 groups, respectively. Strong correlation of TK1 level with IGHV mutations (r = 0.412, P < 0.001) or ZAP-70 (r = 0.263, P = 0.024) was observed. According to receiver operating characteristic curve analysis for serum TK1 concentration and IGHV mutational status, area under the curve was 0.757 (P = 0.001) and the optimal cut-off value of serum TK1 concentration level was 1.75 pM, with a 87.8% specificity, a 63.6% sensitivity. It was showed that serum TK1 concentration could be a predictive marker of IGHV mutational status, 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.

Clinical importance of different calreticulin gene mutation types in wild-type JAK2 essential thrombocythemia and myelofibrosis patients.

The Janus kinase 2 (JAK2) V617F mutation (JAK2 V617F), JAK2 exon 12 mutations and myeloproliferative leukemia virus oncogene W515L/K mutation (MPL W515L/K) have become three major molecular diagnosis criteria for myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) from 2005.1 However, diagnosing MPNs with non-mutated JAK2 and MPL remains a major diagnostic challenge.2–4 Some recent studies have reported calreticulin (CALR) gene mutations in patients with non-mutated JAK2 V617F MPNs.5–7 There are some distinct mutation types in MPN subtypes, but the differences in the clinical significance and prognosis among the different mutation types are obscure.8–10 Here, we report our data on CALR mutation in wild-type (wt) JAK2 MPN on patients. It should also be mentioned that this is undoubtedly the first report regarding CALR mutations in Chinese MPN patients. From January 2008 to December 2013, bone marrow or peripheral blood samples from 301 MPNs patients were collected in the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province, China, including ET (n=222), PV (n=37), PMF (n=33), post-ET MF (PET-MF; n=6), and post-PV MF (PPV-MF; n=3). We also obtained bone marrow samples from 174 patients with other myeloid neoplasms including: myelodysplastic syndrome (MDS; n=8), chronic myelogeneous leukemia (CML; n=55), acute myeloid leukemia (AML; n=104), and chronic myelomonocytic leukemia (CMML; n=7), as well as peripheral blood samples from 121 healthy controls. All participants provided their informed consent. Genomic PCR combined with direct and cloning sequencing was applied to screen CALR mutations. A total of 24.3% (73 of 301) patients with MPNs were found harboring CALR mutations. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of patients with ET and PMF, respectively (Figure 1A). Moreover, CALR mutations were found in 57.0% (69 of 121) ET patients with wt JAK2 and 30.8% (4 of 13) PMF patients with wt JAK2. No CALR mutation in patients with PV, PET-MF, PPV-MF (Figure 1A) was found. The CALR mutations have multiple deletions or insertions including: L367fs*46 (33 of 74; 44.6%), K385fs*47 (25 of 74; 33.8%), K368fs*51 (3 of 74; 4.1%), Q365fs*50 (3 of 74; 4.1%), E364fs*49 (2 of 74; 2.7%), K374fs*56 (2 of 74; 2.7%), L367fs*48 (1 of 74; 1.4%), Q365fs*48 (1 of 74; 1.4%), E364fs*55 (1 of 74; 1.4%), K375fs*48 (1 of 74; 1.4%), K375fs*55 (1 of 74; 1.4%), and K377fs*50 (1 of 74; 1.4%). Figure 1. (A) Frequency of CALR mutations in myeloid neoplasms and healthy control. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of ET and PMF patients, respectively. Approximately 1% of patients (1 of 104) with AML were found to harbor ... These patients with MPNs were simultaneously examined for the presence of other gene mutations. PV patients were screened for JAK2 V617F and JAK2 exon 12 mutations, while ET and MF patients were screened for JAK2 V617F and MPL W515L/K mutation. Among the total 301 patients with MPNs, 52.2% (157 of 301) were found to harbor JAK2 V617F mutation. Among the 222 patients with ET and 37 patients with PV, 0.9% (2 of 222) were found to harbor MPL W515L/K mutations and 2.7% (1 of 37) to harbor JAK2 exon 12 mutation, respectively (Figure 1B). JAK2 V617F, JAK2 exon 12 mutation, MPL W515L/K mutations and CALR mutations were found exclusively in these MPNs patients. We also screened CALR mutations in 104 AML patients, 55 CML patients, 7 CMML patients, and 8 MDS patients (including 5 refractory cytopenia with multilineage dysplasis, 2 refractory anemia with excess blasts, and one refractory anemia) to investigate whether CALR mutations were present in other myeloid neoplasms. Although most of these patients had negative results, one AML patient (59-years old, male, M2 subtype) was found to harbor CALR mutation (L367fs*46) without JAK2 V617F and MPL W515L/K mutations (Figure 1A). This patient had no previous history of MPN or MDS, Fms-related tyrosine kinase 3 internal tandem duplication, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog mutation, nucleophosmin mutation. CCAAT/enhancer binding protein alpha mutation was all negative and cytogenetics analysis showed normal karyotype. In addition, no CALR mutation was detected in the 121 healthy controls (Figure 1A). For mutation types, a total of 12 distinct variants of CALR mutation, including 11 deletions and one insertion, were identified in our patients. L367fs*46, which resulted from a 52-bp deletion, and K385fs*47, which resulted from a 5-bp insertion, were the most frequent CALR mutations. The two mutations accounted for 44.6% (33 of 74) and 33.8% (25 of 74) in all cases with mutant CALR, respectively. For ET patients, the two mutations were 14.0% (31 of 222) and 10.4% (23 of 222), respectively. For PMF patients, the two mutations were 3.0% (1 of 33) and 6.1% (2 of 33), respectively. There was no significant difference in the two mutation types between patients with ET and PMF (P=0.137 and P=0.645, respectively). Moreover, the two mutations were 44.9% (31 of 69) and 33.3% (23 of 69) in CALR mutation positive ET patients (Figure 1C), as well as 25% (1 of 4) and 50% (2 of 4) in CALR mutation positive PMF patients (P=0.793 and P=0.888, respectively). There were few other mutation types in the CALR-mutated samples (Figure 1C). ET patients with mutant CALR were significantly younger (P<0.001) and had lower white blood cell (WBC) counts (P<0.001), lower hemoglobin (Hb) levels (P=0.002), and higher platelet (PLT) counts (P<0.001) than patients with JAK2 V617F. No significant difference can be identified between ET patients with mutant CALR and JAK2 V617F in terms of sex and thrombotic events (Table 1). Similarly, PMF patients with mutant CALR showed lower Hb level (P=0.001) than JAK2 V617F. There was no significant difference in sex, age, WBC count, PLT counts or thrombotic events between PMF patients with mutant CALR and JAK2 V617F (Table 1). For different CALR mutations in ET patients, younger age (P=0.020), lower WBC count (P<0.001), and lower Hb level (P=0.002) were observed in CALR L367fs*46 than JAK2 V617F. In addition, ET patients with CALR K385fs*47 showed lower age (P<0.001), lower WBC counts (P<0.001), lower Hb levels (P=0.025) and higher PLT counts (P=0.005) than JAK2 V617F (Table 2). Table 1. Clinical features of essential thrombocythemia and primary myelofibrosis patients with CALR and JAK2 mutation. Table 2. Clinical characteristics of essential thrombocythemia patients with different types of CALR and JAK2 mutation. The overall survival (OS) rates of patients with ET and PMF were analyzed using the Kaplan-Meier curve. Longer OS was observed in ET and PMF patients with mutant CALR, but not wt CALR (P=0.511 and P=0.729, respectively) (Table 1 and Figure 1D). According to the risk stratification system in ET,11 there was a significant difference between patients with ET in the CALR-mutated group and JAK2 V617F mutant group or wt CALR group (both P<0.001) (Table 1). In summary, our data from this large cohort of Chinese patients with MPNs confirmed CALR mutations were novel molecular markers in wt JAK2 MPNs. It should always be noted that the combination of CALR, JAK2, and MPL W515L/K mutation analysis could contribute to the diagnosis of MPNs.5–7 Different CALR mutations of patients with MPNs had distinct clinical characteristics. Patients with the L367fs*46 and K385fs*47 mutations have shown a favorable prognosis, but further research is required to confirm this result. Given the relative proportion of MPN patients without JAK2/MPL/CALR mutations in our patient group, further investigation should be carried out to find novel molecular aberrations.

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.

STAT3 mutations are frequent in T-cell large granular lymphocytic leukemia with pure red cell aplasia

T-cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder and can cooccur in the context of pure red cell aplasia (PRCA). The aim of the current study was to analyze the signal transducer and activator of transcription 3 (STAT3) mutation status and its clinical significance in T-LGLL. We found STAT3 mutations in 21.4% of patients with T-LGLL. High β2-MG (β2-microglobulin) levels (P = 0.005), neutropenia (P = 0.018) and PRCA (P = 0.001) all displayed a significant association with STAT3 mutations. In univariate analysis, treatment-free survival (TFS) was affected by STAT3 mutation status (P = 0.008) and β2-MG (P = 0.006). Our results demonstrate the remarkable correlation of STAT3 mutation with PRCA, neutropenia and β2-MG.

Identification of the STAT5B‐RARα fusion transcript in an acute promyelocytic leukemia patient without FLT3, NPM1, c‐Kit and C/EBPα mutation

T(15;17) is the most common chromosomal aberration in patients with acute promyelocytic leukemia (APL), leading to the formation of PML‐RARα fusion gene. In a small subset of patients with APL, the RARα gene is fused with different partners. Here, we report a rare APL case with STAT5B‐RARα fusion transcript. Cytomorphologic and immunophenotypic analyses showed typical features of APL. However, cytogenetic analysis showed normal karyotype, and interphase fluorescence in situ hybridization (FISH) showed PML‐RARα negative. Quantitative RT‐PCR also showed PML‐RARα negative but STAT5B‐RARα positive and sequencing analysis confirmed the result. Molecular markers including FLT3, NPM1, c‐Kit and C/EBPα mutation were all negative. To our knowledge, this is the first APL patient with STAT5B‐RARα in Chinese population and the fifth patient around the world according to published paper.

Clinical significance of CSF3R , SRSF2 and SETBP1 mutations in chronic neutrophilic leukemia and chronic myelomonocytic leukemia

Chronic neutrophilic leukemia (CNL) and chronic myelomonocytic leukemia (CMML) are rare hematologic neoplasms. We performed CSF3R, SRSF2 and SETBP1 mutational analyses in 10 CNL and 56 CMML patients. In this sample cohort, 80% of CNL patients harbored CSF3R mutations, of which the CSF3R T618I mutation was dominant. Mutations in CSF3R and SETBP1 were found in 7.1% and 5.3% CMML patients respectively, while 25% of CMML patients carried SRSF2 mutations. Strikingly, we identified that all of the CSF3R mutations detected in CMML patients were represented by a P733T mutation. The CSF3R P733T mutation represents a novel CSF3R mutation. In addition, none of the four CSF3R P733T mutated patients carried SRSF2 mutations [0/14 (0%) patients with combined CSF3R P733T and SRSF2 mutations vs. 4/42 (9.5%) with CSF3R P733T and wt SRSF2, P < 0.001]. Both mut SRSF2 and mut SETBP1 patients had shorter overall survival (OS) and progression-free survival (PFS) compared to patients with wt SRSF2 (P < 0.001 both) and wt SETBP1 (P < 0.001 and P = 0.02, respectively). While we found no significant differences in OS and PFS as a consequence of CSF3R mutation status, our work suggest that the CSF3R T618I mutation is a diagnostic marker with good specificity and sensitivity for CNL. In conclusion, our study highlights effective diagnostic and prognostic markers of CNL and CMML patients in the Chinese population.

Heterogeneous leukemic clones identified by NPM1 mutation analysis in patient with acute monocytic leukemia

Abstract NPM1 mutation is the most common molecular abnormality in patients with acute myeloid leukemia (AML), especially normal karyotype AML (NK-AML), and is associated with a favorable prognosis in the absence of concomitant FLT3-ITD. Like other molecular abnormalities such as FLT3-ITD, C/EBPα and c-Kit mutation, NPM1 mutation normally presents as a recurrent molecular abnormality. The NPM1 mutation is generally used as a molecular marker in the prognosis evaluation of a patient with AML. Here, we report a different case. He was first diagnosed with NPM1 mutation-positive acute monocytic leukemia. However, he achieved no remission, but the NPM1 mutation dramatically became negative after induction chemotherapy. Finally, he achieved complete remission after salvage chemotherapy and the NPM1 mutation was still negative. To our knowledge, this is a rare case according to the worldwide published literature.