Guo-Rui Ruan

Expression patterns of WT1 and PRAME in acute myeloid leukemia patients and their usefulness for monitoring minimal residual disease.

Both WT1 and PRAME are highly expressed in acute myeloid leukemia (AML) patients. To assess the efficacy of their simultaneous detection for the purpose of monitoring minimal residual disease (MRD), we used real-time quantitative RT-PCR to quantify both WT1 and PRAME transcript levels in the bone marrow of 204 newly diagnosed AML patients, and 21 patients were serially monitored for a median of 11 months. The 22 normal bone marrow samples which served as controls collectively expressed low levels of WT1 and PRAME. An increase of >1-log over the upper limit of normal bone marrow was defined as positive. The positive rates of WT1 and PRAME for all patients were 79.2% and 55.4%, respectively. For the 108 patients lacking a specific fusion gene, the positive rate of WT1 was significantly higher than that of PRAME (76.9% vs. 35.2%, P<0.001). PRAME was positive in 9/25 WT1 (-) patients and the log increase of PRAME was higher than that of WT1 in 12/83 WT1 (+) patients. Dynamic patterns of WT1 and PRAME during follow-up showed that a consistent elevation or a rise over time to exceed the normal range predicted clinical relapse. The exception was that one patients WT1 significantly decreased at relapse compared to diagnosis. Therefore, a simultaneous detection of WT1 and PRAME might not only provide AML patients with either a positive or a more sensitive molecular marker for MRD monitoring. Moreover, it might also avoid false negativity in the case of expression alteration.

Different kinetic patterns of BCR-ABL transcript levels in imatinib-treated chronic myeloid leukemia patients after achieving complete cytogenetic response

Bone marrow BCR‐ABL transcript levels were monitored serially by real‐time quantitative PCR in 46 imatinib‐treated chronic myeloid leukemia patients after achieving complete cytogenetic response (CCyR) for a median of 42 months (range: 9–53). Of 41 patients in continuous CCyR, 32 and nine could achieve a ≥3‐log (MMoR group) or 2‐ to 3‐log reduction (non‐MMoR group), respectively. The MMoR group had a significantly lower recurrence rate of Ph+ metaphase than the non‐MMoR group (6/32 vs. 7/9, P = 0.002), which was not significantly different between patients first achieving CCyR within or after 12 months of imatinib treatment (7/27 vs. 6/14, P = 0.086). Five patients suffered cytogenetic or hematological relapse. For all 46 patients, a >2‐log reduction but not time when CCyR was first achieved was related to a lower relapse rate (1/42 vs. 4/4, P < 0.001). We concluded that the depth of BCR‐ABL reduction after CCyR is more critical than when CCyR is first achieved. The kinetic pattern of BCR‐ABL transcript is a good predictor of disease stability.

Two Single-Nucleotide Polymorphisms with Linkage Disequilibrium in the Human Programmed Cell Death 5 Gene 5′ Regulatory Region Affect Promoter Activity and the Susceptibility of Chronic Myelogenous Leukemia in Chinese Population

Purpose: Chronic myelogenous leukemia (CML) is a disease characterized cytogenetically by the presence of the Philadelphia chromosome. Recent studies suggested that altered PDCD5 expression may have significant implications in CML progression. The aim of this study was to identify single-nucleotide polymorphisms (SNP) within the programmed cell death 5 (PDCD5) promoter region and show their functional relevance to PDCD5 expression as well as their genetic susceptibility to CML. Experimental Design: One hundred twenty-nine CML subjects and 211 healthy controls were recruited for identification of SNPs and subsequent genetic analysis. Luciferase reporter assays were carried out to show the functional significance of the SNPs located in the promoter region to PDCD5 expression. Real-time quantitative PCR and Western blot analysis were done to determine the expression differences of PDCD5 in CML patients with different genotypes. Results: Two SNPs were identified within the PDCD5 promoter. They are −27A>G and −11G>A (transcription start site as position 1), respectively. The complete linkage disequilibrium was found between these two polymorphisms. The frequencies of −27G+/−11A+ genotype and −27G/−11A allele were significantly higher in CML patients than in healthy controls (genotype: 26.36% versus 11.85%, χ2=11.75, P < 0.01; allele: 13.57% versus 6.40%, χ2 = 9.48, P < 0.01). Luciferase reporter assays revealed that the promoter with −27G/−11A had significantly lower transcriptional activity and could not be up-regulated after apoptotic stimulations compared with the promoter with −27A/−11G. PDCD5 expression analysis in mononuclear cells derived from CML patients and cell lines with different −27/−11 genotypes showed consistent results with the reporter assays. Conclusions: These data suggest that −27G/−11A is associated with reduced PDCD5 promoter activity and increased susceptibility to CML.

Effect of stealthy liposomal topotecan plus amlodipine on the multidrug-resistant leukaemia cells in vitro and xenograft in mice

Background  Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy as the over‐expressed MDR protein acts as an efflux pump, which leads to a reduction in the uptake of the anticancer agent by tumour cells. We combined topotecan and amlodipine together into the stealthy liposomes, in which amlodipine was applied as a MDR reversing agent to overcome the resistance.

MPL W515L/K mutations in 343 Chinese adults with JAK2V617F mutation-negative chronic myeloproliferative disorders detected by a newly developed RQ-PCR based on TaqMan MGB probes.

Acquired mutations in the juxtamembrane region of MPL (W515L or W515K), the receptor for thrombopoietin, have been reported in patients with primary essential thrombocythemia (ET) or primary myelofibrosis (PMF). The mutations were detected by the newly developed real‐time quantitative PCR (RQ‐PCR) with TaqMan MGB probes and followed by the sequencing analysis. DNA samples were from 343 Chinese adults with JAK2V617F mutation‐negative chronic myeloproliferative disorders (cMPDs). Reference curves were obtained using cloned fragments of MPL containing either the wild‐type or MPL W515L or MPL W515K mutated sequence; the predicted sensitivity level was at least 0.5%(0.1–0.5%) for MPL W515L and 0.5%(0.2–0.5%) for MPL W515K mutant allele in a wild‐type background. The detection rates of MPL W515 mutations were 3.5% in 199 ET patients (7/199), 12.5% in 24 PMF patients (3/24) and 5.6% in 36 cMPD‐unclassed patients (2/36), respectively. No MPL W515 mutations were detected in 32 polycythemia vera (PV) patients, 40 chronic myeloid leukaemia (CML) patients, 12 hypereosinophilic syndrome (HES) patients and 29 normal volunteers. The mean calculated burden of MPL mutant alleles using RQ‐PCR for MPL W515L/K was 24.88 ± 14.80% (range, 1.10–56.32%). MPL W515L/K patients presented lower haemoglobin levels, compared with the patients with JAK2V617F mutation‐positive cMPDs (p < 0.01). The results demonstrated that RQ‐PCR was a reliable and sensitive method for large‐scale screening of the MPL W515L/K mutation in patients suspected to have a cMPD. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy. Copyright

CD34 expression on bone marrow blasts is a novel predictor of poor prognosis independent of FlT3-ITD in acute myeloid leukemia with the NPM1-mutation

To explore the prognostic value of CD34 expression in NPM1-mutated acute myeloid leukemia (NPM1m+AML), seventy-one NPM1m+AML patients were retrospectively analyzed. The patients with >7% CD34 expression (according to the ROC analysis) had a lower complete remission (CR) rate after 1 course of induction, disease-free survival (DFS), and overall survival (OS) compared to those with ≤7% CD34 expression (p=0.0038; p=0.001; p<0.0001). A multivariate analysis revealed that CD34 expression is a prognostic factor that is independent of FlT3-ITD for relapse, DFS and OS. We established a novel prognostic model based on the CD34 and FLT3 status at diagnosis, which could facilitate the segregation of patients into three prognostically different subgroups. We demonstrate that CD34 expression on blasts is a novel, poor predictor independent of FlT3-ITD in NPM1-mutated patients and established a new prognostic model based on the CD34 and FLT3 status at diagnosis, which may facilitate immediate clinical utility for prognostic stratification and risk-adapted therapeutic choices.

Aberrant expression of CKLF-like MARVEL transmembrane member 5 (CMTM5) by promoter methylation in myeloid leukemia

CMTM5 has been shown to exhibit tumor suppressor activities, however, its role in leukemia is unclear. Herein we firstly reported the expression and function of CMTM5 in myeloid leukemia. CMTM5 was down-regulated, or undetectable, in leukemia cell lines and bone marrow cells from leukemia patients with promoter methylation. Ectopic expression of CMTM5-v1 strongly inhibited the proliferation of K562 and MEG-01 cells. In addition, significant negative correlations were observed between CMTM5 and three leukemia-specific fusion genes (AML1-ETO, PML-RARα and BCR/ABL1). CMTM5 expression was up-regulated in patients who had undergone treatment. Therefore, CMTM5 may be involved in the pathomechanism of myeloid leukemias.

NPM1-mutated acute myeloid leukemia of monocytic or myeloid origin exhibit distinct immunophenotypes.

Acute myeloid leukemia with mutated nucleophosmin (NPM1m+AML) is a heterogeneous entity. We investigated whether NPM1m+AML with monocytic or myeloid differentiation have distinct immunophenotype. The study included 160 NPM1m+AMLpatients and 178 AML patients without NPM1 mutation and recurrent cytogenetic abnormality (NPM1wt-AML). We analyzed the immunophenotype by flow cytometry. NPM1 mutation was detected by PCR. Compared with NPM1wt-AML patients, NPM1m+AML patients showed higher positive rates of CD33 and CD9 and lower positive rates of CD34, HLA-DR, CD7, CD15 and CD117 (all P<0.05). HLA-DR, CD64, CD14, CD11b, CD15, CD4, CD9 and CD10 were higher (P<0.001) and CD117 was lower (P<0.01) in monocytic NPM1m+AML compared with myeloid NPM1m+AML. Similar rates of lymphoid antigen (CD19, CD2, and CD7) and myeloid antigen (CD13, CD33) positivity were detected in monocytic and myeloid NPM1m+AML. Compared with NPM1wt-AML, CD34 expression was lower both in myeloid and monocytic NPM1m+AML subgroups, although HLA-DR was lower in NPM1m+AML compared with NPM1wt-AML only in myeloid subgroup. Comparisons of NPM1m+AML and NPM1wt-AML showed no differences in monocyte-associated markers such as CD14 and CD11b in myeloid and monocytic subgroup. Myeloid NPM1m+AML correlated with the female gender (P=0.001), lower WBC counts (P=0.04) and higher WT1 transcripts (P=0.006) compared with monocytic NPM1m+AML.These results suggested monocytic and myeloid-derived NPM1m+AML exhibit distinct immunophenotypes.

Frequency and allele burden of CALR mutations in Chinese with essential thrombocythemia and primary myelofibrosis without JAK2V617F or MPL mutations

CALR mutations are detected in about 50% of persons of predominately European descent with essential thrombocythemia (ET) or primary myelofibrosis (PMF) with wild-type alleles of JAK2 and MPL. We studied 1088 Chinese with diverse myeloproliferative neoplasms including ET (N=234) and PMF (N=50) without JAK2(V617F) or MPL exon 10 mutations. CALR mutation was detected in 53% (95% CI, 46-60%) of subjects with ET and 56% (95% CI, 41-70%) of subjects with PMF. 152 CALR mutations were identified clustering into 15 types including deletions (N=8), insertions (N=3) and complex indels (N=4). We also identified 9 new mutations. Mean (±SD) mutant allele burden was 31±12% (range, 0.5-69%). Persons with PMF had higher CALR mutant allele burdens than those with ET (38±8% vs. 29±12%; P<0.001). Amongst persons with CALR mutations, those with PMF had different clinical features from those with ET. These data may be useful for diagnosing ET and PMF in Chinese who are about 40% of all persons with ET and PMF and for monitoring therapy-response. They also highlight similarities and differences in CALR mutations between Chinese and persons of predominately European descent with these diseases.

A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms

Abstract Background Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed. Methods We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. Results We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5–5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5–5%) in a wild-type background. Conclusions PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.