In JoVE (1)

Other Publications (29)

Articles by Wanlong Ma in JoVE

Other articles by Wanlong Ma on PubMed

Plasma RNA As an Alternative to Cells for Monitoring Molecular Response in Patients with Chronic Myeloid Leukemia

Haematologica. Feb, 2007  |  Pubmed ID: 17296565

Quantitation of BCR-ABL mRNA is emerging as the standard of care to monitor the status of chronic myeloid leukemia (CML). Peripheral blood plasma was analyzed in this study because of previous detection of nucleic acids and proteins from tumor cells in plasma samples.

Bi-clonal Disease in Patients with Chronic Lymphocytic Leukaemia As Detected by Analysing IGHV Mutation Status

British Journal of Haematology. Nov, 2007  |  Pubmed ID: 17868045

Enzymatic Activity of Circulating Proteasomes Correlates with Clinical Behavior in Patients with Chronic Lymphocytic Leukemia

Cancer. Mar, 2008  |  Pubmed ID: 18224667

The ubiquitin-proteasome pathway has been implicated in the pathogenesis of many hematologic malignancies.

Higher Detection Rate of JAK2 Mutation Using Plasma

Blood. Apr, 2008  |  Pubmed ID: 18362222

BCR-ABL Alternative Splicing As a Common Mechanism for Imatinib Resistance: Evidence from Molecular Dynamics Simulations

Molecular Cancer Therapeutics. Dec, 2008  |  Pubmed ID: 19056677

Rare cases of chronic myelogenous leukemia (CML) express high levels of alternatively spliced BCR-ABL mRNA with a 35-bp insertion (35INS) between ABL kinase domain exons 8 and 9. This insertion results in a frameshift leading to the addition of 10 residues and truncation of 653 residues due to early termination. Sensitive PCR-based testing showed that 32 of 52 (62%) imatinib-resistant CML patients in chronic phase and 8 of 38 (21%) in accelerated or blast crisis expressed varying levels of the alternatively spliced BCR-ABL mRNA. A three-dimensional structural model of the 35INS ABL kinase domain complexed with imatinib was built using homology modeling, followed by molecular dynamics simulations. Simulation results showed that the new residues cause a significant global conformational change, altering imatinib binding in a way similar to that of the T315I mutation and, therefore, providing resistance to imatinib that depends on the level of expression.

Mutation Profile of JAK2 Transcripts in Patients with Chronic Myeloproliferative Neoplasias

The Journal of Molecular Diagnostics : JMD. Jan, 2009  |  Pubmed ID: 19074595

Here, we describe the JAK2 mutation profile in a series of approximately 20,000 blood samples from patients with clinically suspected myeloproliferative neoplasias. Using a sensitive reverse transcription-PCR and direct sequencing approach on RNA rather than DNA, we detected JAK2 mutations in exons 12-15 in approximately 20% of these patients. We identified new mutations in addition to the known V617F and exon 12 mutations, which were the most common. Most of the novel mutations are located in the pseudokinase domain and therefore are expected to relieve the autoinhibitory function of this domain on JAK2 kinase activity. Our data suggest that molecular testing of JAK2 mutations should not be restricted to the V617F and exon 12 mutations, but perhaps should extend to most of the pseudokinase domain coding region as well. Furthermore, mutation screening using RNA is highly sensitive and could replace DNA-based testing because of the relative abundance of target transcripts and the ease in detecting deletion of the entire exon.

Mechanisms of Constitutive Activation of Janus Kinase 2-V617F Revealed at the Atomic Level Through Molecular Dynamics Simulations

Cancer. Apr, 2009  |  Pubmed ID: 19195039

The tyrosine kinase Janus kinase 2 (JAK2) is important in triggering nuclear translocation and regulation of target genes expression through signal transducer and activator of transcription pathways. The valine-to-phenylalanine mutation at amino acid 617 (V617F), which results in the deregulation of JAK2, has been implicated in the oncogenesis of chronic myeloproliferative disease. However, both the mechanism of JAK2 autoinhibition and the mechanism of V617F constitutive activation remain unclear.

B-cell Clones As Early Markers for Chronic Lymphocytic Leukemia

The New England Journal of Medicine. Feb, 2009  |  Pubmed ID: 19213679

Otherwise healthy persons with a small number of B-cell clones circulating in the peripheral blood have been designated as having monoclonal B-cell lymphocytosis (MBL). Hospital-based series indicate an excess risk of progression from MBL to chronic lymphocytic leukemia (CLL). In this prospective cohort study, we tested the hypothesis that CLL is always preceded by MBL.

K-ras Mutations and Cetuximab in Colorectal Cancer

The New England Journal of Medicine. Feb, 2009  |  Pubmed ID: 19238675

Detection of Nucleophosmin Gene Mutations in Plasma from Patients with Acute Myeloid Leukemia: Clinical Significance and Implications

Cancer Biomarkers : Section A of Disease Markers. 2009  |  Pubmed ID: 19242062

Roughly one-third of acute myeloid leukemia (AML) patients exhibit mutations in the nucleophosmin (NPM1) gene, and multiple studies have linked these mutations with a more favorable clinical outcome. We developed an assay for the detection of NPM1 mutations in peripheral blood plasma, and compared the results with clinical outcomes from a single institution. Analyzing plasma from previously untreated AML patients revealed NPM1 insertion mutations in 24 of 98 (24%) patients, with greater sensitivity than existing peripheral blood cell-based tests which showed positivity in only 22 of the 24 patients. Plasma testing allowed the detection of a novel 4 bp deletion in NPM1 in one patient. Analysis of clinical data corroborated previous data linking NPM1 mutations with better clinical outcome. These data underline the significance of NPM1 in the biology and clinical behavior of AML, and demonstrate the reliability and efficacy of plasma-based testing for NPM1 mutations.

BCR-ABL Truncation Due to Premature Translation Termination As a Mechanism of Resistance to Kinase Inhibitors

Acta Haematologica. 2009  |  Pubmed ID: 19332983

Proteasome Enzymatic Activities in Plasma As Risk Stratification of Patients with Acute Myeloid Leukemia and Advanced-stage Myelodysplastic Syndrome

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Jun, 2009  |  Pubmed ID: 19458051

Cytogenetic abnormalities are currently the most important predictors of response and clinical outcome for patients with acute myeloid leukemia (AML) or advanced-stage myelodysplastic syndrome (MDS). Because clinical outcomes vary markedly within cytogenetic subgroups, additional biological markers are needed for risk stratification.

Structural Effects of Clinically Observed Mutations in JAK2 Exons 13-15: Comparison with V617F and Exon 12 Mutations

BMC Structural Biology. Sep, 2009  |  Pubmed ID: 19744331

The functional relevance of many of the recently detected JAK2 mutations, except V617F and exon 12 mutants, in patients with chronic myeloproliferative neoplasia (MPN) has been significantly overlooked. To explore atomic-level explanations of the possible mutational effects from those overlooked mutants, we performed a set of molecular dynamics simulations on clinically observed mutants, including newly discovered mutations (K539L, R564L, L579F, H587N, S591L, H606Q, V617I, V617F, C618R, L624P, whole exon 14-deletion) and control mutants (V617C, V617Y, K603Q/N667K).

Circulating Ki-67 Protein in Plasma As a Biomarker and Prognostic Indicator of Acute Lymphoblastic Leukemia

Leukemia Research. Feb, 2010  |  Pubmed ID: 19679351

Tissue-based determination of Ki-67, a marker of cellular proliferation, has shown prognostic value in solid tumors and hematological malignancies. We developed and validated an electrochemiluminescence-based method for sensitive measurement of circulating Ki-67 in plasma (cKi-67). This assay demonstrated significantly higher levels of cKi-67 in patients with newly diagnosed acute lymphoblastic leukemia (ALL) (n=27; median, 762; range, 0-4574U/100 microL) than in healthy control subjects (n=114; median, 399; range, 36-2830U/100 microL). Moreover, elevated plasma cKi-67 was associated with significantly shorter survival in ALL patients (P=0.05). These findings suggest that Ki-67 can be detected in circulation and has potential for use as a biomarker for predicting clinical behavior in ALL.

Circulating Ki-67 Index in Plasma As a Biomarker and Prognostic Indicator in Chronic Lymphocytic Leukemia

Leukemia Research. Oct, 2010  |  Pubmed ID: 20362333

Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle, except G(0), and is widely used as a marker of cellular proliferation in human tumors. We recently showed that elevated levels of Ki-67 circulating in plasma (cKi-67) are associated with shorter survival in patients with acute lymphoblastic leukemia. The current study included 194 patients with CLL and 96 healthy control subjects. cKi-67 levels in plasma were determined using an electrochemiluminescent immunoassay. We normalized the cKi-67 level to the absolute number of lymphocytes in the patient's peripheral blood to establish the plasma cKi-67 index. The cKi-67 index showed significant correlation with lymph node involvement and Rai stage (P=0.05). Higher cKi-67 index values were significantly associated with shorter survival. Multivariate Cox proportional hazards regression analysis demonstrated that the association of the cKi-67 index with shorter survival was independent of IgV(H) mutation status. In a multivariate model incorporating the cKi-67 index with B2M and IgV(H), only cKi-67 index and B2M levels remained as independent predictors of survival. The results of this study suggest that the plasma cKi-67 index, along with B2M level, is a strong predictor of clinical behavior in CLL.

JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

PloS One. Aug, 2010  |  Pubmed ID: 20730051

The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Deltaexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing.

Significant Association Between Polymorphism of the Erythropoietin Gene Promoter and Myelodysplastic Syndrome

BMC Medical Genetics. Nov, 2010  |  Pubmed ID: 21078205

Myelodysplastic syndrome (MDS) may be induced by certain mutagenic environmental or chemotherapeutic toxins; however, the role of susceptibility genes remains unclear. The G/G genotype of the single-nucleotide polymorphism (SNP) rs1617640 in the erythropoietin (EPO) promoter has been shown to be associated with decreased EPO expression. We examined the association of rs1617640 genotype with MDS.

Ubiquitin-proteasome System Profiling in Acute Leukemias and Its Clinical Relevance

Leukemia Research. Apr, 2011  |  Pubmed ID: 20951430

The ubiquitin-proteasome system (UPS) plays a major role in the homeostasis of cellular protein. We demonstrate that each of the major hematologic diseases (AML, ALL, and MDS) has a specific and different plasma profile of UPS protein and enzymatic activities. While high levels of proteasome and ubiquitin proteins and enzymatic activities are detected in the plasma samples from patients, normalizing enzymatic activities, show that each proteasome has lower enzymatic activities in these diseases as compared with normal controls. Proteasome protein levels in AML are strong predictor of survival independently of cytogenetics, performance status and age. The Ch-L activity when normalized to the level of proteasome protein show significant negative correlation with survival in ALL.

MPL Mutation Profile in JAK2 Mutation-negative Patients with Myeloproliferative Disorders

Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B. Mar, 2011  |  Pubmed ID: 21326037

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Ubiquitin-proteasome Profiling for Enhanced Detection of Hepatocellular Carcinoma in Patients with Chronic Liver Disease

Journal of Gastroenterology and Hepatology. Apr, 2011  |  Pubmed ID: 21418304

A reliable test for the detection of hepatocellular carcinoma (HCC) could improve disease management. Recent reports suggested a link between abnormalities in the ubiquitin-proteasome system (UPS) and HCC. We investigated the potential of using UPS markers, along with HCC markers, to differentiate HCC from chronic liver disease (CLD).

Effects of Clinically Relevant MPL Mutations in the Transmembrane Domain Revealed at the Atomic Level Through Computational Modeling

PloS One. 2011  |  Pubmed ID: 21858098

Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations.

BCR-JAK2 Fusion As a Result of a Translocation (9;22)(p24;q11.2) in a Patient with CML-like Myeloproliferative Disease

Molecular Cytogenetics. May, 2012  |  Pubmed ID: 22549126

Translocation (9;22)(q34;q11.2) resulting in BCR/ABL1 fusion at the molecular level is the hallmark of chronic myelogenous leukemia (CML). Variants of the Philadelphia translocation and complex translocations involving BCR have been reported in myeloproliferative disorders (MPD). A rare translocation, t(9;22)(p24;q11.2), resulting in a novel BCR-JAK2 fusion has been reported in a handful of cases of CML and acute myelogenous leukemia (AML). We present clinical-pathological and cytogenetic evaluation of a patient with Philadelphia-chromosome negative CML/MPD harboring a t(9;22)(p24;q11.2) resulting in BCR-JAK2 fusion. Fluorescence in situ hybridization and molecular characterization of the translocation confirmed a BCR-JAK2 fusion and helped delineate the breakpoints upstream of exon 1 of minor cluster region of BCR gene and likely intron 18 of the JAK2 gene, resulting in an in-frame transcript This case provides convincing support, along with two previous case-reports, for a role for activation of the Janus kinase 2 in evolution of myeloproliferative disease. The recurrent, albeit rare, nature of the breakpoints within BCR and JAK2 suggests a potential new diagnostic target that should be interrogated in Ph-negative CML/MPD patients.

Plasma Ubiquitin-proteasome System Profile in Patients with Multiple Sclerosis: Correlation with Clinical Features, Neuroimaging, and Treatment with Interferon-beta-1b

Neurological Research. Jul, 2012  |  Pubmed ID: 22709658

Interferon-beta-1b (IFN-beta-1b) reduces relapses in multiple sclerosis (MS) and improves magnetic resonance imaging (MRI) outcomes. Mechanism of action of IFN-beta-1b is only marginally understood. The roles and plasma levels of factors within the ubiquitin-proteasome system (UPS) and the plasma proteasome enzymatic activity of MS patients have not been explored. We hypothesized that pharmacologic double inhibition of the UPS by IFN-beta-1b occurs in MS patients and contributes to improvement of clinical course and reduction in MRI activity.

Diagnostic and Prognostic Scoring System for Prostate Cancer Using Urine and Plasma Biomarkers

Genetic Testing and Molecular Biomarkers. Mar, 2014  |  Pubmed ID: 24512523

To avoid relying solely on serum prostate-specific antigen (sPSA) in screening for prostate cancer (PCa), we developed a scoring system for detecting PCa and the prediction of aggressiveness. We analyzed urine and plasma specimens from 121 patients with PCa or benign prostatic hyperplasia (BPH) for the levels of UAP1, PDLIM5, IMPDH2, HSPD1, PCA3, PSA, TMPRSS2, ERG, GAPDH, and B2M genes. Patient age, sPSA level, and polymerase chain reaction data were entered through multiple algorithms to determine models most useful for the detection of cancer and predicting aggressiveness.

Synonymous Polymorphisms in HOXB13 As a Protective Factor for Prostate Cancer

Journal of Cancer. 2015  |  Pubmed ID: 25874003

Genomic association and linkage studies, as well as epidemiological data have implicated both the HOXB13 gene and single nucleotide polymorphisms (SNPs) in the development of prostate cancer (PCa). The recent association between the G84E polymorphism in the HOXB13 gene and PCa has been shown to result in a more aggressive cancer with an earlier onset of the disease. We examined the frequency of this mutation and other recurrent HOXB13 SNPs in patients with PCa and those with benign prostatic hyperplasia (BPH) or no cancer.

Predicting Prostate Biopsy Results Using a Panel of Plasma and Urine Biomarkers Combined in a Scoring System

Journal of Cancer. 2016  |  Pubmed ID: 26918043

Determining the need for prostate biopsy is frequently difficult and more objective criteria are needed to predict the presence of high grade prostate cancer (PCa). To reduce the rate of unnecessary biopsies, we explored the potential of using biomarkers in urine and plasma to develop a scoring system to predict prostate biopsy results and the presence of high grade PCa.

Deep Sequencing of Cell-Free Peripheral Blood DNA As a Reliable Method for Confirming the Diagnosis of Myelodysplastic Syndrome

Genetic Testing and Molecular Biomarkers. Jul, 2016  |  Pubmed ID: 27248906

Demonstrating the presence of myelodysplastic syndrome (MDS)-specific molecular abnormalities can aid in diagnosis and patient management. We explored the potential of using peripheral blood (PB) cell-free DNA (cf-DNA) and next-generation sequencing (NGS).

Resistance to Imatinib in Patients with Chronic Myelogenous Leukemia and the Splice Variant BCR-ABL1(35INS)

Leukemia Research. Oct, 2016  |  Pubmed ID: 27658269

In patients with chronic myelogenous leukemia (CML), point mutations in the BCR-ABL1 kinase domain are the most common cause of treatment failure with a tyrosine kinase inhibitor (TKI). It is not clear whether the splice variant BCR-ABL1(35INS) is also associated with treatment failure.

Using High-sensitivity Sequencing for the Detection of Mutations in BTK and PLCγ2 Genes in Cellular and Cell-free DNA and Correlation with Progression in Patients Treated with BTK Inhibitors

Oncotarget. Feb, 2017  |  Pubmed ID: 28212557

Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

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