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1.  Circulating heat shock protein 70 and progression in patients with chronic myeloid leukemia 
Leukemia research  2008;33(2):212-217.
We evaluated the association of circulating levels of heat shock protein 70 (Hsp70) in plasma with clinical behavior and progression in 139 chronic myeloid leukemia (CML) patients. Circulating Hsp70 levels did not differ significantly between CML patients in the chronic phase (n = 93; median 33.24 ng/mL, range 3.89–128.2 ng/mL) and those in the accelerated/blast phase (n = 46; median 26.57 ng/mL, range 4.5–114.7 ng/mL). However, overall CML patients had significantly higher levels of Hsp70 than healthy subjects (n = 95, median 4.17 ng/mL, range 1.75–24.7 ng/mL) (P < 0.001). In chronic phase CML patients, Hsp70 levels above the median were associated with a higher rate of progression to the accelerated/blast phase and a tendency toward shorter survival. Plasma Hsp70 thus could be a potential marker for predicting disease progression in patients with chronic phase CML.
PMCID: PMC4163801  PMID: 18715642
Circulating heat shock protein; Chronic myeloid leukemia; Imatinib resistance
2.  Clinical Correlation of Circulating Heat Shock Protein 70 in Acute Leukemia 
Leukemia research  2009;34(5):605-609.
The heat shock protein 70 (HSP70) is one of the molecular chaperone family involved in the protection of cells upon exposure to various types of stresses. Plasma circulating HSP70 (cHSP70) is believed to play a role in the anti-tumor immune responses and its levels may reflect the levels of severity or the disease condition. Using electrochemiluminescence protein detection immunoassay, we measured the cHSP70 levels in the plasma of patients with acute myeloid leukemia (AML) (n=96), myelodysplastic syndrome (MDS) (n=28), and acute lymphoblastic leukemia (ALL) (n=40) and compared with those in normal individuals (n=99). cHSP70 levels were significantly higher in AML (median: 10.71 ng/mL, range: 1.93-79.0 ng/mL) and ALL (median: 27.59 ng/mL, range: 5.09-129.6 ng/mL) as compared to those in MDS (median: 4.54 ng/mL, range: 1.35-58.3 ng/mL) or healthy controls (median: 4.13 ng/mL, range: 1.75-13.6 ng/mL). Levels of cHSP70 showed significant positive correlation with lactate dehydrogenase (LDH) and white blood cells (WBC) in AML and ALL patients, which may reflect overall tumor load. Furthermore, patients with higher levels of cHSP70 had significantly shorter survival in AML (P=0.04) and ALL (P=0.05), suggesting that in these two acute diseases, cHSP70 is an indicator for poor prognosis. Our data support the potential of using free cHSP70 as a biomarker in leukemias and potentially other types of cancers.
PMCID: PMC4127889  PMID: 19800118
circulating heat shock protein; acute leukemia; survival
3.  Effects of Clinically Relevant MPL Mutations in the Transmembrane Domain Revealed at the Atomic Level through Computational Modeling 
PLoS ONE  2011;6(8):e23396.
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.
Transmembrane (TM) domain predictions, homology modeling, ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL's kinase partner, JAK2.
Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation (azimuthal) angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems.
PMCID: PMC3157383  PMID: 21858098
4.  Significant association between polymorphism of the erythropoietin gene promoter and myelodysplastic syndrome 
BMC Medical Genetics  2010;11:163.
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.
We genotyped the EPO rS1617640 SNP in 189 patients with MDS, 257 with acute myeloid leukemia (AML), 106 with acute lymphoblastic leukemia, 97 with chronic lymphocytic leukemia, 353 with chronic myeloid leukemia, and 95 healthy controls.
The G/G genotype was significantly more common in MDS patients (47/187; 25.1%) than in controls (6/95; 6.3%) or in patients with other leukemias (101/813; 12.4%) (all P < 0.001). Individuals with the G/G genotype were more likely than those with other genotypes to have MDS (odd ratio = 4.98; 95% CI = 2.04-12.13). Clinical and follow up data were available for 112 MDS patients and 186 AML patients. There was no correlation between EPO promoter genotype and response to therapy or overall survival in MDS or AML. In the MDS group, the GG genotype was significantly associated with shorter complete remission duration, as compared with the TT genotype (P = 0.03). Time to neutrophils recovery after therapy was significantly longer in MDS patients with the G/G genotype (P = 0.02).
These findings suggest a strong association between the rs1617640 G/G genotype and MDS. Further studies are warranted to investigate the utility of screening for this marker in individuals exposed to environmental toxins or chemotherapy.
PMCID: PMC2992491  PMID: 21078205
5.  Detection, Analysis and Clinical Validation of Chromosomal Aberrations by Multiplex Ligation-Dependent Probe Amplification in Chronic Leukemia 
PLoS ONE  2010;5(10):e15407.
Current diagnostic screening strategies based on karyotyping or fluorescent in situ hybridization (FISH) for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are laborious, time-consuming, costly, and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA) can simultaneously detect copy number changes of multiple loci in one simple PCR reaction, making it an attractive alternative to FISH. To enhance the clinical robustness and further harness MLPA technology for routine laboratory operations, we have developed and validated a protocol for comprehensive, automatic data analysis and interpretation. A training set of 50 normal samples was used to establish reference ranges for each individual probe, for the calling of statistically significant copy number changes. The maximum normal ranges of 2 and 3 standard deviations (SD) are distributed between 0.82 and 1.18 (Mean ± 2SD, 95% CI, P = 0.05), and between 0.73 and 1.27 (Mean ± 3SD, 99% CI, P = 0.01), respectively. We found an excellent correlation between MLPA and FISH with 93.6% concordance (P<0.0001) from a testing cohort of 100 clinically suspected CLL cases. MLPA analyses done on 94/100 patients showed sensitivity and specificity of 94.2% and 92.9%, respectively. MLPA detected additional copy number gains on 18q21.1 and chromosome 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci in six samples. Three FISH-failed samples were tested positive by MLPA, while three 13q- cases with a low percentage of leukemia cells (7%, 12% and 19%) were not detected by MLPA. The improved CLL MLPA represents a high-throughput, accurate, cost-effective and user-friendly platform that can be used as a first-line screening test in a clinical laboratory.
PMCID: PMC2963645  PMID: 21049055
6.  JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms 
PLoS ONE  2010;5(8):e12165.
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 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing.
Methodology/Principal Findings
We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects.
These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for testing.
PMCID: PMC2921382  PMID: 20730051

Results 1-7 (7)