Effects of clinically relevant MPL mutations in the transmembrane domain revealed at the atomic level through computational modeling.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Multiplex ligation-dependent probe amplification for detection of chromosomal abnormalities in myelodysplastic syndrome and acute myeloid leukemia.

Current strategies for detecting chromosome abnormalities in MDS/AML include FISH or traditional cytogenetics. MLPA detects abnormalities in multiple loci simultaneously, with higher resolution and throughput. Peripheral blood from 50 healthy subjects was used to establish probe-specific reference ranges, increasing MLPA sensitivity and specificity. MLPA was then performed on 110 FISH-tested blood or bone marrow samples from suspected leukemia patients. Our novel MLPA analysis system combined maximum stringency with sensitive detection of low-frequency abnormalities. Accuracy/specificity of MLPA were excellent compared to FISH. Our MLPA analysis/interpretation method provides a clinically robust, high-throughput, high-resolution option for detection of abnormalities associated with MDS/AML.

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

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.

Significant association between polymorphism of the erythropoietin gene promoter and myelodysplastic syndrome.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Detection, analysis and clinical validation of chromosomal aberrations by multiplex ligation-dependent probe amplification in chronic leukemia.

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.

JAK2 exon 14 deletion in patients with chronic myeloproliferative neoplasms.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the possibility that MPN patients may express the JAK2 Deltaexon14 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 Deltaexon14 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 Deltaexon14 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 Deltaexon14 mRNA expressed a corresponding truncated JAK2 protein. The Deltaexon14 variant was not detected in the 46 control subjects. CONCLUSIONS/SIGNIFICANCE: These data suggest that expression of the JAK2 Deltaexon14 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.

Quantification of intracellular proteins and monitoring therapy using flow cytometry.

Here we review phospho-specific, quantitative flow cytometry approach as a rapid and reliable tool for measuring intracellular signaling proteins with potential applications in monitoring efficacy of targeted therapy. The single cell, multiparameter nature of flow cytometry allows simultaneous investigation of specific cell type and the corresponding intracellular markers. Peripheral blood can be directly stained with surface markers to delineate cell populations of interest, followed by fixation, permeabilization, and immunostaining with specific antibodies to the cellular targets. By using calibrated standardized phycoerythrin (PE)-conjugated beads for signal quantification, an informative Index value can be generated for each sample by multiplication of percentages of positive cells with fluorescence intensity per cell. This technique can yield both qualitative and quantitative information on effects of cellular markers upon targeted therapy, thereby providing another layer of advantages over the conventional flow cytometry analysis. Advances in this technology: high-throughput capability and automation, making it a valuable platform in modern drug discovery.

Circulating Ki-67 index in plasma as a biomarker and prognostic indicator in chronic lymphocytic leukemia.

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.

Circulating CD33 and its clinical value in acute leukemia.

OBJECTIVE: CD33 is a cell surface antigen for committed myelomonocytic lineage. We explored the potential of detecting CD33 as cell-free circulating protein in patients with leukemia. MATERIALS AND METHODS: We developed a quantitative bead-based immunoflow cytometry assay to measure cell-free circulating CD33 (cCD33) levels in the plasma of patients with acute leukemia, and correlated these results with corresponding clinical behavior. We measured cCD33 levels in the plasma of 48 healthy subjects and in patients with acute myelogenous leukemia (n = 98), acute lymphoblastic leukemia (n = 46), myelodysplastic syndrome (MDS) (n = 50), and myeloproliferative disorder (n = 49). RESULTS: Patients with acute myeloid leukemia and myeloproliferative disorders had significantly higher concentrations of cCD33 than the other patient groups and normal individuals (p = 0.0001), and among these groups, MDS patients displayed the lowest cCD33 levels (p = 0.02). Circulating CD33 values correlated positively with the CD33(+) blast cell counts in these patients. While there was no correlation between cCD33 levels and survival in acute myelogenous leukemia and MDS, higher cCD33 plasma concentrations did correlate with shorter survival in acute lymphoblastic leukemia (p = 0.03), and with shorter complete remission duration in acute myelogenous leukemia (p = 0.04) and MDS (p = 0.03). CONCLUSION: Circulating CD33 can be detected in the plasma from patients with leukemias, and cCD33 levels may have clinical implication, e.g., predictive and prognostic value, in these patients.

Plasma tyrosine kinase activity as a potential biomarker in BCR-ABL1-targeted therapy.

In targeted therapy using tyrosine kinase inhibitors (TKIs), measurement of TK activities could be beneficial for diagnosis, identification of potential responders, and monitoring treatment efficacy. Here we evaluated the utility of measuring circulating TK (cTK) activity directly from plasma in leukemia patients positive for the BCR-ABL1. Plasma cTK activity was measured from 46 patients with newly diagnosed chronic myelogenous leukemia (CML), 24 with multidrug-resistant CML, 24 with BCR-ABL1-positive acute lymphocytic leukemia (ALL), and 38 healthy donors. Circulating TK activity was significantly higher in CML (median 801.93 U/mL, range 18.10-3932.30 U/mL) and BCR-ABL1-positive ALL patients (median 659.55 U/mL, range 0-1626.90 U/mL) than in healthy donors (median 82.85 U/mL, range 0.63-852.80 U/mL) (P < 0.001). Plasma cTK activity was closely correlated with cellular BCR-ABL1 kinase activation as indicated by phosphorylation of the downstream signaling proteins CRKL (P < 0.001) and STAT-5 (P= 0.003). However, cTK activity was not associated with BCR-ABL1 transcript level and was independent of BCR-ABL1 mutation type. Ex vivo inhibition of imatinib and dasatinib on plasma cTK activity was severely diminished in patients harboring T315I mutation. Ex vivo testing measuring the effect of TKIs on plasma cTK activity thus hold promise as drug sensitivity tests for predicting and monitoring response to specific TKIs.

Circulating Ki-67 protein in plasma as a biomarker and prognostic indicator of acute lymphoblastic leukemia.

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.

Clinical correlation of circulating heat shock protein 70 in acute leukemia.

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.

Plasma-based detection of clonality in lymphoid malignancies.

OBJECTIVES: Plasma has been found to be enriched with tumor-specific DNA, RNA, and protein in patients with hematologic disease. We assessed the utility of plasma as a DNA source for detection of genetic abnormalities in patients with suspected B- or T-cell lymphoproliferative disorders. METHODS: DNA was extracted from paired peripheral blood (PB) cells and plasma for polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) and T-cell receptor gamma chain (TCR-gamma) rearrangements, and B-cell leukemia/lymphoma (BCL)-1/IgH and BCL-2/IgH translocations. RESULTS: Concordance between plasma and PB cell analysis was 100% for IgH (n = 57), TCR-gamma (n = 57), and BCL-1/IgH (n = 37) rearrangements, and 94% (60/64) for BCL-2/IgH; four of 11 plasma samples positive for BCL-2/IgH tested negative in paired cells. No plasma or PB cell samples from 195 healthy donors showed genetic abnormalities. CONCLUSIONS: These findings indicate that plasma is a reliable sample type for detection of abnormalities associated with B- and T-cell lymphoproliferative disorders, providing sensitivity equal to or greater than that of PB cells.

Circulating heat shock protein 70 and progression in patients with chronic myeloid leukemia.

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.

Mutation profile of JAK2 transcripts in patients with chronic myeloproliferative neoplasias.

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.

Liquid-based fluorescence in situ hybridization assay for detection of ERBB2 gene amplification in patients with breast cancer.

BACKGROUND: Current reference methods for evaluating gene amplification and expression of ERBB2 (also known as HER-2)--cell-based fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC)--are subjective and influenced by methods of tissue preparation and fixation. We developed and evaluated a novel, quantitative liquid-based FISH (L-FISH) assay that uses flow cytometry to detect ERBB2 gene amplification in breast cancer patients. METHODS: DNA was extracted from serum or tissue, biotinylated, hybridized to differentially labeled probes for ERBB2 and a chromosome 17-specific single-copy sequence (17-SSC), and immobilized to streptavidin-coated microspheres. The ERBB2/17-SSC signal ratio measured by flow cytometry was used to evaluate ERBB2 amplification. We used L-FISH to test 122 stored formalin-fixed, paraffin-embedded (FFPE) tissue samples and 22 serum samples from randomly selected breast cancer patients; results were compared with those obtained with conventional FISH and IHC. RESULTS: The inter- and intraassay imprecisions were 3.7%-18.9% for FFPE tissue and 2.8%-6.3% for serum. Overall, L-FISH analyses of FFPE tissues demonstrated 84.4% concordance with results obtained with conventional FISH (P < 0.001) and 78.8% concordance with IHC results (P < 0.001). L-FISH analyses of serum samples showed 91% concordance with tissue-based IHC/FISH results (P = 0.038). CONCLUSIONS: Our data indicate that this PCR-free L-FISH method can be used to evaluate ERBB2 amplification in both cell-containing (paraffin-embedded tissue) and cell-free (serum) samples. This approach provides more objective results and is amenable to automation and quantitative measurement.

Neutral sphingomyelinase activation in endothelial and glial cell death induced by amyloid beta-peptide.

We have explored the molecular mechanism underlying amyloid beta-peptide (Abeta)-mediated cytotoxicity in vitro. Exposure of murine cerebral endothelial cells (CECs) or C6 glioma cells to Abeta25-35 resulted in dose-dependent cell death. Ceramide is a pro-apoptotic lipid mediator. Forced elevation of cellular ceramide levels, either by application of an exogenous C2 ceramide analogue or bacterial sphingomyelinase that induces endogenous ceramide release from sphingomyelin, mimicked Abeta25-35 cytotoxicity in both CECs and C6 glioma cells. Abeta25-35-induced synthesis of ceramide was selectively mediated by activation of neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase (aSMase) or ceramide synthase. Both 3-O-Me-SM and N-acetyl-L-cysteine, the selective and nonselective pharmacological inhibitors of nSMase, respectively, suppressed nSMase activation, ceramide production, and cytotoxic action induced by Abeta25-35 in CECs. Furthermore, genetic knockdown of nSMase by an antisense strategy rendered C6 glioma cells specifically resistant to Abeta25-35 cytotoxicity without affecting their vulnerability to serum deprivation. Together, nSMase activation with subsequent ceramide production may contribute, at least partially, to Abeta25-35 cytotoxicity in cell types with cerebral endothelial and glial lineage.