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.

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.

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.

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.

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.