Abstract
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.
Highlights
Advances in cancer genomics allow us to determine and quantify disease-associated genetic profiles, and to improve clinical diagnosis/prognosis, tumor classification and cancer therapy [1]
Instead of using an arbitrary ratio range (e.g., 0.75–1.25, 0.8–1.2 or 0.95–1.05) as a single universal cutoff value for all probes, which is applied in most Multiplex ligation-dependent probe amplification (MLPA) studies [13,14], we set out to establish a normal range for each MLPA probe to provide a more appropriate baseline from which any copy number variation (CNV) will be confidently identified in Chronic lymphocytic leukemia (CLL) patients
The reference range data showed a normal distribution in each case, and a narrow variation in the mean and standard deviations (SD) between these normals, with the Mean 6 2SD value ranging at maximum from 0.82 to 1.18, and Mean 6 3SD value from 0.73 to 1.27 (Table 1)
Summary
Advances in cancer genomics allow us to determine and quantify disease-associated genetic profiles, and to improve clinical diagnosis/prognosis, tumor classification and cancer therapy [1]. Chromosomal alterations in leukemia have been shown to have prognostic and predictive value, and are important markers of minimal residual disease in the follow-up of leukemia patients [2]. The complex process that drives the development of leukemia could rise from several clonal molecular abnormalities, including copy number gains and losses in the genome leading to activation of proto-oncogenes and silencing (or deletion) of tumor suppressor genes, respectively [3,4]. Specific chromosome copy number alterations characteristic of CLL, such as loss of the 13q14 region (with a frequency of 50–60%), trisomy of chromosome 12 (15– 25%), and deletions of 11q22 (10–20%) and 17p13 (5–10%), have been shown to provide clinically relevant prognostic information and help identify more aggressive disease [5,6]. Chromosome translocations are relatively rare in CLL [8]
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