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

Abdool A, Donahue AC, Wohlgemuth JG, Yeh CH - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Quest Diagnostics Nichols Institute, San Juan Capistrano, California, United States of America.

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.

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Representative MLPA analysis on CLL patients.(a) Normalized ratio plot (relative copy number, RCN) from a CLL patient with trisomy 12 and 19, and 13q14 deletions. The data were normalized to those of normal controls. The reference ranges of 2 SD (Mean ± 2SD, 95% CI, P = 0.05) and 3 SD (Mean ± 3SD, 99% CI, P = 0.01) for each probe are shown by diamonds and squares, respectively. (b) MLPA analysis of copy number changes on multiple CLL samples are called and highlighted on a heatmap as blue (gain, ≥3SD), light blue (gain, ≥2SD), red (loss, ≤3SD) and pink (loss, ≤2SD) blocks that lie outside of reference ranges of each probe. Note that the high resolution of MLPA in several 13q- and 11q- cases can pinpoint deletion region down to a single gene level.
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pone-0015407-g001: Representative MLPA analysis on CLL patients.(a) Normalized ratio plot (relative copy number, RCN) from a CLL patient with trisomy 12 and 19, and 13q14 deletions. The data were normalized to those of normal controls. The reference ranges of 2 SD (Mean ± 2SD, 95% CI, P = 0.05) and 3 SD (Mean ± 3SD, 99% CI, P = 0.01) for each probe are shown by diamonds and squares, respectively. (b) MLPA analysis of copy number changes on multiple CLL samples are called and highlighted on a heatmap as blue (gain, ≥3SD), light blue (gain, ≥2SD), red (loss, ≤3SD) and pink (loss, ≤2SD) blocks that lie outside of reference ranges of each probe. Note that the high resolution of MLPA in several 13q- and 11q- cases can pinpoint deletion region down to a single gene level.

Mentions: A testing set of 100 samples from suspected CLL patients was analyzed for chromosomal abnormalities by MLPA. MLPA probes target regions commonly associated with CLL prognosis, such as 13q14, ATM, and TP53, and trisomy 12. Additionally, the probe mix also targets other genomic loci with imbalances in CLL, e.g., 10q23, 18q21 and chromosome 19 [15]. Figure 1 shows a representative bar graph (A) and a heatmap (B) of relative copy number changes in CLL patients by MLPA. Although peripheral blood samples were not subjected to B-cell purification before the MLPA assay, cases with low percentages of cells carrying genomic alteration can be reliably detected and scored by our analysis. The key criteria and guidelines for final call on chromosome copy number loss or gain are detailed in Table 2. In the validation samples, deletions in 13q14 with >20% of cells carrying this aberration, deletions in 11q23 with >12% abnormal cells, or trisomy 12 with at least 25% leukemia cells were detected and called by our system. In addition, multiallelic abnormalities from the same patient, e.g., 13q-/11q-, 13q-/17q-, 13q-/19q13.43-, 12tri/17p- or 12tri/13q-/19+ were identified. In 52 of the 100 testing samples (52%), chromosomal abnormalities were detected by MLPA: 25 cases (25%) showed loss of the 13q14 region; 8 cases (8%) showed trisomy of chromosome 12; 3 cases (3%) showed loss of the ATM gene; 3 cases (3%) showed loss of the TP53 gene; and 13 cases (13%) showed multiallelic imbalance (Table 3). Overall, a good correlation was found between MLPA and FISH results, and all abnormalities observed by FISH were also identified by MLPA, with the exception of three 13q- cases with a prohibitively low percentage of leukemia cells (7%, 12% and 19% by FISH).


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

Abdool A, Donahue AC, Wohlgemuth JG, Yeh CH - PLoS ONE (2010)

Representative MLPA analysis on CLL patients.(a) Normalized ratio plot (relative copy number, RCN) from a CLL patient with trisomy 12 and 19, and 13q14 deletions. The data were normalized to those of normal controls. The reference ranges of 2 SD (Mean ± 2SD, 95% CI, P = 0.05) and 3 SD (Mean ± 3SD, 99% CI, P = 0.01) for each probe are shown by diamonds and squares, respectively. (b) MLPA analysis of copy number changes on multiple CLL samples are called and highlighted on a heatmap as blue (gain, ≥3SD), light blue (gain, ≥2SD), red (loss, ≤3SD) and pink (loss, ≤2SD) blocks that lie outside of reference ranges of each probe. Note that the high resolution of MLPA in several 13q- and 11q- cases can pinpoint deletion region down to a single gene level.
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Related In: Results  -  Collection

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pone-0015407-g001: Representative MLPA analysis on CLL patients.(a) Normalized ratio plot (relative copy number, RCN) from a CLL patient with trisomy 12 and 19, and 13q14 deletions. The data were normalized to those of normal controls. The reference ranges of 2 SD (Mean ± 2SD, 95% CI, P = 0.05) and 3 SD (Mean ± 3SD, 99% CI, P = 0.01) for each probe are shown by diamonds and squares, respectively. (b) MLPA analysis of copy number changes on multiple CLL samples are called and highlighted on a heatmap as blue (gain, ≥3SD), light blue (gain, ≥2SD), red (loss, ≤3SD) and pink (loss, ≤2SD) blocks that lie outside of reference ranges of each probe. Note that the high resolution of MLPA in several 13q- and 11q- cases can pinpoint deletion region down to a single gene level.
Mentions: A testing set of 100 samples from suspected CLL patients was analyzed for chromosomal abnormalities by MLPA. MLPA probes target regions commonly associated with CLL prognosis, such as 13q14, ATM, and TP53, and trisomy 12. Additionally, the probe mix also targets other genomic loci with imbalances in CLL, e.g., 10q23, 18q21 and chromosome 19 [15]. Figure 1 shows a representative bar graph (A) and a heatmap (B) of relative copy number changes in CLL patients by MLPA. Although peripheral blood samples were not subjected to B-cell purification before the MLPA assay, cases with low percentages of cells carrying genomic alteration can be reliably detected and scored by our analysis. The key criteria and guidelines for final call on chromosome copy number loss or gain are detailed in Table 2. In the validation samples, deletions in 13q14 with >20% of cells carrying this aberration, deletions in 11q23 with >12% abnormal cells, or trisomy 12 with at least 25% leukemia cells were detected and called by our system. In addition, multiallelic abnormalities from the same patient, e.g., 13q-/11q-, 13q-/17q-, 13q-/19q13.43-, 12tri/17p- or 12tri/13q-/19+ were identified. In 52 of the 100 testing samples (52%), chromosomal abnormalities were detected by MLPA: 25 cases (25%) showed loss of the 13q14 region; 8 cases (8%) showed trisomy of chromosome 12; 3 cases (3%) showed loss of the ATM gene; 3 cases (3%) showed loss of the TP53 gene; and 13 cases (13%) showed multiallelic imbalance (Table 3). Overall, a good correlation was found between MLPA and FISH results, and all abnormalities observed by FISH were also identified by MLPA, with the exception of three 13q- cases with a prohibitively low percentage of leukemia cells (7%, 12% and 19% by FISH).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Quest Diagnostics Nichols Institute, San Juan Capistrano, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus