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.
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, 0-4574 U/100 μL) than in healthy control subjects (n=114; median, 399; range, 36-2830 U/100 μL). 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.
5510 Background: In 2009, the American Thyroid Association revised its guidelines for patients with thyroid nodules and differentiated thyroid cancers, recommending BRAF, RAS, RET/PTC, and PAX8-PPARγ molecular testing in patients with indeterminate fine-needle aspirate (FNA) cytology. Alterations in any of these markers in thyroid nodules are strongly associated with malignancy. We studied the prevalence of these alterations in thyroid FNA specimens in order to establish a strategy to improve disease management. Methods: DNA and RNA were extracted from thyroid FNA specimens (N=149) and tested for BRAF mutations using allele-specific PCR (V600E and K601E); for RAS (H-, K-, N-) mutations using pyrosequencing (codons 12, 13, and 61); and for RET/PTC1 and RET/PTC3 rearrangements and PAX8-PPARγ translocations using RT-PCR. Results: Overall, 90 (60.4%) of the specimens had alterations in ≥1 of the 4 molecular markers (Table). BRAF V600E mutations (54.4%) were the most prevalent mutations in papillary thyroid cancer (PTC); no K601E mutations were found. RAS mutations were found in PTC, follicular adenoma (FA), and follicular thyroid cancer (FTC) specimens; half of these mutations involved N-RAS Q61. RET/PTC rearrangements were detected in 3.5% of PTC specimens and were evenly distributed between the 2 major subtypes, RET/PTC1 and RET/PTC3. PAX8/PPARγ translocations were detected in 18.8% of FTC but not in FA, probably due to small sample size. Out of 7 indeterminate thyroid nodules, 2 had BRAF mutations and 2 had RAS mutations. The presence of the 4 markers was generally mutually exclusive; only 1 PTC specimen had concurrent RAS and RET/PTC1 alterations. Conclusions: The prevalence of BRAF, RAS, RET/PTC, and PAX8/PPARγ alterations in thyroid cancer specimens highlights the potential for targeted therapeutic strategies. The mutually exclusive pattern of alterations also suggests a hierarchical screening strategy for samples with limited availability. [Table: see text]
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