Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Characterization of BCR-ABL deletion mutants from patients with chronic myeloid leukemia.

The BCR-ABL oncogenic tyrosine kinase causes chronic myeloid leukemia and is the target for imatinib therapy. During imatinib treatment, cells are selected in some patients with BCR-ABL kinase domain mutations that render decreased drug sensitivity. In addition, some patients express deletion mutants of BCR-ABL, apparently due to missplicing. Most commonly these deletion mutants lack a significant portion of the kinase domain that includes the P-loop. We describe a screen for such mutations in patients with CML and demonstrate that they are not oncogenic and are catalytically inactive. We hypothesized that coexpressing BCR-ABL deletion mutants has a dominant-negative effect on the native form through heterocomplex formation. However, upon coexpression of native and deletion mutant BCR-ABL in Ba/F3 cells, growth factor independence is maintained and signaling is activated normally. Despite this, these cells have increased imatinib sensitivity compared to cells expressing only native BCR-ABL. Thus, it will be important to investigate the prognostic impact of coexpression of deletion mutants in CML patients during imatinib treatment.

A genome-wide linkage scan for iron phenotype quantitative trait loci: the HEIRS Family Study.

Iron overload phenotypes in persons with and without hemochromatosis are variable. To investigate this further, probands with hemochromatosis or evidence of elevated iron stores and their family members were recruited for a genome-wide linkage scan to identify potential quantitative trait loci (QTL) that contribute to variation in transferrin saturation (TS), unsaturated iron-binding capacity (UIBC), and serum ferritin (SF). Genotyping utilized 402 microsatellite markers with average spacing of 9 cM. A total of 943 individuals, 64% Caucasian, were evaluated from 174 families. After adjusting for age, gender, and race/ethnicity, there was evidence for linkage of UIBC to chromosome 4q logarithm of the odds (LOD) = 2.08, p = 0.001) and of UIBC (LOD = 9.52), TS (LOD = 4.78), and SF (LOD = 2.75) to the chromosome 6p region containing HFE (each p < 0.0001). After adjustments for HFE genotype and other covariates, there was evidence of linkage of SF to chromosome 16p (LOD = 2.63, p = 0.0007) and of UIBC to chromosome 5q (LOD = 2.12, p = 0.002) and to chromosome 17q (LOD = 2.19, p = 0.002). We conclude that these regions should be considered for fine mapping studies to identify QTL that contribute to variation in SF and UIBC.

Mutations of the BCR-ABL-kinase domain occur in a minority of patients with stable complete cytogenetic response to imatinib.

Residual leukemia is demonstrable by reverse transcriptase-polymerase chain reaction in most patients with chronic myeloid leukemia who obtain a complete cytogenetic response (CCR) to imatinib. In patients who relapse during imatinib therapy, a high rate of mutations in the kinase domain of BCR-ABL have been identified, but the mechanisms underlying disease persistence in patients with a CCR are poorly characterized. To test whether kinase domain mutations are a common mechanism of disease persistence, we studied patients in stable CCR. Mutations were demonstrated in eight of 42 (19%) patients with successful amplification and sequencing of BCR-ABL. Mutation types were those commonly associated with acquired drug resistance. Four patients with mutations had a concomitant rise of BCR-ABL transcript levels, two of whom subsequently relapsed; the remaining four did not have an increase in transcript levels and follow-up samples, when amplifiable, were wild type. BCR-ABL-kinase domain mutations in patients with a stable CCR are infrequent, and their detection does not consistently predict relapse. Alternative mechanisms must be responsible for disease persistence in the majority of patients.

Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

Linked linear amplification: a new method for the amplification of DNA.

BACKGROUND: Linked Linear Amplification (LLA) is a new nucleic acid amplification method that uses multiple cycles of primer extension reactions. The presence of nonreplicable elements in LLA primers renders primer extension products unusable as templates for further amplification, leading to linear accumulation of products. Through the use of nested primers, linear reactions can be "linked", providing total amplification yields comparable to those obtained by PCR. METHODS: The LLA model predicts (a) that amplification yield will approach that of PCR as the number of primers increases and (b) that the unique composition of LLA products will give lower carryover amplification efficiency compared with PCR. To test these hypotheses, the human ss-globin gene was amplified by 10-, 14-, or 18-primer LLA and the yield was compared with PCR. Carryover contamination was simulated by reamplifying a dilution series of LLA or PCR products. To demonstrate the clinical utility of the method, LLA coupled with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in a panel of 111 DNA samples. RESULTS: Fourteen- and 18-primer LLA gave amplification yields comparable to PCR. However, LLA carryover amplification efficiency was four orders of magnitude lower than that of PCR. The LLA-ASO assay detected the correct factor V Leiden genotype in all 111 samples. CONCLUSIONS: LLA is a robust target amplification method that is comparable to PCR in yield. However, LLA is more resistant to false results caused by carryover amplicon contamination.