A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms

Abstract

In Ph− myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or β2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph− MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms.Electronic supplementary materialThe online version of this article (doi:10.1007/s00277-013-1920-0) contains supplementary material, which is available to authorized users.

Relationship betweenJAK2V617Fmutation, allele burden and coagulation function in Ph-negative myeloproliferative neoplasms

“…JAK2V617F mutation would have an influence on blood parameters in MPN, and in ET patients, PLT was lower in JAK2V617F mutated patients [9], conversely, RBC counts and WBC counts were much higher in JAK2V617F positive patients based on Chinese population [11], which was similar to our findings. Moreover, JAK2V617F mutation allele burden might be corrected with the change of the degrees of blood parameters, and in PV patients, a negative statistically significant correlation between the JAK2V617F mutation allele burden and PLT count was observed [26] and no statistically significant correlation was found in the study performed by Edahiro et al [27], in addition, JAK2V617F allele burden was correlated with WBC counts and RBC counts in PV patients based on Chinese population [11]. However, the present data indicated that PLT was positively correlated with JAK2V617F mutation allele burden in PV patients, suggesting that the relationship between blood parameters and JAK2V617F mutation allele burden remained inconsistent.…”

Adverse Prognostic Impact of the KIT D816V Transcriptional Activity in Advanced Systemic Mastocytosis

“…To date, there are only a few reports comparing mutational analysis at DNA and RNA/cDNA level in hematological neoplasms. A discrepancy has been reported regarding the JAK2 V617F mutation in patients with essential thrombocythemia and polycythemia vera, and also regarding the type A mutation of NPM1 in acute myeloid leukemia (AML) [ 20 , 21 , 22 ]. All reports found significantly higher mutation levels at RNA/cDNA level compared to DNA-level highlighting the potential superior sensitivity of RNA-based assays and the possible impact of this discrepancy on disease phenotype and prognosis.…”

Diagnostic performance of the molecular BCR-ABL1 monitoring system may impact on inclusion of CML patients in stopping trials

“…While Beillard et al [ 24 ] recommended ABL1 as control gene for RQ-PCR-based diagnosis and MRD detection in leukemic patients because of more stable, uniform expression and lack of pseudogenes, GUSB transcript levels were later confirmed as a suitable alternative [ 25 ]. Other authors described the suitability of ABL1 , BCR and GUSB for BCR-ABL1 quantification [ 15 ] or even considered beta-glucoronidase ( GUSB ) as the most suitable genes tested [ 26 ] because the expression pattern of beta-glucoronidase ( GUSB ) was found more homogenous than that of ABL1 or β2 microglobulin ( B2M ) [ 27 ]. Not to be neglected the main practical advantage of the use GUSB over ABL 1 as housekeeping genes is the performance of BCR-ABL1 and GUSB quantification in one single well (duplex PCR) avoiding the bias introduced by setting-up two spatially distinct TM qPCR reactions.…”