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

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

QUANTIFICATION OF JAK2WT AND JAK2V617F ALLELE IN mRNA FROM PH(-) MYELOPROLIFERATIVE NEOPLASMS PATIENTS: CORRELATION WITH DNA ALLELE BURDEN AND DESEASE PROGRESSION

INTRODUCTION: Currently, the JAK2V617F allele quantification in Ph- MPN is performed on genomic DNA. However, compared to DNA, mRNA analysis may offer some advantages such as: 1)higher sensitivity; 2) inclusion of platelet mRNA; 3) mRNA transcriptional functionality. Therefore, we developed an absolute allele-specific RQ-PCR method to quantify JAK2WT, V617F and ABL (as housekeeping gene) transcript levels aiming to: 1) verify specificity and sensitivity of this assay; 2)evaluate whether gene expression levels correlate with disease phenotype and may mark disease progression (i.e. ET toward PV). METHODS: To construct reference curves, including five 10-fold serial dilutions, two plasmid standards were manufactured to contain 150 bp of either JAK2WT and JAK2V617F cDNA sequence. To perform an allelic discrimination we used primers described by Merker et al (JMD, 2010): reverse primers are complementary to the WT or the mutant cDNA sequence, respectively. Assay sensitivity was determined by serial 10-fold dilutions of the K562 (JAK2WT) and HEL (V671F homo) cell lines which were included in each experiment as negative and positive controls. RESULTS: We tested cDNA synthesized from peripheral blood buffy coat specimens of 48 MPN patients (16 PV; 25 ET, 7 PMF), 13 patients with secondary polycythemia (SP) and 23 healthy donors (CTRL). Respect to the qualitative PCR method (Baxter el al, Lancet 2005) our RQ-PCR assay shows a 100% concordance rate in identifying the presence of the JAK2V617F. V617F mutation was detected in 16/16 PV (100%), 19/25 ET (76%) and 5/7 (71%) PMF samples. The mean expression of JAK2WT allele, as absolute averaged copies+SEM was: healthy donors 12127+2181; SP 12244+2489; PV 4955+1642, WT ET 10876+1781; V617F ET 5693+830 and V617F PMF 3004+1442. Statistically significant differences were detected between CTRL and PV (p <0.005) and CTRL and V617F ET (p <0.005). The mean expression of V617F allele was PV 27107+12699, ET 6524 +1877 and PMF 29464+14587. A significant difference was found comparing PV and ET (p <0.005) and ET and PMF (p <0.005). Respect to the allele burden quantitative assay (IPSOGEN), the mutational burden (V617F/(V617F+WT) ratio detected by our assay is significantly higher (59% vs 36% burden in RQ-PCR and IPSOGEN, respectively; p <0.005). Three ET patients progressed to PV phase. The sequential analysis of the mutational burden ratio at diagnosis and during disease progression showed an increase in one patient’s sample at both DNA and mRNA level. By contrast, in the remaining 2 cases the ratio remained constant at the DNA level, but significantly increased when the mRNA was analysed. CONCLUSION: Present data demonstrated that our method is as specific but more sensitive than the allele burden determination. In addition, the complementary determination of mRNA transcript levels may help in elucidate the pathogenetic mechanisms of the Ph- MPN diseases