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.

Fantasia, F., Di Capua, E., Cenfra, N., Pessina, G., Mecarocci, S., Rago, A., et al. (2014). 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. ANNALS OF HEMATOLOGY, 93(4), 609-616 [10.1007/s00277-013-1920-0].

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

LO COCO, FRANCESCO;
2014-01-01

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.
2014
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore MED/15 - MALATTIE DEL SANGUE
English
Adult; Aged; Aged, 80 and over; Female; Humans; Janus Kinase 2; K562 Cells; Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative; Male; Middle Aged; Myeloproliferative Disorders; Real-Time Polymerase Chain Reaction; Gene Expression Regulation, Neoplastic
Fantasia, F., Di Capua, E., Cenfra, N., Pessina, G., Mecarocci, S., Rago, A., et al. (2014). 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. ANNALS OF HEMATOLOGY, 93(4), 609-616 [10.1007/s00277-013-1920-0].
Fantasia, F; Di Capua, E; Cenfra, N; Pessina, G; Mecarocci, S; Rago, A; Cotroneo, E; Busanello, A; Equitani, F; LO COCO, F; Nervi, C; Cimino, G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/160521
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