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Concordance of assays designed for the quantification of JAK2V617F: a multicenter study

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study,10.3324/haematol.13486,Haematologica-the Hematology Journal,E.

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study   (Citations: 11)
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E. Lippert, F. Girodon, E. Hammond, J. Jelinek, N. S. Reading, B. Fehse, K. Hanlon, M. Hermans, C. Richard, S. Swierczek, V. Ugo, S. Carillohttp://academic.research.microsoft.com/io.ashx?type=5&id=33467455&selfId1=0&selfId2=0&maxNumber=12&query=
A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F stan- dards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques - one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results out- side the mean±2SD - with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primer- based specificity could detect 0.2% JAK2V617F. Conclusions Techniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility. ABSTRACT
Journal: Haematologica-the Hematology Journal , vol. 94, no. 1, pp. 38-45, 2009
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    • ...These methods include: direct sequencing (DS) or RFLP, pyrosequencing, allele-specific primer extension (ASPE), amplification-refractory mutation sequencing (ARMS), quantitative real time PCR (QRT-PCR) and DNA melting curve analysis [6,11-14]...
    • ...A recent report [11] on the concordance of various assays designed for the quantitative detection of JAK2V617F concluded that the allelic burden as defined by JAK2V617F/total JAK2 produced similar results with variable sensitivity using a common set of standards...

    François W Paradiset al. Quantitative assay for the detection of the V617F variant in the Janus...

    • ...Median age, y (range) 57 (21-88) 62 (21-90) .47 Median follow-up, mo (range) 27 (1-185) 29 (1-237) .72 Females, no. (%) 18 (30.5) 51 (40.1) .23 Median white blood cell count, 109/L (range) 7.70 (1.9-90.78) 10.10 (3.01-16.15) .009*...
    • ...Median age, y (range) 57 (21-88) 62 (21-90) .47 Median follow-up, mo (range) 27 (1-185) 29 (1-237) .72 Females, no. (%) 18 (30.5) 51 (40.1) .23 Median white blood cell count, 109/L (range) 7.70 (1.9-90.78) 10.10 (3.01-16.15) .009*...
    • ...Median age, y (range) 57 (21-88) 62 (21-90) .47 Median follow-up, mo (range) 27 (1-185) 29 (1-237) .72 Females, no. (%) 18 (30.5) 51 (40.1) .23 Median white blood cell count, 109/L (range) 7.70 (1.9-90.78) 10.10 (3.01-16.15) .009*...
    • ...Median JAK2V617F allele burden (range) — 54 (5-100) — Splenomegaly 15 cm from LCM, no. (%) 11 (18.6) 26 (19.7) .89 Constitutional symptoms, no. (%) 15 (25.4) 38 (29.9) .15 Leukemia transformation, no. (%) 6 (10.1%) 9 (7.1) .45 Deaths, no. (%) 8 (13.5) 15 (11.8) .64 Lille score, no. (%) 0 22 (37.2) 80 (63.0) .017* 1 26 (44.0) 38 (30.0) 2 11 (18.8) 9 (7.0)...
    • ...No. 17 43 31 36 Median age, y (range) 58 (38-90) 63 (28-90) 65 (28-77) 60.31 (21-86) .72 Median follow-up, mo (range) 16 (1-46) 24 (1-84) 26 (3-101) 45 (1-237) .001* Females, no. (%) 4 (23.5) 21 (48.8) 12 (38.7) 14 (38.9) .40 Median JAK2 V617F allele burden (range) 15.5 (5-25) 41 (27-50) 66 (52-75) 90.5 (77-100) — Median white blood cell count, 109/L (range) 10.26 (3.01-26.85) 9.47 (3.73-106.15) 9.40 (3.70-40.0) 13.40 (3.40-40.60) .03* ...
    • ...No. 17 43 31 36 Median age, y (range) 58 (38-90) 63 (28-90) 65 (28-77) 60.31 (21-86) .72 Median follow-up, mo (range) 16 (1-46) 24 (1-84) 26 (3-101) 45 (1-237) .001* Females, no. (%) 4 (23.5) 21 (48.8) 12 (38.7) 14 (38.9) .40 Median JAK2 V617F allele burden (range) 15.5 (5-25) 41 (27-50) 66 (52-75) 90.5 (77-100) — Median white blood cell count, 109/L (range) 10.26 (3.01-26.85) 9.47 (3.73-106.15) 9.40 (3.70-40.0) 13.40 (3.40-40.60) .03* ...
    • ...No. 17 43 31 36 Median age, y (range) 58 (38-90) 63 (28-90) 65 (28-77) 60.31 (21-86) .72 Median follow-up, mo (range) 16 (1-46) 24 (1-84) 26 (3-101) 45 (1-237) .001* Females, no. (%) 4 (23.5) 21 (48.8) 12 (38.7) 14 (38.9) .40 Median JAK2 V617F allele burden (range) 15.5 (5-25) 41 (27-50) 66 (52-75) 90.5 (77-100) — Median white blood cell count, 109/L (range) 10.26 (3.01-26.85) 9.47 (3.73-106.15) 9.40 (3.70-40.0) 13.40 (3.40-40.60) .03* ...
    • ...No. 17 43 31 36 Median age, y (range) 58 (38-90) 63 (28-90) 65 (28-77) 60.31 (21-86) .72 Median follow-up, mo (range) 16 (1-46) 24 (1-84) 26 (3-101) 45 (1-237) .001* Females, no. (%) 4 (23.5) 21 (48.8) 12 (38.7) 14 (38.9) .40 Median JAK2 V617F allele burden (range) 15.5 (5-25) 41 (27-50) 66 (52-75) 90.5 (77-100) — Median white blood cell count, 109/L (range) 10.26 (3.01-26.85) 9.47 (3.73-106.15) 9.40 (3.70-40.0) 13.40 (3.40-40.60) .03* ...
    • ...No. 17 43 31 36 Median age, y (range) 58 (38-90) 63 (28-90) 65 (28-77) 60.31 (21-86) .72 Median follow-up, mo (range) 16 (1-46) 24 (1-84) 26 (3-101) 45 (1-237) .001* Females, no. (%) 4 (23.5) 21 (48.8) 12 (38.7) 14 (38.9) .40 Median JAK2 V617F allele burden (range) 15.5 (5-25) 41 (27-50) 66 (52-75) 90.5 (77-100) — Median white blood cell count, 109/L (range) 10.26 (3.01-26.85) 9.47 (3.73-106.15) 9.40 (3.70-40.0) 13.40 (3.40-40.60) .03* ...
    • ...No. 17 43 31 36 Median age, y (range) 58 (38-90) 63 (28-90) 65 (28-77) 60.31 (21-86) .72 Median follow-up, mo (range) 16 (1-46) 24 (1-84) 26 (3-101) 45 (1-237) .001* Females, no. (%) 4 (23.5) 21 (48.8) 12 (38.7) 14 (38.9) .40 Median JAK2 V617F allele burden (range) 15.5 (5-25) 41 (27-50) 66 (52-75) 90.5 (77-100) — Median white blood cell count, 109/L (range) 10.26 (3.01-26.85) 9.47 (3.73-106.15) 9.40 (3.70-40.0) 13.40 (3.40-40.60) .03* ...
    • ...No. 17 43 31 36 Median age, y (range) 58 (38-90) 63 (28-90) 65 (28-77) 60.31 (21-86) .72 Median follow-up, mo (range) 16 (1-46) 24 (1-84) 26 (3-101) 45 (1-237) .001* Females, no. (%) 4 (23.5) 21 (48.8) 12 (38.7) 14 (38.9) .40 Median JAK2 V617F allele burden (range) 15.5 (5-25) 41 (27-50) 66 (52-75) 90.5 (77-100) — Median white blood cell count, 109/L (range) 10.26 (3.01-26.85) 9.47 (3.73-106.15) 9.40 (3.70-40.0) 13.40 (3.40-40.60) .03* ...

    Paola Guglielmelliet al. Identification of patients with poorer survival in primary myelofibros...

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