Peripheral Blood JAK2V617F Quantitative Assessment in Clinical Practice: Correlations Between Allele Burden and Clinical Phenotype

Background: Peripheral blood (PB) JAK2V617F testing is ordered in routine clinical practice in several different clinical scenarios. Interpretation of the mutation level/allele burden information is however challenging given lack of a standardized assay and lack of adequate information to permit data interpretation in a non-research setting.

Objectives: To describe: (i) disease distribution in unselected JAK2V617F-positive patients seen in routine clinical practice: (ii) disease-specific dispersion of JAK2V617F allele burden in these patients; and (iii) correlation of JAK2V617F allele burden with patient clinical characteristics both between and within specific diseases.

Methods: The current study was approved by the Mayo Clinic institutional review board. Myeloid neoplasms were classified as per World Health Organization criteria. Clinical, laboratory and pathology results were centrally reviewed to confirm the reported diagnoses. JAK2V617F testing was done as part of routine clinical care; testing included allele burden measurement by a quantitative allele-specific PCR assay (sensitivity 0.01%) that is in clinical use at our institution (http://www.mayomedicallaboratories.com/test-catalog/Overview/31155); reporting of allele-burden data started in January 2012. The current analysis was restricted to PB samples from adult patients seen at our institution who consented to use of their medical records for research use; results of consecutive samples tested from January 2012 to April 2015 were collected. The PCR assay calibrator is a mixture of DNA from a JAK2V617F-positive cell line (HEL) and a -negative cell line (HL60). The final result is reported as % JAK2 V617F of total JAK2 (ie, [mutated/(mutated + wild-type)] x 100%).

Results: We studied 349 patients (166 female; median age 65 years, range 18-92) who met eligibility criteria. The disease distribution was: polycythemia vera (PV) 146 patients (42%), primary myelofibrosis (PMF) 63 (18%), essential thrombocythemia (ET) 55 (16%) , post-PV MF 34 (10%), post-ET MF 12 (3%), myeloproliferative neoplasm-unclassified (MPN-u) 10 (3%), refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) 8 (2%), post-MPN acute myeloid leukemia (AML) 6 (2%), MPN-myelodysplastic syndrome-unclassified (MPN/MDS-u) 4 (1%), no identified hematological disease 2 (0.5%), and 'other' 9 (2.5%).

The median (range) %JAK2V617F was as follows: PV 10.5% (0.5-86%), PMF 8% (0.5-88%), ET 2% (0.5-21%), post-PV MF 24% (0.8-95%), post-ET MF 14.5% (1-88%), MPN-u 4% (0.7-11%), RARS-T 2.5% (0.5-9%), post-MPN AML 31.5% (9-92%), MPN/MDS-u 14.5% (7-74%), no hematological disease 0.7% (n/a), and other 0.9% (0.5-19%) (p<0.0001).

Distribution of patients in the following 7 JAK2V617Fallele burden strata: <1%, 1-2% , 3-5%, 6-10%, 11-20%, 21-50%, and 51-100% was: 42 patients (12%), 44 (13%), 61 (17%), 61 (17%), 49 (14%), 50 (15%) and 42 (12%), respectively. PV patients were identified across the entire allele burden spectrum, namely 3%, 12%, 16%, 19%, 14%, 20% and 16% in the aforementioned strata, respectively. Disease distribution in the 42 patients with <1% allele burden was: ET 14 patients, PMF 12, PV and 'other' 5 each, RARS-T and no hematological disease 2 each, and post-PV MF and MPN-u 1 each.

JAK2V617F allele burden in ET (peak 21%) was significantly lower than PMF or PV (peak 86-88%) (p<0.0001). Mutation levels in post-ET (p<0.0001) and post-PV (p=0.004) MF were significantly higher than in ET and PV, respectively. Levels in post-PV/post-ET MF were also significantly higher than in PMF (p<0.0001). In contrast, JAK2V617F levels in ET and RARS-T were comparable (p=0.8). Finally, levels in post-MPN AML were significantly higher than in ET (p=0.0001) and PMF (p=0.007), marginally higher relative to PV (p=0.05), but not significantly higher relative to post-ET/post-PV MF.

Conclusions: The current study provides guidance for the interpretation of peripheral blood JAK2 V617F allele burden, in routine clinical practice: i) levels below 1% are not infrequently reported, even in phenotypically classic PV, and do not imply low likelihood of a myeloid neoplasm, ii) allele burden per se is not diagnostically specific, but a threshold of greater than 20% might be used to question a diagnosis of ET or RARS-T, and iii) the highest allele burdens are seen in fibrotic and/or leukemic transformation of chronic-phase myeloproliferative neoplasm.