Diagnostic Challenges in Patients with Clinical Suspicion of Myeloproliferative Neoplasms and JAK2V617F≤1%
Authors: Garcia-Gisbert Nieves , Gomez-Perez Lucia , Velez Patricia , Fernández-Ibarrondo Lierni , Longarón Raquel , Senín Alicia , Fernández Concepción , Gibert Joan , Diez-Feijóo Varela Ramón , Tazon Barbara , Camacho Laura , Botella Irene , Ji Junjie , Lafuente Marta , Salar Antonio , Besses Carlos , Bellosillo Beatriz 
Published: December 2024

Background. JAK2V617F mutation is the most relevant genetic marker for the diagnosis of myeloproliferative neoplasms (MPN). Recommendations of WHO/ICC include the use of highly sensitive techniques for the detection of JAK2V617F with variant allele frequency (VAF) &lt;1%. However, the interpretation of low JAK2V617F burden may be challenging as this mutation has been described in individuals without hematological disease and no cut-off value has been defined for diagnosing MPNs.
Aim. To analyse the clinical and molecular characteristics of a cohort of patients referred with the suspicion of MPN and with JAK2V617F VAF≤1%.
Methods. A total of 40 patients referred to the Hematology Department due to erythrocytosis (n= 21) or thrombocytosis (n=19) with JAK2V617F≤1% were studied (median age 61, range 32-83). JAK2V617F was assessed in 198 granulocyte samples (40 at diagnosis and 158 during follow-up), platelet samples (n=33) and, when available, bone marrow aspirates (n=14) or bone marrow trephines (n=4) using quantitative PCR (Larsen, 2008, LoD 0.02%). Next-generation sequencing was performed in 38 patients with a QIAseq Custom DNA Panel (Qiagen) including the whole codifying region of 26 myeloid-associated genes.
Results. The median JAK2V617F VAF at the time of referral was 0.17%(0.02-1), with a higher value in cases with thrombocytosis compared to erythrocytosis (0.23% vs. 0.08%, p=0.004). JAK2V617F mutation was analyzed in platelet RNA in 33 cases, being positive in 8/15 cases derived for thrombocytosis and in 4/18 cases with erythrocytosis. Bone marrow analysis confirmed the presence of JAK2V617F mutation in all 18 cases analyzed, with allele frequency similar to that of granulocytes (median:0.26%;range:0.02-2.34). Screening for additional driver mutations showed two cases with CALR and 3 cases with MPL mutations among the patients with thrombocytosis and 1 case with MPL in a patient with erythrocytosis. Additional non-driver mutations were identified in 14/38 cases (37%) being DNMT3A (4/38), ASXL1 (4/38), and TET2 (3/38) the most frequently mutated genes. Median VAF of additional mutations was 6.85% (1.3-57%).
Of the 21 cases with erythrocytosis, 2 were finally diagnosed with polycythemia vera (PV), 3 with erythrocytosis without meeting MPN criteria, 11 with secondary erythrocytosis, and in 5 cases, the information was insufficient to establish a diagnosis. The median hematocrit was 52.3% (48-61), 6/21 cases required phlebotomy, and none received cytoreductive therapy. Of the 19 patients with thrombocytosis, 10 were diagnosed with essential thrombocythemia (ET) (5 with JAK2V617F≤1% only, 3 JAK2+MPL, and 2 JAK2+CALR), 1 with masked PV, 3 with thrombocytosis without meeting MPN criteria, and in 3 cases, there was no bone marrow biopsy available for a definitive diagnosis. Median platelet count was 627x109/L (457-1585), and 8/19 received cytoreductive therapy.
Sequential samples were collected in 36/40 cases, and JAK2V617F was confirmed in 34/36 patients. One of the unconfirmed cases had a CALR mutation. With a median follow-up of 36 months, the JAK2V617F clone remained stable over time with VAF&lt;2% in 32/36 cases. Clonal expansion was observed in one ET case (from 1% to 33%, follow-up 14 years) and in one unclassified patient (from 0.9% to 6.5%, follow-up 5 years). In two ET cases, JAK2V617F became negative during follow-up: one was a case positive for JAK2+CALR (being CALR the dominant clone); the other transformed into JAK2V617F-negative acute leukemia (with TP53 biallelic and DNMT3A mut).
Conclusion. Comprehensive molecular and clinical characterization of patients with erythrocytosis and/or thrombocytosis and JAK2V617F VAF≤1% allowed the diagnosis
of MPNs in 33% cases. This approach was particularly valuable for cases with thrombocytosis and low JAK2V617F, achieving an ET diagnosis rate of 58%.
In most patients (89%) with low-burden JAK2V617F mutation, the VAF remained stable during follow-up. These results highlight the need for thorough evaluation of cases with low JAK2V617F VAF and emphasize the importance of assessing the presence of
other driver mutations (CALR/MPL) in these cases.
Acknowledgments. Instituto de Salud Carlos III (ISCIII), PI16/0153, PI19/0005, PI23/00626, co-funded by the European Union; 2021SGR628, PT20/00023, and Xarxa de Banc de Tumors de Catalunya.