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4566 Diagnostic Challenges in Patients with Clinical Suspicion of Myeloproliferative Neoplasms and JAK2V617F≤1%

Program: Oral and Poster Abstracts
Session: 634. Myeloproliferative Syndromes: Clinical and Epidemiological: Poster III
Hematology Disease Topics & Pathways:
Research, MPN, Clinical Research, Chronic Myeloid Malignancies, Diseases, Real-world evidence, Myeloid Malignancies, Biological Processes, Molecular biology, Technology and Procedures, Molecular testing
Monday, December 9, 2024, 6:00 PM-8:00 PM

Nieves Garcia-Gisbert, PhD1,2*, Lucia Gomez-Perez, MD3*, Patricia Velez, MD3*, Lierni Fernández-Ibarrondo, PhD1*, Raquel Longarón1,4*, Alicia Senín, MD3*, Concepción Fernández, PhD1,4*, Joan Gibert, PhD1*, Ramón Diez-Feijóo Varela, MD1,3*, Barbara Tazon, PhD4*, Laura Camacho1,4*, Irene Botella1*, Junjie Ji1*, Marta Lafuente1*, Antonio Salar, MD1*, Carlos Besses, MD1* and Beatriz Bellosillo, PhD1,4*

1Hospital del Mar Research Institute, Barcelona, Spain
2Department of Hematology, Cambridge Stem Cell Institute, Cambridge, United Kingdom
3Department of Hematology, Hospital del Mar, Barcelona, Spain
4Department of Pathology, Hospital del Mar, Barcelona, Spain

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) <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<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.

Disclosures: Velez: Novartis: Speakers Bureau; GSK: Speakers Bureau. Senín: Novartis: Speakers Bureau; GSK: Speakers Bureau. Fernández: Astra-Zeneca: Speakers Bureau. Gibert: Roche: Membership on an entity's Board of Directors or advisory committees; Astra-Zeneca: Speakers Bureau. Tazon: Astellas: Speakers Bureau; Novartis: Speakers Bureau. Salar: Gilead Sciences: Research Funding; Beigene: Consultancy, Speakers Bureau; Astra-Zeneca: Consultancy, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Ipsen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; BeiGene: Consultancy, Speakers Bureau. Bellosillo: Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck-Serono: Speakers Bureau; Novartis: Speakers Bureau; Roche: Research Funding, Speakers Bureau; ThermoFisher: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau.

*signifies non-member of ASH