Barbara Mora,¹ Claudia Siracusa,² Elisa Rumi,^3,4 Margherita Maffioli,¹ Ilaria Carola Casetti,^3,4 Daniela Barraco,¹ Michele Merli,¹ Marianna Rossi,⁵ Marta Ubezio,⁵ Raffaella Accetta,² Laura Libera,² Daniela Pietra,³ Chiara Trotti,⁴ Silvia Uccella,^6,7 Francesco Pallotti,^2,7 Rosario Casalone,² Lorenza Bertù,⁷ Luca Arcaini,^3,4 Matteo Giovanni Della Porta ⁵ and Francesco Passamonti.^1,7

¹Hematology, ASST Sette Laghi, Ospedale di Circolo, Varese, Italy; ²Cytogenetics and Medical Genetics Laboratory, ASST Sette Laghi, Ospedale di Circolo, Varese Italy; ³Division of Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; ⁴Department of Molecular Medicine, University of Pavia, Pavia, Italy; ⁵Cancer Center, IRCCS Humanitas Research Hospital & Humanitas University, Rozzano, Italy; ⁶Pathology, ASST Sette Laghi, Ospedale di Circolo, Varese Italy; ⁷Department of Medicine and Surgery, University of Insubria, Varese, Italy.

Worldwide, for essential thrombocythemia (ET) and primary myelofibrosis (PMF) driver mutations are investigated on the basis of a stepwise approach: JAK2 first, and CALR/MPL only in case of JAK2 negativity.¹ Further molecular investigations are usually not performed, except for those centers that routinely use next generation sequencing (NGS).¹

Recent studies showed that CALR or MPL could co-occur with JAK2 in 5-15% subjects, especially in case of low (<5%) JAK2 allele burden (AB).^2-4 In those papers, information on double mutated (DM) cases is scant.^2-4 Therefore, we studied incidence and phenotype of DM patients within 388 JAK2 mutated ET, PMF and post polycythemia myelofibrosis.  For the purpose of the study, we included also 116 CALR mutated ET and PMF, together with an external validation cohort of 91 low JAK2 AB ET and PMF.

Our study confirmed that is not useful to test for additional driver mutations in case of JAK2 AB higher than 5%. On the contrary, within the low (<5%) JAK2 AB ET and PMF group, DM cases ranged between 30 to 36%, slightly more frequent than previously reported.^2-4 Therefore, the approach of not searching for driver mutations in case of JAK2 positivity might be inappropriate in about one third of low JAK2 AB ET and PMF.

Among DM cases, CALR was the most frequently co-mutated gene, with a variant allelic fraction that clearly identifies CALR clone as dominant.

By applying NGS in low JAK2 AB cases, we did not disclose any significant association between DM status and number of myeloid genes variants, as already reported in larger series of single mutated ET and PMF cases.^5,6 (Figure 1)

Figure 1. Thirty genes-panel DNA sequence variants in 47 patients with essential thrombocythemia and primary myelofibrosis and a low JAK2 allele burden according to double/single mutated status.

Double (DM) and single mutated (SM) cases are distinguished as follow: DM for JAK2 and CALR= light grey; DM for JAK2 and MPL= dark grey; SM= dark dots. Variant/mutations are depicted by representative colored bars. Red= variants with a variant allele frequency (VAF) >5%; blu= variants with a VAF ranging from 2 to 5%; orange= variants with a VAF <2%. ET= essential thrombocythemia; oPMF= overt primary myelofibrosis; pPMF= prefibrotic primary myelofibrosis.

How can practicing physicians recognize low JAK2 mutated ET and PMF patients with the highest probability to be DM? We found and validated the 700 x10⁹/L-platelet count as discriminatory for DM: there was a 70%-probability that DM patients had a platelet count exceeding 700 x10⁹ /L and a 74-94% that single mutated patients had values lower than 700 x10⁹ /L. In the presence of this platelet count, testing other driver mutation is much advised.

REFERENCES

1. Barbui T, Tefferi A, Vannucchi AM, Passamonti F, Silver RT, Hoffman R et al. Philadelphia chromosome-negative classical myeloproliferative neoplasms: revised management recommendations from European LeukemiaNet. Leukemia 2018; 32: 1057-1069.
2. Lim KH, Chang YC, Gon-Shen Chen C, Lin HC, Wang WT, Chianget YT et al. Frequent CALR exon 9 alterations in JAK2 V617F-mutated essential thrombocythemia detected by high resolution melting analysis. Blood Cancer J 2015; 5:
3. Mansier O, Luque Paz D, Ianotto JC, Le Bris Y, Chauveau A, Boyer F et al. Clinical and biological characterization of MPN patients harboring two driver mutations, a French intergroup of myeloproliferative neoplasms (FIM) study. Am J Hematol2018; 93: E84-E86. 
4. Boddu P, Chihara D, Masarova L, Pemmaraju N, Patel KP, Verstovsek S. The co-occurrence of driver mutations in chronic myeloproliferative neoplasms. Ann Hematol 2018; 97: 2071-2080. 
5. Tefferi A, Guglielmelli P, Lasho TL, Coltro G, Finke CM, Loscocco GG et al. Mutation‐enhanced international prognostic systems for essential thrombocythaemia and polycythaemia vera. Br J Haematol 2020; 189: 291-302.
6. Tefferi A, Lasho TL, Finke CM, Elala Y, Hanson CA, Ketterling RP et al. Targeted deep sequencing in primary myelofibrosis. Blood Adv 2016; 1: 105-111.