Clinical and biological characterization of MPN patients harboring two driver mutations, a <scp>F</scp>rench intergroup of myeloproliferative neoplasms (FIM) study
Authors: Olivier Mansier, Damien Luque Paz, Jean‐Christophe Ianotto, Yannick Le Bris, Aurélie Chauveau, Françoise Boyer, Carole Conejero, Olivier Fitoussi, Jérémie Riou, Didier Adiko, Mohamed Touati, Jasmine Chauzeix, Jean‐François Viallard, Marie C. Béné, Stéphane Giraudier, Valérie Ugo, Eric Lippert
Journal: American Journal of Hematology (2017)

Mutations in "driver" genes (JAK2, MPL, or CALR) are found in the vast majority of Philadelphia-negative myeloproliferative neoplasms (MPN): JAK2V617F or exon 12 mutations in polycythemia vera (PV), JAK2V617F, CALR exon 9, or MPL exon 10 mutations in essential thrombocythemia (ET) or primary myelofibrosis (PMF). In the latter, phenotypic presentation and evolutive course vary according to the mutation, eg, higher platelet counts in CALR mutated ET patients and higher hemoglobin, white blood cell counts and higher risk of thrombosis in JAK2-mutated ET.1 Driver mutations were initially described as mutually exclusive, but can co-exist in rare "doubly mutated" (DM) patients.2-4 We have previously shown that double mutations were more frequently encountered in patients with low (<5%) JAK2V617F allelic burdens.5 However, due to the limited reports of DM patients, it is unclear how co-occurrence of driver mutations affects the presentation or evolution of MPN. In order to confirm DM frequency in a multicentric fashion, two additional cohorts of JAK2-mutated MPN were screened for CALR mutations: 134 JAK2V617F-mutated ET from the University Hospital of Créteil (France) and a cohort of 31 MPN with a low JAK2V617F allele burden (<2%) from the University Hospital of Nantes (France). Five of 49 patients with a JAK2V617F allele burden <5% also had a CALR mutation (10.2%), similar to our previous findings (14.3%) versus none of the 119 patients with JAK2V617F > 5%. This confirms the relatively high frequency of DM in patients with low JAK2V617F burden. In addition, clinical and biological data were gathered on 21 DM patients discovered fortuitously in 4 other French centers. Overall, 47 cases of DM patients (Supporting Information Figure 1) were analyzed, of which 28 harbored a JAK2V617F allele burden equal to or below 1% (60%) while only 2 (5%) had a JAK2V617F allele burden above 5%. Among the 47 DM patients, 40 were diagnosed with ET, 5 with PMF, 1 with PV, and 1 with MDS/MPN-RS-T. Thirty-two patients (68%) presented with JAK2V617F and CALR mutations, 11 (23%) with JAK2V617F and MPL mutations, 2 with CALR and MPL mutations, 1 PV patient had JAK2V617F and JAK2 exon 12 mutations, and 1 patient had an association of two different CALR mutations. In order to know whether a double mutation impacts the clinical presentation or the evolutive course, the 40 DM patients with ET were compared to a control cohort comprising 577 "classical" ET patients from OBENE cohort, NCT02897297. Patients' characteristics were compared using Fisher exact test for categorical variables as well as Mann and Whitney and Kruskal–Wallis (followed by Dunn tests for multiples comparisons) tests for continuous variables. For multiple tests, P-values were adjusted with Hochberg's method. Survival estimates were obtained with the Kaplan–Meier method, and statistical analyses were performed with Log-rank test and Cox model. DM-ET patients were more often males (M/F sex ratio 1.4 [23/17] vs. 0.68 [233/344], P = .045) and significantly older than control ET patients (median: 72 vs. 61 years old, P < .001), especially compared to JAK2-singly mutated (SM) (63 years old, P = .006), CALR-SM (59 years old, P < .001) or triple-negative (56 years old, P < .001) ET patients. For clinical comparisons, the 37 DM-ET patients with a JAK2V617F mutation (29 with JAK2-CALR and 8 with JAK2-MPL) were compared to age- and sex-matched ET patients (Supporting Information Table 1): each CALR-JAK2V617F DM patient was matched with 3 JAK2V617F and 2 CALR; MPL-JAK2V617F DM with 3 JAK2V617F and 1 MPL-SM ET patients. At diagnosis, DM-ET and CALR or MPL-SM matched control patients had similar hemoglobin and platelet counts, but lower hemoglobin and higher platelet counts than JAK2-SM controls (Figure 1A,B). Leukocyte counts of JAK2-CALR DM-ET patients were intermediate between those of CALR and JAK2-mutated controls, without statistically significant difference (Figure 1C). Splenomegaly at diagnosis was observed in 17% (4/24) of JAK2-CALR DM-ET patients and none of the 6 JAK2-MPL DM-ET for whom this data was available. These frequencies were not significantly different from those observed in respective JAK2, CALR, or MPL SM control groups (Supporting Information Table 1). A history of thrombosis before or at diagnosis was found in 3/16 (19%) of JAK2-CALR DM-ET, which is equivalent to the proportion observed in CALR mutated controls (21%), but lower than that in JAK2 mutated controls (41%), without reaching statistical significance (P = .27). Biological presentation at diagnosis of doubly mutated ET (DM-ET) compared to matched patients: hemoglobin (A), platelets (B), and leukocytes (C) The JAK2V617F allele burden was much lower in DM-ET patients than in matched controls (median 1% vs. 26%, P < .001, Supporting Information Figure 2A). The relatively frequent discovery of low JAK2V617F burdens in DM patients might be explained by a pre-existing clonal hematopoiesis of indeterminate potential (CHIP), followed by the acquisition of a second oncogenic event (CALR or MPL mutation) leading to the MPN phenotype.6 This would be supported by the fact that DM patients were older. On the other hand, CALR mutations in JAK2-CALR DM-ET patients were also found at a lower allelic burden than in matched CALR-mutated patients: median 40% vs. 47%, P = .014 for fragment analysis (Supporting Information Figure 2B), 30% vs. 45%, and P = .009 for digital PCR (Supporting Information Figure 2C), consistent with a competition between two different clones. The occurrence of thrombosis during follow-up (median 36 months) in the 40 DM-ET patients was not different from JAK2- and CALR-SM matched ET-patients (Supporting Information Figure 3A). The long-term (over 90 months) risk of thrombosis in DM-ET patients appeared to be close to that observed in CALR-mutated patients and lower than in JAK2-mutated patients (Supporting Information Figure 3B), but the population was too small to reach clear statistical significance. Only two patients, one with JAK2-CALR and one with JAK2-MPL co-mutations, evolved to secondary myelofibrosis after 4 and 14 years, respectively. This was not different from the evolution in JAK2- and CALR-SM patients (Supporting Information Figure 3C). Only one patient with JAK2-MPL co-mutation transformed to secondary acute myeloid leukemia 93 months after diagnosis and died 9 months later. No other DM-patient died during the follow-up period. In conclusion, screening for a second driver mutation in patients with a low JAK2V617F allele burden (particularly if <1%) can be helpful to confirm the MPN diagnosis. At least in ET, the disease phenotype of DM patients seems to be dictated by the predominant mutation. Authors acknowledge the physicians who gave medical information: Pr Frédéric Bauduer and Dr. Fanny Menard (CH de Bayonne), Dr. Gabriel Etienne (CLCC Bergonié), Dr. François Lifermann and Dr. Bruno Le Pluart (CH de Dax), Dr. Corinne Dagada (CH de Pau), Dr. Anne Solanilla (Clinique mutualiste du Médoc), Dr. Le Coz Marie-Francoise (CH de Lorient), Dr. Marion Loirat (CH de Saint Nazaire), Dr. Cédric Pastoret (CHU de Rennes). The authors would also like to thank all local tumor biobanks for providing high-quality samples and the French clinical and biological network of myeloproliferative neoplasms (FIMBANK) supported by a grant from the French National Institute of Cancer (INCa BCB 2013). OM was supported by the Ministère de l'Enseignement Supérieur et de la Recherche. Nothing to report. OM, DLP, JCI, YLB, AC, CC, OF, MCB and SG collected the data; OM and DLP analyzed the data; OM, DLP, CC, SG and YLB performed molecular testing; OM, DLP and JR performed statistical analysis; VU and EL designed the study; OM, DLP, VU and EL wrote the paper. Additional Supporting Information may be found online in the supporting information tab for this article. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.