Background: Recent data indicate that driver mutations are prognostically informative in patients (pts) with primary myelofibrosis (PMF). Patients with CALR type 1/like (Ty1) mutation had better survival (OS) compared with pts with CALR type 2/like (Ty2) and JAK2 V617F/ MPL W515 mutation (Tefferi A, Blood 2014; Guglielmelli P, BCJ. 2015); conversely, pts lacking any driver mutation, i.e. "triple negative"(TN), suffered from the worst survival (Rumi E, Blood 2014; Guglielmelli P, Blood 2017). Furthermore, a high molecular risk category (HMR), pointing to pts harboring at least one of ASXL1, EZH2, IDH1/2 and SRSF2 mutated genes, independently predicted for shortened survival and increased risk of leukemia (Vannucchi A, Leukemia 2013; Guglielmelli P, Leukemia 2014).

Aims: To comparatively evaluate the molecular landscape of TN patients in comparison with other driver mutation categories and the possible relationships with the adverse outcome of this pts' group.

Methods: The patient cohort included 680 PMF patients, diagnosed according to WHO-2016 criteria, who provided informed consent for the study. Pts were referred by 6 Italian Centers belonging to the AGIMM group. For all patients driver (JAK2, MPL, CALR) and HMR mutation status was obtained by PCR and NGS, respectively. For 201 of these patients (38 Ty1, 20 Ty2, 110 JAK2/MPL and 32 TN), extended NGS sequencing was performed using Ion Torrent platform in Florence (n=133) and a hybrid-capture NGS-CGP assay [FoundationOne heme® assay (He J et al, Blood, 2016)] in Cambridge (n=68). NGS covered a panel of 20 non-driver genes including: all coding sequence of c-KIT, TET2, RUNX1, NRAS, KRAS, DNAMT3A, IKZF1, EZH2, TP53 ; hot spot of IDH1/2 and SRSF2 and selected exons of CBL, IDH2, ASXL1, SF3B1, NFE2, SH2B3, U2AF1 and SETBP1 . The nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimate of survival and log-rank test were used as appropriate.

Results: Of the 680 pts included, 419 were JAK2 +(70%), 38 MPL + (5.6%), 82 TN (12.1%) and 140 CALR + (22%; 105 (75%) Ty1 and 35 (25%) Ty2). Overall, 81 pts (11.9%) progressed to AML. Death occurred in 325 pts (47.8%) after a median follow up of 4.8y. Median survival of CALR Ty1 was 17.8y vs 8.6y for CALR Ty2, 7.2y for JAK2/MPL and 2.6y for TN (P<.0001). The corresponding hazard ratio, taking CALR Ty1 as reference, was 1.6 (95%CI,1.0-2.9), 2.2 (95%CI, 1.5-3.1) and 4.9 (95%CI, 3.2-7.5) for CALR Ty2, JAK2/MPL and TN pts, respectively. HMR status was associated with shortened survival (HR 2.4, 95%CI 1.9-3.0; P<.001) as it was having >2 HMR mutations (HR 4.1, 95%CI 3.0-5.6; P<.001). Among the mutated genes of the extended panel, those associated with shortened OS, in addition to the 5 HMR genes, were NRAS (P=.002), TP53 (P=.049), U2AF1 (P=.035) and SETBP1 (<.001). Negative impact on survival was maintained irrespective of underlying driver mutation.

TN pts showed enrichment of SRSF2 mutations (28% vs 1% in CALR Ty1, 6% CALR Ty2, 9% JAK2/MPL ; P<.0001) and ASXL1 (37% vs 23% in CALR Ty1, 29% CALR Ty2, 25% JAK2/MPL ; P=0.02). As many as 56% of TN pts were in the HMR category compared with27% in CALR Ty1, 37% CALR Ty2, 36% JAK2/MPL (P=0.02) and >2 HMR mutations were found in 27% of TN pts vs 4% Ty1, 9% Ty2, 9% JAK2/MPL (P<.001). Regarding the additional prognostically meaningful mutations of the extended panel, 69% of TN pts had at least 1 mutated gene compared with 38% Ty1, 39% Ty2, 48% JAK2/MPL (P<.01); furthermore, 2 and more mutated genes were found in 53% of TN pts vs 13% in Ty1, 17% in Ty2, 27% in JAK2/MPL ( P=.01). In particular, NRAS mutations were found in 18% of TN pts vs 8% Ty1, 10% Ty2, 2% JAK2/MPL (P=.006). The median OS was significantly reduced in TN pts having at least one of the additional mutations: 1.9y (range 1.8-2.1y) compared with pts lacking those mutations (OS not reached; HR 5.3; 95%CI, 1.5-18.8). The median age of TN pts (67y, 13-91y) was no different from JAK2/MPL (65y; 19-90) and Ty2 (60y, 18-88y), while Ty1 were significantly younger (48y, 16-96); therefore, accumulation of mutations among TN pts in comparison with other driver mutation categories could not be explained by advanced age, except for CALR Ty1.

Conclusions: These findings suggest that the worse outcome associated with Triple Negativity in PMF might be explained, at least in part, by an underlying greater mutation complexity.


Nahas: Foundation Medicine inc: Employment, Other: stockholder. He: Foundation Medicine Inc: Employment, Other: stockholder. Agarwal: Foundation Medicine Inc: Employment, Other: stockholder. Rambaldi: Novartis, Roche/Genentech, Amgen, Italfarmaco: Consultancy; Novartis, Amgen, Celgene, Sanofi: Other: Travel, Accomodations, Expenses. Mughal: Foundation Medicine Inc: Other: stockholder. Passamonti: Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Vannucchi: Novartis: Honoraria, Speakers Bureau; Shire: Speakers Bureau.

Author notes


Asterisk with author names denotes non-ASH members.