Clinical Resistance To Ruxolitinib Is More Frequent In Patients Without MPN-Associated Mutations and Is Rarely Due To Mutations In The JAK2 Kinase Drug-Binding Domain
Authors: Andreoli Annalisa , Verger Emmanuelle , Robin Marie , Raffoux Emmanuel , Zini Jean-Marc , Rousselot Philippe , Socié Gérard , Rea Delphine , Parquet Nathalie , Giraudier Stephane , Chomienne Christine , Cassinat Bruno , Kiladjian Jean-Jacques 
Published: October 2019

Abstract

Background
Ruxolitinib (RUX) has been recently approved for the therapy of patients (pts) with myelofibrosis (MF). Although various definitions for response and resistance to this drug have been proposed in the setting of clinical trials, none of them has been validated in clinical practice. Based on RUX efficacy, it is assumed that primary resistance is related to absence or minor reduction in spleen size and constitutional symptoms, while spleen regrowth and recurrence of symptoms after a period with good response establish secondary resistance. The extents of all these parameters have not been clearly defined yet for patients’ management in clinical practice. On the other hand, several biological mechanisms of resistance have been described. In particular, acquisition of new mutations in the predicted RUX-binding region (like Y931C, G935R, R938L, I960V and E985K) was previously shown to confer resistance to JAK inhibitors in vitro (Hornakova et al, Haematologica, 2011; Weigert et al, J Exp Med, 2012). Such mutations have not yet been found in pts treated with RUX.

Aims
1) Characterize the parameters leading physicians to conclude that RUX-resistance (RUX-R) was reached in a cohort of consecutive MF pts treated with RUX in clinical practice. 2) Identify a molecular signature of RUX-R.

Methods
Consecutive pts with MF treated with RUX in our center were identified. Clinical characteristics and evolution during and after RUX therapy were collected. Pts were screened for JAK2V617F (PCR), MPL, TET2, and SRSF2 mutations (direct Sanger sequencing). In addition, the last exons of JAK2 corresponding to the RUX-binding domain were sequenced in RUX-R pts.

Results
In all, 41 pts received RUX between Nov 2009 and May 2013, including 21 primary, 8 post-PV and 12 post-ET MF, and full data were available in 39 of them for this analysis. Median age was 64 yrs; IPSS risk score was high in 46%, and int-1 or 2 in 54%; 72% were JAK2V617F positive, 10% MPL515-positive. Overall, 16/39 (41%) pts were considered RUX-R by their physician, only 4/16 being primary resistant with <10% reduction in spleen size after a median RUX exposure of 89 days. Median spleen size reduction (best response by palpation, compared with baseline) was 60% in the whole cohort, 50% in pts who developed secondary resistance, and 80% in non RUX-R pts. Secondary resistance was always due to regrowth of spleen either alone (n=6), or associated with recurrence of symptoms (n=5), or with marked leukocytosis (n=1). 44% of RUX-R pts also had hematological intolerance (35% in non RUX-R pts). Median RUX exposure was 298 days (Q1-Q3: 159-640), longer in RUX-R pts compared to non RUX-R pts (median of 383 vs. 292 days). Median starting dose was similar in both groups (15 mg BID), but a higher proportion of pts in the RUX-R group had to reduce the dose < 10 mg BID during follow-up (29% vs 17% in non RUX-R pts). However, 43% of RUX-R was noticed in pts on stable doses, and in 36% after dose escalation (up to 25 mg BID). Among RUX-R pts, there was a higher proportion of pts with high IPSS (56% vs. 39% in non RUX-R; p=0.06), and of post-ET MF (38% vs. 26%; p=0.08). Molecular profile of pts developing RUX-R showed that 31% of them had no mutation detectable at diagnosis in JAK2, MPL, TET2, and SRSF2, compared to 9% of pts in the non RUX-R group (p=0.003). Sequencing of the JAK2 kinase domain in samples taken at the time of resistance in 14/16 RUX-R pts did not detect any new mutation potentially affecting drug binding.

Conclusion
In this series of consecutive pts treated with RUX in a single center, RUX-R was observed in 41% of pts, mostly as a late event (after median exposure of 1 year) and rarely as primary resistance (10%). RUX-R was always associated with spleen regrowth, and accompanied with recurrence of symptoms in half of the cases. RUX-R was more frequently seen in pts with high risk IPSS score, post-ET MF, smaller spleen response (-50% vs. -80% in non RUX-R), but the only factor significantly associated with RUX-R was the absence of mutation in all of the following genes: JAK2, MPL, TET2, and SRSF2. Furthermore, sequencing of the kinase domain of JAK2 in RUX-R pts didn’t detect any mutation possibly affecting drug binding, suggesting that such mechanism is rarely involved in clinical resistance to RUX. Other mechanisms, like the phenomenon of “persistence” to JAK inhibitor therapy described by Koppikar at al (Nature, 2012) should be investigated in these pts.

Disclosures:
Robin: Novartis: Research Funding. Rea:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Teva: Honoraria; Ariad: Honoraria. Giraudier:NOvartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria. Kiladjian:AOP Orphan: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Research Funding; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.