Background. Ruxolitinib (RUX) is standard of care for most patients (pts) with primary (PMF) and secondary (sMF) myelofibrosis. However, about half of responders lose benefits at 3-5y, associated with clonal progression, and have a dismal outcome thereafter. The molecular bases for response/loss of response to RUX have not been clarified yet.

Aim. To explore genetic variables associated with response/loss of response to RUX in MF pts.

Patients and Methods. All consecutive pts with WHO2016/IWG-MRT diagnosis of PMF/sMF, treated with RUX at our Center, were included. To the purpose of the study, response was stringently defined as achievement of spleen lenght reduction from LCM of >50% at any time point; non-responders (NR) were all the others. Loss-of-response was defined as the reappearance of >5cm splenomegaly, if the nadir under RUX was <5cm, and >50% increase if the nadir was >5cm. A custom NGS panel including complete CDS of 552 genes, selected for putative involvement in RUX response (belonging to JAK/STAT, MAPK, ERK pathways, inflammatory reactions, apoptosis) or associated with myeloid neoplasms, was designed. For RNAseq analysis, we used the R package DOSE and performed Over Representation Analysis (ORA) to identify over-represented biological functions/processes.

Results. Baseline characteristics. A total of 171 pts were included, 52.1% PMF (15.8% prePMF, 36.5% overtPMF) and 82 sMF (46.9%; 29.2% PPV-, 17.7% PET-MF). DIPSS at RUX start was Int-1: 43.8%, Int-2: 39.8%, high: 16.4%. Median FU from RUX start was 3.5y (0.8-17.3y), median time on RUX was 2.4y (0.2-12y). Sixty-eight pts (39.8%) died, 39 (22.8%) while on RUX. AML developed in 13 pts (7.6%), 10 of which while on RUX. OS from RUX start was 5.8y (4.4-7.3y).

JAK2V617F mut in 72.3%, MPLW515 7.6%, CALR 19.3% (12.8% Type1/like, 6.4% Type2/like), TN 2.3%. 68 pts (39.8%) were High Molecular Risk (HMR; ASXL1, EZH2, IDH1, IDH2, SRSF2, U2AF1).

Response to RUX. 93/171 pts (54.4%) were NR. Of the 78 responders (45.6%), 45 (57.7%) lost response (hence, R/L) after a median of 22.7 mo from RUX start, and 33 pts (43.3%) maintained response (hence, R/M) at last FU. Response outcome was influenced by RUX dose: 75.2% of NR and 64.4% of R/L had received <20 mg/die, compared with 63.6% of R/M who had received >30 mg/die. RUX stop (any reason) occurred in 24.2% of R/M, 62.2% R/L, 55% of NR (P=0.02).

Baseline molecular predictors. Baseline molecular variables found to correlate with response/loss of response included: (i) HMR status, found in 51.1% R/L vs 24.2% R/M and 40.2% NR (P=0.04); (ii) >1 RAS pathway mutated gene (CBL, NRAS, KRAS, PTPN11), found in 30% NR, 5% R/L and 13.8% R/M (P=0.016); (iii) isolated CBL (n=8, 10.5%; P=0.02) and U2AF1 (n=5,6.7%; P=0.05) mutation, found exclusively in NR pts. Nor type nor variant allele frequency (VAF) of driver mutations impacted on response/loss of response.

Serial mutational analysis. Among JAK2V617F mut pts, a partial molecular response (PMR, >50% VAF reduction; IWG-MRT criteria) was achieved by 18.6% of pts, after a median of 1.7y (1-5y). Median VAF reduction under RUX of pts with PMR was 71.3% (51-100%). A PMR occurred in 44.4% of R/M vs 14.8% of (R/L+NR), P=0.03. In 14 responder pts who were on stable dose of RUX since >1y and later became R/L, target NGS sequencing for 552 genes was performed using granulocyte DNA, collected at baseline (T0), at the time (+6mo) of response adjudication (T1) and at R/L (T2). A total of 91 mutated genes were acquired at T2 (mean gene number/pt, 2.5, range 1.0-16). Of these, 22 genes belonged to categories of "transcription regulator", 19 "cytokine mediator signaling", 14 "spliceasome/RNA processing", 11 "EGFR/TKI resistance" and 8 "chromatin regulators" (GO, KEGG, Reactome, Wikipathway). Serial, single cell analysis to interpret clonal hierarchy is ongoing, results will be presented at meeting. Pairwise RNAseq was performed in 4 pts with purified PB CD34+ cells collected at T1 and T2. By using a stringent FDR<0.1 value, enriched transcripts belonged to "RHO-GTPase" (n=88), "spliceasome" (n=40), "Type-2 interferon responses" (n=20), "IL12 stimulated gene and proteins" (n=14) categories.

Conclusions. This study adds to the understanding of molecular mechanisms of response/loss of response to RUX by identifying baseline predictors and highlighting a set of involved genes/pathways, that remain to be validated.

Guglielmelli:Novartis, Abbvie: Other: Other member of advisory board, speaker at meeting. Coltro:Novartis: Speakers Bureau. Loscocco:Novartis: Speakers Bureau. Mannelli:Blueprint: Speakers Bureau; Novartis: Speakers Bureau. Sant'Antonio:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vannucchi:Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Author notes

*

Asterisk with author names denotes non-ASH members.

Sign in via your Institution