Molecular Characterization of Response/Loss-of-Response to Ruxolitinib in Patients with Myelofibrosis

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.