Abstract Introduction: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with driver mutations in JAK2, CALR, and MPL genes. Non-driver mutations involved in epigenetic regulation, signaling, and splicing are suggested to affect disease progression and treatment response. Interferon-alpha2 (IFN) induces hematologic and molecular responses in patients (pts) with MPNs. We studied 20 CALR positive pts by targeted next generation sequencing (NGS) of 68 genes and investigated the impact of somatic mutations on the molecular response to IFN. Methods: Twenty JAK2V617F negative CALR mutated pts (type 1 (n=14), type 2 (n=4), and other (n=2)) with ET (n=3), pre-PMF (n=5) and PMF (n=12) treated with IFN participated in the study. Targeted NGS was performed on DNA from peripheral blood at baseline and during IFN therapy. Libraries were prepared using an in-house gene panel covering 68 genes (Table 1). NGS was performed on the Ion Torrent platform and data were analyzed using Biomedical Genomics workbench and VarSeq. Variants with coverage <100x, variant allele frequency (VAF) <3%, introns, germline and synonymous variants, and SNPs with minor allele frequency >1% were excluded. A mutation with VAF <3% in either a pre- or post-treatment sample was retained if VAF was >3% in the paired sample. Statistical analysis was done in R and a p-value <0.05 was considered significant. Results: Mean average coverage per base was 2749 (range:1964-3874). In all pts, median CALR allele burden (%CALR) was 41.5% (range: 30-53) and 40% (range: 5.7-54) at baseline and follow-up, respectively. The median duration of IFN treatment from NGS at baseline to follow-up was 33 months (range: 7-100). Median number of non-driver mutations was 3 (range: 0-9) in all pts. Nineteen (95%) pts had at least one non-driver mutation, 14 (70%) >1, and 12 (60%) ³3. Stratified according to molecular response (MR) and non-MR, 4 (20%) achieved MR and 16 (80%) non-MR. Median %CALR at baseline was 42 (30-53) and 39 (33-49) in non-MR and MR, respectively, and 44 (25-54) and 10 (6-15) in non-MR and MR, respectively, during treatment with IFN. Median number of non-driver mutations was 2 (range: 0-6) in MR and 3 (range: 1-9) in non-MR. In all pts, there were 64 non-driver mutations in 18 genes at baseline and during IFN therapy. Of the 20 pts analyzed, TET2 occurred in 50% of pts, CUX1 in 45%, DNMT3A in 40%, GATA2 in 35%, ASXL1 in 30%, and SH2B3 in 10%. Each of the mutations - CBL, IKZF1, VEGFA and XPC occurred in 10% and EZH2, JAK2S523del, NF1, NFE2, PHIP, SF3B1, SRSF2, and TGFB1 in 5%. Interestingly, the epigenetic regulator genes ASXL1, DNMT3A, and TET2 were frequently mutated. Notably, TET2 occurred exclusively in pre-PMF and PMF. To examine if non-driver mutations were associated with a response to treatment with IFN, %CALR was evaluated in TET2, ASXL1, CUX1, DNMT3A, and GATA2 wild type (wt) and mutated pre- and post-treatment samples. Patients with mutations in CUX1 had a significantly higher post-IFN-treatment %CALR compared to CUX1 wt pts (p<0.03). Moreover, CUX1 wt pts had a significant reduction in %CALR during treatment (p<0.04) (Figure 2). Different scenarios were observed when comparing the evolution of the mutant allele burden in non-driver mutations and the CALR mutation during treatment (Figure 3). An ASXL1 mutation was present in one MR with VAF decreasing from 3 to 0.5 in response to IFN. Five non-MRs carried an ASXL1 mutation with increasing or unchanged VAF in 4 of 5 pts and decreasing VAF in one pt. Discussion and Conclusions: In the present study, ASXL1, DNMT3A, and TET2 were frequently mutated possibly due to the high number of pts with pre-PMF and PMF. An association between poor response to IFN and ASXL1 mutations was recently reported in PMF. Indeed, we found a possible association between ASXL1 mutations and non-MR with increased or unchanged %ASXL1 during IFN therapy. However, in one MR and one non-MR, we recorded a marked decline in %ASXL1 but a sustained decrease or increase in %CALR, respectively. These results might imply a pt specific clonal heterogeneity. CUX1 is a transcription factor regulating TP53. We show that pts without CUX1 mutations had a significant reduction in %CALR and a significantly lower %CALR compared to CUX1 mutated pts during treatment. Our finding of a blunted response to IFN in CALR/CUX1 mutated pts deserves further studies in larger cohorts of CALR mutated MPNs. Disclosures Hasselbalch: Novartis: Research Funding.