Multiclass HCV resistance to direct‐acting antiviral failure in real‐life patients advocates for tailored second‐line therapies

Abstract Background & Aims Despite the excellent efficacy of direct‐acting antivirals ( DAA ) reported in clinical trials, virological failures can occur, often associated with the development of resistance‐associated substitutions ( RAS s). This study aimed to characterize the presence of clinically relevant RAS s to all classes in real‐life DAA failures. Methods Of the 200 virological failures that were analyzed in 197 DAA ‐treated patients, 89 with pegylated‐interferon+ribavirin (Peg IFN + RBV ) and 111 without ( HCV ‐1a/1b/1g/2/3/4=58/83/1/6/24/25; 56.8% treatment experienced; 65.5% cirrhotic) were observed. Sanger sequencing of NS 3/ NS 5A/ NS 5B was performed by home‐made protocols, at failure (N=200) and whenever possible at baseline (N=70). Results The majority of the virological failures were relapsers (57.0%), 22.5% breakthroughs, 20.5% non‐responders. RAS prevalence varied according to IFN / RBV use, DAA class, failure type and HCV genotype/subtype. It was 73.0% in IFN group vs 49.5% in IFN free, with the highest prevalence of NS 5A‐ RAS s (96.1%), compared to NS 3‐ RAS s (75.9% with IFN , 70.5% without) and NS 5B‐ RAS s (66.6% with IFN , 20.4% without, in sofosbuvir failures). In the IFN ‐free group, RAS s were higher in breakthrough/non‐responders than in relapsers (90.5% vs 40.0%, P <.001). Interestingly, 57.1% of DAA IFN ‐free non‐responders had a misclassified genotype, and 3/4 sofosbuvir breakthroughs showed the major‐ RAS ‐S282T, while RAS ‐L159F was frequently found in sofosbuvir relapsers (18.2%). Notably, 9.0% of patients showed also extra target RAS s, and 47.4% of patients treated with ≥2 DAA classes showed multiclass resistance, including 11/11 NS 3+ NS 5A failures. Furthermore, 20.0% of patients had baseline‐ RAS s, which were always confirmed at failure. Conclusions In our failure setting, RAS prevalence was remarkably high in all genes, with a partial exception for NS 5B, whose limited resistance is still higher than previously reported. This multiclass resistance advocates for HCV resistance testing at failure, in all three genes for the best second‐line therapeutic tailoring.