Background: Treatment of chronic hepatitis C virus (HCV) infection with Glecaprevir and Pibrentasvir (G/P) results in high rates of virologic success both in clinical trials and real world. However, the number of patients with virologic failure to G/P in phase II/III studies was limited (n=22), and there is also little information on how patients fail to this combination.. Here we present real world data on failure to G/P from three international cohorts across different genotypes.

Methods: Samples were obtained from resistance databases in Germany, Spain, Italy, Austria and Switzerland containing samples from more than 1200 DAA failure patients. Population sequencing of NS3, NS5A and NSB5 was conducted and RASs conferring a >2-fold increased DAA susceptibility were analyzed. Limited clinical parameters were collected retrospectively.

Results: Altogether 63 patients had a virologic treatment failure to G/P, 93% (52/56) treated for 8 weeks and 5% (3/56) for 12 weeks. 47 patients were male (82,5%) with a mean age of 52 years (IQR, 44-61). The majority of patients was infected with genotype (GT) 3a (n=29, 46%), while 15 individuals had GT1a, 11 had GT2c, 5 had GT1b, 2 had GT2a and 1 was infected with GT4d. Cirrhosis was detected in 6 patients, 2 patients had previous exposure to interferon and one had failed to a previous DAA combination treatment. Overall, 17 patients (26%) failed a G/P regimen without any RAS to the regimen. After failure to G/P, 47% (36/58) patients developed RASs in NS5A, 19.3% (11/57) in NS3, and 13.0% (7/54) in both NS3 and NS5A. The most frequent RASs were Y93H (22/58; 37.9%) in NS5A and Q168L (4/57; 7.0%) in NS3. Interestingly, a combination of two or more RASs in NS5A was frequently detected (one n= 5; two n=26; three n=5). P32 deletion in NS5A was detected in a genotype 1b infected patient.
Conclusion: Even if around one third of patients failed to G/P without resistance, RASs in NS5A were frequent and more prone to develop than in NS3. NS5A RAS were frequently observed as dual and triple patterns, with a high impact on NS5A inhibitor activity. Our results support the use of resistance findings to aid decision making on how to retreat G/P failures.

Background: Up today, there is a limited documentation about the efficacy of retreatment in patients who previously failed a recommended NS5A-containing regimen in Italy.
Materials & Methods: Within the Italian network VIRONET-C, a total of 412 NS5A-failing patients infected with different HCV-genotypes (GT) (GT1a/1b/2a-c/3a-b-g-h/4a-d-n-o-v=96/125/26/127/38) were analyzed. The retreatment of 108 failures was also investigated. Sanger sequencing of NS3-protease and/or NS5A and/or NS5B was performed at the time of virologic failure, with in-house protocols in 14 different Italian centers.
Results: Failures following seven different NS5A-containing regimens were studied: 3D/2D (paritaprevir/ombitasvir+dasabuvir)+ribavirin (N=77/4), daclatasvir/ledipasvir/velpatasvir +sofosbuvir+ribavirin (N=113/131/24), grazoprevir/elbasvir+ribavirin (N=34), glecaprevir/pibrentasvir (N=29). Notably, 19.2% (79/412) of NS5A-failing patients did not show any resistance-associated-substitutions (RAS), while 80.8% (333/412) showed at least one NS5A-RAS, with multiclass-resistance in 33.7% (139/412). A different distribution of NS5A and NS3 RAS was observed at grazoprevir/elbasvir and glecaprevir/pibrentasvir failure, respectively. In particular, NS5A-RAS were more prevalent in grazoprevir/elbasvir failures (94.1%, 32/34) than in glecaprevir/pibrentasvir failures (51.7%, 15/29; p<0.001). Also NS3-RAS were frequently detected in grazoprevir/elbasvir failures (29.4%, 10/34) compared to glecaprevir/pibrentasvir failures (6.9%, 2/29; p=0.02). Notably, all patients with NS3-RAS showed also NS5A-RAS at failure in both these regimes. At velpatasvir/sofosbuvir failure, NS5A-RAS were found in 15/24 (62.5%) patients, particularly in GT1a (28.6%, N=7) and GT3a (90.0%, N=10).
To date, 108 failures have started a retreatment: sofosbuvir/velpatasvir+ribavirin (N=30), sofosbuvir/velpatasvir/voxilaprevir+ribavirin (N=70), glecaprevir/pibrentasvir (N=4), grazoprevir/elbasvir+sofosbuvir+ribavirin (N=3), grazoprevir/elbasvir+ribavirin (N=1).
The majority of patients were cirrhotic (50.9%) and relapsers (93.1%). The prevalence of NS5A-RAS before retreatment was 79.6%, and multiclass-resistance 27.8%. Among patients completing post-retreatment follow-up, a sustained-viral-response at week 12 (SVR12) was observed in 66/74 (89.2%). SVR4 was documented in 76/83 (91.5%). SVR12 was 77.8% with sofosbuvir/velpatasvir+ribavirin (N=27). Differently, SVR12 was 100% with glecaprevir/pibrentasvir for 8/12/16 weeks (N=4), grazoprevir/elbasvir+sofosbuvir+ribavirin for 12/24 weeks (N=4), despite the presence of NS5A-RASs.
Of 70 patients who started sofosbuvir/velpatasvir/voxilaprevir+ribavirin recommended-retreatment for 12 weeks, 56/70 (80.0%) showed at least one baseline NS5A-RAS, 23/70 (32.8%) multiple-NS5A-RASs, and 22/70 (31.4%) multiclass-resistance. Of 39 patients with available outcome, 94.9% had SVR12. Virologic failure was observed in two patients. One was a GT1b infected patient non-responder, without any RAS before and after retreatment. The other failure was a GT1a infected patient relapse, who showed the NS5A resistance pattern Q30R+L31M at baseline of retreatment. This resistance pattern was confirmed at retreatment-failure, without any new additional RAS in NS3 and NS5B genes.

Conclusions: In this Italian real-life experience, NS5A-RASs were frequently detected in NS5A-failing patients, and multiclass-resistance was around 30%. Overall, SVR after resistance-test-guided retreatment was >94%, with the exception of the sofosbuvir/velpatasvir retreatment. Our results show how HCV genotypic resistance test after failure may be useful to optimize the retreatment strategies.

Background: Hepatocellular Carcinoma (HCC) generally arises after a stage of advanced liver fibrosis and cirrhosis with an incidence about 1-8% per year. The HCV protein, NS5A, is known to interact with different cellular proteins, including P53, thus inducing intracellular signaling pathways associated with cell proliferations. In this light, we explored the genetic variations in NS5A associated with HCC.

Materials & Methods: This study includes 188 patients chronically infected with HCV genotype 1b, all cirrhotic and DAA-naïve: 34 diagnosed with HCC and 154 controls without HCC. NS5A domain-1 (amino acid: 1-183) sequences were obtained for all patients by Sanger method from plasma samples. Association of mutations with HCC was assessed by Fisher Exact test. Shannon Entropy (SE) was used to describe residues with significantly higher (P<0.05) variability (SE>0.2) in HCC compared to No-HCC.

Results: HCC patients were characterized by comparable median (IQR) log serum HCV-RNA [5.6 (5.3-6.1) vs 5.8 (5.3-6.1) IU/mL], ALT [65 (37-86) vs 71 (50-112) U/L], and significantly higher liver stiffness [28 (20-33) vs 19 (15-26) KPa, P<0.001] compared to No-HCC patients.
By mutational analysis, four specific NS5A polymorphisms significantly correlated with HCC: S3T (8.8 vs 1.3%, P=0.01), T122M (8.8% vs 0.0%, P<0.001), M133I (20.6 vs 3.9%, P<0.001), and Q181E (11.8 vs 0.6%, P<0.001). By SE, other three residues were more variable in HCC: C13R/S (SE=0.264; P=0.03, located in highly conserved N-terminus NS5A domain), F127L/S (SE=0.225; P=0.03), and N137D/K (SE=0.264; P<0.001). Furthermore, an enrichment of additional mutations is observed at residue 181 (Q181E/G/H/P, SE=0.410; P=0.01). Notably, all the above mentioned residues are localized in regions of NS5A domain-1 known to interact with cellular proteins as P53 (aa:1-149), involved in the apoptosis regulation, and/or with P85-PIK3 (aa:1-112), involved in Wnt/β-catenin signaling pathway regulating the cell growth.

Conclusions: The association of specific NS5A polymorphisms with HCC provide a focus for further investigations aimed at elucidating the molecular basis of HCV-mediated oncogenesis. These viral signatures, if confirmed in a larger population, could play a crucial role as prognostic markers of HCC, especially in cirrhotic-HCV patients, helping to identify patients at higher HCC-risk, deserving more intense liver evaluation and/or early treatment.