Multiclass HCV Resistance to Direct-acting Antiviral Failure in Real-life Patients Advocates for Tailored Second-line Therapies

Velia C. Di Maio; Valeria Cento; Ilaria Lenci; Marianna Aragri; Piera Rossi; Silvia Barbaliscia; Michela Melis; Gabriella Verucchi; Carlo F. Magni; Elisabetta Teti; Ada Bertoli; FrancescoPaolo Antonucci; Maria C. Bellocchi; Valeria Micheli; Chiara Masetti; Simona Landonio; Simona Francioso; Francesco Santopaolo; Adriano M. Pellicelli; Vincenza Calvaruso; Laura Gianserra; Massimo Siciliano; Dante Romagnoli; Raffaele Cozzolongo; Antonio Grieco; Jacopo Vecchiet; Filomena Morisco; Manuela Merli; Giuseppina Brancaccio; Antonio Di Biagio; Elisabetta Loggi; Claudio M. Mastroianni; Valeria Pace Palitti; Pierluigi Tarquini; Massimo Puoti; Gloria Taliani; Loredana Sarmati; Antonino Picciotto; Vincenzo Vullo; Nicola Caporaso; Maurizio Paoloni; Caterina Pasquazzi; Giuliano Rizzardini; Giustino Parruti; Antonio Craxì; Sergio Babudieri; Massimo Andreoni; Mario Angelico; Carlo F. Perno; Francesca Ceccherini-Silberstein


Liver International. 2017;37(4):514-528. 

In This Article


Type of Virological Failure and DAA Regimen Used

The study analysed 200 virological failures which occurred in 197 patients treated with a DAA-containing regimen. Three of them experienced firstly a virological failure to BOC (N=1) or TVR (N=2) with PegIFN+RBV. Patients have been divided according to the type of DAA regimen used, 89 with PegIFN+RBV and 111 without, and whether recommended or not by the European guidelines.[24] The majority of virological failures were relapses (114, 57.0%), while an additional 45 patients (22.5%) had a breakthrough and 41 (20.5%) were non-responders ( Table 1 ). In particular, among the 111 patients failing a DAA IFN-free regimen, 53 (47.8%) patients failed a regimen recommended by guidelines:[24] SMV+SOF±RBV (N=33), DCV or LDV+SOF±RBV (N=8), paritaprevir/r+ombitasvir+dasabuvir±RBV (3D) (N=7) and SOF+RBV (N=5, all HCV-2-infected patients). The majority of them were relapsers (83.0%). The other 58/111 (52.2%) patients failed a suboptimal/not-recommended IFN-free regimen: SOF±RBV (N=47), SMV or ASU+DCV±RBV (N=11). Among the 89 patients that failed a DAA-regimen with PegIFN+RBV, 83 failed a protease inhibitor (PI)-containing regimen (39=TVR, 40=BOC, 4=SMV) and 6 SOF+PegIFN+RBV regimen ( Table 1 ).

Baseline Characteristics of Patients

The clinical and virological characteristics of the 197 HCV-infected patients included in the analysis are reported in Table 2 . The median (IQR) age was 52 (45–58) years in DAA+PegIFN+RBV group and 56 (52–66) years among patients treated with DAA IFN-free regimen. Most of patients were cirrhotic in both groups: 52.8% and 76.6%. At baseline, median (IQR) liver stiffness was 14 (11–26) and 21 (14–31) kPA respectively. The majority of patients were infected with an HCV-1b genotype (33.3% IFN-free versus 52.8% +IFN). Overall, nine patients (4.6%) had an HIV co-infection. Furthermore, among the 111 patients failing a DAA IFN-free regimen, 11.7% were treated after orthotopic liver transplant and 9.0% had a history of hepatocellular carcinoma (HCC). In DAA IFN-free failures, 26.1% of patients were naïve to anti-HCV treatment before the current DAA treatment, 21.6% were non-responders to previous treatment and 6.3% had failed previously a first-generation protease inhibitor (BOC or TVR).

Distribution of RASs According to Patients' Characteristics

Overall, in the DAA IFN-free group, 55/111 patients (49.5%) showed at failure at least one RAS related to the DAA regimen administered. RASs prevalence was significantly higher in breakthrough/non-responders than in relapsers (90.5% vs 40.0%, P<.001) and in patients who did not receive RBV (76.9% vs 41.2%, P=.003) (Figure 1). However, no difference in RAS prevalence was observed according to RBV use when only patients treated with a recommended regimen were considered, for which RBV use was optional (70.0% without vs 63.3% with, P=.855; Figure 1). Interestingly, among patients with available IL-28 genotype data (N=28), RAS prevalence at failure was significantly higher for patients with unfavourable IL-28 CT/TT genotype compared to patients with favourable IL-28 CC genotype (50.0% vs 0.0%, P=.055, Figure 1). In the DAA+PegIFN+RBV group, 65/89 (73.0%) showed at least one RAS related to the DAA regimen which failed (Table S2). In this group, no differences in RAS prevalence were observed according to the type of response at failure (breakthrough/non-response, 74.2% vs relapse, 73.9%).

Figure 1.

Distribution of RASs at failure according to DAA response and RBV use in the DAA regimen. (**) P value was calculated by P was calculated by Chi2 test and Fisher's exact test when was appropriate. (*) In this group were considered as all the patients treated with a RBV-containing regimen (including also all suboptimal SOF+RBV containing regimen). (†) In this group were considered as only patients treated with a recommended regimen (30 treated with a RBV-containing regimen and 20 without)

Distribution of RASs According to DAA Class and HCV Genotype/Subtype

The prevalence of RASs related to the DAA class at failure was: 96.1% NS5A-RASs in NS5A-failures (N=26), 75.9% and 70.5% NS3-RASs in NS3-failures (with [N=83] or without IFN [N=51] respectively), 66.6% and 20.4% NS5B-RASs in SOF failures (with [N=6] or without IFN [N=93] respectively) and 28.6% of NS5B-RASs in DSV failures (N=7) (Figure 2).

Figure 2.

Prevalence of RASs related to each DAA class. NS3, NS5A, NS5B NI/NNI indicate the specific DAA class. The number indicates the patients who failed a regimen containing an inhibitor of the specific DAA class

Furthermore, the prevalence of RASs for each DAA class varied according to HCV genotype/subtype (Figure 3, panel A). In particular, in the DAA+PegIFN+RBV group, where the majority of patients were treated with a PI containing regimen (Table S3), a high prevalence (89%) of NS3 RAS was detected in HCV-1a-infected patients compared to HCV-1b (60%) and HCV-4 (33%)-infected patients (P=.001, Figure 3, panel A, Table S2).

Figure 3.

Distribution of RASs. The graph in panel (A) shows the distribution of RASs according to DAA target and HCV genotype/subtype among DAA+PegIFN+RBV and IFN-free failing patients. For more details regarding the distributions of regimens see Table S3. The histograms in panel (B) show prevalence of specific NS3 RASs according to HCV genotype/subtype among DAA+PegIFN+RBV and IFN-free failing patients. The histograms in panel (C) show prevalence of specific NS5A and NS5B SOF or DAS RASs according to HCV genotype/subtype among DAA+PegIFN+RBV and IFN-free failing patients

Instead, among the DAA IFN-free group (Figure 3, panel A, Table S3), the prevalence of RASs related to a specific treatment was higher among HCV-1b-infected patients compared to HCV-1a and other HCV genotypes, such as HCV-2 and HCV-3, according to the frequent use of only SOF+RBV in these cases.

Furthermore, by analysing the specific NS3, NS5A and NS5B RASs, a different prevalence and distribution of clinically relevant RASs was observed according to HCV genotype/subtype (Figure 3, panels B, C). For example, the NS3 RASs Q80K and R155K were detected only among HCV-1a-infected patients, treated with or without IFN (21.6–67.6% and 25.0–16.7% respectively). Among the NS3-IFN-free treatment group, in which most patients received SMV (81%), different RAS prevalence at position 168 was observed. In particular, the major RAS D168V was the most common across all genotypes, with the exception of HCV-3a (Figure 3, Panel B; Table 3 and Table 4 ).

In NS5A-failing patients, Y93H major NS5A-RAS, as well as RASs at position 30–31, were also detected with different prevalence across all HCV genotypes (Figure 3, Panel C; Table 3 and Table 4 ).

Regarding SOF-RASs, the major S282T was detected in three patients, all breakthroughs to SOF-IFN-free regimen (2 HCV-4a and 1 HCV-3a), and the putative L159F was detected only in HCV-1b and HCV-3 SOF relapsers (Figure 3, Panel C; Table 4 and S2). Finally, NS5B-NNI-RASs were detected only in 2/7 DSV-failing patients, at positions 553 (in HCV-1a) and 556 (in HCV-1b).

Prevalence of RASs According to DAA Class Combinations

Notably, 28/59 (47.4%) patients treated with ≥2 DAA classes showed RASs on ≥2 DAA targets at failure. In particular, all patients treated with a combination of NS3+NS5A inhibitors with low barrier to resistance (SMV/ASU+DCV±RBV, N=11) showed NS3+NS5A-RASs at failure (Figure 4, Table 3 and Table 4 ). A different distribution of RASs was observed among patients failing to respond to DCV or LDV, in combination with SOF (Figure 4, Table 3 and Table 4 ). In particular, the four patients who failed to respond to DCV+SOF±RBV were all relapsers and showed RASs only in NS5A. Differently, among the four patients treated with LDV+SOF±RBV, one showed RASs only in NS5A, two had resistance in NS5A+NS5B and one showed RASs in NS5A+NS3 (extra-target of the specific therapy). Among the 3D-failing patients, 2/7 showed triple-class resistance, while two showed RASs in two targets (always NS3+NS5A) and three showed RASs in only one target (one NS3 and two NS5A) (Figure 4, Table 3 and Table 4 ).

Figure 4.

Prevalence of RASs conferring resistance to different DAA classes. The histogram shows the prevalence of RASs detected in each specific DAA regimen

Interestingly, among failures without specific RASs related to the regimen (N=80, 40%), when all the three genes where tested at failure (N=56), 12.5% of them presented natural "extra-target" RASs: two (one HCV-1b and one HCV-4d) infected patients showed natural resistance in NS3, four HCV-1b-infected patients showed natural resistance in NS5A and one HCV-3a showed natural resistance in NS5B (Table S4).

Virological Failures Caused by Incorrect Genotype Classification

Overall, 6/197 (3.0%) DAA-failing patients had a misclassified genotype from the one presumed before treatment start (obtained by commercial genotyping-assays). In particular, among seven non-responders to a DAA IFN-free regimen, four (57.1%) had a misclassified genotype. Three non-responders to an IFN-free treatment, who were previously classified as HCV-1a or HCV-1b, were instead infected with a non-1 HCV genotype (one HCV-2c and two HCV-3a). All of them failed 3D+RBV regimen (which is approved for the treatment of only HCV-1-infected patients). Moreover, the same three patients also failed with presence of resistance in NS3 or NS5A ( Table 3 ). The other patient, previously defined as infected with HCV-1b, was instead infected with HCV-4d and failed to respond to ASU+DCV+RBV with multiple NS5A+NS3 RASs ( Table 3 ).

In addition, two patients previously classified with HCV-1 were found infected with an HCV-4 strain at BOC/TVR failure, both without RASs (Table S4).

Baseline RASs

Finally, to test if RASs present at failure were already present before starting the treatment, baseline samples available for 70 failing patients (39 treated with and 31 without IFN) were analysed. Nine of 39 DAA+PegIFN+RBV-failing patients (eight HCV-1a and one HCV-1 g) showed baseline RASs, even though, not always specific for the treatment failed. In particular, all were treated with a PI (TVR or BOC or SMV) and all confirmed at failure the RASs detected at baseline; moreover, six of nine patients showed additional RASs at failure (Tables S2–S4).

Among the failures to IFN-free regimens, 5/31 showed baseline RASs specific for the treatment used.

Three HCV-1b patients failed as relapsers to a SOF-containing regimen. In particular, two SOF+RBV failures, showed at baseline and at failure L159F+C316N in NS5B. The third, a SOF+DCV relapser, had at baseline L28M in NS5A and showed additional NS5A-RASs at failure ( Table 4 ). Differently, one HCV-4d and one HCV-1b patient with natural NS3+NS5A-RASs were both breakthrough to low-barrier NS3+NS5A inhibitors, and developed additional RASs in both drug targets ( Table 3 ).