HCV Eradication does not Impact Gut Dysbiosis or Systemic Inflammation in Cirrhotic Patients

POSTER PRESENTATIONS variants in the HCV NS5A region at L31I, S62L, and Y93H were observed. Here, the presence of minor variants in the HCV NS5A and NS5B regions at baseline and at failure were assessed in the 17 patients who failed treatment in the ALLY-3 study using nextgeneration sequencing. Methods: Plasma samples were analysed using next-generation sequencing (Illumina technology; sensitivity ≥1% [DDL, Netherlands]). The HCV NS5A (amino acids 28, 30, 31, and 93) and NS5B (amino acids 159, 282, and 321) regions were surveyed and the results compared with those obtained using direct sequencing (sensitivity cut-off ∼20%). Results: Using next-generation sequencing, minor variants in the HCV NS5A region at M28, A30, or Y93 were observed in three patients. M28V (1.3%) or A30V (1.6%) were no longer detected at relapse in two patients with these respective variants at baseline, while the minor variant Y93H (2%) observed in one patient was enriched (99.4%) at failure. No minor variants associated with resistance to sofosbuvir were observed, including the two patients who had also failed prior treatment with sofosbuvir. At treatment failure, the only detected signature variants in NS5Awere A30K (one patient), L31I (one patient) and Y93H (15 patients; eight were emergent). All of these variants were also detected by direct sequencing. One patient had emergent NS5B S282T together with emergent NS5A Y93H at treatment failure; neither variant was detected at baseline or by post-treatment Week 12 using next-generation sequencing. Conclusions: In general, minor variants in NS5A (at 28, 30, 31, or 93) and NS5B (at 159, 282, or 321) were not detected at baseline by next-generation sequencing in patients with emergent variants associated with resistance to daclatasvir or sofosbuvir. In trying to determine which patients with HCV GT 3 infection would respond to treatment with 12 weeks of daclatasvir + sofosbuvir, next-generation sequencing delivered similar results to those obtained using direct sequencing. From these data, the presence of minor variants in NS5A or NS5B at baseline does not appear to predict response to treatment with daclatasvir + sofosbuvir. FRI-172 NEXT GENERATION SEQUENCING FOR HCV GENOTYPING AND OPTIONAL IDENTIFICATION OF RESISTANCE-ASSOCIATED VARIANTS E. Rakhmanaliev1, Z. Rui1, W. Huang1, K.S. Poon2, W.C. Cui2, J.K. Mui2, E. Koay2, E. Passomsub3, W. Chantratita3, G. Michel1. 1Vela Diagnostics Pte. Ltd.; 2Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Hospital, Singapore; 3Department of Pathology, Faculty of Medicine, Ramathibodi Hospital Mahidol University, Bangkok, Thailand E-mail: [email protected] Background and Aims: Despite the advent of a number of direct acting antiviral drugs interferon treatment is still a valid therapy option, requiring HCV genotype (GT) determination before initiation of therapy. Moreover, preliminary data from several studies indicate that genotype testing, in particular GT3, might be useful for decision making for some novel drug regimens. Therefore accurate detection of HCV GTs is critical and can have significant impact on outcome of therapy. The objective of this study was to compare a widely used line probe-based test (VERSANT HCV Genotype 2.0 LiPA) and a newly developed automated next generation sequencing (NGS)-based integrated workflow, comprised of an upfront robotic liquid handling system and kits for RNA extraction and library preparation (Sentosa SQ HCV Genotyping Assay), instruments and kits for template preparation and Ion Torrent based deep sequencing, as well as bioinformatics analysis and reporting software. The latter gives full data reports on GTs 1a and 1b including known Resistance Associated Variants (RAVs). Methods: A total of 136 RAVs are included in the analysis across GTs and can be retrieved by the user. However, the system does not make

direct treatment recommendations, which are left to the respective investigator. This study included a cohort of 346 patients with chronic HCV, eligible for therapy. Archived serum and plasma samples across all 6 HCV GTs were tested on both platforms. Results: For 47/346 (13.6%) samples GT results by VERSANT were “indeterminate”. In 19/299 (6.4%) of the samples, discordant results between the two methods were obtained. All discordant and indeterminate samples were subjected to Sanger sequencing. The ability to correctly determine HCV genotypes was 93.7% (95%CI: 90.3–95.9%) for VERSANT and 100% (95%CI: 98.7–100%) for Sentosa SQ HCV Genotyping Assay. Among the 19 discordant samples, 10 GT6 were incorrectly classified as GT1b by line probing, 6 GT3 as GT4, 2 GT3 as GT6, and 1 GT1c as GT1a. GT distribution among the 47 samples indeterminate by VERSANT was: 5 GT1a, 1 GT2, 19 GT3, 1 GT4, 20 GT6 and 1 mixed infection (GTs 2 and 3). Clinical sensitivity aggregated was 86.4% (95%CI: 82.4–89.6%) for VERSANT and 100% (95%CI: 98.9–100%) for Sentosa SQ HCV Genotyping Assay. Conclusions: Our data indicate that NGS in combination with thorough bioinformatics data analysis is highly accurate for determination of HCV genotypes and identification of RAVs, both representing essential (GTs) and potentially important (RAVs) information for HCV treatment approaches. FRI-173 HCV ERADICATION DOES NOT IMPACT GUT DYSBIOSIS OR SYSTEMIC INFLAMMATION IN CIRRHOTIC PATIENTS J.S.S. Bajaj1, R. Sterling1, P. Hylemon1, D. Nixon1, M. Fuchs1, D. Heuman1, M. SIkaroodi2, P. Gillevet2. 1Virginia Commonwealth University, Richmond; 2George Mason University, Manassas, United States E-mail: [email protected] Background and Aims: Cirrhotic patients could have several reasons for their inherently harmful systemic pro-inflammatory milieu. This could be partly related to the underlying disease activity or gut dysbiosis. With the ease of HCV eradication, the impact of this sustained virological response (SVR) on the gut microbiota and systemic inflammation in HCV cirrhotic patients is important. Define the impact of HCV eradication on gut microbiota composition and systemic inflammatory milieu in cirrhosis. Methods: Outpatients with cirrhosis with only HCV as the cirrhosis etiology underwent stool and serum collection and a 3-day dietary recall. HCVRNA, genotype and SVR status were evaluated. We excluded subjects with concomitant cirrhosis etiologies, HIV coinfection and with decompensated cirrhosis (use of lactulose, antibiotics, rifaximin, probiotics). Stool microbiota composition using multi-tagged sequencing and serum inflammatory cytokines (IL-1b & TNF using ELISA) and endotoxemia were studied between HCV cirrhotics who had achieved an SVR compared to those who were still viremic. Analysis of microbiota was performed using UNIFRAC and metastats. Results: We included 105 compensated HCV cirrhotics, of which 21 had reached SVR 12 ± 3 months prior to enrollment. There was no difference in age (56 vs 57 years) or MELD score (11 vs. 12) between patients with and without SVR. Viremic patients had a median viral load of 0.9 million copies/mL and were mostly genotype 1 (77%). There was no difference in daily caloric intake (2891 ± 439 SVR vs. 2942 ± 750 no SVR). Microbiota: On UNIFRAC there was no significant differences in gut microbial composition ( p = 0.3) between groups. No changes in specific phyla relative abundance were observed (Firmicutes 45% vs 48%, Bacteroidetes 34% vs 31%, Proteobacteria 5% vs 5% in SVR/no SVR groups). When specific comparisons between families were performed (Figure), no changes in relative abundances of potentially pathogenic families (Enterobacteriaceae) or potentially beneficial ones (Clostridiales XIV,Lachnospiraceae, Ruminococcaceae) were seen.

Journal of Hepatology 2016 vol. 64 | S425–S630

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POSTER PRESENTATIONS Table 1: Comparisons* of HT Grade ≥3 Based on Chemotherapy Received Characteristics, % of HT ≥ 3 Any On Rituximab Not on Rituximab On Rituximab and Steroid On Rituximab, Steroid, and Anthracycline On Anthracycline without Rituximab and Steroid On Other

CTRL1

P (HCV Viremic vs CTRL1)

CTRL2

P (HCV Viremic vs CTRL2)

5.7% 4.5% 5.9% 3.3% 3.7%

0.023 < 0.001 1.000 0.024 0.162

3.5% 2.4% 3.6% 3.1% 2.9%

<0.001 <0.001 0.226 0.016 0.095

16.7%

6.3%

0.187

3.5%

0.027

4.6%

5.9%

1.000

3.6%

1.000

HCV Viremic 10.7% 26.2% 6.5% 27.3% 22.2%

*Multiple comparisons with Bonferroni correction method were used. CTRL1 compared to CTRL2 not shown.

Inflammation: There was no difference in IL-1 (4.3 ± 11 vs 2.5 ± 2 pg/ mL, p = 0.1), TNF-α (7 ± 3 vs 5.4 ± 4 pg/mL, p = 0.5) or endotoxin (0.2 ± 0.3 vs. 0.2 ± 0.1 EU/mL, p = 1.0) between the groups. Conclusions: HCV cirrhotics continue to have systemic inflammation and gut dysbiosis despite successful HCV eradication. Investigation of treatment strategies for amelioration of this pro-inflammatory milieu should be continued despite HCV eradication. FRI-174 HEPATOTOXICITY WITH CHEMOTHERAPY IN PATIENTS INFECTED WITH HEPATITIS C J.-L. Szpakowski1, L.-Y. Tucker2, D. Baer3, M. Pauly4. 1Gastroenterology, Kaiser Permanente, Fremont; 2Kaiser Permanente, Oakland; 3Oncology, Kaiser Permanente, Oakland; 4Gastroenterology, Kaiser Permanente, Sacramento, United States E-mail: [email protected] Background and Aims: The hepatotoxicity (HT) of cancer chemotherapy (CT) in patients with hepatitis C virus (HCV) is not well characterized. Methods: A retrospective observational data only study was conducted at a Northern California integrated health care delivery system examining patients undergoing CT 2000-2010. Grade 3 and 4 HT was determined using the National Cancer Institute Common Toxicity Criteria, with adaptation for baseline abnormal liver function tests. Patients with HIV, past or present Hepatitis B infection (HBV), HBsAb of unclear provenance, and hepatobiliary cancers were excluded. The HCV cohort included those testing positive for HCV (either detectable virus or genotype) on or before day one of initial chemotherapy ( period 1), and were followed for HT up to one year post CT ( period 2). Two control groups were established: CT patients tested negative for HBV and HCV (CTRL1), and those not tested (CTRL2). Cancers were grouped into lymphoma-chronic lymphocytic leukemia (CLL), lung, breast, colon, and other tumors. We examined the impact on HT of the following agents: rituximab, prednisone, anthracycline agents, and other. HT ≥ 3 was considered related to CT if the agent was given in the preceding 14 months. Results: The HCV group (n = 197) was significantly more likely than the control groups (CTRL1 = 6,934, CTRL2 = 28,633) to be male and under 65 year of age and less likely to be Asian. In multivariable logistic regression analysis, the HCV viremic group was more likely to have HT ≥ 3 compared to CTRL1 (OR 2.0, 95% CI 1.3–3.2, p = 0.003) and CTRL2 (OR 3.2, 95% CI 2.0–5.1, p < 0.001). Other risk factors predictive of HT ≥ 3 included female, age < 65, breast cancer, lymphoma-CLL, other cancer, and CT agents received.

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Conclusions: Patients with HCV undergoing CT have an increased HT grade ≥ 3. Factors associated with increased HT include age, gender, diagnosis, and CT agent. FRI-175 LIVER STIFFNESS MEASUREMENT AFTER SUCCESSFUL ANTI-VIRAL THERAPY IN HEPATOCELLULAR CARCINOMA RISK ASSESSMENT FOR CHRONIC HEPATITIS C PATIENTS J.-H. Wang1, Y.-H. Yen1, C.-H. Hung1, C.-H. Chen1, C.-M. Lee1, S.-N. Lu1. 1 Division of Hepato-Gastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung City, Taiwan E-mail: [email protected] Background and Aims: Chronic hepatitis C (CHC) patients after successful antiviral therapy remained at risk of hepatocellular carcinoma (HCC). This study was to determine whether liver stiffness measurement (LSM) was useful in HCC risk assessment and develop a risk-score system for clinical use. Methods: This retrospective study enrolled CHC patients achieved sustained virological response (SVR) after interferon-based therapy with LSM at/after SVR determination. The demographics, clinical characteristics and HCC development were obtained from medical chart reviews. The diagnosis of HCC was based on recommended criteria. Results: A total of 376 (M/F: 185/191, mean age: 54.1 year) patients, including 278 with pre-treatment liver biopsy specimens, were enrolled. The median follow-up period was 5.7 years. Twenty-one patients, including 18 with pre-treatment biopsy, developed HCC. The 5-year cumulative HCC incidence was 5.1%. Multivariate analysis showed advanced fibrosis/cirrhosis (odds ratio: 12.38, 95% confidence interval: 1.56–98.14), diabetes (2.80, 1.03–7.65) and LSM (1.01, 1.04–1.16) were associated with HCC developments. For LSM in HCC prediction, the performance and cutoff was 0.783 and 12 kilopascal (kPa) respectively. A risk-score system (score 0–4) combining advanced fibrosis/cirrhosis, diabetes and LSM>12 kPa was developed for 278 patients with pre-treatment biopsy. With score 0 and 1 as a reference, patients with score 2 and 3 (hazard ratio: 11.67, 95% confidence interval: 1.48–92.13) and score 4 (112.20, 14.02–897.71) carried higher risk of HCC development. Conclusions: For CHC patients in SVR, LSM was useful in HCC risk assessment. Patients with pre-treatment advanced fibrosis/cirrhosis, diabetes and LSM>12 kPa after SVR were at high risk of HCC development. FRI-176 HIGH RESOLUTION ANALYSIS FOR GENOTYPES AND SUBGENOTYPES IDENTIFICATION IN HEPATITIS C REVEALS THE IMPORTANCE OF MIXED INFECTIONS FOR OPTIMAL THERAPY J.A. Del Campo1, M. Parra2, S. Delgado2, M. García-Valdecasas3, J. Gregori4, J. Quer5, S. Bernal6, J.C. Palomares6, L. Grande1, M. Romero-Gómez7. 1Digestive Diseases, Valme University Hospital – CIBERehd; 2Infectious Diseases, Valme University Hospital; 3Digestive

Journal of Hepatology 2016 vol. 64 | S425–S630