Supplementary Appendix

This appendix has been provided by the authors to give readers additional information about their work.

Supplement to: Afdhal N, Reddy KR, Nelson DR, et al. Ledipasvir and sofosbuvir for previously treated HCV genotype 1 infection. N Engl J Med DOI: 10.1056/NEJMoa1316366

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Contents

Page

ION-2 Principal Investigators............................................................................................................................ 3

Statistical Hypothesis for the Primary Efficacy Endpoint................................................................................. 4

Figure S1. Algorithm for Prior Treatment Response Categorization................................................................. 5

Table S1. Reasons for Screen Failure................................................................................................................ 6

Figure S2. Patient Disposition.................................................................................. ........................................ 7

Study Assessments........................................................................................................................................... 8

Criteria for Determining Treatment Duration for Group 1............................................................................... 9

Table S2. Bonferroni-Adjusted Confidence Intervals for the Pairwise Differences in SVR12 Rates.................. 10

Table S3. Sustained Virologic Response by Subgroups..................................................................................... 11

Table S4. Proportion of Patients with ALT >ULN at Baseline Who Had ALT Normalization During Treatment 13

Table S5. Baseline Characteristics of Patients with Virologic Relapse after Treatment.................................... 14

Exact Logistic Regression Analysis for Evaluating Associations between Baseline Characteristics and SVR12. 15

Table S6: Univariate Exact Logistic Regression in Assessing Factors Associated with SVR12........................... 15

Table S7: Final Exact Logistic Regression Model Identifying Factor(s) Associated with SVR12........................ 16

Table S8. Contingency Table of Cirrhosis Status and Treatment Duration with SVR12.................................... 16

Figure S3. Mean HCV RNA in the 1st Weeks of Treatment by Baseline NS5A Resistance-Associated Variants. 17

Figure S4. Proportion of Patients with HCV RNA <LLOQ by Baseline NS5A Resistance-Associated Variants.... 18

Table S9. Proportion of Patients with HCV RNA <25 IU/mL during Treatment by Cirrhosis Status.................. 19

Table S10. SVR12 by Early Viral Response and Cirrhosis Status........................................................................ 21

Table S11. Platelets and Change from Baseline by Visit.................................................................................... 22

Table S12. Albumin and Change from Baseline by Visit..................................................................................... 23

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ION-2 Principal Investigators

Nezam Afdhal, Avanish Aggarwal, Sanjeev Arora, Leslie Bank, Kimberly Beavers, Nicholaos

Bellos, Michael Bennett, David Bernstein, Thomas Boyer, Robert Brown Jr, Mario Chojkier,

Christopher Christensen, James Cooper, Adrian Di Bisceglie, Richard Elion, Robert Emslie,

Kyle Etzkorn, Gregory Everson, Steven Flamm, Todd Frederick, Bradley Freilich, Michael

Galambos, Joseph Galati, Reem Ghalib, Norman Gitlin, Stuart Gordon, David Hassman, Trevor

Hawkins, Robert Herring, Federico Hinestrosa, Charles Howell, Ira Jacobson, Marcelo

Kugelmas, Paul Kwo, Jacob Lalezari, Eric Lawitz, Claudia Martorell, Anthony Mills, Ronald

Nahass, David Nelson, Mindie Nguyen, Keyur Patel, Paul Pockros, Gary Poleynard, John

Poterucha, John Poulos, David Pound, Ronald Pruitt, Bruce Rashbaum, Natarajan Ravendhran,

K. Rajender Reddy, Robert Reindollar, Peter Ruane, Vinod Rustgi, Michael Ryan, Eugene

Schiff, Thomas Sepe, Aasim Sheikh, Mitchell Shiffman, Coleman Smith, Mark Sulkowski, Hugo

Vargas, Kimberly Workowski, David Wyles, Ziad Younes

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Statistical Hypothesis for the Primary Efficacy Endpoint

The 4 primary statistical hypotheses of the study were that the SVR12 rates in each of the 4

treatment groups of the study would be higher than the adjusted historical SVR null rate of 25%.

The 25% null SVR rate was derived from the historical data as follows:

1) For treatment experienced subjects (eg, PEG+RBV) receiving a PI-based triple therapy

regimen, a historical retreatment SVR rate of approximately 65% was calculated from the

telaprevir (REALIZE study) and boceprevir (RESPOND-2 study) data after adjusting for the

expected proportion of subjects with cirrhosis (approximately 20%) in this study. The

weighted average of the telaprevir and boceprevir data provided an estimate of SVR rate to

be approximately 69% in noncirrhotic subjects and 50% in cirrhotic subjects. The

retreatment SVR rate for the historical control in this study (ie, a patient population of 80%

noncirrhotics and 20% cirrhotics) was then calculated to be approximately 65% (ie, 0.8 ×

69% + 0.2 ×50%);

2) For subjects who have had failed treatment with a PI + PEG+ RBV retreatment options

are currently lacking. A conservative retreatment SVR rate of 5% was therefore used.

In this study, the expected proportion of subjects having had prior treatment with a

PI+PEG+RBV was approximately 50%. A 35% null SVR rate was obtained after averaging a

65% retreatment SVR control rate for treatment experienced subjects (eg, PEG+RBV) being

retreated with PI+PEG+RBV (current SOC)and a 5% SVR control rate for subjects who failed

prior treatment of a PI+PEG+RBV, if retreated with a PI+PEG+RBV. In addition, we allowed a

discount of 10 percentage points in efficacy due to the expected improved safety profile and

significantly shorter duration associated with the treatment, which resulted in a null SVR rate for

this study of 25%.

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Figure S1. Algorithm for Prior Treatment Response Categorization

6

Table S1. Reasons for Screen Failure

Of the 551 patients screened, 110 were screen failures (four patients failed for more than one

reason)

Screen failure

patients who did

not meet criteria Inclusion criteria

31 Lab parameters at screening: ALT ≤10xULN, AST ≤10xULN, Hgb ≥12 g/dL (M) & 11 g/dL

(F), Platelets ≥50,000/uL, INR ≤1.5, Albumin ≥3 g/dL, Direct bilirubin ≤1.5xULN, HbA1c

≤8.5%, Creatinine clearance ≥60 mL /min, INR ≤1.5 × ULN

10 Cirrhosis determination:

a) Cirrhosis is defined as any one of the following: i) Liver biopsy showing cirrhosis (e.g.,

Metavir score = 4 or Ishak score ≥ 5), ii) FibroTest® score of > 0.75 AND an AST: platelet

ratio index (APRI) of > 2 during Screening

b) Absence of cirrhosis is defined as any one of the following: i) Liver biopsy within 2 years of

Screening showing absence of cirrhosis, ii) FibroTest® score of ≤ 0.48 AND APRI of ≤ 1

during Screening

c) In the absence of a definitive diagnosis of presence or absence of cirrhosis by the above

criteria, a liver biopsy is required; liver biopsy results will supersede blood test results and be

considered definitive.

5 Liver imaging within 6 months of Baseline/Day 1 to exclude hepatocellular carcinoma is

required in patients with cirrhosis

3 Prior virologic failure after treatment with a pegylated interferon (PEG) and ribavirin (RBV)

regimen, including those who have failed treatment with a NS3/4A protease inhibitor (PI) plus

PEG/RBV regimen. Subjects must not have discontinued prior therapy due to an adverse event.

2 Willing and able to provide written informed consent

2 HCV RNA 104 IU/mL at Screening

1 HCV genotype 1a, 1b, or mixed 1a/1b at Screening as determined by the Central Laboratory.

1 Not pregnant or nursing

1 Subject must be of generally good health, with the exception of chronic HCV infection

Exclusion criteria

8 Clinically-relevant drug abuse within 12 months of screening.

2 History of clinically-significant illness or any other major medical disorder that may interfere

with treatment, assessment, or compliance with the protocol

1 Chronic liver disease of a non-HCV etiology

1 Prohibited concomitant medication

Screen failure

patients who did

meet criteria Reason for non-enrollment

3 Subject withdrew consent

16 Study enrollment closed

27 Other

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Figure S2. Patient Disposition

109 were assigned to receive SOF + LDV for 12 weeks

N = 109

111 were assigned to receive SOF + LDV + RBV for 24

weeks N = 111

111 were assigned to receive SOF + LDV +

RBV for 12 weeks N = 111

109 were assigned to receive SOF + LDV for 24 weeks

N = 109

109 included in the efficacy and safety analyses

111 included in the efficacy and safety analyses

111 included in the efficacy and safety analyses

109 included in the efficacy and safety analyses

109 completed treatment

110 completed treatment

111 completed treatment

107 completed treatment

551 screened

441 randomized

1 randomized patient was never dosed

110 screen failures 64 did not meet eligibility criteria 16 due to close of enrollment period 3 withdrew consent 27 due to other reasons

440 randomized and treated

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Study Assessments

HCV genotype and subtype were determined using the Siemens VERSANT HCV Genotype

INNO-LiPA 2.0 assay. IL28B genotype was determined by PCR amplification and sequencing of

the rs12979860 single-nucleotide polymorphism.

On-treatment assessments included laboratory testing, serum HCV RNA, vital signs,

electrocardiography, and symptom-directed physical examinations. All adverse events were

recorded and graded according to a standardized scale (see Appendix 3 of study protocol,

available at NEJM.org).

Plasma samples for viral sequence analysis were collected at Baseline/Day 1 and every

subsequent visit (as appropriate).

Patients with virologic failure underwent resistance testing. Analyses of samples for HCV NS5A

and NS5B substitutions at baseline and virologic failure time points were conducted. DDL

Diagnostics Laboratory (Rijswijk, The Netherlands, EU) performed amplification and population

sequencing, and WuXi Apptec (Shanghai, China) performed deep sequencing assays to identify

treatment-emergent virologic resistance.

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Criteria for Determining Treatment Duration for Group 1

The duration of treatment for the first group depended on emerging results from the LONESTAR

trial (clinicaltrials.gov number NCT01726517). One treatment group in LONESTAR was made

up of patients who had previously received unsuccessful treatment with a protease-inhibitor

regimen (50% of whom could have cirrhosis). This group received 12 weeks of treatment with

the sofosbuvir-ledipasvir fixed-dose combination. If at least 75% of those patients had HCV

RNA <LLOQ 4 weeks after the end of treatment, patients randomized in the present study to

receive sofosbuvir-ledipasvir for the pending treatment duration (ie12 or 24 weeks) in the current

study would stop treatment with sofosbuvir-ledipasvir after 12 weeks; if not, they would

continue to 24 weeks.

The rate of sustained virologic response in the group of 19 patients receiving sofosbuvir-

ledipasvir in the LONESTAR trial was 95% (18 of 19 patients).1 Therefore, patients in group 1

of ION-2 stopped treatment after 12 weeks.

1. Lawitz E, Poordad FF, Pang PS, et al. Sofosbuvir and ledipasvir fixed-dose combination with and without

ribavirin in treatment-naïve and previously treated patients with genotype 1 hepatitis C virus infection

(LONESTAR): an open-label, randomised, phase 2 trial. Lancet 2014;383:515-23.

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Table S2. Bonferroni-Adjusted Confidence Intervals for the Pairwise Differences in SVR12 Rates

SOF/LDV

12 Weeks

(N = 109)

SOF/LDV + RBV

12 Weeks

(N = 111)

SOF/LDV

24 Weeks

(N = 109)

SOF/LDV + RBV

24 Weeks

(N = 111)

SVR12

95% CI

102/109 (93.6%)

87.2% to 97.4%

107/111 (96.4%)

91.0% to 99.0%

108/109 (99.1%)

95.0% to 100.0%

110/111 (99.1%)

95.1% to 100.0%

SOF/LDV 12 weeks vs SOF/LDV + RBV 12 weeks

Prop Diff

(99.17% CI)

-2.7%

(-12.4%, 7.0%)

SOF/LDV 24 weeks vs SOF/LDV + RBV 24 weeks

Prop Diff

(99.17% CI)

-0.04%

(-7.6%, 7.5%)

SOF/LDV 12 weeks vs SOF/LDV 24 weeks

Prop Diff

(99.17% CI)

-5.5%

(-14.6%, 3.6%)

SOF/LDV + RBV 12 weeks vs SOF/LDV + RBV 24 weeks

Prop Diff

(99.17% CI)

-2.7%

(-11.0%, 5.5%)

SOF/LDV 12 weeks vs SOF/LDV + RBV 24 weeks

Prop Diff

(99.17% CI)

-5.5%

(-14.6%, 3.6%)

SOF/LDV + RBV 12 weeks vs SOF/LDV 24 weeks

Prop Diff

(99.17% CI)

-2.8%

(-11.1%, 5.5%)

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Table S3. Sustained Virologic Response by Subgroups

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Table S3. Sustained Virologic Response by Subgroups (continued)

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Table S4. Proportion of Patients with ALT >ULN at Baseline Who Had ALT

Normalization During Treatment

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Table S5. Baseline Characteristics of Patients with Virologic Relapse after Treatment

12-Week Treatment

Group Age

(years) Race Sex HCV

Genotype

HCV RNA (log10 IU/mL)

Platelets (×10

3/µL) IL28B

Cirrhosis (yes/no)

Baseline NS5A RAV

(yes/no) Prior Treatment Prior Treatment Response

SOF-LDV 64 White M 1b 6.6 75 CT Yes No PI+Peg-IFN+RBV Relapse/ Breakthrough

SOF-LDV 62 White M 1b 6.7 101 CT Yes No Peg-IFN+RBV Null Responder

SOF-LDV 64 White M 1a 5.9 53 CT Yes No PI+Peg-IFN+RBV Relapse/ Breakthrough

SOF-LDV 61 White M 1b 6.7 237 CT No Yes Peg-IFN+RBV Null Responder

SOF-LDV 58 White F 1a 7.2 253 CT No Yes PI+Peg-IFN+RBV Non-Responder

SOF-LDV 57 White F 1a 5.9 126 CT No Yes PI+Peg-IFN+RBV Relapse/ Breakthrough

SOF-LDV 54 White M 1a 7.2 262 CT No Yes Peg-IFN+RBV Non-Responder: Partial Responder

SOF-LDV+RBV 60 White M 1a 6.7 174 TT Yes Yes PI+Peg-IFN+RBV Non-Responder

SOF-LDV+RBV 65 White M 1a 7.1 249 CT Yes No Peg-IFN+RBV Relapse/ Breakthrough

SOF-LDV+RBV 63 Black M 1a 7.2 122 CT Yes No PI+Peg-IFN+RBV Non-Responder

SOF-LDV+RBV 60 White M 1a 7.1 105 CT Yes Yes Peg-IFN+RBV Relapse/ Breakthrough

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Exact logistic regression analysis for evaluating associations between baseline

characteristics and SVR12

Univariate exact logistic regression analysis was performed to assess the relationship between sustained

virologic response and 15 baseline demographic and clinical factors: treatment with or without RBV, 12

or 24 weeks of treatment, age (<65 or ≥65 years old), sex, race (black or non-black), ethnicity

(Hispanic/Latino or not Hispanic/Latino), HCV genotype (1a or 1b), cirrhosis (yes or no), response to

prior HCV therapy (relapse/breakthrough or non-responder), prior HCV therapy (PEG+RBV or

PI+PEG+RBV), baseline HCV RNA viral load (<800,000 IU/mL or ≥800,000 IU/mL), BMI (<30 or ≥30

kg/m2), IL28B alleles (CC or non-CC), GGT, and platelets (<125 or ≥125 ×10

3/µL ). Factors found to be

significant according to in the univariate analysis (p value <0.05) were then evaluated in a multivariate

exact logistic regression model using a forward selection procedure to identify the factors most predictive

of sustained virologic response. Factors found to be significant (p value <0.05) using this forward

selection procedure were included in the final exact logistic regression analysis.

Table S6. Univariate Exact Logistic Regression in Assessing Factors Associated with SVR12

Variable Odds Ratio

95%

Confidence Limit

2-Sided

P Value

Treatment: with ribavivin 1.651 (0.468, 6.524) 0.5524

Treatment: 24 weeks 5.718 (1.226, 53.702) 0.0206

Age group (years): <65 1.103 (0.025, 7.939) 1.0000

Sex: female 3.003 (0.643, 28.233) 0.2257

Race: black 1.171 (0.248, 11.093) 1.0000

Ethnicity: Hispanic or Latino 1.933 (0.313, Infinity) 0.5453

HCV genotype: 1a 1.123 (0.195, 4.484) 1.0000

Cirrhosis: no 4.958 (1.386, 18.378) 0.0124

Response to prior HCV therapy: relapse/breakthrough 1.482 (0.419, 5.433) 0.6709

Prior HCV therapy: PEG+RBV 1.047 (0.296, 3.837) 1.0000

Baseline HCV RNA (IU/mL): <800,000 1.554 (0.221, 67.836) 1.0000

Baseline BMI (kg/m2): ≥30 1.624 (0.410. 9.329) 0.6857

IL28B: CC 1.735 (0.247, 75.621) 1.0000

Baseline GGT (U/L) (continuous) 0.998 (0.994, 1.003) 0.3928

Baseline platelets (×103/µL): ≥125 5.144 (1.269, 18.724) 0.0216

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Table S7. Final Exact Logistic Regression Model Identifying Factor(s) Associated with

SVR12

Variable Odds Ratio

95%

Confidence Limit

2-Sided

P Value

Cirrhosis: no 5.103 (1.406, 19.194) 0.0117

Treatment: 24 weeks 5.864 (1.243, 55.526) 0.0195

Table S8. Contingency Table of Cirrhosis Status and Treatment Duration with SVR12

SVR12 SVR12 Rate

(95% CI) Yes No

Cirrhosis

yes 81 7 92.1%

(84.3%, 96.7%)

no 346 6 98.3%

(96.3%, 99.4%)

Treatment

Duration

12 weeks 209 11 95.0%

(91.2%, 97.5%)

24 weeks 218 2 99.1%

(96.8%, 99.9%) Note: The exact 95% CI of SVR12 rate is based on the Clopper-Pearson method.

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Figure S3. Mean HCV RNA in the 1st Weeks of Treatment by Baseline NS5A Resistance-Associated Variants

All randomized subjects who received at least 1 dose of any study drug and had determined baseline NS5A are

included. One subject who had failed baseline NS5A is excluded.

Each error bar is constructed using 1 standard deviation from the mean.

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Figure S4. Proportion of Patients with HCV RNA <LLOQ by Baseline NS5A Resistance-Associated Variants

All randomized subjects who received at least 1 dose of any study drug and had determined baseline NS5A are

included. One subject who had failed baseline NS5A is excluded.

19

Table S9. Proportion of Patients with HCV RNA <25 IU/mL during Treatment by

Cirrhosis Status

20

Table S9. Proportion of Patients with HCV RNA <25 IU/mL during Treatment by

Cirrhosis Status (continued)

21

Table S10. SVR12 by Early Viral Response and Cirrhosis Status

22

Table S11. Platelets and Change from Baseline by Visit

23

Table S12. Albumin and Change from Baseline by Visit