Clinical trial NCT01368497
Conditions: Hepatitis B
Keywords: Hepatitis B HBV
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Start date: 2012-09-01; Completion date: 2016-12-23
Enrollment: 60 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to determine the safety and efficacy of treatment using a combination of drugs (entecavir and pegylated interferon) in children ages 3-\<18 years old with immunotolerant chronic hepatitis B.

Detailed description:
This single arm treatment study was conducted by the pediatric centers within the National Institute of Diabetes Digestive and Kidney Diseases (NIDDK)-sponsored Hepatitis B Research Network (HBRN). Children age 3-\<18 years with immunotolerant chronic hepatitis B (CHB) infection who fulfilled the entry criteria received entecavir as monotherapy for 8 weeks and then combination therapy with entecavir and pegylated interferon by weekly subcutaneous injection until week 48. Children were to be followed for 48 weeks after discontinuation of therapy (week 96 for those who completed 48 weeks of treatment).

Assessment was undertaken at baseline, weeks 4, 8, 10, 12, 14, \& 16, then every 4 weeks until week 48, and then 4, 8, 12, 24,36, and 48 weeks following treatment discontinuation corresponding to weeks 52, 56, 60, 72, 84 and 96 for those who received treatment for 48 weeks. Blood work was drawn to measure markers of viral and liver disease status and for research biospecimen banking.

Participants were to receive therapy until week 48 and enter 48 weeks of follow-up thereafter. Participants who experienced a sustained elevation of alanine aminotransferase (ALT) were eligible to receive treatment as recommended by their hepatologist and continued to complete the study protocol.

Arms: arm 1: Entecavir for 8 weeks followed by 40 weeks of both entecavir and peginterferon
Interventions: intervention 1: Entecavir and peginterferon
Intervention descriptions: intervention 1: Entecavir 0.015 mg/kg/day (up to 0.5 mg maximum daily dose) once daily for 48 weeks and Peginterferon alfa-2a 180 µg/1.73m2 subcutaneously once weekly for 40 weeks beginning 8 weeks after entecavir monotherapy).

Protocol documents:
Prot_SAP_000.pdf: 
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Combination Entecavir and Peginterferon alfa-2a Therapy in HBeAg-Positive Immune 
Tolerant Adults with Chronic Hepatitis B  
Synopsis 
 
Study aims: 
• 
To determine the safety and efficacy of treatment with 8 weeks of entecavir followed by 
40 weeks of both entecavir and peginterferon alfa-2a in the treatment of chronic hepatitis 
B in HBeAg positive adults who are in the immune tolerant phase. 
• 
To evaluate “off treatment” sustained responses after treatment with entecavir and 
peginterferon alfa-2a in the treatment of chronic hepatitis B in HBeAg positive adults who 
are in the immune tolerant phase. 
 
Type of study 
• 
Single arm treatment study 
 
Clinical trial 
A  single arm pilot study of 8 weeks of entecavir followed by 40 weeks of both entecavir and 
peginterferon alfa-2a in adults with HBeAg-positive chronic hepatitis B with normal or near 
normal ALT levels and high serum levels of HBV DNA (“immune tolerant” HBeAg-positive 
chronic hepatitis B). All participants will be followed until week 96 (48 weeks after 
discontinuation of therapy) at which time the primary outcome will be measured. 
  
1. Background   
1.1. “Immune tolerant” HBV in adults 
Chronic hepatitis B virus infection (CHB) affects 360 million people worldwide, including 
approximately 2.0 million in North America. Chronic hepatitis B may result in progressive liver 
disease that leads to cirrhosis, end-stage liver disease and hepatocellular carcinoma (HCC). 
Indications and approaches to therapy of hepatitis B are based upon knowledge of the features 
of its natural history. The natural history of chronic hepatitis B, particularly if acquired in early 
childhood, runs through several stages or phases that have different clinical manifestations and 
implications for long-term hepatic injury (1, 2). The initial “immune tolerant” phase is variable in 
duration, but typically evolves during childhood or early adulthood to the “immune clearance” 
phase, marked by rises in serum alanine aminotransferase (ALT) and aspartate 
aminotransferase (AST), increased inflammation and hepatocellular necrosis on liver biopsy, 
and an increase in cytoplasmic hepatitis B core antigen (HBcAg) expression. As the term 
‘immune tolerant’ (IT) suggests this is a state where the host appears to exert no overt immune 
response to a chronic infection with Hepatitis B. The state is characterized by the presence of 
hepatitis B e antigen (HBeAg) and high levels of hepatitis B virus (HBV) DNA (typically HBV 
DNA ≥108 IU/mL), but minimal or no elevations in serum ALT and AST levels and minimal 
activity or injury on liver biopsy. Most but not all individuals with ‘Immune Tolerant’ CHB are 
children or young adults, with a mean age of transition to the immune clearance phase of ~30 
years. Thus, many adults with CHB who acquired their infection at birth or in early childhood 
may be in the immune tolerant phase of the infection up to the age of 30 or 40 years, or longer.  
 
Adults in the immune tolerant CHB phase have variable histologic findings, largely dependent 
upon the definition of ALT used to define the population.  For studies using the more stringent 
ALT criteria of ≤30 U/L for males and ≤19 U/L for females, histologic disease is minimal 
(majority fibrosis <2/4 and necroinflammation 0-1/4) (3-5). Natural history studies of adults in the 
immune tolerant phase have shown that the ALT levels were significantly higher in patients with 


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subsequent loss of tolerance. For inclusion criterion, we propose to use ALT upper limits of 30 
U/L for females and 45 U/L for males, and avoid the need for liver biopsy. 
  
It has been hypothesized that the mother-to-child HBV transmission leads to transplacental 
transfer of maternal HBeAg that may induce a specific unresponsiveness of helper T cells to 
HBeAg in neonates. Because HBeAg and HBcAg are highly cross-reactive at the T-cell level, 
deletion of the helper T-cell response to HBeAg results in an ineffective cytotoxic T-lymphocyte 
response to HBcAg (6). During the immune tolerant phase of chronic infection, the continued 
secretion of HBeAg (the tolerogen) is necessary to maintain the tolerant state. Little is known 
about the mechanisms that regulate the loss of immune tolerance in chronic HBV infection. 
Recent studies have emphasized the importance of low expression of transporter associated 
with antigen processing (TAP1) and low molecular weight protein 2 (LMP2) compared with CHB 
patients in the immune clearance phase, suggesting a deficiency in antigen processing and 
transport to major histocompatibility complex class I molecules in IT adults (7). Point mutations 
and deletions in HBV epitopes that occur prior to or concurrent with the onset of the 
immunoactive phase during chronic HBV infection may contribute to a significant decrease in 
both viral replicative competence and HBV-specific CD8(+) T cell responses, and in turn, lead to 
maintenance of viral persistence (8).  The proposed study will provide an opportunity to study 
the immunologic features and correlates of immune tolerant HBV infection in adults as well as 
the immune markers associated with breaking of tolerance in the context of antiviral therapy.  
 
1.2 Treatment of CHB in adults 
The known natural history of chronic hepatitis B underlies the current criteria for therapy and the 
endpoints of treatment. Treatment of adults with CHB targets those with active disease, 
characterized by elevated ALT levels and/or evidence of significant inflammatory activity or 
fibrosis on biopsy in conjunction with elevated HBV DNA levels. Treatment of adults in the 
immune tolerant phase of infection is not recommended (9). Reasons for not recommending 
treatment include the low frequency of histologic disease and complications and the lack of 
established effective therapies. Serum HBV DNA level has been found to be directly correlated 
with the development of HCC and cirrhosis independent of serum ALT level, HBV genotype, 
and HBeAg status (10,11). This has led to promotion of the use of serum HBV DNA 
concentration as an indicator for treatment regardless of serum ALT level, and even patients 
with normal serum ALT levels are to be treated with antiviral therapy with the primary aim of 
suppressing HBV DNA.  In this context, the risks and benefits of treatment of immune tolerant 
adults is an important issue – not addressed by treatment studies to date. 
 
Currently, there are two broad categories of HBV therapies available: pegylated interferon 
(peginterferon) and nucleos(t)ide analogues, The advantages of nucleos(t)ide analogue therapy 
is the ease of administration, the lack of significant side effects and the consistent suppression 
of HBV DNA that can be achieved, particularly with the newer agents such as tenofovir and 
entecavir. While emergence of drug resistance can limit the efficacy of oral agents, entecavir 
and tenofovir are characterized by great potency and higher genetic barrier to resistance 
leading to a low rate of antiviral resistance (1% or less even with prolonged therapy for 4 to 5 
years). As a consequence, 80-90% of patients treated long-term with these agents achieve 
rates of marked suppression of HBV DNA levels and normal ALT levels. The advantage of 
peginterferon therapy is that it is administered for a limited period (6 to 12 months) and results in 
loss of HBsAg in a significant proportion of patients. The latter feature may be particularly 
relevant in the treatment of immune tolerant adults, the target population for this study. Previous 
studies have shown that a one-year course of combination therapy of peginterferon and 
nucleos(t)ide analogue does not yield a higher response rate than a one-year course of either 
agent alone.  However, these studies have been limited by the use of a nucleos(t)ide analogue 


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with high risk of resistance (e.g. lamivudine)  and may have been affected by the concurrent use 
of peginterferon and nucleos(t)ide analogue rather than sequential use.  
 
1.3 Considerations in the treatment of immune-tolerant adults 
There are no prior studies of treatment of immune tolerant adults.  Data from the pediatric 
literature indicated that treatment with IFN alone during the immune tolerant phase of infection 
is rarely effective and loss of HBV DNA or anti-HBe seroconversion, with less than 10% of 
treated children achieving this outcome (12).  However, the combination of a nucleoside 
analogue and IFN was examined in a pilot study of 23 children and appears encouraging (13). 
Lamivudine was given for 8 weeks and then combined IFN and lamivudine therapy for a further 
44 weeks.  HBV DNA by PCR was negative in 48% at end of treatment, and 5 (22%) children 
seroconverted to anti-HBe.  Four (17%) children cleared HBsAg and seroconverted to anti-HBs.  
Six months after treatment, HBV DNA was again positive in all but the children who had 
seroconverted to anti-HBe and (presumably) anti-HBs. 
 
Based upon these stimulating results, we propose to evaluate the safety and efficacy of a short 
lead-in course (8 weeks) of entecavir followed by combination of entecavir plus peginterferon 
alfa-2a for 40 weeks. We hypothesize that using a potent nucleos(t)ide analogue will provide a 
higher rate of HBeAg loss and suppression of HBV DNA. The primary measure of outcome will 
be HBeAg loss and suppression of HBV DNA to ≤1000 IU/mL in serum 48 weeks after stopping 
all antiviral therapy (sustained off-treatment response). 
 
2. Study objectives 
2.1 Primary objectives 
• 
To determine the safety and efficacy of treatment with 8 weeks of entecavir followed by 
40 weeks of both entecavir and peginterferon alfa-2a in the treatment of chronic hepatitis 
B in HBeAg positive adults who are in the immune tolerant phase. 
• 
To evaluate “off treatment” safety and sustained responses after treatment with 
entecavir and peginterferon alfa-2a in the treatment of chronic hepatitis B in HBeAg 
positive adults who are in the immune tolerant phase. 
 
2.2. Primary endpoint 
2.2.1 Safety: Adverse Events and Serious Adverse Events through the end of treatment and 
through the end of follow-up 
 
2.2.2 Efficacy: HBeAg loss (lack of detectable HBeAg) AND HBV DNA ≤1,000 IU/mL 48 weeks 
after stopping therapy. 
 
2.3. Secondary endpoints  
2.3.1. At the end of treatment: 
a. Cumulative HBsAg loss 
b. HBeAg loss 
c. HBeAg seroconversion 
d. HBsAg seroconversion  
e. ALT <45 U/L for men, <30 U/L for women (approximately 1.5 ULN)  
f. ALT normalization (men <30 U/L, women <20 U/L) 
g. HBV DNA ≤1,000 IU/mL 
h. HBV DNA <20 IU/mL (LLOQ of COBAS Ampliprep/COBAS TaqMan HBV v2.0 test) 
i. 
Absence of detectable antiviral drug-resistance HBV mutations  
 
2.3.2.  Sustained end of follow-up responses 48 weeks after end-of-treatment (week 96) 


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a. Cumulative HBsAg loss 
b. HBeAg loss 
c. HBeAg seroconversion  
d. HBsAg seroconversion 
e. ALT <45 U/L for men, <30 U/L for women (approximately 1.5 ULN)  
f. ALT normalization (men <30 U/L, women <20 U/L) 
g. HBV DNA ≤1000 IU/mL 
h. HBV DNA <20 IU/mL (LLOQ of COBAS Ampliprep/COBAS TaqMan HBV v2.0 test) 
 
3. Study design 
A single arm pilot study of 8 weeks of entecavir followed by 40 weeks of both entecavir and 
peginterferon alfa-2a in adults with HBeAg-positive chronic hepatitis B with normal or near 
normal ALT levels and high serum levels of HBV DNA (“immune tolerant” HBeAg-positive 
chronic hepatitis B). Participants will be followed until week 96 (48 weeks after discontinuation 
of therapy). Screening can include historical values up to 52 weeks prior to the baseline visit, 
but there must be a screening visit within 6 weeks of the baseline visit. 
 
4. Study population 
4.1. Inclusion criteria 
2. 1. Enrolled in the Hepatitis B Research Network (HBRN) Cohort Study  or completed 
the necessary components of the Cohort baseline evaluation by the end of the 
baseline visit for this study>18 years of age at the baseline visit (day 0). Patients >50 
years of age at the baseline visit need to have a liver biopsy as standard of care with 
HAI ≤3 and Ishak fibrosis score ≤1 within 96 weeks prior to the baseline visit. 
3. Documented chronic HBV infection as evidenced by detection of HBsAg in serum for 
≥24 weeks prior to the baseline visit OR at least one positive HBsAg and negative anti-
HBc IgM within 24 weeks prior to the baseline visit OR at least one positive HBsAg 
and two positive HBV DNA over a period of ≥24 weeks prior to the baseline visit.   
4. Presence of HBeAg in serum at the last screening visit within 6 weeks of the baseline 
visit . 
5. Serum HBV DNA level >107 IU/mL on at least two occasions at least 12 weeks apart 
during the 52 weeks before the baseline visit. One of the two HBV DNA levels must be 
within 6 weeks of the baseline visit. 
6.   ALT levels persistently ≤45 U/L in males, ≤30 U/L in females (approximately   
      1.5 times the upper limit of normal [ULN] range) as documented by at least three 
7.  values: one taken 28-52 weeks before the baseline visit, one taken 6 to 28 weeks 
before the baseline visit, and the final value within 6 weeks prior to the baseline visit. 
No evidence of HCC based upon alpha-fetoprotein (AFP) ≤20 ng/mL at the screening 
visit (up to 6 weeks prior to the baseline visit): 
a. Participants who meet AASLD criteria for HCC surveillance must have negative 
liver imaging as shown by ultrasound (US), computerized tomography (CT) or 
magnetic resonance imaging (MRI) within 28 weeks prior to the baseline visit as 
part of standard of care. 
b. Participants with AFP >20 ng/mL must be evaluated clinically with additional 
imaging and shown not to have HCC on CT or MRI before they can be enrolled. 
8. Provide informed consent and agree to adhere to the requirements of the study.  
 
4.2. Exclusion criteria 
1. History of hepatic decompensation, including but not limited to ascites, variceal bleeding, 
or hepatic encephalopathy. 


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2. Evidence of decompensated liver disease prior to or during screening, including direct 
bilirubin >0.5 mg/dL, INR >1.5, or serum albumin <3.5 g/dL. 
3. Platelet count <120,000/mm3, hemoglobin <13 g/dL (males) or <12 g/dL (females), 
absolute neutrophil count < 1500 /mm3 (<1000/mm3 for African-Americans) at the last 
screening visit. 
4. Previous treatment with medications that have established activity against HBV 
including, but not limited to, interferon and nucleos(t)ide analogs ≥24 weeks. Patients 
with <24 weeks of prior HBV treatment and a wash-out period >24 weeks are not 
excluded. Brief and episodic use of famciclovir or valacyclovir for herpes infection is not 
exclusionary. 
5. Known allergy or intolerance to any of the study medications. 
6. Females who are pregnant or breastfeeding. 
7. Females of childbearing potential unable or unwilling to use a reliable method of 
contraception during the treatment period.  
8. Renal insufficiency with calculated creatinine clearance <50 mL/min at the last screening 
visit.  
9. History of alcohol or drug abuse within 48 weeks of the baseline visit. 
10. Previous liver or other organ transplantation including engrafted bone marrow transplant. 
11. Any other concomitant liver disease, including hepatitis C or D. Non-alcoholic fatty liver 
disease (NAFLD) with steatosis and/or mild to moderate steatohepatitis is acceptable 
but NALFD with severe steatohepatitis is exclusionary.  
12. Presence of anti-HDV or anti-HCV (unless HCV RNA negative) in serum on any 
occasion in the 144 weeks prior to the baseline visit. 
13. Presence of anti-HIV (test to be completed within 6 weeks prior to the baseline visit). 
14. Pre-existing psychiatric condition(s), including, but not limited to: 
a. Current moderate or severe depression as determined by the study physician 
b. History of depression requiring hospitalization within past 10 years 
c. History of suicidal or homicidal attempt within the past 10 years 
d. History of severe psychiatric disorders including, but not limited to schizophrenia, 
psychosis, bipolar disorder as determined by a study physician.  
15. History of immune-mediated or cerebrovascular disease, chronic pulmonary or cardiac 
disease associated with functional limitation, retinopathy, uncontrolled thyroid disease, 
poorly controlled diabetes or uncontrolled seizure disorder, as determined by a study 
physician. 
16. Any medical condition that would, in the opinion of a study physician, be predicted to be 
exacerbated by therapy or that would limit study participation. 
17. Any medical condition requiring, or likely to require, chronic systemic administration of 
corticosteroids or other immunosuppressive medications during the course of this study. 
18. Evidence of active or suspected malignancy, or a history of malignancy within the 144 
weeks prior to the baseline visit (except adequately treated carcinoma in situ or basal 
cell carcinoma of the skin). 
19. Expected need for ongoing use of any antivirals with activity against HBV during the 
course of the study. 
20. Concomitant use of complementary or alternative medications purported to have antiviral 
activity.  
21. Participation in any other clinical trial involving investigational drugs within 30 days of the 
baseline visit or intention to participate in another clinical trial involving investigational 
drugs during participation in this study. 
22. Any other condition or situation that in the opinion of a study physician would make the 
participant unsuitable for enrollment or could interfere with the participant participating in 
and completing the study. 


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5. Study drugs and drug management 
5.1 Dosage and administration 
 
 Treatment Entecavir 0.5 mg daily orally for 48 weeks plus peginterferon alfa-2a 180 µg 
subcutaneously (sq) weekly during weeks 9-48 of treatment. After treatment discontinuation, 
follow-up off treatment will be for 48 weeks. 
 
Participants will be instructed to take one tablet of entecavir daily on an empty stomach at least 
2 hours prior to or after meals. Participants will be instructed to take 180 µg of peginterferon 
alfa-2a subcutaneously once weekly. The participant will be instructed to inject the peginterferon 
alfa-2a on the same day each week.  
 
 
5.2 Entecavir 
5.2.1 Mechanism of action 
Entecavir is a novel carbocyclic 2’-deoxyguanosine analogue with potent and selective anti HBV 
activity.  Entecavir blocks three phases of HBV replication: (1) priming of the HBV polymerase, 
(2) reverse transcription of the negative strand DNA synthesis, and (3) DNA polymerase activity 
responsible for positive strand DNA synthesis. Entecavir functionally inhibits episomal viral 
replication rather than genomic transcriptional events mediated by cellular polymerases. 
Entecavir is converted to the triphosphate, which then acts as an inhibitor of hepadnaviral 
polymerase.  Entecavir is not recognized by mitochondrial DNA polymerase and cannot be 
incorporated into (human) DNA or inhibit a polymerase assay system even when tested at very 
high doses (14).  EC50 falls by 50% with wild type HBV DNA but there is a reduction 8-30 fold in 
entecavir phenotypic susceptibility with LAM resistance.  In treatment naïve patients the mean 
log fall in HBV DNA in patients on entecavir is 5-6 logs.   
 
Entecavir is not recommended in patients with HIV who are not on HAART as it has weak 
activity against HIV.  All study participants will be tested for HIV within the 6 weeks prior to the 
baseline visit. Participants who self-report acquiring HIV will be removed from the study and 
referred to an HIV specialist. 
 
5.2.2 Metabolism 
The intracellular half-life is about 15 hrs.  Oral entecavir given to subjects with CHB in doses 
ranging from 0.05 to 1mg daily over 28 days lead to a mean log fall in HBV DNA of 2.8.  
Absorption appears to be rapid – oral bioavailability estimated to be >70%.  Entecavir is not 
affected by the co-administration of agents that are metabolized by inhibit or induce CYP450 
system. 
 
5.2.3 Safety of entecavir 
 
In animal studies life time entecavir administration gave rise to an increased incidence of lung 
tumors when given at low multiples of human exposure and more with high exposure multiples.  
These tumors may be species specific and not relevant to humans. Clinical experience from 
1755 patients treated with entecavir is available and does not indicate an increased risk of 
malignancies. There is no evidence in animal models for either genotoxicity or carcinogenicity. 
 
Studies in rats and rabbits given in high dose during pregnancy indicated no embryotoxicity or 
maternal toxicity. 
 
5.2.4 Entecavir resistant mutations   


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Entecavir has a very high genetic barrier when given in treatment naïve patients in a dose of 
0.5mg/d.  Resistance to entecavir after 5 yrs of therapy is reported to be 1.5%.  When given to 
patients with lamivudine-resistance, a higher dose of 1mg/day is recommended and the 
cumulative probability of entecavir resistance is detailed below: 
 
 
1 yr 
2 yr 
3 yr 
4 yr 
5 yr 
Naïve to entecavir 
0.2% 
0.5% 
1.2% 
1.2% 
1.2% 
Previous lamivudine 
resistance 
6% 
15% 
36% 
47% 
52% 
 
5.3 Peginterferon alfa-2a 
5.3.1 General description 
Peginterferon alfa-2a is a covalent conjugate of recombinant alfa-2a interferon with a single 
branched molecule of polyethylene glycol with a molecular weight of approximately 40,000 
daltons. Peginterferon alfa-2a is produced using recombinant DNA technology and contains 180 
µg/1.0 mL in vial form and the same amount per 0.5 ml in a prefilled syringe. The drug must be 
kept refrigerated at +4C until use.  
 
Interferon inhibits viral replication by inducing an antiviral state in cells.  Interferon does not 
enter the hepatocyte, but rather binds to specific receptors on the cell surface, which initiates 
intracellular signaling that leads to rapid activation of multiple “interferon-stimulated genes 
(ISGs); the encoded proteins of these genes inhibit viral replication in infected hepatocytes by 
multiple mechanisms (including inhibition of viral protein synthesis and breakdown of viral RNA).  
Peginterferon alfa-2a also has immunomodulatory activity that is thought to be important in 
obtaining a virological response. Steady-state serum concentrations of peginterferon alfa-2a are 
reached within 5 to 8 weeks using once weekly dosing.  The mean terminal half-life after 
subcutaneous dosing is 160 hours compared to 5 hours for standard, non-peginterferon alfa-2a. 
 
5.3.2 Pharmacokinetic considerations  
In patients with end-stage-renal-disease undergoing hemodialysis, there is a 25% to 45% 
reduction in clearance of peginterferon alfa-2a. The effect of milder renal impairment has not 
been studied but it is advised that in patients with impaired renal function, signs and symptoms 
of interferon toxicity should be closely monitored.  It is also recommended that peginterferon 
alfa-2a be used with caution in patients with creatinine clearance < 50 mL/min. 
 
There is no known effect of peginterferon alfa-2a on the pharmacokinetics of drugs metabolized 
by the cytochrome P-450 system. There are no known clinically significant interactions with 
nucleos(t)ide analogue therapy, but a phase IV registration trial of peginterferon alfa-2a and 
telbivudine was prematurely discontinued due to a higher than anticipated incidence of 
peripheral neuropathy.  
 
5.3.3. Safety 
As with standard alpha interferon, treatment with peginterferon alfa-2a is associated with many 
troublesome and occasionally serious, or even life-threatening, side effects. Dose 
discontinuation has been reported in 6% to 9% and dose modification in 31% to 47% of patients 
treated in the peginterferon alfa-2a registration trials for hepatitis B.  The most frequent causes 
of dose adjustment were laboratory abnormalities such as thrombocytopenia or leucopenia.  
These side effects are particularly common in patients with advanced liver disease and 
hypersplenism. Peginterferon alfa-2a is absolutely contraindicated in patients with 
decompensated cirrhosis due to the possibility of serious infections, flares of disease and 
worsening decompensation. 


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The majority of clinical experience in the use of peginterferon alfa-2a has been in chronic 
hepatitis C in which peginterferon alfa-2a is combined with ribavirin. In the few studies of 
peginterferon in chronic hepatitis B it has appeared that side effects are less frequent than in 
chronic hepatitis C. The overall incidence of serious adverse events was less in studies in 
hepatitis B (4%-5%) than in those with hepatitis C (7% to 16%), and fewer drug withdrawals 
were reported (6% to 8% versus 17% to 33%, respectively) despite similar doses and durations 
of therapy. Depression was also less frequently reported in studies done in hepatitis B (4%) 
than hepatitis C (22%, p < 0.001). 
 
Peginterferon alfa-2a should be avoided in patients with other serious co-morbid illnesses, 
including, but not limited to, coronary artery disease, cerebrovascular disease, serious 
autoimmune conditions and severe depression.  
 
Pregnancy: Peginterferon alfa-2a is a Pregnancy Category C drug that has not been 
adequately evaluated in humans for its teratogenic effect. Standard interferon has been shown 
to increase the rate of abortion in Rhesus monkeys when given approximately 20 to 500 times 
the human weekly dose.  Interferon, however, is highly species specific in its effects and animal 
studies may not reliably reflect the potential of side effects in humans. There have been no 
adequate and well-controlled studies of peginterferon alfa-2a in pregnant women. Therefore, in 
this study, women of childbearing potential will be enrolled only if they agree to use effective 
contraception during therapy and pregnancy testing will occur at every visit during the treatment 
phase. 
 
5.4. Administration and drug accountability 
Study drugs (peginterferon alfa-2a and entecavir) will be shipped to study sites from a drug 
distribution center. Staff at each site will be responsible for drug accountability.  This will include 
the documentation of drugs received from the drug distribution center, of study drug dispensed 
to study participants, of used and unused study drug returned, and an accounting of any drug 
destroyed by the site. An accurate and up-to-date accountability log will be maintained by each 
site.    
 
5.5. Concomitant medications   
All concomitant medications (prescription and non-prescription) being taken by each participant 
will be queried and recorded by the study coordinator at each visit.   
 
5.6. Prohibited medications  
Use of the following medications is prohibited while participants are on study drugs: 
1. Antivirals with activity against HBV, including valacyclovir.  The need for ongoing use of 
these antivirals would be exclusionary at study entry, but would be recorded as a 
concomitant medication if medically indicated and prescribed during the course of the 
study.   
2. Systemic chemotherapeutic agents (i.e., cancer treatment medications). 
3. Systemic corticosteroids. The need for ongoing use of systemic corticosteroids would be 
exclusionary at study entry, but would be recorded as a concomitant medication if 
medically indicated and prescribed during the course of the study.   
4. Any investigational agents. 
5. Growth factors such as GCSF or epotein alfa. 
 
6. Study procedures 
6.1. Study enrollment  


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Participants thought to be eligible for participation in this trial will be asked to give their consent 
to participate after discussions with study investigators and by signing a consent form approved 
by the IRB/REB at that institution. Patients who are not enrolled in the HBRN Cohort study may 
be enrolled concurrently with the screening visit for this trial, or if they choose not to enroll into 
the Cohort Study the necessary components of the Cohort baseline evaluation will be 
completed prior to enrolling in this study at the screening or baseline visit. They will then 
undergo the screening procedures, including medical history, physical examination and blood 
tests as summarized in the Schedule of Events (Appendix I).   
 
6.2.  Screening assessment 
The following assessments must be obtained within 6 weeks (42 days) prior to the baseline visit 
(start of treatment)  
1. Written informed consent  
2. Detailed medical history 
3. Physical examination  
4. Depression assessment  
5. Blood tests: CBC with differential, routine biochemical tests (including ALT, AST, total 
bilirubin, albumin, alkaline phosphatase, glucose, total protein, creatinine, [including 
estimated creatinine clearance], TSH, PT/INR, alphafetoprotein (AFP), HBsAg, HBeAg, 
quantitative HBV DNA, and anti-HIV  (see Appendix I).   
6. Anti-HCV and anti-HDV are to be tested unless results within 144 weeks of the baseline 
visit are available.  
7. Urinalysis  
8. Pregnancy test in women with reproductive potential (urine or blood) 
9. Abdominal imaging as necessary 
10. Plasma and serum samples will be stored at each visit. 
11. Anti-HBs/HBe will be done at the next testing time point if HBsAg/HBeAg becomes 
negative 
  
 
6.3. Baseline visit Baseline assessments (day 0) must be obtained prior to the initiation of 
study drugs. Participants will have a limited clinical evaluation and have blood drawn for 
complete blood count, hepatic panel, renal panel, PT/INR and HBV DNA.  Urine for urinalysis 
will be obtained. A sample of blood will be stored for eventual assessment of HBeAg and 
HBsAg concentration, anti-HBs, anti-HBe, full HBV DNA sequence and presence of pre-core 
and core mutations as well as polymerase gene antiviral resistance mutations, and DNA 
extraction (if sample not already obtained as part of Cohort Study, and sample may be obtained 
at future visit if not obtained at baseline) (See Schedule of Events, Appendix 1).  QOL and 
health behaviors will be assessed. A pregnancy test will be performed in women with 
reproductive potential prior to start of treatment. At the end of the baseline visit, study drug 
(Entecavir) will be provided to the participant with instructions on its use. 
 
 
6.4. On-treatment assessments 
After starting treatment, visits will occur at weeks 8, 12, 16, 20, 24, 30, 36, 42, and 48. Each visit 
will include assessment of compliance, adverse events reporting, vital signs, weight, symptoms 
evaluation, record of concomitant medications, and blood testing as detailed in Schedule of 
Events (Appendix I).  Participants  will be dispensed study medications.  
1. At the Week 8 visit, peginterferon alfa-2a will be provided to subjects with instructions on 
its use, including education on self-injection, if appropriate. Subjects will have a limited 
clinical evaluation as well as blood and urine tests.  


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2. Pregnancy test will be done in women with reproductive potential at each visit until the 
end of treatment. 
3. Depression assessment will occur at weeks 8, 12, 24, 36 and 48. 
4. QOL assessments will occur at day 0, weeks 8 and 48.  Health behaviors will be 
included at day 0 and week 48. 
5. Fatigue questionnaire will occur at day 0. 
6. CBC (with differential), hepatic panel (ALT, AST, total bilirubin), renal panel (Cr, CrCl) 
and HBV DNA quantitation at each visit. 
7. TSH at week 12, 24, 36 and 48. 
8. Albumin, alkaline phosphatase, glucose and total protein will be obtained at week 48  
9. HBeAg at weeks 8, 12, 24, 36 and 48. 
10. HBsAg at week 48. 
11. Blood samples for serum/plasma at each visit. 
12. HBsAg and HBeAg quantification at each visit. 
13. Anti-HBs/HBe will be done at day 0 and week 48. Also at the next visit if HBsAg/HBeAg 
becomes negative. 
 
6.5 End of treatment assessment 
At week 48, all participants will complete the treatment phase. Laboratory testing will be 
performed as outlined in Appendix 1 Schedule of Events.  A sample of blood will be stored for 
eventual assessment of HBeAg and HBsAg concentration. 
 
6.6. Post-treatment assessments  
1. Visits will occur at weeks 52, 60, 72, and 96 .  
2. Depression assessment will occur at weeks 60 and 96. 
3. QOL and health behaviors assessments will occur at week 96. 
4. Fatigue questionnaire will be obtained at week 96. 
5. CBC (without differential), hepatic panel (ALT, AST, total bilirubin), renal panel (Cr, 
CrCl), and HBV DNA quantitation at each visit. 
6. TSH at week 60. 
7.  Albumin, alkaline phosphatase, total protein, glucose, INR/PT, and urinalysis at week 
96. 
8. HBeAg at weeks 60, 72, and 96. 
9. HBsAg at weeks 72 and 96. 
10. HBsAg and HBeAg quantification every visit. 
11. Blood samples for serum/plasma at each visit. 
12. Anti-HBs/HBe will be done at week 96. Also at the next visit if HBsAg/HBeAg becomes 
negative. 
 
6.7 Assessment of adherence 
During each visit, study personnel will review the study drug dosing schedule with the 
participant and counseling on adherence will be provided. At each study visit while on therapy, 
participants will be asked to return all bottles of entecavir dispensed and used vials of 
peginterferon to document how much study medications were used. Serum drug levels may be 
measured on stored serum samples. 
6.8 Stopping rules 
Therapy will only be stopped for the following indications: 
• 
Grade IV toxicity (except hematologic, see appendix III.) 
• 
Death 
• 
Pregnancy 


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• 
Non-response per section 6.9. 
• 
Virologic breakthrough per section 6.10.   
• 
Hepatic decompensation defined by: 
o Total bilirubin ≥2.0 mg/dL and direct bilirubin ≥1.0 mg/dL 
o Any clinical measure of decompensation 
• 
Participant refusal to continue treatment  
 
Participants who discontinue treatment due to grade IV toxicity or hepatic decompensation will 
continue to be followed until these events are resolved or stabilized, according to the post-
treatment follow-up schedule. Managing these participants including more frequent 
assessments and alternative treatment will be at the discretion of the investigator.  
 
Participants who become pregnant while receiving treatment will stop treatment and subsequent 
management will be at the discretion of the investigator. The outcome of the pregnancy will be 
recorded.   
 
6.9. Management of non-response  
Definition: Primary non-response is defined as a failure to achieve at least >1-log10 IU/mL 
decline in HBV DNA after the first 24 weeks of treatment by the quantitative HBV DNA assay, 
which will be confirmed on re-testing 4 weeks later. Compliance with study medications will be 
assessed and documented. Primary non-response is considered treatment failure. In the event 
of non-response, the following will be implemented: 
 
6.9.1. Reinforce compliance upon initial determination of non-response. 
6.9.2. Treatment (both entecavir and peginterferon alfa-2a) will be discontinued if non-response 
is confirmed on re-testing 4 weeks later.   
6.9.3. Participants will be assessed at week 4 after treatment discontinuation and then be 
followed at weeks 12, 24, and 48 post-discontinuation . 
 
6.10. Management of virological breakthrough 
Definition: Virological breakthrough is defined as a > 1-log10 IU/mL increase in HBV DNA level 
from nadir in a participant with an initial virological response or redetection of HBV DNA after 
becoming undetectable, and will be confirmed on re-testing 4 weeks later in a participant with 
either an initial or partial virological response and who has been compliant with study 
medications. 
• 
Virologic breakthrough is considered a treatment failure. 
 
Management algorithm if virological breakthrough6.10.1. Reinforce compliance. The 
importance of compliance with study medication will be reinforced with study participants at 
each visit. 
6.10.2. Resistance testing will be performed but will not be used to guide decisions (as results 
will not be available in real-time). Participants meeting the definition for virologic breakthrough 
on repeat testing will stop all treatment.   
6.10.3. Participants will be assessed at weeks 4, 12, 24, and 48 post-discontinuation. 
 
6.11 Management algorithm for participants with ALT flare during the treatment phase  
6.11.1. If ALT values increase to ≥300 U/L for males and ≥200 U/L for females, retest the 
hepatic panel within 4 weeks. Continue testing of hepatic panel every 4 weeks as long as the 
ALT remains >300 U/L for males and >200 U/L for females.  


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6.11.2. If ALT values decline below these levels, the frequency of testing the hepatic panel can 
return to the usual schedule. 
6.11.3. Participants will remain on treatment until week 48 unless: 
i. 
there are signs of clinical decompensation or  
ii. 
total bilirubin ≥2.0 mg/dL and direct bilirubin ≥1.0 mg/dL or 
iii. 
ALT >450 U/L for males or >300 U/L for females for more than 12 weeks  
Participants meeting any of these criteria will continue to be followed in the study but with any 
subsequent management at the discretion of the study physician.  
 
6.12. Management algorithm for participants with ALT elevation during the follow-up 
phase (weeks 48-96) 
6.12.1. If ALT increases to >300 U/L for males and >200 U/L for females, retest the hepatic 
panel in 4 weeks. Continue testing of hepatic panel every 4 weeks as long as the ALT >300 U/L 
for males and >200 U/L for females. 
6.12.2. If ALT decline below these levels, the frequency of testing the hepatic panel can return 
to the usual schedule. 
6.12.3. For participants with ALT persistently >150 U/L in males and >100 U/L in females for 
≥24 weeks and HBV DNA ≥10,000 IU/mL, they are eligible for  retreatment with entecavir (up to 
one year or by December 31, 2015 (whichever comes first), or peginterferon alfa-2a or both 
drugs for one year if desired by participants and provider.   
 
6.13.  Participants previously randomized to control group 
Participants enrolled in this trial prior to amendment 7.0 of this protocol who were randomized to 
the control (no treatment) group will be offered treatment if they still meet the entry criteria for 
this trial (i.e.  HBV DNA and ALT results from the most recent IT trial visit meet the inclusion and 
exclusion criteria listed in sections 4.1 and 4.2), and are willing to receive treatment. These 
participants will be reconsented prior to starting treatment and follow the visit schedule and 
assessment beginning with baseline (Day 0) visit.  
 
Participants who no longer meet entry criteria for this trial or are not interested in receiving 
treatment will complete a Termination Visit at the next scheduled visit for the control (no 
treatment) group. The procedures to be followed for this Termination Visit will be those specified 
for the Week 48 visit. 
 
7. Adverse events and toxicity management 
7.1. Managing peginterferon alfa-2a side effects 
Common side effects of peginterferon alfa-2a include influenza-like symptoms, particularly with 
the first few injections, fatigue, neuropsychiatric symptoms such as depression and reduction in 
white blood cell counts and platelet counts.  Hyper- or hypothyroidism has been noted to occur 
in up to 5% of patients receiving peginterferon alfa-2a. 
 
Some rare but potentially serious side effects include: 
• 
neuropsychiatric complications, which may include suicide, suicidal ideation and severe 
depression 
• 
serious and severe bacterial infections, some fatal have been reported during treatment 
with peginterferon alfa-2a 
• 
pancreatitis 
• 
interstitial pneumonitis, fatal cases have been reported with peginterferon alfa 2a use  
 
Other possible but uncommon adverse events associated with peginterferon alfa-2a include: 


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• 
development or exacerbation of existing autoimmune disorders such as rheumatoid 
arthritis, systemic lupus erythematous, idiopathic thrombocytopenic purpura, psoriasis 
etc 
• 
other pulmonary disorders including dyspnea, pulmonary infiltrates, and sarcoidosis 
• 
exacerbations of inflammatory bowel disease  
• 
eye disorders, including decrease or loss of vision, retinal hemorrhages 
• 
hypersensitivity including sometimes severe skin reactions in the spectrum of Stevens 
Johnson syndrome 
 
7.1.1. Managing depression  
When possible, the CES-D will be used along with the investigators judgment, to screen 
participants for depression at the screening visit (within 6 weeks of the baseline visit) and to 
monitor for onset or worsening of symptoms during the treatment and follow-up phases visits in 
all participants (See Schedule of Events). If a participant has a CES-D score >15 at the 
screening visit (within 6 weeks of the baseline visit), evaluation by a mental health provider 
should be considered before enrollment (start of treatment). During the treatment phase, if a 
participant has a CES-D score >15, the investigator will determine whether referral to a mental 
health provider is indicated.  
 
If the management of the depression is not successful after eight weeks or if the participant 
develops suicidal or homicidal ideation, the peginterferon alfa-2a will be stopped but entecavir 
continued until the end of the treatment period (see section 7.5.4) and the participant will be 
referred to a mental health provider.  
 
7.2. Peginterferon alfa-2a dose reduction guidelines 
Factors that may lead to a reduction in the dose of peginterferon alfa-2a include: 
1. Disabling symptoms, which, in the opinion of the investigator, are related to 
Peginterferon alfa-2a treatment and prevent the participant from performing 
his/her occupation or daily tasks. 
2. A rash consistent with allergic reaction or vasculitis. 
3. Reductions in the platelet count according to the guidelines in Appendix III.  
4. A reduction in neutrophil count according to the guidelines in Appendix III. 
5. Any adverse reaction, which, in the opinion of the study physician, places the participant 
at increased risk. 
 
The dose of study medications may be decreased due to adverse events or laboratory 
abnormalities.  In most instances the dose of peginterferon alfa-2a will be reduced in graduated 
steps as follows: 
• 
Level 1 decrease from 180 µg to 135 µg  
• 
Level 2 decrease from 135 µg to 90 µg 
• 
Level 3 decrease from 90 µg to 45µg  
 
For participants who require a downward dose adjustment for neutropenia or thrombocytopenia, 
laboratory values should be repeated, if practical, prior to the next dose of peginterferon alfa-2a 
to confirm the result and dictate the course of action.  Participants who remain with a neutrophil 
count below 500/mm3 (but above 250/mm3) or a platelet count below 50,000/mm3 (but above 
25,000/mm3) should receive stepwise dose reductions until levels of neutrophils and platelets 
are ≥500/mm3 or ≥50,000/mm3, respectively, or until a dose of 45 µg of peginterferon alfa-2a is 
reached. 
 


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7.2.1. Guidelines for subsequent peginterferon dose adjustments 
Once a participant’s dose has been decreased for laboratory abnormalities or adverse events, 
the study physician may attempt to increase the dose back to or toward the previous stable level 
according to the following guidelines: 
1. The event or circumstance responsible for the dosage adjustment has resolved or 
improved; 
2.   The participant has been at the lower dose for ≤4 consecutive doses; 
3. The participants had ≤6 total doses administered at the lower level during the entirety of 
the treatment period.  
 
If four or more consecutive doses of peginterferon alfa-2a are held or otherwise not 
administered (i.e., the participant has not received test medication for more than 28 days), the 
participant will be considered intolerant of the test medication or non-compliant, whichever is 
more appropriate to the clinical situation. In such cases, the study physician will discontinue 
peginterferon alfa-2a but entecavir treatment will continue (see section 7.5.4). 
 
Every attempt will be made to keep participants on treatment with peginterferon alfa-2a on 
therapy by dose reduction.  The use of hematological growth factors during the study will not be 
allowed. 
7.2.2. Peginterferon alfa-2a dosing and creatinine clearance (CrCL) 
Creatinine clearance will be calculated in all participants prior to initiating therapy and at each 
study visit. The peginterferon alfa-2a dose does not require adjustment unless the patient is on 
dialysis. However, in this study, peginterferon alfa-2a will be discontinued if CrCl is ≤30 ml/min 
to be consistent with criteria for entecavir discontinuation (see below). 
 
7.3. Managing adverse effects of entecavir 
7.3.1. Adverse effects of entecavir 
• 
Headache, fatigue, dizziness, nausea, upper abdominal pain, upset stomach, diarrhea, 
vomiting, trouble sleeping or sleepiness, changes to liver and pancreas enzymes.  
These side effects were generally mild to moderate in severity. 
• 
Lactic Acidosis:  Patients taking drugs similar to entecavir have experienced lactic 
acidosis.  Symptoms of lactic acidosis include weakness/tiredness, muscle pain, trouble 
breathing, stomach pain with nausea and vomiting, feeling cold (particularly in 
extremities), dizziness/lightheadedness, and increased heart rate.   
• 
Hepatitis “flares”:  In patients with chronic hepatitis B, liver enzyme changes are 
common.   
• 
Laboratory studies have shown that entecavir does not interact with the enzymes in the 
liver that are responsible for most drug interactions.  However, because the body gets rid 
of entecavir in the urine, it is possible that drugs which affect kidney function could 
increase the levels of entecavir when a patient uses both drugs.  These types of drugs 
are not allowed in this study. 
• 
When mice and rats were given entecavir for their entire adult life, these animals 
developed more tumors, including cancerous tumors, than are normally expected. Male 
mice developed non-cancerous lung tumors when they were exposed to entecavir at 
levels greater than or equal to 4 times higher what participants will be exposed to in this 
study. Cancerous lung tumors were also observed in male and female mice when they 
were given high levels of entecavir. Further animal studies have been performed to 
better understand these findings. These additional studies suggest that the lung findings 
in mice are unique for mice. The lung findings in mice did not occur in rats, dogs or 


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monkeys. It is not known whether these tumor findings in animals are relevant to 
humans who are treated with Entecavir. 
 
7.3.2. Entecavir and renal function 
Entecavir dosing is based upon CrCL: Creatinine clearance will be calculated for all participants 
prior to initiating therapy and at each study visit to determine Entecavir dose.  
• 
Participants whose calculated creatinine clearance decreases to <50 ml/min should have 
this value confirmed within 72 hrs.   
• 
Entecavir may be continued if the CrCL is ≥30-50 ml/min at the adjusted dose of 0.5 mg 
every 48 hours.  
• 
Entecavir will be discontinued if the CrCL is <30 ml/min.  
 
7.4. Pregnancy 
Participants will be asked to practice effective birth control while they are taking antiviral agents 
in this study.  Additionally, following precautions will be taken: 
• 
All female participants with reproductive potential will undergo pregnancy test on 
enrollment and will have pregnancy tests performed at each visit during treatment. 
• 
If a participant on entecavir or peginterferon alfa-2a becomes pregnant, the study 
physician should be notified immediately and treatment will be discontinued immediately. 
Subsequent management will be at the discretion of the study physician. The study will 
collect data on the outcomes of any pregnancies that occur in women who conceived 
while taking study medication.  
 
7.5. Adverse events – definition and management 
7.5.1. Definitions 
An adverse event (AE) is any adverse change from the participant’s baseline (pre-treatment) 
condition, including intercurrent illness which occurs during the course of the trial, after the 
consent form has been signed, whether the event is considered related to treatment or not. 
 
The modified World Health Organization (WHO) grading system will be used for grading severity 
of AEs.  For AEs not outlined in the modified WHO grading system, study specific definitions will 
be identified in the Manual of Operation (MOP).  
 
A serious adverse event is an untoward medical occurrence that results in any 
of the following: 
1. Death 
2. Is life threatening (risk of death at the time of the event) 
3. Requires in-patient hospitalization or prolongation of existing hospitalization 
4. Results in persistent or significant disability/incapacity 
5. Congenital abnormality or birth defect 
 
Important medical events that do not result in one of the events listed above may be considered 
a serious adverse event when, based upon appropriate medical judgment, they may jeopardize 
the participant and may require medical or surgical intervention to prevent one of the outcomes 
listed above. 
 
A serious adverse event which is unexpected and is related will require expedited reporting to 
the DCC and the NIDDK and appropriate oversight committees or entities. 
 
Disease related outcomes, such as the following, will not be considered to be serious adverse 


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events: 
1. Development of HCC  
2. Hepatic decompensation 
 
Although pregnancy, overdose, cancer (excluding HCC), and potential drug induced liver injury 
(DILI) are not always serious by regulatory definition, these events must be handled as SAEs.   
 
7.5.2. Data collection procedures for adverse events 
Participants will be interviewed regarding medical conditions, medication changes, and 
symptoms that have occurred at each study visit. An Adverse Event form will be completed if 
any adverse event is reported. If the study coordinator or a study physician learns of any 
hospitalizations or other adverse events between study visits, an Adverse Event form will be 
completed. All adverse events and Serious Adverse Events from time of study entry (consent) 
up to the end of follow-up (week 96) will be reported to the DCC. 
 
A Serious Adverse Event form will be completed for all adverse events rated as 
serious. 
 
Participants will be followed for all ongoing unresolved adverse events until they are either 
resolved, or in the opinion of the study physician, the participant is medically stable. 
 
A study physician will assess the relationship of each adverse event to the use of study drug, 
based on available information, using guidelines outlined in the MOP.  
 
7.5.3 Reporting procedures 
All serious adverse events that are unexpected and related to study drug(s) will be reported to 
the DCC within 24 hours of knowledge of the event, via the Serious Adverse Event form. This 
reporting includes serious adverse events that occur from the time the participant has signed 
the clinical trial consent. 
 
The DCC will distribute the expedited report to the NIDDK, the appropriate oversight committees 
or entities, and clinical centers. Status reports on serious adverse events will be generated by 
the DCC and will include the relationship of the adverse event to trial medications, the severity 
of the event and if the event is resolved or ongoing.   
 
All deaths will be reported to the DCC within 24 hours of knowledge of the death via the Serious 
Adverse Event form. This reporting begins at the time the participant has signed the informed 
consent up to the last scheduled participant visit. The report will include the relationship of the 
death to trial medications. A Clinical Outcome form will also be completed and sent to DCC for 
distribution. Deaths will be reported immediately to the NIDDK, the appropriate oversight 
committees or entities, and the clinical centers. A death will be reported as an expedited report 
only if it is unexpected and drug related. A death must also be reported in accordance with local 
law and regulations. 
 
The HBRN will comply with Genentech/Roche and Bristol-Myers Squibb SAE reporting 
requirements (see Manual of Operations). 
 
 
7.5.4. Management if either entecavir or peginterferon alfa-2a is discontinued due to 
adverse events 
If entecavir is discontinued due to adverse event, then peginterferon alfa-2a will be continued 
until the end of the treatment period. If peginterferon alfa-2a is discontinued due to adverse 


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event, then entecavir will be continued until the end of the treatment period.  
 
8. Statistical considerations 
8.1. Statistical analysis 
Summary statistics will be generated to describe the study sample at baseline.  Descriptive 
statistics, including measures of central tendency (e.g. means, medians) and estimates of 
spread (e.g., standard deviations, quartiles) will be used for continuous variables such as age 
and baseline HBV DNA and HBsAg levels.  Frequency distributions will be used for categorical 
variables such as HBV genotype and HBeAg status.  Ninety-five percent confidence intervals 
will be calculated for all point estimates of continuous data.  Graphical displays (e.g. histograms, 
box plots) will also be used to describe the data.  
 
8.1.1. Analysis of primary endpoints 
The primary endpoints include safety, measured by the rate of adverse and Serious Adverse 
Events, and efficacy, defined as loss of HBeAg and HBV DNA ≤1,000 IU/mL at the end of 
follow-up (week 96). That is, only participants achieving HBeAg loss and having HBV DNA 
≤1000 IU/mL at the end of follow-up will be considered as achieving the primary efficacy 
endpoint.   
Safety 
 
Summaries of adverse events (number of adverse events, number and percentage of 
participants with at least one adverse event, and for each adverse event, rate per person-years) 
will be provided. Events will be summarized based on the date of onset for the event. A 
treatment emergent adverse event will be defined as an adverse event that begins on or after 
the date of first dose of study drug.  
Summaries of the following are planned:  
• 
all adverse events recorded between screening and first dose of study medication,  
• 
all treatment emergent adverse events,  
• 
all emergent and related adverse events,  
• 
all treatment emergent renal adverse events, 
• 
all treatment emergent and related renal adverse events 
• 
combined Grade 2, 3 and 4 treatment emergent adverse events,  
• 
combined Grade 2, 3 and 4 related treatment and emergent adverse events,  
• 
combined Grade 3 and 4 treatment emergent adverse events,  
• 
combined Grade 3 and 4 related treatment and emergent adverse events,  
• 
all adverse events that caused permanent discontinuation of study drug,  
• 
all adverse events that caused temporary interruption of study drug,  
• 
all serious adverse events, and  
• 
all serious and related adverse events.  
 
We will provide 95% confidence intervals for the rate of adverse and serious adverse events.  
 
Efficacy 
 
The percentage of participants achieving the primary efficacy endpoint will be summarized with 
the point estimate and 95% confidence interval.  
8.1.2. Analysis of secondary endpoints 
8.1.2.1. Analysis of secondary endpoint listed in 2.3.1.: Estimate the cumulative proportion of 
HBsAg loss over time during and after treatment  


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Cumulative proportion of HBsAg loss over time will be calculated using the product limit 
(Kaplan-Meier) method and presented with point-wise 95% confidence interval.  
 
8.1.2.2. Analyses of other secondary endpoints  
For all of the secondary endpoints listed in sections 2.3.1. – 2.3.4, we will provide frequencies 
and percentages with corresponding 95% confidence intervals. Time to such events may also 
be of interest (e.g. cumulative HBeAg loss). For time-to event analysis, Kaplan-Meier curves will 
be used to plot the cumulative proportion of events over time.  
The second group of endpoints is continuous variables such as changes in ALT, HBV DNA 
levels from baseline at designated treatment or follow-up time points. Descriptive statistics, 
including measures of central tendency (e.g. means, medians) and estimates of spread (e.g., 
standard deviations, quartiles) will be used to characterize such changes.  . 
 
8.2. Missing data 
Primary analyses will be performed using all enrolled participants (intention-to-treat analysis). 
Participants who do not have primary endpoint data due to drop-out or other reasons at the end 
of follow-up will be considered as treatment failures. Since this is a pilot study with a small 
sample size, formal procedures for handling missing data such as inverse-weighting and 
multiple imputation will not have enough power, nevertheless, we will apply the following 
methods to assess the sensitivity of our primary analysis results to different missing data 
assumptions: 
1. Single LOCF Imputation: In this case the primary endpoint analysis will be repeated by 
replacing missing endpoint data at follow-up using data from prior visits. 
2. Inverse-probability-weighting: This analysis will be carried out in two stages. In the first stage, 
for each subject we will estimate the probability of having complete data at week 96 based on 
the data collected at prior visits using logistic regression. In the second stage, the primary 
endpoint analyses will be repeated by weighting each subject with complete data by the inverse 
of the corresponding estimated probability from the first stage. 
3. Multiple Imputations: The missing values at week 96 will be imputed by Markov-Chain Monte-
Carlo algorithm, based on the data collected prior to week 96. Five such imputations will be 
used and the results of the primary endpoints analyses will be repeated on each of these 
imputed datasets and averaged. 
 
    8.3. Sample size 
This pilot study will enroll 40 participants into treatment.  This sample size is considered to be 
achievable. We have calculated precision estimates based on various rates of primary endpoint 
of efficacy [loss of HBeAg and HBV DNA ≤1,000 IU/mL at the end of follow-up (week 96)]. For 
example, if the expected proportion achieving primary efficacy endpoint is 25%, then we would 
be able to estimate it with 95% Clopper-Pearson confidence to ensure that the expected width 
of the interval does not exceed 29% (or equivalently, the estimated 95% confidence limits will be 
within ± 15% of the estimate). Below are some further precision estimates: 
 
Table:  Precision (Width) Estimates for 95% Confidence Intervals under Different Primary 
Endpoint Proportions  
 
Expected proportion with 
primary efficacy endpoint 
95% 
lower 
95% 
upper 
Width 
0.15 
0.057 
0.298 
0.24 
0.20 
0.091 
0.356 
0.27 
0.25 
0.127 
0.412 
0.29 


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0.30 
0.166 
0.465 
0.30 
    
 
 
 
9. Data and safety monitoring board 
Data and safety will be monitored by the National Institute of Diabetes and Digestive and Kidney 
Diseases (NIDDK) in conjunction with an NIDDK-appointed Data and Safety Monitoring Board 
(DSMB).  This board serves in a consultative capacity to inform the NIDDK decisions regarding 
conduct of the study.  The description of DSMB activities is included in the DSMB Charter. 
 
9.1. Data and Safety Monitoring  
This trial aims to examine the safety and efficacy of treatment with 8 weeks of entecavir 
followed by 40 weeks of both entecavir and peginterferon alfa-2a  in the treatment of chronic 
hepatitis B in HBeAg positive adults who are in the immune tolerant phase. The individual drugs 
(peginterferon alfa-2a and entecavir) are FDA-approved and have been well-studied except in 
the immune tolerant participants. The risks to participants are outlined in sections 5.2.3 and 
5.3.3 of this protocol.  Section 7 of the protocol defines the toxicities associated with these drugs 
and outlines dose adjustment and discontinuation guidelines due to toxicity. The data and safety 
monitoring plan (DSMP) for this study focuses on close monitoring by the principal investigators 
(PI) and prompt reporting of excessive adverse events and all serious adverse events to the 
DCC and the appropriate oversight committees or entities such as the NIDDK, the Data and 
Safety Monitoring Board and to the participating centers’ IRBs. 
 
The Data Coordinating Center (DCC) will monitor clinical center performance (e.g., recruitment, 
retention, data completeness, timeliness of data collection and submission) and protocol 
compliance.  These reports, with summaries of adverse event data, will be provided to the 
DSMB for their quarterly reports and biannual calls or meetings, and to the Steering Committee 
at its annual meeting.   
 
The DCC will work with the HBRN Safety Officer and the Steering Committee to maintain a 
cumulative summary of adverse events (overall and stratified by serious/non-serious status) that 
will be forwarded to the DSMB every three months. Safety reports will also be sent to the 
Principal Investigators and the NIDDK Project Officers.  The Project Coordinator will be 
responsible for distributing these reports and assuring that all parties obtain copies of these 
reports. 
 
The frequency of data review for this study differs according to the type of data and can be 
summarized in the following table: 
 
Data type 
Frequency of review 
Recruitment 
Retention 
Protocol adherence(e.g., meeting 
inclusion/exclusion criteria, treatment 
compliance) 
Pregnancy in female subjects receiving 
treatment 
Quarterly reports for NIDDK and the 
appropriate oversight committees 
Adverse events and Serious Adverse Events 
(SAE) that do not meet expedited reporting 
Quarterly reports for NIDDK and the 
appropriate oversight committees 


Adult Immune Tolerant Protocol 
 
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criteria 
Serious Adverse Events that meet expedited 
reporting criteria 
As they occur for NIDDK, DSMB, 
appropriate industry partner(s), and 
cumulative reports quarterly 
Laboratory data  
Yearly for DSMB 
 
Definitions of adverse and serious adverse events and their management guidelines are 
provided in section 7.5 of this protocol along with the reporting procedures and in the MOP. No 
interim analysis for efficacy is planned. 
 
9.2 Participant confidentiality 
The central database of the study is on a server at the Epidemiology Data Center (EDC) in the 
Graduate School of Public Health at the University of Pittsburgh secured behind locked doors 
and an alarm with password access provided only to authorized personnel.  Backups are 
performed daily to guard against data loss due to an equipment or power failure.  Scheduled 
backups and archives at the EDC protect central and local information from hard disk failures.  
Tape backup volumes and CD-ROM copies of critical project files are located in a secured off-
site storage area to prevent data loss due to catastrophic events.  Routine virus detection is also 
enforced for all EDC computers involved in the study.  All critical information regarding database 
transactions is logged and stored in journal files.  In the event of accidental corruption of the 
project database, a previous database state may be restored from backup volumes or journal 
files.  All servers used for this project are connected to uninterrupted power supplies to protect 
equipment against electrical surges and outages.  A secured, computer room in an area with a 
burglar alarm houses all project server equipment.  
 
Participant confidentiality is preserved by encoding subject names into alphanumeric IDs at the 
clinical centers.  Data sent to the DCC are identified by alphanumeric ID only. No reports of this 
study will use names or other identifying information such as social security numbers or 
addresses.  Data, with alphanumeric ID only, will be stored at the DCC indefinitely.  In addition, 
following the completion of the study data and information, with alphanumeric ID only, will be 
submitted in the NIDDK repository by a suitable date agreed upon by NIDDK and the steering 
committee.   
 
 
 


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References 
 
1. 
Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B: summary 
of a clinical research workshop. Hepatology 2007;45(4):1056-75. 
2. 
Yim HJ, Lok AS. Natural history of chronic hepatitis B virus infection: what we knew in 1981 
and what we know in 2005. Hepatology 2006;43(2 Suppl 1):S173-81. 
3. 
Wang C, Lim L, Deubner H, et al. Factors predictive of significant hepatic fibrosis in adults 
with chronic hepatitis B and normal serum ALT. J Clin Gastroenterol 2008;42(7):820-6. 
4. 
Lai M, Hyatt BJ, Nasser I, Curry M, Afdhal NH. The clinical significance of persistently 
normal ALT in chronic hepatitis B infection. J Hepatol 2007;47(6):760-7. 
5. 
Andreani T, Serfaty L, Mohand D, et al. Chronic hepatitis B virus carriers in the 
immunotolerant phase of infection: histologic findings and outcome. Clin Gastroenterol Hepatol 
2007;5(5):636-41. 
6. 
Milich D, Liang T. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus 
infection. Hepatology 2003;38(5):1075-86. 
7. 
Sukriti S, Pati N, Bose S, Hissar S, Sarin S. Impaired antigen processing and presentation 
machinery is associated with immunotolerant state in chronic hepatitis B virus infection. J Clin 
Immunol 2010;30(3):419-25. 
8. 
Ohkawa K, Takehara T, Tatsumi T, et al. Alterations in hepatitis B virus nucleotide 
sequences in a chronic virus carrier from immunotolerant to immunoactive phase. Biochem 
Biophys Res Commun 2010;394(3):574-80. 
9. 
Lok A, McMahon B. Chronic hepatitis B: update 2009. Hepatology 2009;50(3):661-2. 
10. Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular carcinoma across a biological gradient 
of serum hepatitis B virus DNA level. Jama 2006;295(1):65-73. 
11. Iloeje UH, Yang HI, Su J, Jen CL, You SL, Chen CJ. Predicting cirrhosis risk based on the 
level of circulating hepatitis B viral load. Gastroenterology 2006;130(3):678-86. 
12. Lok A, McMahon B. Chronic hepatitis B. AASLD Practice Guidelines. Hepatology 
2001;34:1225-41. 
13. D'Antiga L, Aw M, Atkins M, Moorat A, Vergani D, Mieli-Vergani G. Combined 
lamivudine/interferon-alpha treatment in "immunotolerant" children perinatally infected with 
hepatitis B: a pilot study. J Pediatr 2006;148(2):228-33. 
14. Mazzucco CE, Hamatake RK, Colonno RJ, Tenney DJ. Entecavir for treatment of hepatitis 
B virus displays no in vitro mitochondrial toxicity or DNA polymerase gamma inhibition. 
Antimicrobial Agents and Chemotherapy 2008; 52:598-605 
15. Panel on Handling Missing Data in Clinical Trials; National Research Council ISBN. The 
Prevention and Treatment of Missing Data in Clinical Trials 
(http://www.nap.edu/catalog/12955.html). 
16. Marcellin P, Lau GKK, Zeusem S, et al. Comparing the safety, tolerability and quality of life 
in patients with chronic hepatitis B vs chronic hepatitis C treated with peginterferon alpha-2a. 
Liver Int 2008;28:477-85. 
 
 
 
 
 
 
 
 
 
 
 


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Appendix I:  Schedule of events for treated participants  
 
 
Phase of Study 
Screening 
Treatment 
Follow-up 
Weeks of Study 
-28 to 
-52 
-6 to    
-28 
up to 
-6 
0
1 
8 
1
2 
1
6 
2
0 
2
4 
3
0 
3
6 
4
2 
4
8 
5
2 
6
0 
7
2 
9
6 
Informed consent 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical exam 
 
 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
X 
Drug dispensing 
 
 
 
X X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Adherence assessment/ Clinical evaluation 
including AEs, concomitant meds 
 
 
 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Anthropometric measures 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
Questionnaires (QOL) 
 
 
 
X X 
 
 
 
 
 
 
 
X 
 
 
 
X 
Health behaviors 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
Depression assessment  
 
 
X 
 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
Symptom questionnaire 
 
 
 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Fatigue questionnaire 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
CBC, including platelets and differential2 
 
 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hepatic tests (AST, ALT, and bilirubin; albumin 
and ALKP day 0 and weeks 48 and 96 only)3 
X4 
X4 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Other chemistries (glucose, total protein) 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
TSH 
 
 
X  
 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
 
Renal tests (Creatinine and CrCl calculated 
MDRD) 
 
 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis 
 
 
X 
X X 
 
 
 
 
 
 
 
X 
 
 
 
X 
PT/INR 
 
 
X 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
AFP 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test (urine or blood)5 
 
 
X 
X X
X
X
X 
X 
X 
X 
X 
X 
 
 
 
 


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Phase of Study 
Screening 
Treatment 
Follow-up 
Weeks of Study 
-24 to 
-52 
-6 to    
-24 
up to 
-6 
0
1 
8 
1
2 
1
6 
2
0 
2
4 
3
0 
3
6 
4
2 
4
8 
5
2 
6
0 
7
2 
9
6 
HBsAg 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
X 
HBsAg quant 
 
 
 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
HBeAg 
 
 
X 
X X 
X 
 
 
X 
 
X 
 
X 
 
X 
X 
X 
HBeAg quant 
 
 
 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Anti-HBe6 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
Quant HBV DNA 
X4 
 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Anti-HBs6 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
HBV genotype and subtype7 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBV precore/BCP 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Antiviral drug resistance mutation testing98 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Anti-HCV9 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anti-HDV9 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anti-HIV 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Abdominal imaging10 
 
 
X 
 
 
 
 
 
X 
 
 
 
X 
 
 
X 
X 
Plasma/serum banking11 
 
 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
DNA Banking12 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1. 
Day 0 is the Baseline visit, and start of study drug. 
2. 
CBC to include differential count up to week 48. 
3. 
If total bilirubin elevated, obtain direct bilirubin at next blood draw. 
4. 
Historical data acceptable–ALT (2 values, one 28-52 weeks before screening, one 6-28 weeks before screening) and HBV DNA (1 value, within 12-52 
weeks before screening).  
5. 
.   
5. 
All women with reproductive potential prior to baseline visit; women on treatment with reproductive potential after start of treatment . 
6. 
Anti-HBe/Anti-HBs to be done at next visit if HBeAg/HBsAg becomes negative.  
7. 
For those who did not have genotype performed for the cohort study 
8. 
Also if meets criteria for virologic breakthrough (results not available in real-time).  
9. 
Anti-HCV and anti-HDV, if results within 144 weeks of baseline visit are available, tests do not need to be repeated. 
10. Patients with AFP >20 should be further evaluated by imaging (US/CT/MRI) as clinically needed to rule out HCC before entry into the study. Patients 
meeting AASLD criteria for HCC surveillance should have follow-up imaging per standard of care.  
11. 24mL of whole blood (for serum) and 8mL of whole blood (for plasma) at screening visit, day 0, 8, 48 and 96, and 8mL of whole blood (for serum) and 8mL 
of whole blood (for plasma) at other time points. These volumes include blood that will be collected for central testing. Patients who will be enrolled in HBRN 
Cohort Study concurrent with screening visit for this trial will have an additional 8 mL serum and 8 mL plasma at the baseline visit for future research.  
12. 5 mL of whole blood if DNA sample (with consent) not previously collected.  May be collected at any study visit if not at baseline. 


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Appendix II: Dosing of peginterferon if missed doses 
 
Dose delayed 1 or 2 days: administer on usual dosing day of the week (e.g., if 
Monday is the usual dosing day and the dose is delayed until Wednesday, the next 
dose may be administered as usual on Monday). 
Dose delayed 3-5 days:  administer subsequent doses every 5th or 6th day until the 
participant is back to his or her original schedule (e.g., if Monday is the usual dosing 
day and the dose is delayed until Saturday, the next dose should be administered on 
Thursday, the following dose on Tuesday, then the dose after that as usual on 
Monday). 
Dose delayed 6 days:  hold the dose for that week then continue on the usual 
schedule the following week (e.g., if Monday is the usual dosing day but the 
participant is not ready to be dosed until the following Sunday, the dose is 
considered to have been held and the next injection should be for the following 
weeks; dose on Monday). 
Dose delayed 7 or more days: the investigator may reintroduce study drug at any 
time and, if necessary, dose the participant every 5th or 6th day until the participant 
resumes weekly dosing on their usual scheduled day. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix III: Peg-Interferon Dose Reduction  
 
Peginterferon alfa-2a dose adjustment guidelines 
Specific dose adjustment guidelines for peginterferon alfa-2a are provided in the tables 
below for neutropenia, and thrombocytopenia. For other adverse effects considered to 
be possibly related to peginterferon, including laboratory abnormalities, adverse events, 
and vital signs changes, investigators should utilize the table below labeled “General 
Dose Reduction Guidelines.” When practicable, abnormal laboratory results should be 
confirmed as soon as possible following notification of the investigator. If appropriate, 
downward adjustments in one level reductions should be considered. The lowest dose of 
peginterferon alfa 2a that should be administered is 45 µg weekly. It should be kept in 
mind that whereas these guidelines should be generally followed to promote consistency 
across centers, other responses by an investigator may be more appropriate in some 
circumstances. 
 
a. General dose reduction guidelines 
 
For Peginterferon alfa-2a, 1 dose reduction = 135 µg; 2 dose = 90 µg; 3 dose = 45 µg 
 
Number of Dose Reduction Levels 
Mild 
Moderate 
Limited 
Moderate 
Persistent 
Severe 
Limited 
Severe 
Persistent 
Life- 
Threatening 
0 
0 
0-1 
0-1 
1-3 
Stop drug 
 
 
b. Dose adjustments for low absolute neutrophil and platelet counts  
Parameter 
Downward Dose Adjustment or Delay in Dosing 
ANC (cells/mm3) 
 
≥500 
No change 
250-499 
I step initially; potentially up to 3 steps 
<250 
Stop drug 
Platelet Count 
(cells/mm3) 
 
>50,000  
No change 
25,000-50,000 
1 step initially; potentially up to 3 steps 
<25,000 
Stop drug 


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Appendix IV: Participating centers 
 
Bethesda, MD: National Institutes of Health (NIH) Clinical Center 
 
Boston, MA:  Beth Israel Deaconess Medical Center, Massachusetts General Hospital 
 
Los Angeles, CA:  UCLA, Cedars Sinai Medical Center 
 
Michigan Consortium:  University of Michigan, Queen’s Medical Center, Honolulu, HI 
 
Minnesota:  Mayo Clinic Rochester, University of Minnesota 
 
North Carolina: University of North Carolina, Duke University Medical Center 
 
Pittsburgh, PA: University of Pittsburgh Graduate School of Public Health (DCC) 
 
San Francisco, CA:  UCSF, California Pacific Medical Center 
 
St. Louis, MO:  Saint Louis University, Washington University School of Medicine 
 
Texas:  University of Texas Southwestern, Baylor University Medical Center 
 
Toronto, Ontario, Canada:  University of Toronto 
 
Virginia:  Virginia Commonwealth University 
 
Washington: University of Washington Medical Center, Virginia Mason Medical Center