Clinical trial NCT02364336
Conditions: Chronic Hepatitis B
Keywords: Chronic Hepatitis B
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Start date: 2015-02-14; Completion date: 2018-05-21
Enrollment: 14 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Background:

\- Chronic hepatitis B is caused by a virus that infects the liver. Cure is not possible but the virus can be controlled with the use of antiviral medicines,. Researchers think that adding a second antiviral medicine might help.

Objective:

\- To understand how peginterferon might help treat people with chronic hepatitis B. Also, to see if peginterferon is safe to use with other antiviral medications.

Eligibility:

\- Adults age 18 and older who have chronic hepatitis B and had therapy with 1 or more oral medicines for hepatitis B for at least 4 years.

Design:

* Participants will be screened with physical exam and medical history. They will complete health questionnaires about their levels of fatigue and pain. They will have blood and urine tests. They may have an eye exam.
* Participants also will have a Fibroscan. A test to measure how stiff your liver is.
* Eligible participants will have a liver biopsy. Blood will be drawn.
* Participants will be admitted to the NIH Clinical Center. They will be injected with the study drug. Then they will have a second liver biopsy. They will be discharged 24 hours later.
* Participants will give themselves study drug injections under the skin weekly for 24 weeks.
* Participants will have 5 clinic visits during the 24-week treatment period. Then they will have follow-up visits every 12 weeks for 48 weeks.
* During visits, participants may have a physical exam and medical history. They may have blood and urine tests. They may have a Fibroscan and complete questionnaires. At the final visit, they will also have a Fibroscan.

Detailed description:
Chronic hepatitis B virus (HBV) infection is a leading cause of liver associated morbidity and mortality. Currently available first-line therapies for treatment of chronic hepatitis B include pegylated interferon-alpha and the nucleos(t)ide analogues (NUCs) entecavir and tenofovir. These were shown to effectively suppress viral replication, but their ability to induce durable off-treatment response is limited to a small subset of patients. Combination treatment with peginterferon and NUCs has been attempted in several randomized controlled trials, with no apparent advantage over either agent given alone. In these studies however, treatment with peginterferon was initiated either simultaneously or shortly after NUCs administration. The efficacy of peginterferon following long-term viral suppression with NUCs was only tested in one small pilot study, nevertheless showing 60% hepatitis B s antigen (HBsAg) loss rate.

The underlying mechanisms responsible for improved efficacy of peginterferon in this setting are unknown and warrant further investigation. In this single arm study we propose to evaluate the efficacy and mechanisms associated with response to peginterferon add-on therapy following a minimum of 192 weeks of viral suppression induced by NUCs in a group of chronic HBV infected patients. Sixty patients with either hepatitis B e antigen (HBeAg) positive (n=30) or negative (n=30) chronic HBV infection will be enrolled to this study. After medical evaluation and pretreatment liver biopsy, treatment with subcutaneous injections of pegylated interferon alpha-2a 180 g per week will be given for a total of 24 weeks, followed by an off-treatment evaluation period of 48 weeks. A second liver biopsy will be performed six hours following the first peginterferon injection. Primary end-point for this study will be the change in interferon-stimulated-genes response before and after first interferon injection in responders versus non-responders to treatment. The responsiveness to IFN-based therapy of treatment responders vs nonresponders will additionally be evaluated by studying intrahepatic and peripheral blood natural killer cells. The study will also assess HBeAg and HBsAg loss and seroconversion rates in comparison to historical controls treated with either peginterferon or NUCs monotherapy. Finally, we will assess whether treatment responders develop an HBV-specific T cell response similar in quantity and quality to that of patients who spontaneously resolve HBV infection.

Arms: arm 1: Patients who have been treated with one or more nucleos(t)ides for at least 192 weeks who have known serum HBsAg and HBeAg positivity arm 2: Patients who have been treated with one or more nucleos(t)ides for at least 192 weeks who have known serum HBsAg positivity and HBeAg negativity
Interventions: intervention 1: Peginterferon alfa-2a
Intervention descriptions: intervention 1: None

Protocol documents:
Prot_SAP_000.pdf: 
 
1
CLINICAL RESEARCH PROTOCOL 
1 
 
2 
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES 
3 
 
4 
DATE: August 3, 2016 
 
 
CLINICAL PROTOCOL NO.: 15-DK-0082 
5 
 
6 
         
 
 
 
 
 
IND NO: xx,xxx  
 
  
7 
        
 
  
 
 
 
IND Name: xx 
8 
         
 
 
 
 
 
IND HOLDER:  
9 
 
10 
TITLE:  Mechanisms Associated with Favorable Response to Peginterferon-Alpha Add-on Therapy 
11 
Following Long-term Nucleos(t)ide Analogue Treatment in Patients with Chronic Hepatitis B  
12 
SHORT TITLE: Add on PegIFN to NUC Therapy for CHB 
13 
 
14 
IDENTIFYING WORDS: Peginterferon alfa-2a Nucleos(t)ide analogues, Chronic Hepatitis B, Liver 
15 
Biopsy, HBeAg loss, HBsAg loss, Interferon stimulating gene.  
16 
 
17 
PRINCIPAL INVESTIGATOR: Marc Ghany, M.D., MHSc, Liver Diseases Branch (LDB), National 
18 
Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)* 
19 
 
20 
ASSOCIATE SENIOR INVESTIGATORS:   
21 
T Jake Liang, Chief, Liver Diseases Branch (LDB), National Institute of Diabetes and Digestive and 
22 
Kidney Diseases (NIDDK)* 
23 
Barbara Rehermann, M.D., Senior Investigator, LDB, NIDDK 
24 
Edward Doo, M.D., LDRB, Division of Digestive Diseases and Nutrition, NIDDK* 
25 
Theo Heller, M.D., Investigator, LDB, NIDDK* 
26 
Yaron Rotman, M.D. Investigator, LDB, NIDDK* 
27 
Christopher Koh, M.D. Staff Physician, LDB, NIDDK* 
28 
Elizabeth Wright, Ph.D. OD, NIDDK 
29 
David Kleiner, M.D., PhD., Laboratory of Clinical Pathology, NCI 
30 
 
31 
ASSOCIATE STAFF INVESTIGATORS: 
32 
Ahmad Alawad, M.D. Medical Staff Fellow, LDB, NIDDK* 
33 
Devika Kapuria, M.D. Medical Staff Fellow, LDB, NIDDK* 
34 
Gil Ben-Yakov, M.D., Medical Staff Fellow, LDB, NIDDK* 
35 
Elenita Rivera, R.N., Research Nurse, LDB, NIDDK 
36 
Amy Huang R.N. Research Nurse, LDB, NIDDK 
37 
 
38 
Non-NIH Investigators 
39 
Gavin Cloherty, PhD, Abbott Diagnostics 
40 
 
41 
*Investigators with this designation will obtain informed consent for this protocol. 
42 
 
43 
ESTIMATED DURATION OF STUDY: 3 Years 
44 
NUMBER AND TYPE OF PATIENTS: 60 patients with chronic hepatitis B, ages above 18 years, both 
45 
male and female 
46 
 
47 
SUBJECTS OF STUDY:     
48 
Number of patients: 60     Sex:  Male & Female        
 Age Range: Above 18 years 
49 
Volunteers: None 
50 
 
51 
PROJECT USES IONIZING RADIATION:  Yes, for medical indications only. 
52 
 
53 
PROJECT USES "DURABLE POWER OF ATTORNEY":  No 
54 


 
2
 
1 
OFF-SITE PROJECT: No       MULTI-INSTITUTIONAL PROJECT:  No 
2 
 
3 
 
4 
 
5 
 
6 
 
 
7 


 
3
Précis 
1 
 
2 
Chronic hepatitis B virus (HBV) infection is a leading cause of liver associated morbidity and 
3 
mortality. Currently available first-line therapies for treatment of chronic hepatitis B include 
4 
pegylated interferon-alpha and the nucleos(t)ide analogues (NUCs) entecavir and tenofovir. These 
5 
were shown to effectively suppress viral replication, but their ability to induce durable off-treatment 
6 
response is limited to a small subset of patients.  Combination treatment with peginterferon and 
7 
NUCs has been attempted in several randomized controlled trials, with no apparent advantage over 
8 
either agent given alone. In these studies however, treatment with peginterferon was initiated either 
9 
simultaneously or shortly after NUCs administration. The efficacy of peginterferon following long-
10 
term viral suppression with NUCs was only tested in one small pilot study, nevertheless showing 
11 
60% HBsAg loss rate.  
12 
The underlying mechanisms responsible for improved efficacy of peginterferon in this setting are 
13 
unknown and warrant further investigation. In this single arm study we propose to evaluate the 
14 
efficacy and mechanisms associated with response to peginterferon add-on therapy following a 
15 
minimum of 192 weeks of viral suppression induced by NUCs in a group of chronic HBV infected 
16 
patients. Sixty patients with either HBeAg positive (n=30) or negative (n=30) chronic HBV infection 
17 
will be enrolled to this study. After medical evaluation and pretreatment liver biopsy, treatment with 
18 
subcutaneous injections of pegylated interferon alpha-2a 180 μg per week will be given for a total of 
19 
24 weeks, followed by an off-treatment evaluation period of 48 weeks. A second liver biopsy will be 
20 
performed six hours following the first peginterferon injection. Primary end-point for this study will 
21 
be the change in interferon-stimulated-genes response before and after first interferon injection in 
22 
responders versus non-responders to treatment.  The responsiveness to IFN-based therapy of 
23 
treatment responders vs nonresponders will additionally be evaluated by studying intrahepatic and 
24 
peripheral blood natural killer cells. The study will also assess HBeAg and HBsAg loss and 
25 
seroconversion rates in comparison to historical controls treated with either peginterferon or NUCs 
26 
monotherapy.  Finally, we will assess whether treatment responders develop an HBV-specific T cell 
27 
response similar in quantity and quality to that of patients who spontaneously resolve HBV infection. 
28 
 
29 
 
 
30 


 
4
Table of Contents 
1 
Précis ................................................................................................................................................................................ 3 
2 
BACKGROUND ............................................................................................................................................................... 5 
3 
HYPOTHESIS .................................................................................................................................................................. 8 
4 
SPECIFIC AIMS ............................................................................................................................................................... 8 
5 
PROTOCOL OVERVIEW .............................................................................................................................................. 8 
6 
INCLUSION/EXCLUSION CRITERIA ........................................................................................................................ 9 
7 
Inclusion criteria: HBeAg positive group ..................................................................................................... 9 
8 
Inclusion criteria: HBeAg negative group .................................................................................................... 9 
9 
Exclusion criteria (for both eAg positive and negative patients) .................................................... 10 
10 
STUDY DESIGN AND PROCEDURES .................................................................................................................... 11 
11 
Screening ................................................................................................................................................................ 11 
12 
Admission for pretreatment liver biopsy .................................................................................................. 12 
13 
Initiation of Treatment ..................................................................................................................................... 13 
14 
On-treatment liver biopsy ............................................................................................................................... 13 
15 
Monitoring ............................................................................................................................................................. 14 
16 
Table 1: List of study related procedures ............................................................................................................... 17 
17 
ASSESSMENT OF RESPONSE .................................................................................................................................. 18 
18 
Clinical Definitions ............................................................................................................................................. 18 
19 
Primary end-point .............................................................................................................................................. 19 
20 
Secondary endpoints ......................................................................................................................................... 19 
21 
DOSE CHANGES .......................................................................................................................................................... 20 
22 
Stopping peginterferon therapy: .................................................................................................................. 20 
23 
Stopping NUCs therapy ..................................................................................................................................... 21 
24 
Peginterferon Dose Modifications ................................................................................................................ 21 
25 
Table 2: Peg-Interferon Alfa-2a Dose Modification Guidelines .................................................................... 21 
26 
STATISTICAL ANALYSIS.......................................................................................................................................... 23 
27 
Power calculation ............................................................................................................................................... 23 
28 
Demographics and Baseline Characteristics ............................................................................................ 23 
29 
Analysis plan ......................................................................................................................................................... 23 
30 
Analysis methodology ....................................................................................................................................... 24 
31 
HAZARDS AND DISCOMFORTS ............................................................................................................................. 24 
32 
RISK IN RELATION TO BENEFIT .......................................................................................................................... 28 
33 
ADVERSE EVENT REPORTING .............................................................................................................................. 29 
34 
DATA AND SAFETY MONITORING ...................................................................................................................... 28 
35 
CLINICAL MONITORING PLAN .............................................................................................................................. 30 
36 
ENROLLMENT OF CHILDREN, WOMEN AND MINORITY INDIVIDUALS ................................................ 30 
37 
RESEARCH USE, STORAGE AND DISPOSITION OF HUMAN SAMPLES AND DATA ............................. 31 
38 
RECRUITMENT PLAN ............................................................................................................................................... 32 
39 
CONSENT PROCESS ................................................................................................................................................... 33 
40 
COMPENSATION ........................................................................................................................................................ 33 
41 
REFERENCES ............................................................................................................................................................... 34 
42 
 
43 
 
 
44 


 
5
 
1 
BACKGROUND 
2 
 
3 
 
4 
It is estimated that there are over 350 million persons with chronic hepatitis B virus (HBV) infection 
5 
worldwide [1] Globally, chronic HBV infection is the leading cause of cirrhosis and hepatocellular 
6 
carcinoma accounting for ~620,000 deaths annually [2] Epidemiological studies have demonstrated 
7 
a strong association between high viral levels and worse outcome of chronic HBV infection  [3, 4]. 
8 
Thus, the goal of therapy is long-term, sustained suppression of HBV replication. Current first line 
9 
recommendations for treatment of chronic HBV infection include entecavir, tenofovir (nucleos(t)ide 
10 
analogues) or peginterferon-alpha [1, 5, 6].  
11 
 
12 
 NUCs with potent activity against the HBV reverse transcriptase and high barrier to resistance have 
13 
been very effective at suppressing viral replication and were shown to be associated with improved 
14 
clinical outcomes including reversal of cirrhosis [7, 8]. However, discontinuing therapy after one year 
15 
is associated with high relapse rates and therefore most patients require long-term therapy to 
16 
maintain suppression of viral replication [9-11]. Even with long-term use of NUCs, only about 50% of 
17 
persons who were initially HBeAg positive undergo HBeAg seroconversion (the endpoint of therapy 
18 
for HBeAg positive persons) [12, 13]and among those who were initially HBeAg negative, only about 
19 
1%-2% experience HBsAg seroconversion (the endpoint of therapy for HBeAg negative persons) [14, 
20 
15] Unfortunately, there are substantial costs associated with long-term therapy and there is the 
21 
potential for serious side effects including renal toxicity and possibly loss of bone mass [16-20] Thus, 
22 
new agents or approaches are needed to manage patients with chronic HBV infection. One approach 
23 
that has been taken to improve HBsAg loss was to combine the two most potent NUCs. In one notable 
24 
trial, the efficacy and safety of entecavir monotherapy was compared to the combination of entecavir 
25 
and tenofovir in persons with HBeAg positive and negative chronic HBV infection. After 96 weeks of 
26 
therapy, a comparable proportion of patients in each study arm achieved the primary end point of a 
27 
level of HBV DNA<50 IU/mL (83.2% vs 76.4%, respectively), indicating no benefit of a combination 
28 
NUC approach over potent monotherapy [21]. Another approach was to combine a nucleoside 
29 
analogue with peginterferon. This strategy is theoretically appealing as the combined effect of 
30 
peginterferon, an immunomodulator, and a nucleos(t)ide analogue, a potent inhibitor of viral 
31 
replication, may result in a synergistic anti-viral effect. However, several trials comparing the efficacy 
32 
of combination peginterferon-NUCs therapy to either agent alone failed to show a clear advantage of 
33 
combination therapy over either agent alone. Four randomized controlled trials comparing 
34 
peginterferon alfa and NUCs (either lamivudine or adefovir) to peginterferon alone or NUCs alone 
35 
have been conducted in persons with HBeAg positive and negative chronic HBV infection. [22-25]. All 
36 


 
6
studies reported greater on-treatment viral suppression with the combination regimen compared to 
1 
either agent alone, but higher rates of sustained off-treatment viral suppression with the 
2 
combination regimen were only achieved in comparisoin to NUCs alone but not to peginterferon 
3 
alone. Thus, there is really no advantage of combination peginterferon and lamivudine or adefovir to 
4 
peginterferon alone, when treatment with the two agents is initiated simultaneously. 
5 
 
6 
Studies assessing baseline predictors of response to peginterferon have shown that high viral levels 
7 
are associated with a less favorable response in both HBeAg positive and negative HBV infected 
8 
patients [26, 27]. In this context, it may be postulated that high viral titers may inhibit the action of 
9 
peginterferon and that combination peginterferon plus a nucleos(t)ide analogue may be more 
10 
effective if the peginterferon is preceded by a lead-in period of the nucleos(t)ide analogue. In one 
11 
study testing this strategy, 185 persons with HBeAg positive chronic hepatitis B were randomized to 
12 
receive either entecavir monotherapy for 48 weeks or a lead-in period of 24 weeks of entecavir 
13 
followed by 24 weeks of combination entecavir plus peginterferon [28]. At end-of-treatment more 
14 
patients in the combination arm, 19%, achieved the primary endpoint of HBeAg seroconversion 
15 
compared to the entecavir monotherapy arm, 10%. This difference however was not statistically 
16 
significant (p=0.095), and there was no peginterferon monotherapy arm. However, in a prespecified 
17 
multivariate analysis adjusted for differences in HBV DNA at the start of the randomized therapy 
18 
phase, add-on peginterferon was significantly associated with a 4.78-fold increase in response rate 
19 
(P = .004). It is possible though that a longer period of viral suppression is required to improve the 
20 
efficacy of peginterferon therapy in combination with a NUCs. Indeed in a small pilot study in which 
21 
peginterferon was added after a minimum period of 3 years of entecavir therapy with complete viral 
22 
suppression, showed that 60% of persons developed HBsAg loss [29]. These provocative results 
23 
suggest that a peginterferon add-on strategy following a substantial period of nucleos(t)ide induced 
24 
viral suppression, may result in high rates of HBsAg seroconversion. The underlying mechanisms 
25 
responsible for improved efficacy of peginterferon in this setting, are unknown, or for that matter the 
26 
mechanism of action of peginterferon in chronic HBV infection and warrant further investigation. 
27 
 
28 
IFN-α is a naturally occurring cytokine with broad anti-viral activity. Its anti-viral effect is mediated 
29 
through induction of a diverse set of target genes, referred to as IFN-stimulated genes (ISGs)[30]. 
30 
This results in inhibition of HBV replication through a variety of mechanisms including a block of 
31 
RNA-containing core particle formation, an accelerated decay of replication-competent core particles, 
32 
and degradation of the pre-genomic RNA [31-33]. Recently, IFN-α was also shown to inhibit HBV 
33 
replication by decreasing RNA transcription from cccDNA. This effect was mediated through 
34 
epigenetic repression of cccDNA (via histone hypoacetylation), as well as by active recruitment to the 
35 
cccDNA of transcriptional co-repressors [34]. 
36 


 
7
In addition to its direct anti-viral activity, IFN-α is also a potent immunomodulator with robust 
1 
effects on cells of the innate immune system, such as natural killer cells (NK). NK cells consistitute 
2 
the largest lymphocyte population in the liver and can exert antiviral function via direct cytotoxic 
3 
mechanisms and via indirect cytokine (e.g. IFN-gamma mediated) mechanisms.  NK cells respond to 
4 
IFN-a and we have previously shown that the response of peripheral blood NK cells to Peg-interferon 
5 
correlates well with the overal response and clinical outcome of PegIFN-based regimens in chronic 
6 
HCV infection [35, 36].  Interferon also modulates the adaptive, T cell-mediated response.  T cells are 
7 
typically impaired in their antiviral function  in chronic HBV infection  This impairment has been 
8 
attributed to upregulation of inhibitory molecules such as PD-1, Tim-3, CTLA-4 and others due to 
9 
chronic T cell receptor stimulation by HBV antigens resulting in downregulation of virus-specific T 
10 
cell response of infected patients [37].  In this protocol we will determine whether a decrease in HBV 
11 
titer by NUCs improves the innate and adaptive immune response to PegIFN. 
12 
 
13 
We postulate that in patients with chronic HBV infection, who have long-term viral suppression due 
14 
to NUCs therapy, the addition of peginterferon will lead to HBeAg or HBsAg seroconversion. We plan 
15 
to explore the clinical response and possible mechanisms involved during therapy with 
16 
peginterferon alfa when added to long-term nucleoside therapy in patients with chronic HBV 
17 
infection (both HBeAg-positive and -negative). We hypothesize that the extended pretreatment with 
18 
NUCs will modify the responsiveness to peginterferon. These effects will be studied by analyzing the 
19 
expression pattern of ISGs, the response of NK cells prior to and after peginterferon injection 
20 
resulting in improved transcriptional control of cccDNA , the induction of antibodies against HBe and 
21 
HBsAg and T cell responses. 
22 
Primary endpoints of this trial will be the changes in expression levels of ISG’s, intrahepatic total HBV 
23 
DNA and cccDNA content , changes in NK cell effector cell function measured before and following a 
24 
single dose of peginterferon stratified by treatment response and the rates of HBeAg and HBsAg loss 
25 
and seroconversion in HBeAg positive patients, HBsAg loss and seroconversion rate in HBeAg 
26 
negative patients. Secondary endpoints will be the induction of HBV-specific memory T cell 
27 
responses. A post-hoc analysis will stratify reponse rates upon available viral genotype data and 
28 
duration of NUC therapy prior to study enrollment. Other secondary end-points will include changes 
29 
in quantitative HBeAg and HBsAg levels during therapy and safety of the combination regimen. 
30 
Serological response rates, qHBsAg kinetics and safety end-points will be compared to historical 
31 
controls of published multicenter randomized trials comparing the efficacy of peginterferon and 
32 
NUCs in eAg positive and negative HBV carriers  
33 
 
34 


 
8
HYPOTHESIS 
1 
 
2 
In patients with chronic hepatitis B infection, under long-term viral suppression maintained by NUCs 
3 
treatment, peginterferon add-on therapy will result in a greater proportion of subjects achieving 
4 
serological response compared to before peginterferon was added. We hypothesize that response to 
5 
treatment will be accompanied by measurable changes in host-response gene transcription profile, 
6 
immune cell activation and function, and quantitative viral biomarker levels.  
7 
 
8 
SPECIFIC AIMS 
9 
 
10 
1. To assess the changes in ISG profile and NK cell function after a single dose of peginterferon 
11 
in patients receiving long-term NUCs with maintained viral suppression 
12 
2. To determine rates of HBeAg and HBsAg loss with or without seroconversion and induction 
13 
of HBV-specific memory T cells to peginterferon add-on treatment in eAg positive and 
14 
negative HBV carriers on long-term NUC treatment. 
15 
3. To identify pre- and on-treatment predictors of serological response to peginterferon add-
16 
on/nucleos(t)ide combination therapy.   
17 
4. To determine if there are differences in epigenetic regulation of cccDNA between HBeAg 
18 
positive and negative CHB at baseline and following a single dose of peginterferon treatment 
19 
 
20 
PROTOCOL OVERVIEW 
21 
 
22 
We intend to enroll a total of 60 patients with HBeAg positive (n=30) and HBeAg negative (n=30) 
23 
chronic HBV infection, who have been on-treatment with one or more nucleos(t)ide analogues for at 
24 
least 192 weeks prior to study enrollment with maintained viral suppression (HBV DNA level <100 
25 
IU/ml). After a thorough medical evaluation and liver biopsy, participants will receive therapy with 
26 
subcutaneous injections of peginterferon-alpha 2a at a dose of 180mcg per week for a total of 24 
27 
weeks. A second liver biopsy will be performed 6 hours following the first peginterferon injection for 
28 
the purpose of assessing ISGs, intrahepatic NK cell quantity and function and intrahepatic viral 
29 
biomarker response patterns. Most interferon induced genes achieve maximal response in the liver 
30 
and periphery within 4 hours, begin to decline by 8 hours, and return to baseline levels by 24 hours 
31 
post-administration of interferon. Because peginterferon is slower to be absorbed we have selected 
32 
the six hour rather that a four hour time point.[38] 
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9
Patients will be followed carefully on weeks 0, 2, 4, 8, 12 and 24 during treatment and at 12 week 
1 
intervals thereafter until 48 weeks post-treatment.  
2 
During study visits patients will be assessed for compliance with study medications, as well as the 
3 
development of any treatment related side effects and/or adverse reactions. In addition, blood will 
4 
be drawn periodically for the assessment of routine blood tests, quantitative viral biomarker levels, 
5 
serological response markers and immune cell functional status.  
6 
 
7 
INCLUSION/EXCLUSION CRITERIA 
8 
 
9 
Inclusion criteria: HBeAg positive group 
10 
 
11 
1) Age >18 years and older, male or female. 
12 
2) Known serum HBsAg and HBeAg positivity at the time of screening. 
13 
3) Ongoing treatment with one or more NUCs for at least 192 weeks before study entry. 
14 
Subjects may have a brief interruption of treatment for medical reasons (e.g. breast feeding) 
15 
not to exceed 8 weeks and none within the 48 weeks before study entry.  
16 
4) HBV DNA levels <100 IU/mL, measured at least 12 months prior to, and upon enrollment to 
17 
the study.  
18 
5) ALT level ≤2 ULN based on at least two determinations taken at least one month apart 
19 
during the 24 weeks before study entry with the second being at time of screening 
20 
6) Written informed consent 
21 
 
22 
 Inclusion criteria: HBeAg negative group 
23 
 
24 
1) Age >18 years and older, male or female. 
25 
2) Known serum HBsAg positivity and HBeAg negativity at the time of screening. 
26 
3) Ongoing treatment with one or more NUCs for at least 192 weeks before study entry. 
27 
Subjects may have a brief interruption of treatment for medical reasons (e.g. breast feeding) 
28 
not to exceed 8 weeks and none within the 48 weeks before study entry. 
29 
4) HBV DNA levels <100 IU/mL, measured at least 12 months prior to, and upon enrollment to 
30 
the study 
31 
5) ALT level ≤2 ULN based on at least two determinations taken at least one month apart 
32 
during the 24 weeks before study entry with the second being at time of screening 
33 
6) Written informed consent 
34 
 
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10
Exclusion criteria (for both eAg positive and negative patients) 
1 
 
2 
1) Co-infection with HDV as defined by the presence of anti-HDV in serum and/or HDV antigen 
3 
in the liver. 
4 
2) Co-infection with HCV as defined by the presence of HCV RNA in serum. 
5 
3) Co-infection with HIV as defined by the presence of anti-HIV in serum. 
6 
4) Decompensated liver disease as defined by serum bilirubin >2.5 mg/dL (with direct bilirubin 
7 
> 0.5 mg/dL), prothrombin time of greater than 2 seconds prolonged, a serum albumin of 
8 
less than 3 g/dL, or a history of ascites, variceal bleeding or hepatic encephalopathy. 
9 
5) Presence of other causes of liver disease, (i.e. hemochromatosis, Wilson disease, alcoholic 
10 
liver disease, severe nonalcoholic steatohepatitis defined as the presence of marked 
11 
ballooning injury on liver biopsy, alpha-1-anti-trypsin deficiency). 
12 
6) A history of organ transplantation or in the absence of organ transplantation, any 
13 
immunosuppressive therapy requiring the use of more than 5 mg of prednisone (or its 
14 
equivalent) daily. 
15 
7) Significant systemic illness other than liver diseases including congestive heart failure, renal 
16 
failure, chronic pancreatitis and diabetes mellitus with poor control, that in the opinion of 
17 
the investigator may interfere with therapy. 
18 
8) Pregnancy or inability to practice contraception in patients capable of bearing or fathering 
19 
children  
20 
9) Lactating women. 
21 
10) Hepatocellular carcinoma (HCC), or the presence of a mass on imaging studies of the liver 
22 
that is suggestive of HCC, or an alpha-fetoprotein level of greater than 500 ng/mL. 
23 
11) eGFR < 50 ml/min, serum creatinine > 1.3 mg/dl or urine protein >1 gram/24-hours 
24 
12) History of hypersensitivity to pegylated interferon-alpha 
25 
13) Platelet count <70 mm3/dL 
26 
14) Hgb <12 g/dL for males and <11 g/dL for females 
27 
15) Active ethanol/drug abuse/psychiatric problems such as major depression, schizophrenia, 
28 
bipolar illness, obsessive-compulsive disorder, severe anxiety, or personality disorder that, 
29 
in the investigator’s opinion, might interfere with participation in the study. 
30 
16) History of malignancy or treatment for a malignancy within the past 3 years (except 
31 
adequately treated carcinoma in situ or basal cell carcinoma of the skin). 
32 
17) Any medical condition requiring, or likely to require, chronic systemic administration of 
33 
corticosteroids or other immunosuppressive medications during the course of this study. 
34 
18) History of immune-mediated disease, or cerebrovascular, chronic pulmonary or cardiac 
35 
disease associated with functional limitation, retinopathy, uncontrolled thyroid disease, 
36 


 
11
poorly controlled diabetes or uncontrolled seizure disorder, as determined by a study 
1 
physician. 
2 
19) Presence of conditions that, in the opinion of the investigators, would not allow the patient 
3 
to be followed in the current study for at 1 year.  
4 
20) For subjects who interrupt therapy, documentation of a viral load >1,000 IU/ml while off 
5 
therapy. 
6 
 
7 
STUDY DESIGN AND PROCEDURES 
8 
 
9 
Screening  
10 
 
11 
Eligible patients will be seen in the outpatient liver clinic of the NIH Clinical Center. At the screening 
12 
visit, potential benefits and risks associated with the study will be explained to the study participants 
13 
by an investigator in laymen’s language. Written informed consent will be obtained and documented 
14 
in the participant’s chart. Whenever possible, the informed consent form will be provided to the 
15 
potential participants in advance.  Only the principal and associate investigators from the Liver 
16 
Disease Branch, NIDDK, will be eligible to obtain consent for this study. 
17 
 
18 
The following screening procedures will be performed: 
19 
(1) Obtain signed informed consent 
20 
(2) History and physical examination 
21 
(3) Symptom questionnaire 
22 
(4) Fatigue questionnaire (Promise 7) 
23 
(5) Visual analogue scale 
24 
(6) Routine blood panel: Chemistry 20 (sodium, chloride, potassium, bicarbonate, calcium, 
25 
phosphate, magnesium, glucose, blood urine nitrogen, creatinine, uric acid, ALT, AST, 
26 
LDH, CPK, Alkaline Phosphatase, total and direct bilirubin, albumin, total protein), CBC 
27 
(hematocrit, hemoglobin, white blood cell count and differential, platelet count). 
28 
Testing will also be done for anti-HAV, HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, 
29 
anti-HCV, and stored sample for IL-28b testing.  Baseline HBV DNA level by quantitative 
30 
PCR (COBAS Ampliprep/COBAS Taqman HBV v2.0 Test: Roche Diagnostics, lower limit 
31 
of quantification 116 copies /ml or 20 IU/ml, lower limit of detection 6 IU/ml)  
32 
(7) Extended blood panel: alpha fetoprotein, gGT, immunoglobulins, rheumatoid factor, 
33 
ANUCs, SMA, lipid panel, thyroid panel, INR, reticulocyte count, ESR. 
34 


 
12
(8) Research blood- one 10 ml serum and one 10 ml plasma tube stored frozen in 0.5-1.0 
1 
mL aliquots). The stored serum and plasma will be used to measure selected cytokines, 
2 
interferon-stimulated gene products [including IP10], quantitative HBsAg level (by 
3 
using the Roche Elecsys HBsAg automated quant assay) and for future research. 
4 
(9) Fifty milliliters of whole blood (green top tubes) for baseline immunological studies:  NK 
5 
cell responses, CD4+ and CD8+ T cell HBV specific responses (peripheral blood 
6 
mononuclear cell [PBMC] studies) 
7 
(10) 
Urine tests: routine urinalysis and a pregnancy test for females of childbearing 
8 
potential 
9 
(11) 
Transient elastography 
10 
 
11 
Admission for pretreatment liver biopsy 
12 
 
13 
Once patients are deemed eligible to participate in the study based on results of the screeing 
14 
labs, they will have 12 weeks to complete the liver biopsy and to begin add-on therapy with 
15 
peginterferon. Eligible patients will be admitted to 5NW to undergo a percutaneous liver biopsy. 
16 
The initial liver biopsy will be mostly for research purposes to measuring baseline levels of ISG 
17 
expression, pregenomic and cccDNA concentration and frequency and activity of liver infiltrating 
18 
HBV-specific NK cells but will also be used for the purpose of grading and staging of liver disease 
19 
before treatment and will provide useful information to the clinician and patient on whether the 
20 
liver disease is improving on current therapy. Grading and staging of the liver biopsy will 
21 
performed by an expert hepatopathologist blinded to the clinical information using the modified 
22 
Ishak scoring system. This includes the histological activity index (HAI) for inflammation and 
23 
necrosis (scores ranging from 0 to 18). Fibrosis will be staged using the Ishak scoring system (0-
24 
6, 0=no fibrosis, 6=cirrhosis). Slides will also be immunostained for HBsAg, HBcAg, and HDV 
25 
antigen by peroxidase-antiperoxidase techniques and scored as 0, 1, 2 or 3+ based upon the 
26 
percent of cells reactive for each viral antigen (0=none, 1=10%, 2=11% to 50% and 3=51% of 
27 
hepatocytes).  
28 
Liver-infiltrating lymphocytes will be assessed for frequency and  function of NK cells.  m-RNA 
29 
levels of  ISG’s, immune cell markers and chemokines/cytokines will be quantitated by real-time 
30 
PCR or nanostring. Intrahepatic pregenomic and cccDNA will also be quantitated by Southern 
31 
blotting or real-time PCR.  
32 
 
33 
The following will be obtained on admission: 
34 
1) Brief clinical evaluation 
35 
2) Review patient’s eligibility for study 
36 


 
13
3) Symptom questionnaire 
1 
4) Visual analogue scale 
2 
5) Fatigue questionnaire (Promise 7) 
3 
6) Abdominal ultrasound, CXR and 12 lead ECG (if not done within previous 6 months) 
4 
7) Chemistry 20 (sodium, chloride, potassium, bicarbonate, calcium, phosphate, magnesium, 
5 
glucose, blood urine nitrogen, creatinine, uric acid, ALT, AST, LDH, CPK, Alkaline 
6 
Phosphatase, total and direct bilirubin, albumin, total protein), CBC (hematocrit, hemoglobin, 
7 
white blood cell count and differential, platelet count).  
8 
8) HBV DNA viral load, qualitative and quantitative HBeAg and HBsAg measurement 
9 
9) 50 mL of blood in heparinized green top tubes for NK and T cell analysis. 
10 
10) Blood sample for storage (5 mL of blood separated into serum and stored frozen in 0.5-1.0 
11 
mL aliquots)  
12 
11) Liver biopsy cut into four sections, one (at least 1.5 cm) placed in formalin for routine light 
13 
microscopy and the other 3 (.3cm, .5cm and 1 cm) placed in RNA later and flash frozen in 
14 
liquid nitrogen 24 hours later for RNA and protein analyses and flow cytometry.  A liver 
15 
biopsy fragment of > 5 mm will be used to assess the frequency and  function of NK cells. 
16 
12) Discharge from hospital  
17 
 
18 
 
19 
Initiation of Treatment 
20 
 
21 
Patients will be stratified on the basis of HBeAg status into 2 groups of 30 patients each, to receive 
22 
peginterferon-alpha 2a (PEGASYS®, Roche), 180 mcg per week administered by subcutaneous 
23 
injection for a total of 24 weeks. Patients will continue treatment with their previous nucleosi(t)ide 
24 
analogue regimen during the treatment and follow-up phases of this study.  
25 
Patients will be seen in the outpatient clinic at weeks 2, 4, 8, 12, and 24, during treatment phase, at 
26 
12 week intervals thereafter up to 48 weeks post-therapy 
27 
Compliance will be monitored by medication diaries and counting of the residual vials returned at 
28 
outpatient visits. The side effects will be monitored regularly at each visit during the treatment phase 
29 
of the study by symptom questionnaires. Peginterferon alfa-2a will be dose reduced or stopped per 
30 
the dose reduction and stopping rules for peginterferon alfa-2a. Peginterferon alfa-2a treatment can 
31 
be restarted if the adverse event resolves and the patient is willing to re-start the medication.  
32 
 
33 
On-treatment liver biopsy 
34 
 
35 


 
14
At day 0, all patients will be readmitted to the NIH Clinical Center to start therapy with peginterferon 
1 
alfa-2a and to undergo a repeat liver biopsy.  Biopsy will be scheduled at 6 hours following the first 
2 
injection of peginterferon, for the purpose of assessing ISG response pattern following the initial 
3 
exposure to peginterferon. Liver tissue will also be subjected to routine histopathological grading 
4 
and staging as well as for immune cell, quantitative viral kinetic and cccDNA epigenetic studies as 
5 
previously described.  
6 
 
7 
Since the hypothesis to be tested in this study is that response to treatment peginterferon (i.e. loss of 
8 
HBeAg and HBsAg or both) is mediated by changes in hepatocyte gene transcription profile, it is 
9 
necessary to obtain liver tissue at two time points-before and 6 hours after the first dose of 
10 
interferon. Performing two liver biopsies within 12 weeks or even as short as 24 hours has been 
11 
performed in other Liver Disease Branch studies and has not been associated with an increase in the 
12 
complication rate or lower patient acceptance. 
13 
 
14 
 
15 
Monitoring  
16 
 
17 
Day 0 
18 
 
19 
1) Brief clinical evaluation 
20 
2) Review patient’s eligibility for study 
21 
3) Symptom questionnaire 
22 
4) Visual analogue scale 
23 
5) Fatigue questionnaire (Promise 7) 
24 
6) Chemistry 20 (sodium, chloride, potassium, bicarbonate, calcium, phosphate, magnesium, 
25 
glucose, blood urine nitrogen, creatinine, uric acid, ALT, AST, LDH, CPK, Alkaline 
26 
Phosphatase, total and direct bilirubin, albumin, total protein), CBC (hematocrit, hemoglobin, 
27 
white blood cell count and differential, platelet count).  
28 
7) Females of child bearing potential: Pregnancy test 
29 
8) HBV DNA , qualitative and quantitative HBeAg, HBsAg, measurements 
30 
9) 30 mL of blood in heparinized green top tubes for NK and T cell analysis prior to PegIFN 
31 
injection and 30 mL of blood in heparinized green top tubes for NK and T cell analysis 6h 
32 
after PegIFN injection. 
33 
10) Blood sample for storage (10 mL of blood separated into serum and stored frozen in 0.5-1.0 
34 
mL aliquots) 
35 
11) Start peginterferon 180 ug SQ 
36 


 
15
12) Repeat liver biopsy 6 hours after first dose of peginterferon. Liver biopsy cut into four 
1 
sections, one (at least 1.5 cm) placed in formalin for routine light microscopy and the other 3 
2 
(.3cm, .5cm and 1 cm) placed in RNAlater and flash frozen in liquid nitrogen 24 hours later 
3 
for RNA and protein analyses and flow cytometry. 
4 
 
5 
 
6 
 
7 
Day1 
8 
1) 24h after the first PegIFN injection: 50 mL of blood in heparinized green top tubes for NK.  
9 
This is the time point where we expect the maximal response of NK cells to PegIFN based on 
10 
our previous studies [35, 36] 
11 
2) Discharge from hospital  
12 
 
13 
 
14 
Week 2,4, 8 and 12 ( 3 days):  Outpatient visit 
15 
Each outpatient visit will include assessment of compliance, adverse events reporting, vital signs, 
16 
weight, symptoms evaluation, record of concomitant medications, drug dispensing 
17 
1) Symptom questionnaire 
18 
2) Visual analogue scale 
19 
3) Fatigue questionnaire (Promise 7) (week 2 only) 
20 
4) Chemistry 20 (sodium, chloride, potassium, bicarbonate, calcium, phosphate, magnesium, 
21 
glucose, blood urine nitrogen, creatinine, uric acid, ALT, AST, LDH, CPK, Alkaline 
22 
Phosphatase, total and direct bilirubin, albumin, total protein), CBC (hematocrit, hemoglobin, 
23 
white blood cell count and differential, platelet count).  
24 
5) HBV DNA viral load, HBeAg, anti HBe, HBsAg and anti HBs measurment 
25 
6) Blood sample for storage (10 mL of blood separated into serum and plasma stored frozen in 
26 
0.5-1.0 mL aliquots) 
27 
7) 50 mL of blood in heparinized green top tubes for NK and T cell analysis. 
28 
8) Females of child bearing potential: Pregnancy test 
29 
9) TSH and quantitative HBe and HBs antigen measurement at week 12. 
30 
 
31 
 
32 
End of Add-on Peginterferon Treatment (Week 24  3 days) 
33 
At week 24, all subjects will discontinue peginterferon alfa-2a add-on therapy (but continue the 
34 
NUCs and undergo the following evaluation: 
35 


 
16
1) Physical examination 
1 
2) Symptom questionnaire 
2 
3) Fatigue questionnaire (Promise 7) 
3 
4) Visual analogue scale 
4 
5) Assessment of adverse events, concomitant medications and adherence 
5 
6) Blood will be drawn for Chemistry 20 (sodium, chloride, potassium, bicarbonate, calcium, 
6 
phosphate, magnesium, glucose, blood urine nitrogen, creatinine, uric acid, ALT, AST, LDH, 
7 
CPK, Alkaline Phosphatase, total and direct bilirubin, albumin, total protein), CBC 
8 
(hematocrit, hemoglobin, white blood cell count and differential, platelet count). 
9 
7) 50 mL of blood in heparinized green top tubes for NK and T cell analysis. 
10 
8) Urine tests: routine urinalysis and creatinine, phosphate and beta-2-microglobulin 
11 
9) HBV DNA viral load, HBeAg, anti-HBe, HBsAg, anti-HBs and quantitative HBe and HBs 
12 
measurement  
13 
10) Blood sample for storage (10 mL of blood separated into serum and plasma stored frozen in 
14 
0.5-1.0 mL aliquots) 
15 
11) Females of child bearing potential: Pregnancy test 
16 
12) TSH 
17 
 
18 
Post-treatment weeks 12, 24, 36 ( 5 days) 
19 
1) Limited clinical evaluation 
20 
2) Symptom questionnaire 
21 
3) Visual analogue scale 
22 
4) Routine blood panel: Chemistry 20 (sodium, chloride, potassium, bicarbonate, calcium, 
23 
phosphate, magnesium, glucose, blood urine nitrogen, creatinine, uric acid, ALT, AST, LDH, 
24 
CPK, Alkaline Phosphatase, total and direct bilirubin, albumin, total protein), CBC 
25 
(hematocrit, hemoglobin, white blood cell count and differential, platelet count). 
26 
5) HBV DNA viral load, HBeAg, anti HBe, HBsAg and anti HBs levels 
27 
6) Blood sample for storage (10 mL of serum and 10 ml of plasma stored frozen in 0.5-1.0 mL 
28 
aliquots) 
29 
7) 50 mL of blood in heparinized green top tubes for NK and T cell analysis. 
30 
 
31 
 
32 
Final study visit week 48 post-treatment ( 5 days) 
33 
1) Limited clinical evaluation 
34 
2) Symptom questionnaire 
35 


 
17
3) Fatigue questionnaire (Promise 7) 
1 
4) Visual analogue scale 
2 
5) Routine blood panel: Chemistry 20 (sodium, chloride, potassium, bicarbonate, calcium, 
3 
phosphate, magnesium, glucose, blood urine nitrogen, creatinine, uric acid, ALT, AST, LDH, 
4 
CPK, Alkaline Phosphatase, total and direct bilirubin, albumin, total protein), CBC 
5 
(hematocrit, hemoglobin, white blood cell count and differential, platelet count). 
6 
6) Extended blood panel: alpha fetoprotein, gGT, immunoglobulins, rheumatoid factor, ANA, 
7 
SMA, lipid panel, INR, reticulocyte count, ESR. 
8 
7) 50 mL of blood in heparinized green top tubes for NK and T cell analysis. 
9 
8) HBV DNA viral load, HBeAg, anti HBe, HBsAg, and anti HBs levels  and quantitative HBe and 
10 
HBs measurement  
11 
9) Blood sample for storage (10 mL of serum and 10 ml of plasma stored frozen in 0.5-1.0 mL 
12 
aliquots) 
13 
10)  Fibroscan 
14 
 
15 
Table 1: List of study related procedures 
16 
 
17 
Procedure 
Screen 
Pre-Rx 
biopsy 
Day 0 
Wk 2, 4, 
8 
Wk 
12 
Week 
24 
End 
of Rx 
Post-
Rx wk 
12, 24, 
36 
Post-Rx 
wk 48, 
End of 
Study 
H&P/breif clinical 
evaluation, compliance, 
AE, concommit. drugs, 
etc 
X 
X 
X 
X 
X 
X 
X 
X 
Symptom questionnaire 
X 
X 
X 
X 
X 
X 
X 
X 
Visual Analog Scale 
X 
X 
X 
X 
X 
X 
X 
X 
Promis 7 
X 
X 
X 
wk 2 
X 
X 
Chem 20 
X 
X 
X 
X 
X 
X 
X 
X 
CBC 
X 
X 
X 
X 
X 
X 
X 
X 
Anti-HAV 
X 
HBsAg 
X 
X 
X 
X 
X 
X 
X 
X 
HBsAg quantitative 
X 
X 
X 
X 
anti-HBs 
X 
HBeAg 
X 
X 
X 
X 
X 
X 
X 
X 
HBeAg quantitative 
X 
X 
X 
X 
anti-HBe 
X 
 
 
 
 
 
 
 
anti-HBc 
X 


 
18
anti-HCV 
X 
IL28B 
X 
HBV DNA 
X 
X 
X 
X 
X 
X 
X 
X 
AFP 
X 
X 
gGT 
X 
X 
Immunoglobulins 
X 
X 
rheumatoid factor 
X 
X 
ANA 
X 
X 
SMA 
X 
X 
Lipid panel 
X 
X 
thyroid panel 
X 
TSH 
TSH 
INR 
X 
X 
reticulocyte 
X 
X 
ESR 
X 
X 
storage - 5mL serum and 
5mL plasma 
S&P 
Serum 
Seru
m 
S&P 
S&
P 
S&P 
S&P 
S&P 
Immunology - 50mL 
X 
X 
X 
X 
X 
X 
X 
Urine pregnancy 
X 
X 
X 
X 
X 
UA 
X 
X 
Urine creatinine, 
phosphate, beta-2-
microglubulin 
X 
Liver Biopsy 
X 
Time
d 
Abdominal u/s 
X 
CXR 
X 
EKG 
X 
Fibroscan 
X 
X 
 
1 
 
2 
 
3 
ASSESSMENT OF RESPONSE 
4 
 
5 
Clinical Definitions 
6 
 
7 
a) Loss of HBeAg (HBeAg-positive patients only): loss of detectable HBeAg with or without 
8 
anti-HBe seroconversion confirmed on 2 consecutive visits at least 12 weeks apart.   
9 
b) Loss of HBsAg:  Loss of HBsAg, with or without anti HBs seroconversion confirmed on 2 
10 
consecutive visits at least 12 weeks apart.   
11 


 
19
c) Sustained response: virological, serological, biochemical, or combined response present at 
1 
the time of last observation and at least six months after stopping therapy.   
2 
 
3 
 
4 
The primary goals of this study are centered on understanding the mechanisms, and identification of 
5 
predictive factors associated with a favorable response to peginterferon therapy when added on to 
6 
long-term NUCs treatment, in patients with HBeAg positive and negative chronic HBV infection.  
7 
 
8 
Primary end-points 
9 
 
10 
1) The primary end-point for this study will be the change in level of ISG expression and NK cell 
11 
responses before and after 1st peginterferon injection in responders versus non-responders 
12 
to peginterferon add-on therapy. 
13 
2) Proportion of eAg positive patients showing eAg loss with or without seroconversion at end 
14 
of treatment and 24 and 48 weeks off therapy. 
15 
3) Proportion of eAg positive patients showing sAg loss with or without seroconversion at end 
16 
of treatment and 24 and 48 weeks off therapy 
17 
4) Proportion of eAg negative patients showing sAg loss with or without seroconversion at at 
18 
end of treatment and 24 and 48 weeks off therapy 
19 
 
20 
Secondary endpoints  
21 
 
22 
1. Level of qHBsAg decline at treatment week 12, at end of treatment and 24 and 48 weeks off 
23 
therapy, compared to pretreatment levels in eAg positive and negative patients  
24 
2. Level of qHBeAg decline at treatment week 12, at end of treatment and 24 and 48 weeks off 
25 
therapy, compared to pretreatment levels in eAg positive and negative patients  
26 
3. Correlation of qHBsAg levels at initial evaluation, week 12,24,36 and 48 with intrahepatic 
27 
pregenomic and cccDNA concentration before and after initiation of peginterferon in 
28 
responders versus non-responders to treatment.   
29 
4. Intrahepatic pregenomic and cccDNA concentration before and after starting treatment with 
30 
peginterferon.  
31 
5. Epigenetic alterations in HBeAg positive and negative patients following first 1st 
32 
peginterferon injection compared to pretreatment levels as measured by chromatin 
33 
immunoprecipitation (ChIp) assays  
34 


 
20
6. Frequency and functional activity of circulating and liver infiltrating, HBV-specific T-cells, 
1 
measured before and during treatment in responders versus non-responders to 
2 
peginterferon add-on therapy. 
3 
 
4 
DOSE CHANGES 
5 
 
6 
Stopping peginterferon therapy: 
7 
 
8 
Add-on peginterferon will be discontinued early for any of the following reasons: 
9 
a) Toxicity- the dose of peginterferon will be modified or discontinued according to Table 1: 
10 
Peg-Interferon Alfa-2a Dose Modification Guidelines (see below). 
11 
b) Withdrawal of informed consent (subject’s decision to withdraw for any reason. 
12 
c) Any clinical adverse event, laboratory abnormality, or intercurrent illness, which in the 
13 
opinion of the investigator indicates that continued participation in the study is not in the 
14 
best interest of the subject. If any of the following laboratory or clinical criteria is obtained 
15 
for any subject, the result must be repeated/confirmed within 72 hours. If the results are 
16 
confirmed, the subject must discontinue treatment. Clinical criteria must have Principal 
17 
Investigator or Sub-Investigator assessment prior to permanent discontinuation. 
18 
− Evidence of confirmed hepatic decompensation (Child-Pugh Class B or C, Score > 6) 
19 
(discontinue all treatment); 
20 
− 
Platelets < 25 x 109 cells/L; 
21 
− 
Hemoglobin <8.5 g/dL 
22 
− 
Absolute neutrophil count < 250 mm3/ml 
23 
− Severe neuropsychiatric signs and symptoms (including depression) for both new and 
24 
worsening events that are considered clinically significant by the investigator. 
25 
 
26 
d) Pregnancy women of childbearing potential must contact the investigator or study staff 
27 
immediately if they suspect they might be pregnant (eg, missed or late menstrual period) at 
28 
any time during study participation. If subjects become pregnant the research staff will stop 
29 
peginterferon, but subjects may continue taking their antiviral oral agent at the discretion of 
30 
the study doctor. The investigator will discuss possible management options with the 
31 
subject. Subjects who do become pregnant will continue to be followed for the outcome of 
32 
the pregnancy. The NIH will not cover any aspects of obstetrical, child or related care.  
33 
 
34 


 
21
e) Loss of ability to freely provide consent through imprisonment or involuntary 
1 
incarceration for treatment of either a psychiatric or physical (eg, infectious disease) illness. 
2 
 
3 
 
4 
Stopping NUCs therapy 
5 
 
6 
Treatment with NUCs will be stopped under the following conditions 
7 
a) HBeAg loss, with or without anti HBe seroconversion, in eAg positive patients, that is 
8 
confirmed on 2 separate tests, at least 6 months apart.  
9 
b) HBsAg loss, with or without anti HBs seroconversion, that is confirmed in 2 separate tests, at 
10 
least 6 months apart. 
11 
 
12 
 
13 
 
14 
Peginterferon Dose Modifications 
15 
 
16 
Dose modification of peginterferon α-2a should be managed according to Table 2. This table is based 
17 
on recommendations from the peginterferon α-2a package insert, and have been modified as 
18 
necessary for this study. As Tables 2 is a guideline, investigators may modify the dose based on 
19 
clinical judgment. 
20 
 
21 
Table 2: Peg-Interferon Alfa-2a Dose Modification Guidelines 
22 
 
23 
Laboratory Value/Clinical 
Criteria: 
Dose Modification: 
Additional Instructions: 
 
ANC: 
 
 
≥  0.75 ×  109  cells/L 
Maintain at 180 μ g 
 
≥  0.25 ×  109  to < 0.75 ×  109  
cells/L 
Reduce to 135 μ g 
 
< 0.25 ×  109  cells/L 
All study drugs should be 
interrupted until ANC values 
return to more than 
1000/mm3 
Reinstitute at 90 μ g and 
monitor 
ANC 
Platelets: 
 
 


 
22
≥  50 ×  109  cells/L 
Maintain at 180 μ g 
 
≥  25 ×  109  to < 50 ×  109  
cells/L 
Reduce to 90 μ g 
 
 
< 25 ×  109  cells/L 
Discontinue all study drugs if 
value is confirmed 
Treatment should not be 
reinstituted 
ALT: 
 
 
ALT > 10 ×  ULN 
Reduce to 135 μ g or interrupt 
all study drugs if repeated ALT 
results are > 10 ×  ULN 
If ALT increases are 
progressive or there is 
evidence of hepatic 
decompensation therapy 
should be interrupted  
New Ocular symptom(s): 
 
 
New decrease or loss of vision 
or 
other clinical significant ocular 
sign or symptom 
Interrupt all study treatment 
 
Complete eye examination 
must be performed.  
 
New or Worsening 
Neuropsychiatric Signs or 
Symptoms (Including 
Depression): 
Mild No change Evaluate 
weekly by visit or phone 
until symptoms improve. 
Evaluate weekly (with visit at 
least every other week). 
Consider psychiatric 
consultation if no 
 mprovement. If improved and 
stable for 4 weeks, may 
resume normal dosing. 
 
Moderate 
Reduce dose to 135 μ g 
 
Severe 
Discontinue all study drug 
treatment 
Psychiatric therapy necessary. 
Treatment should not be 
reinstituted. 
Creatinine Clearance (CrCl): 
 
 
< 30 mL/min 
Reduce to 135 μ g 
 
Hemodialysis 
Reduce to 135 μ g 
 
 
 
 
Source: adapted from Pegasys® package insert. 
1 
 
2 
 
3 


 
23
STATISTICAL ANALYSIS 
1 
 
2 
Power calculation 
3 
 
4 
It is not possible to calculate power for the primary endpoints.  We have calculated power for the 
5 
secondary endpoints of the rates of HBeAg and HBsAg loss. Response rates in patients in both groups 
6 
will be compared to historical controls of HBeAg positive and negative. It is assumed that if these 
7 
patients were followed for an additional year without peginterferon, only 10% of the HBeAg positive 
8 
patients would become HBeAg negative and 5% of the HBeAg negative patients would become 
9 
HBsAg negative. A sample size of 29 HBeAg positive and 27 HBeAg negative patients will provide the 
10 
study with a statistical power of 80% to detect a difference of 25% (35% versus 10% for HBeAg loss 
11 
and 30% versus 5% for HBsAg loss). The sample size was increased to 30 patients in each arm to 
12 
allow for withdrawals. 
13 
 
14 
Variables to be analyzed in association with a serological response will include: baseline ALT and 
15 
HBV DNA level, liver histology, HBV genotype, IL28B genotype, duration of NUC therapy, and ISG 
16 
levels. Patients with missing data or who drop out of the study will be considered as treatment 
17 
failures on an intention to treat basis. For comparison of means, Student’s t-test will be used. All 
18 
reported p-values will be 2-sided.  
19 
 
20 
Demographics and Baseline Characteristics 
21 
 
22 
The following will be summarized for treated subjects: 
23 
• Demographics: age, race, gender, geographic region, ethnicity; 
24 
• Disease characteristics at baseline: HBV DNA level, HBV genotype, IL28B SNP genotype and 
25 
cirrhosis status, duration of NUC therapy; 
26 
• Physical measurements at baseline; height, weight, body mass index, hip and waist circumference; 
27 
• Laboratory tests at baseline; 
28 
 
29 
Analysis plan 
30 
 
31 
To address the primary hypothesis that ISG levels are related to virological response to 
32 
peginterferon, the ISG analysis of the on treatment liver biopsy at 6 hours after the first dose of 
33 
interferon will be correlated with off-peginterferon serological outcome (HBeAg loss/seroconversion 
34 
and HBsAg loss). To address the hypothesis that ISG levels are related to HBsAg levels, the ISG 
35 
analysis of the on treatment liver biopsy will be correlated with HBsAg levels at weeks 4, 12, 24.. The 
36 


 
24
analyses will be stratified by HBV genotype and duration of NUC therapy. 
1 
 
2 
Analysis methodology 
3 
 
4 
The analysis will be based on standard approaches to analysis of microarray data. Since the software 
5 
and approaches change frequently in this field, we will provide a broad outline of the approach 
6 
rather than detailed, specific steps.  
7 
a) We will perform quality control and background normalization of the gene array data.  
8 
b) Expression value estimates will be obtained and quantile normalization and median scaling 
9 
of the genes will be performed.  
10 
c) A list of genes up- or down-regulated >2-fold with p>0.05 will be prepared using the 
11 
following procedure: 
12 
1. For each patient, genes up- or down-regulated >2-fold in the second biopsy 
13 
compared to the first will be identified. 
14 
2. Lists will be prepared of genes that are up- or down-regulated >2-fold for any of the 
15 
subjects. 
16 
3. A paired t-test will be used to identify genes from this list that have a p value <0.05 
17 
(Difference is not equal to zero). 
18 
4. The subset of the genes in (3) that are up- or down-regulated in >50% of patients in 
19 
groups defined relapse and/or presence of cirrhosis will be identified. 
20 
5. This procedure will be repeated with p < 0.01, but the primary analysis will be 
21 
based on the genes selected using p < 0.05. 
22 
d) The false discovery rate (FDR) of the t test will be estimated. 
23 
A more detailed statistical analysis plan will be developed prior to the start of data analysis. 
24 
 
25 
 
26 
HAZARDS AND DISCOMFORTS 
27 
 
28 
The risks and discomforts of frequent phlebotomy.  
29 
To document stable levels of biochemical and serologic markers of chronic hepatitis and to monitor 
30 
the effects and toxicities of the combination of peginterferon alfa-2a and the nucleos(t)ide analogue 
31 
frequent blood sampling will be required. Patients will have between 11 and 12 venipunctures 
32 
during the study. Each venipuncture will be for approximately 20 to 75 cc of blood. The total amount 
33 
of blood drawn during an 8 week period will not exceed 10.5 ml/Kg or 550 mls, whichever is smaller. 
34 
 
35 
The risks and discomforts of HIV testing.  
36 


 
25
Patients will have blood tested for anti-HIV at entry.  Mention of anti-HIV is made in the consent form 
1 
which includes the exact language used in the standard consent form used for anti-HIV testing at the 
2 
Clinical Center. It is important to test for HIV infection as many of the drugs used to treat hepatitis B 
3 
(tenofovir, tenofovir and emtricitabine, lamivudine and entecavir) have activity against HIV and a 
4 
treatment regimen using only two drugs against HIV would not be considered standard of care. 
5 
 
6 
The risks and discomfort of percutaneous liver biopsy.  
7 
Patients will undergo up to two liver biopsies in this protocol: one before therapy and a second six 
8 
hours after the initial injection of peginterferon alfa-2a. Patients with inadequate platelet counts 
9 
(<70,000/mm3) or coagulation parameters (prothrombin time > 15 seconds, INR>1.7) will undergo 
10 
transjugular liver biopsy after correction of the laboratory abnormalities as a part of this protocol. 
11 
Patients requiring liver biopsy will be admitted to the Clinical Center for two days for this procedure 
12 
and other blood testing. The major side effects of liver biopsy are pain, bacteremia, puncture of 
13 
another organ and bleeding. Local pain and discomfort at the liver biopsy site occurs in about 20% of 
14 
persons undergoing percutaneous liver biopsy. This is transient (lasting one to twelve hours) and is 
15 
usually mild, rarely requiring analgesics.  Bacteremia occurs in 1-2% of persons undergoing liver 
16 
biopsy.  In the absence of bile duct obstruction, this is almost always self-limited and is rarely 
17 
symptomatic.  Significant bleeding after liver biopsy is the most serious side effect of this procedure.  
18 
In the absence of a blood coagulation defect or hepatic malignancy, significant bleeding is rare, 
19 
occurring in less than one in a thousand cases of liver biopsy.  Death due to bleeding after liver 
20 
biopsy has been reported in less than 1/10,000 cases.  At the NIH Clinical Center, the Liver Diseases 
21 
Branch has performed approximately 150 liver biopsies each year for the last 20 years. During this 
22 
time, only two patients died as a complication of biopsy.  One of these patients had cirrhosis and 
23 
advanced hepatocellular carcinoma and the second had severe coagulation disorders. Both bled from 
24 
the liver biopsy site and died after surgical attempts to stop the bleeding were unsuccessful. 
25 
 
26 
The risks and hazards of Peginterferon therapy  
27 
Local pain and erythema at the site of subcutaneous injection. This is usually mild and is no greater 
28 
than the pain and irritation associated with other subcutaneous injections. Severe local reactions 
29 
have been described but are rare 
30 
A syndrome of fever, malaise, headaches and muscle aches. These influenza-   like symptoms occur 
31 
typically after the first injection and decrease in severity with subsequent treatments. Fever can be as 
32 
high as 40oC. The fatigue often persists or reappears with prolonged therapy with interferon and is 
33 
often the dose-limiting factor in therapy. These symptoms abate 
34 
with discontinuation of treatment, but may last for up to 2 weeks after the    final injection of 
35 
interferon. There is considerable variation in the occurrence and severity of these symptoms. 
36 
Gastrointestinal symptoms of nausea, vomiting, diarrhea and anorexia are common with high doses 
37 


 
26
of interferon. These are rarely severe enough to warrant discontinuation or alteration in interferon 
1 
therapy. 
2 
 
3 
Hair loss is noted by 20-25% of patients treated with interferon for more than two months. This is 
4 
always temporary. 
5 
 
6 
Bone marrow suppression occurs with the dose of interferon used to treat chronic hepatitis. 
7 
Leukopenia and granulocytopenia are the most frequent findings and a 20-50% decrease in white 
8 
blood cell counts is typical during therapy. This effect is always temporary and is dose related. 
9 
Folliculitis and sinusitis, as well as more serious infections such as dissemination of cellulitis and 
10 
bacterial peritonitis have occurred in patients receiving interferon. Indeed, among the more than 400 
11 
patients with chronic viral hepatitis treated with alpha interferon at the NIH, two died of infections 
12 
(spontaneous bacterial peritonitis) during therapy. Both patients had hepatitis B and end-stage liver 
13 
disease. Spontaneous bacterial peritonitis is a common complication of end-stage liver disease. Other 
14 
instances of severe and rare forms of infection have been reported in patients receiving interferon. It 
15 
appears that most of these severe infections are more related to the underlying condition than the 
16 
leukopenia induced by interferon, but interferon may increase susceptibility to infections and severe 
17 
outcomes. For these reasons, all patients will be cautioned, about infectious complications and 
18 
advised to contact their physician or the investigators if they develop fever. Thrombocytopenia also 
19 
occurs with interferon treatment, the platelet count falling by 13-52% of the pretreatment values. 
20 
Serious bleeding episodes have not been reported with interferon therapy. Finally, mild anemia with 
21 
a decrease in hematocrit and hemoglobin of 3-5% is also seen commonly with interferon therapy. 
22 
The effects of interferon on the bone marrow have been temporary and rapidly reversible with 
23 
discontinuation of therapy. 
24 
 
25 
Significant cardiac side effects have included idiopathic and reversible heart failure. This is reversible 
26 
with conventional management. This occurs with a frequency of <1%. 
27 
  
28 
Pulmonary disorders: Dyspnea, pulmonary infiltrates, pneumonia, bronchiolitis obliterans, 
29 
interstitial pneumonitis and sarcoidosis, some resulting in respiratory failure and/or patient deaths, 
30 
may be induced or aggravated by interferon therapy.  Pulmonary complications are rare and occur 
31 
with a frequency of <1%. 
32 
 
33 
Altered liver enzymes: Interferon can cause mild elevations in serum aminotransferases in patients 
34 
who do not have liver disease. In patients with chronic viral hepatitis, these possible elevations in 
35 
aminotransferases may be obscured by the pre-existing liver disease. Transient ALT elevations are 
36 
common during hepatitis B therapy with interferon. Twenty-five percent and 27% of subjects 
37 


 
27
experienced elevations of 5 to 10 x ULN and 12% and 18% had elevations of greater than 10 x ULN 
1 
during treatment of HBeAg negative and HBeAg positive disease, respectively. Flares have been 
2 
accompanied by elevations of total bilirubin and alkaline phosphatase and less commonly with 
3 
prolongation of PT and reduced albumin levels. Eleven percent of subjects had dose modifications 
4 
due to ALT flares and less than 1% of subjects were withdrawn from treatment. Of note these data 
5 
refer to patients not receiving concomitatnt oral antiviral agents. We believe the chance of significant 
6 
ALT flares (>10x ULN) would be rare in the setting of combination therapy. 
7 
 
8 
 
9 
Renal effects: Interferon may have minor renal toxicities. A case of interstitial nephritis during alpha 
10 
interferon therapy has been reported. We have had one patient develop acute renal failure while on 
11 
alpha interferon. This was only partially reversible. We have treated many patients with renal 
12 
disease using alpha interferon without any adverse effects on renal function. Renal complications are 
13 
rare and occur with a frequency of <1%. 
14 
 
15 
CNS Effects: Confusion has been noted with very high doses of interferon (>50 mu)69. At the doses 
16 
used in our trials (180 mcg per injection) the only central nervous system side effects have been mild 
17 
headaches, irritability, depression and psychological changes. Many patients complain of being short-
18 
tempered and irritable while on interferon. Approximately 18% of patients develop emotional 
19 
lability and depression. These side effects are closely monitored and reverse with decrease in the 
20 
interferon dosage. Another 5% of patients treated with interferon in our studies developed more 
21 
severe psychiatric side effects including paranoia, delusional thinking and marked anxiety. We have 
22 
found this side effect largely in patients with pre-existing psychiatric or neurological problems6. 
23 
These side effects reverse within one to two weeks after discontinuing interferon therapy. 
24 
 
25 
Seizures may occur in patients receiving alpha interferon. These have been described as grand mal 
26 
seizures that were self-limited and stopped once interferon was discontinued. We have reported that 
27 
there is a 1-2% incidence of seizures during interferon therapy. These seizures occurred near the end 
28 
of therapy and did not recur. Several patients were hospitalized for the seizures and evaluated; no 
29 
abnormalities were detected on neurological examination or imaging studies, and none are currently 
30 
receiving anti-epileptic medication. 
31 
 
32 
Neuropsychiatric: Life-threatening or fatal neuropsychiatric reactions may manifest in all patients 
33 
receiving therapy with interferon and may include suicide, suicidal ideation, homicidal ideation, 
34 
relapse of drug addiction, and drug overdose. These adverse effects occur in <1% of treated subjects. 
35 
These reactions may occur in patients with and without previous psychiatric illness.  
36 


 
28
  
1 
Autoimmune phenomena: Development or exacerbation of autoimmune disorders including 
2 
myositis, hepatitis, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, 
3 
psoriasis, rheumatoid arthritis, interstitial nephritis, thyroiditis, and systemic lupus erythematosus 
4 
have been reported in patients receiving alpha interferon. These occur in less than 1% of patients.  
5 
 
6 
RISK/DISCOMFORTS IN RELATION TO BENEFIT 
7 
 
8 
Protection against risks 
9 
The risks of study medication regimen administration can be generalized into 3 areas, teratogenicity, 
10 
changes in hematological parameters, and non-specific side effects.  To protect subjects from 
11 
teratogenicity, pregnancy tests will be performed every 2 weeks for the first 2 months, then every 
12 
month thereafter. Additionally, women should practice 2 forms of contraception.  CBC’s will be 
13 
performed every 2 weeks to monitor for hematologic changes, and symptoms or side effects will be 
14 
monitored at every visit, which may result in a study medication regimen dose modification.  Another 
15 
major risk to subjects who agree to participate in this study is the risk of undergoing 2 liver biopsies 
16 
(as outlined in Risks and hazards #3). Since a major endpoint of the study is to understand how 
17 
peginterferon may lead to HBeAg or HBsAg loss, the timing of the biopsies were chosen to maximize 
18 
our ability to demonstrate changes in interferon stimulated genes in the liver-up to 12 weeks before 
19 
and 6 hours after the first dose of peginterferon. Knowledge gained from this analysis may inform on 
20 
how best to use peginterferon in patients with chronic hepatitis B and identify new approaches to 
21 
induce HBeAg and HBsAg loss-highly desirable endpoints that may permit cessation of treatment.  
22 
 
23 
Potential benefits 
24 
For patients with chronic hepatitis B, the strategy of add-on peginterferon offers a potential benefit 
25 
of loss of either HBeAg or HBsAg or both with the possibility of discontinuing all therapy and removal 
26 
of the risks associated with long term nucleoside analogue use-predominantly renal toxicity and 
27 
bone loss with tenofovir and cancer risk with entecavir.  Thus, there is a prospect of overall clinical 
28 
benefit from participation in this study.   
29 
 
30 
DATA AND SAFETY MONITORING 
31 
 
32 
The adequacy of data acquisition and storage of samples and the monitoring of safety in this trial will 
33 
be performed by the principal investigator in collaboration with the associate investigators. Data and 
34 
safety are reviewed weekly in clinical research rounds by the Liver Diseases Branch, NIDDK.  These 
35 
rounds are separate from regular clinical rounds and consist of review of all study patients including 
36 


 
29
flow sheets of major safety and efficacy measurements without knowledge of which therapy is used.  
1 
Monitoring of this study will also be conducted by the statistical department of NIDDK who will have 
2 
knowledge of the patient allocation. Reports of serious adverse events are made to the Clinical 
3 
Director NIDDK, and the IRB as outlined below.   
4 
 
5 
ADVERSE EVENT REPORTING 
6 
 
7 
Definitions 
8 
 
9 
Protocol Deviation: Any change, divergence, or departure from the IRB approved research protocol.  
10 
Unanticipated Problem: Any incident, experience or outcome that meets all of the following criteria: 
11 
 
1. Unexpected (in terms of nature, severity or frequency) given (a) the research procedures 
12 
that are described in the protocol-related documents, such as the IRB approved research protocol 
13 
and informed consent document; and (b) the characteristics of the subject population being studied. 
14 
 
2. Related or possibly related to participation in the research (possibly related means there 
15 
is a reasonable possibility that the incident, experience or outcome may have been caused by the 
16 
procedures involved in the research); and 
17 
 
3. Suggests that the research places subjects or others at a greater risk of harm (including 
18 
physical, psychological, economic or social harm) than was previously known or recognized). 
19 
Expected Adverse Events become unanticipated problems when they occur at a greater frequency or 
20 
severity than was previously known. 
21 
 
22 
Adverse events, protocol deviations, unanticipated problems (UP), Unanticipated Adverse Device 
23 
Effects (UADEs), serious adverse events, sponsor and serious, are defined as described in NIH HRPP 
24 
SOP 16 (“Reporting Requirements for Unanticipated Problems, Adverse Events and Protocol 
25 
Deviations.”).  All adverse events at least possibly related to the patient’s participation in the 
26 
research protocol, including those observed by or reported to the research team, will be recorded.  
27 
Serious unanticipated problems and serious protocol deviations will be reported to the IRB and CD 
28 
as soon as possible but not more than 7 days after the PI first learns of the event.  Not serious 
29 
unanticipated problems will be reported to the IRB and CD as soon as possible but not more than 14 
30 
days after the PI first learns of the event. 
31 
 
32 
Non-serious protocol deviations will only be reported to the IRB (within 14 days after the PI first 
33 
learns of the event) if they represent a departure from NIH policies for the conduct of human subjects 
34 
research, adversely affect the health care of the subject(s) or compromise the interpretation or 
35 
integrity of the research.  Non-serious protocol deviations that result from normal subject scheduling 
36 


 
30
variations or technical issues associated with sampling that do not impact the health of the subject or 
1 
the interpretation of the study data will not be reported. 
2 
 
3 
Adverse events that are expected as a part of treatment or procedures outlined in the protocol will 
4 
not be reported unless they occur at a rate or severity greater than known to occur in patients 
5 
undergoing the treatment or procedures. If AEs associated with the treatment and procedures in the 
6 
study occur at a rate or severity greater than known to occur, they will be reclassified as a UP and 
7 
reported as such. AEs with known relation to the natural history of chronic hepatitis B or to other 
8 
pre-existing conditions will not be reported unless they occur at a rate or severity greater than 
9 
known to occur in patients with hepatitis B or the subject’s other pre-existing conditions. AEs that 
10 
are unrelated to the research will not be reported. The PI is responsible for summarizing all 
11 
reportable serious adverse events and adverse events at least possibly related at the time of 
12 
Continuing Review. Deaths will be reported to the Clinical Director and IRB within 7 days after the PI 
13 
first learns of the event.  
14 
 
15 
 
16 
 
17 
 
18 
 PROTOCOL MONITORING PLAN 
19 
 
20 
Study procedures will be subject to audits and/or monitoring visits to ensure compliance 
21 
with the protocol and applicable regulatory requirements consistent with the Liver 
22 
Diseases Branch quality assurance program plan.  Audit and/or monitoring visits results 
23 
will be reported to the Principal Investigator for further reporting as appropriate.  Study 
24 
documents and pertinent hospital or clinical records will be reviewed to verify that the 
25 
conduct of the study is consistent with the protocol plan. 
26 
 
27 
ENROLLMENT OF CHILDREN, WOMEN AND MINORITY INDIVIDUALS 
28 
 
29 
The current protocol excludes children (age limit > 18 years) as the Liver Diseases Branch does not 
30 
currently treat children. The number of children on long-term nuclos(t)ide analogue therapy would 
31 
expected to be small and the requirement for two liver biopsies may be prohibitive. 
32 


 
31
Chronic HBV infection is about twice as common among men than women.  We expect to include 
1 
women in the proportions that occur in this disease.  Thus, we expect 30% to 40% of patients to be 
2 
women.  The exclusion of women interested in getting pregnant or who are not able to practice 
3 
adequate birth control would be the only possible bias introduced in attaining adequate 
4 
representation of women in this study.    
5 
The distribution of minority individuals in our ongoing hepatitis B study (07-DK-0207) are as 
6 
follows: 73% of subjects were considered minorities, 82% of whom were Asian and 14% African-
7 
American based on self-reported race and ethnicity and NIH, HHS guidelines for defining minority 
8 
race. Thus, we believe minority individuals will be adequately represented in this study. 
9 
 
10 
Additional Potentially Vulnerable Subject Populations 
11 
This study does not plan to accrue subjects who are prisoners nor adults who are or may be unable 
12 
to provide consent. These populations are excluded as this is a more than minimal risk study due to 
13 
the requirement for two liver biopsies. NIH employees are not categorically recruited, but in the 
14 
event that an eligible subject presents who happens to be an NIH employee, we will follow the 
15 
guidelines outlined in the inclusion of employees in NIH intramural research studies. 
16 
RESEARCH USE, STORAGE AND DISPOSITION OF HUMAN SAMPLES AND DATA 
17 
 
18 
Patients will have serum stored from selected time points during this study.  These specimens will be 
19 
used for repeat virological testing and special tests as needed (such as for viral levels, or 
20 
measurement of serum levels of cytokines or interferon induced genes).  Samples may be used to 
21 
assess factors associated with response or non-response to antiviral therapy.  Liver biopsy tissue 
22 
may be stored if residual tissue is available after samples are taken for routine histological staining 
23 
and evaluation, ISG determination and immunological testing.  ISG and immunological determination 
24 
will be evaluated in the Liver Diseases Branch and the routine histological evaluation by the surgical 
25 
pathology services of the Clinical Center. Patient participation in this study will be kept confidential.  
26 
No one other than study personnel at the clinic will be given the subject’s name, address, and other 
27 
personal identifying information. Some study-related forms such as the consent form will be kept in 
28 
the medical record and have subject name and other personal identifying information on it. Other 
29 
research forms such as study diaries and flowsheets will also contain subject name and personal 
30 
identifying information. These records will be kept in locked files or computers that are password 
31 
protected. Liver tissue samples will be stored in the pathology department and will have personal 
32 
identifying information on it. However, other study material such as blood samples will be labeled 
33 
with only an identifying number and code. Any study material that may be shared with investigators 
34 
outside the NIH will not contain any personal identifying information. Only the study personnel at 
35 


 
32
the Liver Diseases Branch, Clinical Center can link your name or other personal identifiers to this 
1 
code. We plan to collaborate with Dr. Gavin Cloherty, Abbott Diagnostics, to obtain the quantitative 
2 
HBsAg testing, specified as a secondary aim of this study. Dr. Cloherty will receive ten to eleven 250 
3 
ul aliquots of sera from each subject for quantitative HBsAg testing. The results will be used for 
4 
research and as part of a planned publication. 
5 
 
6 
Research records and data as well as liver biopsy slides, biopsy reports, liver tissue and sera with the 
7 
patient’s name and a unique identifier will be stored indefinitely in our locked offices and freezers, 
8 
the medical record department and the pathology department.  These materials will be protected and 
9 
tracked by standard operating procedures in the medical record and pathology departments as well 
10 
as a compulsive filing system in our locked offices and freezers.  There will be redundant storage of 
11 
clinical information in the medical record department and our offices. Likewise, there will be 
12 
redundant storage of biopsy information and materials in the pathology department and our offices.  
13 
This should minimize the risk of loss or destruction of information and specimens. If that were to 
14 
occur we would report it to the IRB.  We do not plan to destroy this personal medical information or 
15 
the liver biopsy specimens or research subject sera after completion of the study because it may be 
16 
critically important for physicians (here or elsewhere) to have access them when caring for these 
17 
patients in the future.  
18 
 
19 
RECRUITMENT PLAN 
20 
 
21 
Potentially eligible HBeAg positive and HBeAg negative subjects evaluated under protocol 91-DK-
22 
0214 and 07-DK-0207 will be informed of this trial and offered enrollment in this study.  It is 
23 
anticipated that the majority of subjects (~85%) will be recruited from within the Liver Diseases 
24 
Branch Outpatient Clinic. For the remaining subjects, we will plan to send a recruitment letter to area 
25 
Internists and Gastroenterologists to refer suitable candidates for the trial. Finally, we will advertise 
26 
through the NIH patient recruitment office. All recruitment material will be submitted to the IRB 
27 
review prior to dissemination.   
28 
 
29 
PRIVACY AND CONFIDENTIALITY 
30 
Potentially eligible subjects will be identified through their enrolment in 91-DK-0214 and 07-DK-
31 
0207 and will be informed of this study at the time of a scheduled visit or by phone to notify them of 
32 
their potential eligibility.  Subjects will discuss protocol eligibility with the principal investigator or 
33 
an associate investigator, and associate investigators will continue to be involved in research 
34 
activities throughout subjects’ participation in this study. Information collected about subjects will be 
35 
obtained through routine clinical interactions including documentation in the subjects’ medical 
36 


 
33
charts and through questionnaire completion, patient diaries and telephone calls. We will verbally 
1 
verify with subjects their partner’s compliance with subject reported reproductive contraception use, 
2 
but this information will not be documented in the subjects’ medical charts as these individuals are 
3 
not considered subjects under this study.  The information collected from subjects is primarily 
4 
required to manage the subjects’ clinical care under the current study.  Data collected for research 
5 
only is the minimal information required to complete analysis for primary and secondary aims. 
6 
 
7 
 
8 
CONSENT PROCESS 
9 
 
10 
Potential study subjects will be informed of study rationale, design, participation burden, risks, 
11 
benefits and side effects of therapy during routine LDB clinic visits and will have an opportunity to 
12 
ask questions about the proposed study. They will be provided a copy of the study consent form to 
13 
take home and review in more detail. .  Consent will be obtained by the Principal Investigator, or an 
14 
Associate Investigator following the subject’s review of the provided consent documentation during a 
15 
subsequent clinic visit. If a subject indicates a desire to participate in the research study, he/she will 
16 
have all screening laboratories, radiologic imaging and if necessary, liver biopsy, performed through 
17 
this protocol. The initial liver and second liver biopsy for participation in this study will be for 
18 
research purposes. Patients who refuse either liver biopsy will be excluded from further 
19 
participation in the study, since one of the primary endpoints of the trial is based on analysis of 
20 
change in ISG levels comparing the second to the first liver biopsy. Potential study subjects who meet 
21 
study inclusion and exclusion criteria will sign the consent form to participate in this study during a 
22 
scheduled LDB clinic visit prior to screening.   
23 
 
24 
We do not plan to enroll non-English speaking subjects; however they are not excluded from 
25 
participation either.  Should a non-English speaking subject be enrolled, IRB approval will be 
26 
obtained to use the short form process in the absence of a fully translated consent 
27 
document.  Requests for IRB approval will be obtained prior to implementing the short form consent 
28 
process.  The short form consent process will be incompliance with SOP 12.9.1 and 45 CFR 
29 
46.117(b)(2). 
30 
 
31 
COMPENSATION 
32 
 
33 


 
34
Patients enrolled in this trial will not be offered compensation since they will be receiving study 
1 
medications and all required monitoring at no personal cost. In addition patients stand to derive 
2 
clinical benefit from participation in the study. 
3 
 
4 
REFERENCES 
5 
 
6 
1. 
Lok, A.S. and B.J. McMahon, Chronic hepatitis B: update 2009. Hepatology, 
7 
2009. 50(3): p. 661-2. 
8 
2. 
Goldstein, S.T., et al., A mathematical model to estimate global hepatitis B 
9 
disease burden and vaccination impact. Int J Epidemiol, 2005. 34(6): p. 1329-
10 
39. 
11 
3. 
Chen, C.J., et al., Risk of hepatocellular carcinoma across a biological gradient 
12 
of serum hepatitis B virus DNA level. JAMA, 2006. 295(1): p. 65-73. 
13 
4. 
McMahon, B.J., Natural history of chronic hepatitis B. Clin Liver Dis, 2010. 
14 
14(3): p. 381-96. 
15 
5. 
EASL clinical practice guidelines: Management of chronic hepatitis B virus 
16 
infection. J Hepatol, 2012. 57(1): p. 167-85. 
17 
6. 
Liaw, Y.F., et al., Asian-Pacific consensus statement on the management of 
18 
chronic hepatitis B: a 2005 update. Liver Int, 2005. 25(3): p. 472-89. 
19 
7. 
Chang, T.T., et al., Long-term entecavir therapy results in the reversal of 
20 
fibrosis/cirrhosis and continued histological improvement in patients with 
21 
chronic hepatitis B. Hepatology, 2010. 52(3): p. 886-93. 
22 
8. 
Marcellin, P., et al., Regression of cirrhosis during treatment with tenofovir 
23 
disoproxil fumarate for chronic hepatitis B: a 5-year open-label follow-up 
24 
study. Lancet, 2013. 381(9865): p. 468-75. 
25 
9. 
Hadziyannis, S.J., et al., Efficacy of long-term lamivudine monotherapy in 
26 
patients with hepatitis B e antigen-negative chronic hepatitis B. Hepatology, 
27 
2000. 32(4 Pt 1): p. 847-51. 
28 
10. 
Hadziyannis, S.J., et al., Long-term therapy with adefovir dipivoxil for HBeAg-
29 
negative chronic hepatitis B. N Engl J Med, 2005. 352(26): p. 2673-81. 
30 
11. 
Shouval, D., et al., Relapse of hepatitis B in HBeAg-negative chronic hepatitis B 
31 
patients who discontinued successful entecavir treatment: the case for 
32 
continuous antiviral therapy. J Hepatol, 2009. 50(2): p. 289-95. 
33 
12. 
Chang, T.T., et al., Entecavir treatment for up to 5 years in patients with 
34 
hepatitis B e antigen-positive chronic hepatitis B. Hepatology, 2010. 51(2): p. 
35 
422-30. 
36 
13. 
Marcellin, P., et al., Long-term efficacy and safety of adefovir dipivoxil for the 
37 
treatment of hepatitis B e antigen-positive chronic hepatitis B. Hepatology, 
38 
2008. 48(3): p. 750-8. 
39 
14. 
Liaw, Y.F., et al., 2-Year GLOBE trial results: telbivudine Is superior to 
40 
lamivudine in patients with chronic hepatitis B. Gastroenterology, 2009. 
41 
136(2): p. 486-95. 
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15. 
Marcellin, P., et al., Tenofovir disoproxil fumarate versus adefovir dipivoxil for 
1 
chronic hepatitis B. N Engl J Med, 2008. 359(23): p. 2442-55. 
2 
16. 
Fung, J., et al., Extrahepatic effects of nucleoside and nucleotide analogues in 
3 
chronic hepatitis B treatment. J Gastroenterol Hepatol, 2014. 29(3): p. 428-
4 
34. 
5 
17. 
Lu, J., et al., Direct economic burden of hepatitis B virus related diseases: 
6 
evidence from Shandong, China. BMC Health Serv Res, 2013. 13: p. 37. 
7 
18. 
Rifkin, B.S. and M.A. Perazella, Tenofovir-associated nephrotoxicity: Fanconi 
8 
syndrome and renal failure. Am J Med, 2004. 117(4): p. 282-4. 
9 
19. 
Toy, M., et al., The cost-effectiveness of treating chronic hepatitis B patients in a 
10 
median endemic and middle income country. Eur J Health Econ, 2012. 13(5): 
11 
p. 663-76. 
12 
20. 
Tsai, N., et al., Cost-effectiveness of entecavir versus adefovir for the treatment 
13 
of chronic hepatitis B in patients with decompensated cirrhosis from a third-
14 
party US payer perspective. Clinicoecon Outcomes Res, 2012. 4: p. 227-35. 
15 
21. 
Lok, A.S., et al., Efficacy of entecavir with or without tenofovir disoproxil 
16 
fumarate for nucleos(t)ide-naive patients with chronic hepatitis B. 
17 
Gastroenterology, 2012. 143(3): p. 619-28 e1. 
18 
22. 
Janssen, H.L., et al., Pegylated interferon alfa-2b alone or in combination with 
19 
lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet, 
20 
2005. 365(9454): p. 123-9. 
21 
23. 
Lau, G.K., et al., Peginterferon Alfa-2a, lamivudine, and the combination for 
22 
HBeAg-positive chronic hepatitis B. N Engl J Med, 2005. 352(26): p. 2682-95. 
23 
24. 
Marcellin, P., et al., Peginterferon alfa-2a alone, lamivudine alone, and the two 
24 
in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J 
25 
Med, 2004. 351(12): p. 1206-17. 
26 
25. 
Piccolo, P., et al., A randomized controlled trial of pegylated interferon-alpha2a 
27 
plus adefovir dipivoxil for hepatitis B e antigen-negative chronic hepatitis B. 
28 
Antivir Ther, 2009. 14(8): p. 1165-74. 
29 
26. 
Bonino, F., et al., Predicting response to peginterferon alpha-2a, lamivudine 
30 
and the two combined for HBeAg-negative chronic hepatitis B. Gut, 2007. 
31 
56(5): p. 699-705. 
32 
27. 
Tseng, T.C., et al., Effect of host and viral factors on hepatitis B e antigen-
33 
positive chronic hepatitis B patients receiving pegylated interferon-alpha-2a 
34 
therapy. Antivir Ther, 2011. 16(5): p. 629-37. 
35 
28. 
al, S.M.J.X.Q.Z.N.P.e., Adding peginterferon alfa-2a to entecavir increases HBsAg 
36 
decline and HBeAg clearance - first results from a global randomized trial 
37 
(ARES study). Hepatology, 2012. 56(S1): p. 199A. 
38 
29. 
Ouzan, D., et al., Add-on peg-interferon leads to loss of HBsAg in patients with 
39 
HBeAg-negative chronic hepatitis and HBV DNA fully suppressed by long-term 
40 
nucleotide analogs. J Clin Virol, 2013. 58(4): p. 713-7. 
41 
30. 
Sadler, A.J. and B.R. Williams, Interferon-inducible antiviral effectors. Nat Rev 
42 
Immunol, 2008. 8(7): p. 559-68. 
43 
31. 
Li, J., et al., Inhibition of hepatitis B virus replication by MyD88 involves 
44 
accelerated degradation of pregenomic RNA and nuclear retention of pre-S/S 
45 
RNAs. J Virol, 2010. 84(13): p. 6387-99. 
46 


 
36
32. 
Wieland, S.F., L.G. Guidotti, and F.V. Chisari, Intrahepatic induction of 
1 
alpha/beta interferon eliminates viral RNA-containing capsids in hepatitis B 
2 
virus transgenic mice. J Virol, 2000. 74(9): p. 4165-73. 
3 
33. 
Xu, C., et al., Interferons accelerate decay of replication-competent 
4 
nucleocapsids of hepatitis B virus. J Virol, 2010. 84(18): p. 9332-40. 
5 
34. 
Belloni, L., et al., IFN-alpha inhibits HBV transcription and replication in cell 
6 
culture and in humanized mice by targeting the epigenetic regulation of the 
7 
nuclear cccDNA minichromosome. J Clin Invest, 2012. 122(2): p. 529-37. 
8 
35. 
Ahlenstiel, G., et al., Early changes in natural killer cell function indicate 
9 
virologic response to interferon therapy for hepatitis C. Gastroenterology, 
10 
2011. 141(4): p. 1231-9, 1239 e1-2. 
11 
36. 
Edlich, B., et al., Early changes in interferon signaling define natural killer cell 
12 
response and refractoriness to interferon-based therapy of hepatitis C patients. 
13 
Hepatology, 2012. 55(1): p. 39-48. 
14 
37. 
Rehermann, B., Pathogenesis of chronic viral hepatitis: differential roles of T 
15 
cells and NK cells. Nat Med, 2013. 19(7): p. 859-68. 
16 
38. 
Lanford, R.E., et al., Genomic response to interferon-alpha in chimpanzees: 
17 
implications of rapid downregulation for hepatitis C kinetics. Hepatology, 
18 
2006. 43(5): p. 961-72. 
19 
 
20 
 
21 
 
 
22 


 
37
 Appendix 1. 
GSI Grading Scale for Severity of Adverse Events and Laboratory Abnormalities 
1 
HEMATOLOGY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hemoglobin 
HIV POSITIVE 
 
Adult and Pediatric 
 57 Days 
 
8.5 to 10.0 g/dL  
 
85 to 100 g/L 
 
7.5 to  8.5 g/dL  
 
75 to  85 g/L 
 
6.5 to  7.5 g/dL  
 
65 to  75 g/L 
 
 6.5 g/dL 
 
 65 g/L 
HIV NEGATIVE 
 
Adult and Pediatric 
 57 Days  
10.0 to 10.9 g/dL 
 
100 to 109 g/L 
 
OR 
 
Any decrease 
from Baseline 
 
2.5 to  3.5 g/dL 
 
25 to  35 g/L 
9.0 to  10.0 g/dL 
 
90 to  100 g/L 
 
OR 
 
Any decrease 
from Baseline 
 
3.5 to  4.5 g/dL 
 
35 to  45 g/L 
7.0 to  9.0 g/dL 
 
70 to  90 g/L 
 
OR 
 
Any decrease 
from Baseline 
 
 4.5 g/dL 
 
 45 g/L 
 7.0 g/dL 
 
 70 g/L 
Infant, 36–56 Days 
(HIV POSITIVE OR 
NEGATIVE) 
8.5 to 9.4 g/dL 
 
85 to 94 g/L 
7.0 to  8.5 g/dL 
 
70 to  85 g/L 
6.0 to  7.0 g/dL 
 
60 to < 70 g/L 
 6.0 g/dL 
 
< 60 g/L 
Infant, 22–35 Days 
(HIV POSITIVE OR 
NEGATIVE) 
9.5 to 10.5 g/dL 
 
95 to 105 g/L 
8.0 to  9.5 g/dL 
 
80 to  95 g/L 
7.0 to  8.0 g/dL 
 
70 to  80 g/L 
 7.0 g/dL 
 
 70 g/L 
Infant, 1–21 Days 
(HIV positive or 
negative) 
12.0 to 13.0 g/dL 
 
120 to 130 g/L 
10.0 to 
 12.0 g/dL 
 
100 to  120 g/L 
9.0 to  10.0 g/dL 
 
90 to  100 g/L 
 9.0 g/dL 
 
 90 g/L 
Absolute 
Neutrophil Count  
(ANC) 
 
1000 to 1300/m
m3 
 
750 to  
1000/mm3 
 
500 to  
750/mm3 
 
 500/mm3 


 
38
HEMATOLOGY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
 
Adult and Pediatric, 
 7 Days 
1.00 to 1.30 GI/L 
0.75 to  1.00 
GI/L 
0.50 to  0.75 
GI/L 
 0.50 GI/L 
Infant, 2 –  7 Days 
1250 to 
1500/mm3 
 
1.25 to 1.50 GI/L 
1000 to  
1250/mm3 
 
1.00 to < 1.25 
GI/L 
750 to  
1000/mm3 
 
0.75 to < 1.00 
GI/L 
 750/mm3 
 
< 0.75 GI/L 
Infant, 1 Day 
4000 to 
5000/mm3 
 
4.00 to 5.00 GI/L 
3000 to  
4000/mm3 
 
3.00 to < 4.00 
GI/L 
1500 to  
3000/mm3 
 
1.50 to < 3.00 
GI/L 
 1500/mm3 
 
< 1.50 GI/L 
Absolute CD4+ 
Count 
 
HIV NEGATIVE 
ONLY 
 
Adult and Pediatric 
> 13 Years 
 
 
 
 
300 to 400/mm3 
 
300 to 400/μL 
 
 
 
 
200 to  300/mm
3 
 
200 to  300/μL 
 
 
 
 
100 to  200/mm
3 
 
100 to  200/μL 
 
 
 
 
 100/mm3 
 
 100/μL 
Absolute 
Lymphocyte Count 
 
HIV NEGATIVE 
ONLY 
 
Adult and Pediatric 
 13 Years 
 
 
 
 
600 to 650/mm3 
 
0.60 to 0.65 GI/L 
 
 
 
 
500 to  
600/mm3 
 
0.50 to  0.60 
GI/L 
 
 
 
 
350 to  
500/mm3 
 
0.35 to  0.50 
GI/L 
 
 
 
 
 350/mm3 
 
 0.35 GI/L 
Platelets  
100,000 to 
< 125,000/mm3 
 
100 to  125 GI/L 
50,000 to 
 100,000/mm3 
 
50 to  100 GI/L 
25,000 to 
 50,000/mm3 
 
25 to  50 GI/L 
 25,000/mm
3 
 
 25 GI/L 


 
39
HEMATOLOGY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
WBCs  
2000/mm3 to 
2500/mm3 
1,500 to 
 2,000/mm3 
1000 to 
 1,500/mm3 
 1000/mm3 
2.00 GI/L to 
2.50 GI/L 
1.50 to 
 2.00 GI/L 
1.00 to 
 1.50 GI/L 
 1.00 GI/L 
Hypofibrinogenemi
a 
100 to 200 mg/dL 
75 to 
 100 mg/dL 
50 to  75 mg/dL 
 50 mg/dL 
1.00 to 2.00 g/L 
0.75 to  1.00 g/L 
0.50 to  0.75 g/L 
 0.50 g/L 
Hyperfibrinogenem
ia 
 ULN to 
600 mg/dL 
 600 mg/dL 
— 
— 
 ULN  to 6.0 g/L 
 6.0 g/L 
— 
— 
Fibrin Split Product  
20 to 40 μg/mL 
 40 to 50 μg/mL 
 50 to 60 μg/mL 
 60 μg/mL 
20 to 40 mg/L 
 40 to 50 mg/L 
 50 to 60 mg/L 
 60 mg/L 
Prothrombin Time 
(PT) 
 1.00 to 1.25  
ULN 
 1.25 to 1.50  
ULN  
 1.50 to 3.00  
ULN  
 3.00  ULN  
International 
Normalized Ratio of 
prothrombin time 
(INR) 
1.1 to 1.5 x ULN 
>1.5 to 2.0 x ULN 
>2.0 to 3.0 x ULN 
>3.0 x ULN 
Activated Partial 
 
Thromboplastin 
Time (APTT) 
 
 
 1.00 to 1.66  
ULN 
 
 
 1.66 to 2.33  
ULN 
 
 
 2.33 to 3.00  
ULN 
 
 
 3.00  ULN 
Methemoglobin  
5.0 to 10.0% 
 10.0 to 15.0% 
 15.0 to 20.0% 
 20.0%  
 
1 
CHEMISTRY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hyponatremia  
130 to <LLN 
mEq/L 
125 to 
 130 mEq/L 
121 to 
 125 mEq/L 
 121 mEq/L 
130 to <LLN 
mmol/L 
125 to 
 130 mmol/L 
121 to 
 125 mmol/L 
 121 mmol/L 
Hypernatremia  
146  to 150 
mEq/L 
 150 to 154 
mEq/L 
 154 to 159 
mEq/L 
 159 mEq/L 


 
40
CHEMISTRY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
146  to 150 
mmol/L 
 150 to 154 
mmol/L 
 154 to 159 
mmol/L 
 159 mmol/L 
Hypokalemia  
3.0 to 3.4 mEq/L 
2.5 to 
 3.0 mEq/L 
2.0 to 
 2.5 mEq/L 
 2.0 mEq/L 
3.0 to 3.4 
mmol/L 
2.5 to 
 3.0 mmol/L 
2.0 to 
 2.5 mmol/L 
 2.0 mmol/L 
Hyperkalemia  
5.6 to 6.0 mEq/L 
 6.0 to 
6.5 mEq/L 
 6.5 to 
7.0 mEq/L 
 7.0 mEq/L 
5.6 to 6.0 
mmol/L 
 6.0 to 
6.5 mmol/L 
 6.5 to 
7.0 mmol/L 
 7.0 mmol/L 
Hypoglycemia 
 
Adult and Pediatric 
 
 1 Month 
 
 
55 to 64 mg/dL 
 
 
40 to 
 55 mg/dL 
 
 
30 to 
 40 mg/dL 
 
 
 30 mg/dL 
3.03 to 
3.58 mmol/L 
2.20 to 
 3.03 mmol/L 
1.64 to 
 2.20 mmol/L 
 1.64 mmol/L 
Infant,  1 Month 
50 to 54 mg/dL 
 
2.8 to 
3.0 mmol/L 
40 to 
 50 mg/dL 
 
2.2 to 
 2.8 mmol/L 
30 to 
 40 mg/dL 
 
1.7 to 
 2.2 mmol/L 
 30 mg/dL 
 
 1.7 mmol/L 
Hyperglycemia, 
Nonfasting  
116 to 
160 mg/dL 
 160 to 
250 mg/dL 
 250 to 
500 mg/dL 
 500 mg/dL 
6.42 to 
8.91 mmol/L 
 8.91 to 
13.90 mmol/L 
 13.90 to 
27.79 mmol/L 
 27.79 
mmol/L 
Hyperglycemia, 
Fasting 
110 to 125 
mg/dL 
 
6.08 to 6.96 
mmol/L 
>125 to 250 
mg/dL 
 
>6.96 to 13.90 
mmol/L 
>250 to 500 
mg/dL 
 
>13.90 to 27.79 
mmol/L 
>500 mg/dL 
 
>27.79 
mmol/L 


 
41
CHEMISTRY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hypocalcemia  
 
(corrected for 
albumin if 
appropriate*) 
 
Adult and Pediatric 
 
 7 Days 
 
 
7.8 to 8.4  
mg/dL 
 
 
7.0 to  7.8 
mg/dL 
 
 
6.1 to 
 7.0 mg/dL 
 
 
 6.1 mg/dL 
1.94 to 2.10 
mmol/L 
1.74 to  1.94 
mmol/L 
1.51 to  1.74 
mmol/L 
 1.51 mmol/L 
Infant,  7 Days 
6.5 to 7.5 mg/dL 
 
1.61 to 1.88  
mmol/L 
6.0 to  6.5 
mg/dL 
 
1.49 to < 1.61 
mmol/L 
5.5 to  6.0 
mg/dL 
 
1.36 to < 1.49 
mmol/L  
 5.5 mg/dL 
 
< 1.36 mmol/L 
Hypercalcemia 
(corrected for 
albumin if 
appropriate*) Adult 
and Pediatric  
7 Days 
 
 
>ULN to 11.5 
mg/dL 
 
 
 11.5 to 12.5 
mg/dL 
 
 
 12.5 to 13.5 
mg/dL 
 
 
 13.5 mg/dL 
>ULN to 2.88 
mmol/L 
 2.88 to 3.13 
mmol/L 
 3.13 to 3.38 
mmol/L 
 3.38 mmol/L 
Infant,  7 Days 
11.5 to 12.4 
mg/dL 
 
2.86 to 3.10  
mmol/L 
 12.4 to 12.9 
mg/dL 
 
> 3.10 to 3.23  
mmol/L 
 12.9 to 13.5 
mg/dL 
 
> 3.23 to 3.38  
mmol/L 
 13.5 mg/dL 
 
> 3.38 mmol/L 
Hypocalcemia 
(ionized) 
3.0 mg/dL to  
LLN 
2.5 to < 3.0 
mg/dL 
2.0 to  2.5 
mg/dL 
 2.0 mg/dL 
0.74 mmol/L to 
 LLN 
0.62 to  0.74 
mmol/L 
0.49 to  0.62 
mmol/L 
 0.49 mmol/L 
Hypercalcemia 
(ionized) 
 ULN to 6.0 
mg/dL 
 6.0 to 6.5 
mg/dL 
 6.5 to 7.0 
mg/dL 
 7.0 mg/dL 
 ULN to 1.50 
mmol/L 
 1.50 to 1.63 
mmol/L 
 1.63 to 1.75 
mmol/L 
 1.75 mmol/L 


 
42
CHEMISTRY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hypomagnesemia 
1.40 to  <LLN 
mg/dL 
 
1.2 to <LLN 
mEq/L 
1.04 to  1.40 
mg/dL 
 
0.9 to   1.2 
mEq/L 
0.67 to  1.04 
mg/dL 
 
0.6 to  0.9 
mEq/L 
 0.67 mg/dL 
 
 0.6 mEq/L 
0.58 to <LLN 
mmol/L 
0.43 to  0.58 
mmol/L 
0.28 to  0.43 
mmol/L 
 0.28 mmol/L 
Hypophosphatemia 
 
Adult and Pediatric 
 
 14 Years 
 
 
2.0 to < LLN 
mg/dL 
 
 
1.5 to  2.0 
mg/dL 
 
 
1.0 to  1.5 
mg/dL 
 
 
 1.0 mg/dL 
0.63  to  LLN 
mmol/L 
0.47 to  0.63 
mmol/L 
0.31 to  0.47 
mmol/L 
 0.31 mmol/L 
Pediatric 1 Year–14 
Years  
3.0 to 3.5 mg/dL 
 
0.96 to 1.14 
mmol/L 
2.5 to  3.0 
mg/dL 
 
0.80 to 
< 0.96 mmol/L 
1.5 to  2.5 
mg/dL 
 
0.47 to < 0.80 
mmol/L 
 1.5 mg/dL 
 
< 0.47 mmol/L 
Pediatric  1 Year 
3.5 to 4.5 mg/dL 
 
1.12 to 1.46 
mmol/L 
2.5 to 
 3.5 mg/dL 
 
0.80 to 
< 1.12 mmol/L 
1.5 to  2.5 
mg/dL 
 
0.47 to < 0.80 
mmol/L 
 1.5 mg/dL 
 
< 0.47 mmol/L 
Hyperbilirubinemia  
 
Adult and Pediatric 
> 14 Days 
 
 
 1.0 to 1.5  
ULN  
 
 
 1.5 to 2.5  
ULN 
 
 
 2.5 to 5.0  
ULN  
 
 
 5.0  ULN  
Infant,  14 Days 
(non-hemolytic) 
NUCs 
20.0 to 25.0 
mg/dL 
 
342 to 428 
μmol/L 
 25.0 to 30.0 
mg/dL 
 
> 428 to 513 
μmol/L 
 30.0 mg/dL 
 
> 513 μmol/L 


 
43
CHEMISTRY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Infant,  14 Days 
(hemolytic) 
NUCs 
NUCs 
20.0 to 25.0 
mg/dL 
 
342 to 428 
μmol/L 
 25.0 mg/dL 
 
> 428 μmol/L 
Blood Urea Nitrogen 
1.25 to 2.50  
ULN  
 2.50 to 5.00  
ULN  
 5.00 to 10.00  
ULN  
 10.00  ULN 
Hyperuricemia 
>ULN to 10.0 
mg/dL  
 10.0 to 12.0 
mg/dL  
 12.0 to 15.0 
mg/dL  
 15.0 mg/dL 
>ULN to 597 
μmol/L 
 597 to 716 
μmol/L 
 716 to 895 
μmol/L 
 895 μmol/L 
Hypouricemia 
1.5 mg/dL to  
LLN 
1.0 to  1.5 
mg/dL 
0.5 to  1.0 
mg/dL 
 0.5 mg/dL 
87 μmol/L to  
LLN 
57 to  87 
μmol/L 
27 to  57 
μmol/L 
 27 μmol/L 
Creatinine  
 1.50 to 2.00 
mg/dL 
 2.00 to 3.00 
mg/dL 
 3.00 to 6.00 
mg/dL  
 6.00 mg/dL 
 133 to 177 
μmol/L 
 177 to 265 
μmol/L 
 265 to 530 
μmol/L 
 530 μmol/L 
Bicarbonate 
16.0 mEq/L to 
 LLN  
11.0 to  16.0 
mEq/L 
8.0 to  11.0 
mEq/L 
 8.0 mEq/L 
16.0 mmol/L to 
 LLN 
11.0 to  16.0 
mmol/L 
8.0 to  11.0 
mmol/L 
 8.0 mmol/L 
Triglycerides 
 
(Fasting) 
NUCs 
500 to 750 
mg/dL 
 750 to 1200 
mg/dL 
 1200 mg/dL 
 
5.64–8.47 
mmol/L 
 8.47–13.55 
mmol/L 
 13.55 
mmol/L 
LDL 
 
(Fasting) 
130 to 160 
mg/dL 
>160 to 190 
mg/dL 
> 190 mg/dL 
NUCs 
3.35 to 4.15 
mmol/L 
>4.15 to 4.92 
mmol/L 
>4.92 mmol/L 
 
Pediatric >2 to <18 
years 
110 to 130 
mg/dL 
>130 to 190 
mg/dL 
> 190 mg/dL 
NUCs 


 
44
CHEMISTRY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
2.84 to 3.37 
mmol/L 
>3.37 to  4.92 
mmol/L 
>4.92 mmol/L 
 
Hypercholesterolemia 
 
(Fasting) 
200 to 239 
mg/dL 
 239 to 300 
mg/dL 
 300 mg/dL 
NUCs 
5.16 to 6.19 
mmol/L 
 6.19 to 7.77 
mmol/L 
 7.77 mmol/L 
 
Pediatric  18 Years 
170 to 199 
mg/dL 
 
4.39 to 5.15 
mmol/L 
 199 to 300 
mg/dL 
 
 5.15 to 7.77 
mmol/L 
 300 mg/dL 
 
 7.77 mmol/L 
NUCs 
Creatine Kinase 
3.0 to  6.0  
ULN 
6.0 to  10.0  
ULN 
10.0 to  20.0  
ULN 
 20.0  ULN 
* 
Calcium should be corrected for albumin if albumin is < 4.0 g/dL 
1 
 
2 
ENZYMES 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
AST (SGOT)  
1.25 to 2.50  
ULN 
 2.50 to 5.00  
ULN 
 5.00 to 10.00  
ULN 
 10.00  ULN 
ALT (SGPT)  
1.25 to 2.50  
ULN 
 2.50 to 5.00  
ULN 
 5.00 to 10.00  
ULN 
 10.00  ULN 
GGT 
1.25 to 2.50  
ULN 
 2.50 to 5.00  
ULN 
 5.00 to 10.00  
ULN 
 10.00  ULN 
Alkaline 
Phosphatase  
1.25 to 2.50  
ULN 
 2.50 to 5.00  
ULN 
 5.00 to 10.00  
ULN 
 10.00  ULN 
Total Amylase 
 1.0 to 1.5  
ULN 
 1.5 to 2.0  ULN 
 2.0 to 5.0  
ULN 
 5.0  ULN 
Pancreatic 
Amylase 
 1.0 to 1.5  
ULN 
 1.5 to 2.0  ULN 
 2.0 to 5.0  
ULN 
 5.0  ULN 
Lipase  
 1.0 to 1.5  
ULN 
 1.5 to 3.0  ULN 
 3.0 to 5.0  
ULN 
 5.0  ULN 


 
45
ENZYMES 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Albumin 
3.0 g/dL to  
LLN 
 
30 g/L to  LLN 
2.0 to  3.0 g/dL 
 
20 to  30 g/L 
 2.0 g/dL 
 
 20 g/L 
NUCs 
 
1 
URINUCsLYSIS 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hematuria 
(Dipstick)  
1+ 
2+ 
3-4+ 
NUCs 
Hematuria 
(Quantitative)  
 
See Note below 
 
                              
Females 
 
 
 
 
>ULN - 
10 RBC/HPF 
 
 
 
 
 10-75 RBC/HPF 
 
 
 
 
> 75 RBC/HPF 
 
 
 
 
NUCs 
                              
Males 
6-10 RBC/HPF 
 10-75 RBC/HPF 
> 75 RBC/HPF 
NUCs 
Proteinuria 
(Dipstick) 
1+ 
2–3+ 
4+ 
NUCs 
Proteinuria, 24 
Hour Collection 
 
 
 
 
Adult and 
Pediatric  
 10 Years 
200 to 999 
mg/24 h 
>999 to 1999 
mg/24 h 
>1999 to 3500 
mg/24 h 
 3500 mg/24 
h 
Pediatric  3 Mo to 
< 10 Years 
201 to 499 
mg/m2/24 h 
>499 to 799 
mg/m2/24 h 
>799 to 1000 
mg/m2/24 h 
 
 1000 mg/ 
m2/24 h 
Glycosuria 
(Dipstick) 
1+ 
2-3+ 
4+ 
NUCs  
Notes:  
2 
Toxicity grades for Quantitative and Dipstick Hematuria will be assigned by Covance Laboratory, 
3 
however for other laboratories, toxicity grades will only be assigned to Dipstick Hematuria. 
4 


 
46
With the exception of lipid tests, any graded laboratory test with a result that is between the LLN and 
1 
ULN should be assigned Grade 0. 
2 
If the severity of a clinical AE could fall under either one of two grades (e.g., the severity of an AE 
3 
could be either Grade 2 or Grade 3), select the higher of the two grades for the AE. 
4 
 
5 
CARDIOVASCULAR 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Cardiac Arrhythmia 
(general) 
 
(By ECG or physical 
exam) 
Asymptomatic 
AND No 
intervention 
indicated 
Asymptomatic 
AND Non-
urgent medical 
intervention 
indicated 
Symptomatic, 
non-life-
threatening 
AND Non-
urgent medical 
intervention 
indicated  
Life-threatening 
arrhythmia OR 
Urgent 
intervention 
indicated 
Cardiac-
ischemia/Infarction 
NUCs 
NUCs 
Symptomatic 
ischemia 
(stable angina) 
OR Testing 
consistent with 
ischemia  
Unstable angina 
OR Acute 
myocardial 
infarction 
Hemorrhage 
(significant acute 
blood loss) 
NUCs 
Symptomatic 
AND No 
transfusion 
indicated 
Symptomatic 
AND 
Transfusion of 
 2 units 
packed RBCs 
(for children 
 10 cc/kg) 
indicated  
Life-threatening 
hypotension OR 
Transfusion of 
 2 units packed 
RBCs indicated 
(for children 
 10 cc/kg) 
indicated 
Hypertension (with 
repeat testing at same 
visit) 
140–159 mmHg 
systolic  
OR  
90–99 mmHg 
diastolic 
 159–179 
mmHg systolic  
OR  
 99–109 
mmHg diastolic 
 179 mmHg 
systolic  
OR  
 109 mmHg 
diastolic 
Life-threatening 
consequences 
(eg, malignant 
hypertension) 
OR 
Hospitalization 
(other than ER 
visit) indicated 


 
47
CARDIOVASCULAR 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Pediatric  17 Years 
(with repeat testing at 
same visit) 
NUCs 
91st–94th 
percentile 
adjusted for 
age, height, and 
gender (systolic 
and/or 
diastolic) 
≥ 95th 
percentile 
adjusted for 
age, height, and 
gender (systolic 
and/or 
diastolic) 
Life-threatening 
consequences 
(eg, malignant 
hypertension) 
OR 
Hospitalization 
indicated (other 
than emergency 
room visit) 
Hypotension 
NUCs 
Symptomatic, 
corrected with 
oral fluid 
replacement 
Symptomatic, 
IV fluids 
indicated 
Shock requiring 
use of 
vasopressors or 
mechanical 
assistance to 
maintain blood 
pressure 
Pericardial Effusion 
Asymptomatic, 
small effusion 
requiring no 
intervention 
Asymptomatic, 
moderate or 
larger effusion 
requiring no 
intervention 
Effusion with 
non-life-
threatening 
physiologic 
consequences 
OR Effusion 
with nonurgent 
intervention 
indicated 
Life-threatening 
consequences 
(eg, tamponade) 
OR Urgent 
intervention 
indicated 
Prolonged PR Interval 
PR interval  
0.21 to 0.25 sec 
PR interval 
 0.25 sec 
Type II 2nd 
degree AV 
block OR 
Ventricular 
pause  3.0 sec 
Complete AV 
block  


 
48
CARDIOVASCULAR 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Pediatric  16 Years 
1st degree AV 
block 
(PR > normal 
for age and 
rate) 
Type I 2nd 
degree AV block  
Type II 2nd 
degree AV 
block  
Complete AV 
block 
Prolonged QTc 
Asymptomatic, 
QTc interval 
0.45 to 0.47 sec 
OR Increase 
interval  0.03 
sec above 
baseline 
Asymptomatic, 
QTc interval 
0.48 to 0.49 sec 
OR Increase in 
interval 0.03 to 
0.05 sec above 
baseline 
Asymptomatic, 
QTc interval 
 0.50 sec OR 
Increase in 
interval 
 0.06 sec 
above baseline 
Life-threatening 
consequences, 
eg, Torsade de 
pointes or other 
associated 
serious 
ventricular 
dysrhythmia 
Pediatric  16 Years 
Asymptomatic, 
QTc interval 
0.450 to 0.464 
sec  
Asymptomatic, 
QTc interval 
0.465 to 0.479 
sec  
Asymptomatic, 
QTc interval 
 0.480 sec  
Life-threatening 
consequences, 
eg, Torsade de 
pointes or other 
associated 
serious 
ventricular 
dysrhythmia 
Thrombosis/Embolism 
NA 
Deep vein 
thrombosis 
AND No 
intervention 
indicated (eg, 
anticoagulation, 
lysis filter, 
invasive 
procedure)  
Deep vein 
thrombosis 
AND 
Intervention 
indicated (eg, 
anticoagulation, 
lysis filter, 
invasive 
procedure)  
Embolic event 
(eg, pulmonary 
embolism, life-
threatening 
thrombus)  
Vasovagal Episode 
(associated with a 
procedure of any kind) 
Present without 
loss of 
consciousness  
Present with 
transient loss of 
consciousness 
NA 
NA 


 
49
CARDIOVASCULAR 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Ventricular 
Dysfunction 
(congestive heart 
failure, CHF) 
NA 
Asymptomatic 
diagnostic 
finding AND 
intervention 
indicated 
New onset with 
symptoms OR 
Worsening 
symptomatic 
CHF 
Life-threatening 
CHF 
 
1 
RESPIRATORY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Bronchospasm 
(acute) 
FEV1 or peak 
flow reduced to 
70% to 80% 
FEV1 or peak flow 
50% to 69% 
FEV1 or peak 
flow 25% to 
49%  
Cyanosis OR FEV1 
or peak flow 
 25% OR 
Intubation 
Dyspnea or 
Respiratory 
Distress 
Dyspnea on 
exertion with no 
or minimal 
interference with 
usual social & 
functional 
activities  
Dyspnea on 
exertion causing 
greater than 
minimal 
interference with 
usual social & 
functional 
activities  
Dyspnea at rest 
causing inability 
to perform 
usual social & 
functional 
activities  
Respiratory failure 
with ventilatory 
support indicated 
Pediatric  14 
Years 
Wheezing OR 
minimal increase 
in respiratory 
rate for age 
Nasal flaring OR 
Intercostal 
retractions OR 
Pulse oximetry 
90% to 95% 
Dyspnea at rest 
causing inability 
to perform 
usual social & 
functional 
activities OR 
Pulse oximetry 
 90% 
Respiratory failure 
with ventilatory 
support indicated 
 
2 
OCULAR/VISUAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 


 
50
Uveitis 
Asymptomatic 
but detectable on 
exam 
Symptomatic 
anterior uveitis OR 
Medical 
intervention 
indicated 
Posterior or 
pan-uveitis OR 
Operative 
intervention 
indicated 
Disabling visual 
loss in affected 
eye(s)  
Visual 
Changes 
(from 
baseline) 
Visual changes 
causing no or 
minimal 
interference with 
usual social & 
functional 
activities 
Visual changes 
causing greater 
than minimal 
interference with 
usual social & 
functional 
activities 
Visual changes 
causing inability 
to perform usual 
social & 
functional 
activities 
Disabling visual 
loss in affected 
eye(s) 
 
1 
SKIN 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Alopecia 
Thinning 
detectable by 
study 
participant or  
caregiver (for 
disabled adults) 
Thinning or 
patchy hair loss 
detectable by 
health care 
provider 
Complete hair loss 
NA 


 
51
Cutaneous 
Reaction – Rash 
Localized 
macular rash  
Diffuse macular, 
maculopapular, 
or morbilliform 
rash OR Target 
lesions  
Diffuse macular, 
maculopapular, or 
morbilliform rash 
with vesicles or 
limited number of 
bullae OR 
Superficial 
ulcerations of 
mucous 
membrane limited 
to one site 
Extensive or 
generalized 
bullous lesions 
OR Stevens-
Johnson 
syndrome OR 
Ulceration of 
mucous 
membrane 
involving two 
or more 
distinct 
mucosal sites 
OR Toxic 
epidermal 
necrolysis 
(TEN) 
Hyperpigmentation 
Slight or 
localized 
Marked or 
generalized 
NA 
NA 
Hypopigmentation 
Slight or 
localized  
Marked or 
generalized 
NA 
NA 
Pruritis (itching – 
no skin lesions)  
 
(See also Injection 
Site Reactions: 
Pruritis associated 
with injection) 
Itching causing 
no or minimal 
interference 
with usual 
social & 
functional 
activities 
Itching causing 
greater than 
minimal 
interference with 
usual social & 
functional 
activities 
Itching causing 
inability to 
perform usual 
social & functional 
activities  
NA 
 
1 


 
52
GASTROINTESTINAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Anorexia 
Loss of 
appetite 
without 
decreased oral 
intake 
Loss of appetite 
associated with 
decreased oral 
intake without 
significant weight 
loss  
Loss of appetite 
associated with 
significant weight 
loss  
Life-
threatening 
consequences 
OR 
Aggressive 
intervention 
indicated [eg, 
tube feeding 
or total 
parenteral 
nutrition] 
Ascites 
Asymptomatic 
Symptomatic AND 
Intervention 
indicated (eg, 
diuretics or 
therapeutic 
paracentesis) 
Symptomatic 
despite 
intervention 
Life-
threatening 
consequences 
Cholecystitis 
NA 
Symptomatic AND 
Medical 
intervention 
indicated 
Radiologic, 
endoscopic, or 
operative 
intervention 
indicated 
Life-
threatening 
consequences 
(eg, sepsis or 
perforation)  
Constipation  
NA 
Persistent 
constipation 
requiring regular 
use of dietary 
modifications, 
laxatives, or 
enemas  
Obstipation with 
manual evacuation 
indicated 
Life-
threatening 
consequences 
(eg, 
obstruction) 


 
53
GASTROINTESTINAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Diarrhea  
 
Adult and Pediatric 
 1 Year 
 
 
Transient or 
intermittent 
episodes of 
unformed 
stools OR 
Increase of 
 3 stools over 
baseline/24 hr 
 
 
Persistent 
episodes of 
unformed to 
watery stools OR 
Increase of 4–6 
stools over 
baseline per 
24 hrs.  
 
 
Bloody diarrhea 
OR Increase of 
 7 stools per 
24-hour period OR 
IV fluid 
replacement 
indicated 
 
 
Life-
threatening 
consequences 
(eg, 
hypotensive 
shock) 
Pediatric  1 Year 
Liquid stools 
(more 
unformed 
than usual) 
but usual 
number of 
stools 
Liquid stools with 
increased number 
of stools OR Mild 
dehydration 
Liquid stools with 
moderate 
dehydration 
Liquid stools 
resulting in 
severe 
dehydration 
with 
aggressive 
rehydration 
indicated OR 
Hypotensive 
shock 
Dysphagia-
Odynophagia  
Symptomatic 
but able to eat 
usual diet 
Symptoms causing 
altered dietary 
intake without 
medical 
intervention 
indicated 
Symptoms causing 
severely altered 
dietary intake with 
medical 
intervention 
indicated 
Life-
threatening 
reduction in 
oral intake 


 
54
GASTROINTESTINAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Mucositis/Stomatitis  
(clinical exam) 
 
See also Proctitis, 
Dysphagia-
Odynophagia 
Erythema of 
the mucosa 
Patchy 
pseudomembranes 
or ulcerations  
Confluent 
pseudomembranes 
or ulcerations OR 
Mucosal bleeding 
with minor trauma 
Tissue 
necrosis OR 
Diffuse 
spontaneous 
mucosal 
bleeding OR 
Life-
threatening 
consequences 
(eg, 
aspiration, 
choking) 
Nausea  
Transient 
( 24 hours) 
or 
intermittent 
nausea with 
no or minimal 
interference 
with oral 
intake 
Persistent nausea 
resulting in 
decreased oral 
intake for 24–48 
hours 
Persistent nausea 
resulting in 
minimal oral 
intake for  48 
hours OR 
Aggressive 
rehydration 
indicated (eg, 
IV fluids) 
Life-
threatening 
consequences 
(eg, 
hypotensive 
shock) 
Pancreatitis 
NA 
Symptomatic AND 
Hospitalization not 
indicated (other 
than ER visit) 
Symptomatic AND 
Hospitalization 
indicated (other 
than ER visit) 
Life-
threatening 
consequences 
(eg, sepsis, 
circulatory 
failure, 
hemorrhage) 


 
55
GASTROINTESTINAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Proctitis (functional- 
symptomatic) 
 
Also see Mucositis/ 
Stomatitis for 
Clinical Exam 
Rectal 
discomfort 
AND No 
intervention 
indicated 
Symptoms causing 
greater than 
minimal 
interference with 
usual social & 
functional 
activities OR 
Medical 
intervention 
indicated  
Symptoms causing 
inability to 
perform usual 
social/ functional 
activities OR 
Operative 
intervention 
indicated  
Life-
threatening 
consequences 
(eg, 
perforation) 
Vomiting 
Transient or 
intermittent 
vomiting with 
no or minimal 
interference 
with oral 
intake 
Frequent episodes 
of vomiting with 
no or mild 
dehydration 
Persistent 
vomiting resulting 
in orthostatic 
hypotension OR 
Aggressive 
rehydration 
indicated 
Life-
threatening 
consequences 
(eg, 
hypotensive 
shock) 
 
1 
NEUROLOGICAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Alteration in 
Personality-Behavior 
or in Mood (eg, 
agitation, anxiety, 
depression, mania, 
psychosis) 
Alteration 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities  
Alteration 
causing greater 
than minimal 
interference 
with usual social 
& functional 
activities  
Alteration 
causing 
inability to 
perform usual 
social & 
functional 
activities  
Behavior 
potentially 
harmful to self or 
others (eg, 
suicidal/homicidal 
ideation or 
attempt, acute 
psychosis) OR 
Causing inability 
to perform basic 
self-care functions 


 
56
NEUROLOGICAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Altered Mental Status  
 
For Dementia, see 
Cognitive and 
Behavioral/Attentiona
l Disturbance 
(including dementia 
and ADD) 
Changes 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities 
Mild lethargy or 
somnolence 
causing greater 
than minimal 
interference 
with usual social 
& functional 
activities 
Confusion, 
memory 
impairment, 
lethargy, or 
somnolence 
causing 
inability to 
perform usual 
social & 
functional 
activities 
Delirium OR 
obtundation, OR 
coma 
Ataxia  
Asymptomatic 
ataxia 
detectable on 
exam OR 
Minimal 
ataxia causing 
no or minimal 
interference 
with usual 
social & 
functional 
activities 
Symptomatic 
ataxia causing 
greater than 
minimal 
interference 
with usual social 
& functional 
activities 
Symptomatic 
ataxia causing 
inability to 
perform usual 
social & 
functional 
activities  
Disabling ataxia 
causing inability to 
perform basic self-
care functions 
Cognitive and 
Behavioral/Attentiona
l Disturbance 
(including dementia 
and Attention Deficit 
Disorder)  
Disability 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities OR 
Specialized 
resources not 
indicated 
Disability 
causing greater 
than minimal 
interference 
with usual social 
& functional 
activities OR 
Specialized 
resources on 
part-time basis 
indicated 
Disability 
causing 
inability to 
perform usual 
social & 
functional 
activities OR 
Specialized 
resources on a 
full-time basis 
indicated 
Disability causing 
inability to 
perform basic self-
care functions OR 
Institutionalizatio
n indicated 


 
57
NEUROLOGICAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
CNS Ischemia 
(acute) 
NA 
NA 
Transient 
ischemic 
attack 
Cerebral vascular 
accident (CVA, 
stroke) with 
neurological 
deficit  
Developmental delay – 
Pediatric  16 Years 
Mild 
developmenta
l delay, either 
motor or 
cognitive, as 
determined by 
comparison 
with a 
developmenta
l screening 
tool 
appropriate 
for the setting  
Moderate 
developmental 
delay, either 
motor or 
cognitive, as 
determined by 
comparison 
with a 
developmental 
screening tool 
appropriate for 
the setting  
Severe 
developmenta
l delay, either 
motor or 
cognitive, as 
determined by 
comparison 
with a 
developmenta
l screening 
tool 
appropriate 
for the setting  
Developmental 
regression, either 
motor or 
cognitive, as 
determined by 
comparison with a 
developmental 
screening tool 
appropriate for 
the setting 
Headache  
Symptoms 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities 
Symptoms 
causing greater 
than minimal 
interference 
with usual social 
& functional 
activities 
Symptoms 
causing 
inability to 
perform usual 
social & 
functional 
activities 
Symptoms causing 
inability to 
perform basic self-
care functions OR 
Hospitalization 
indicated (other 
than ER visit) OR 
Headache with 
significant 
impairment of 
alertness or other 
neurologic 
function  


 
58
NEUROLOGICAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Insomnia 
NA 
Difficulty 
sleeping causing 
greater than 
minimal 
interference 
with usual 
social/functiona
l activities 
Difficulty 
sleeping 
causing 
inability to 
perform usual 
social & 
functional 
activities 
Disabling 
insomnia causing 
inability to 
perform basic self-
care functions 
Neuromuscular 
Weakness  
(including myopathy & 
neuropathy)  
Asymptomatic 
with 
decreased 
strength on 
exam OR 
Minimal 
muscle weak-
ness causing 
no or minimal 
interference 
with usual 
social & 
functional 
activities 
Muscle 
weakness 
causing greater 
than minimal 
interference 
with usual social 
& functional 
activities 
Muscle 
weakness 
causing 
inability to 
perform usual 
social & 
functional 
activities  
Disabling muscle 
weakness causing 
inability to 
perform basic self-
care functions OR 
Respiratory 
muscle weakness 
impairing 
ventilation 


 
59
NEUROLOGICAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Neurosensory 
Alteration (including 
paresthesia and 
painful neuropathy) 
Asymptomatic 
with sensory 
alteration on 
exam or 
minimal 
paresthesia 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities  
Sensory 
alteration or 
paresthesia 
causing greater 
than minimal 
interference 
with usual social 
& functional 
activities  
Sensory 
alteration or 
paresthesia 
causing 
inability to 
perform usual 
social & 
functional 
activities 
Disabling sensory 
alteration or 
paresthesia 
causing inability to 
perform basic self-
care functions 
Seizure: (new onset)  
NA 
1 seizure  
2–4 seizures  
Seizures of any 
kind that are 
prolonged, 
repetitive (eg, 
status epilepticus), 
or difficult to 
control (eg, 
refractory 
epilepsy) 


 
60
NEUROLOGICAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Seizure: (pre-existing)  
 
For Worsening of 
Existing Epilepsy the 
Grades Should Be 
Based on an Increase 
from Previous Level of 
Control to Any of 
These Levels 
NA 
Increased 
frequency of 
pre-existing 
seizures (non-
repetitive) 
without change 
in seizure 
character OR 
infrequent 
breakthrough 
seizures while 
on stable meds 
in a previously 
controlled 
seizure disorder 
Change in 
seizure 
character 
from baseline 
either in 
duration or 
quality (eg, 
severity or 
focality)  
Seizures of any 
kind that are 
prolonged, 
repetitive (eg, 
status epilepticus), 
or difficult to 
control (eg, 
refractory 
epilepsy) 
Seizure  
– Pediatric  18 Years 
Seizure, 
generalized 
onset with or 
without 
secondary 
generalization
, lasting 
< 5 minutes 
with 
< 24 hours 
post ictal state 
Seizure, 
generalized 
onset with or 
without 
secondary 
generalization, 
lasting 5–20 
minutes with  
< 24 hours post 
ictal state 
Seizure, 
generalized 
onset with or 
without 
secondary 
generalization
, lasting  
> 20 minutes  
Seizure, 
generalized onset 
with or without 
secondary 
generalization, 
requiring 
intubation and 
sedation 
Syncope (not 
associated with a 
procedure) 
NA 
Present 
NA 
NA 


 
61
NEUROLOGICAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Vertigo 
Vertigo 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities 
Vertigo causing 
greater than 
minimal 
interference 
with usual social 
& functional 
activities  
Vertigo 
causing 
inability to 
perform usual 
social & 
functional 
activities 
Disabling vertigo 
causing inability to 
perform basic self-
care functions 
 
1 
MUSCULOSKELETAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Arthralgia 
 
See also 
Arthritis 
Joint pain 
causing no or 
minimal 
interference 
with usual social 
& functional 
activities 
Joint pain causing 
greater than 
minimal 
interference with 
usual social & 
functional activities 
Joint pain 
causing inability 
to perform usual 
social & 
functional 
activities 
Disabling joint 
pain causing 
inability to 
perform basic 
self-care 
functions 
Arthritis 
 
See also 
Arthralgia 
Stiffness or joint 
swelling causing 
no or minimal 
interference 
with usual social 
& functional 
activities 
Stiffness or joint 
swelling causing 
greater than 
minimal 
interference with 
usual social & 
functional activities 
Stiffness or joint 
swelling causing 
inability to 
perform usual 
social & 
functional 
activities 
Disabling joint 
stiffness or 
swelling causing 
inability to 
perform basic 
self-care 
functions 
Bone Mineral 
Loss 
BMD t-score or 
z-score 
–2.5 to –1.0  
BMD t-score or z-
score  –2.5 
Pathological 
fracture 
(including loss 
of vertebral 
height) 
Pathologic 
fracture causing 
life-threatening 
consequences  


 
62
MUSCULOSKELETAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Pediatric 
 21 Years 
BMD z-score  
–2.5 to –1.0 
BMD z-score < –2.5 
Pathological 
fracture 
(including loss 
of vertebral 
height) 
Pathologic 
fracture causing 
life-threatening 
consequences  
Myalgia 
(non-injection 
site) 
Muscle pain 
causing no or 
minimal 
interference 
with usual social 
& functional 
activities 
Muscle pain causing 
greater than 
minimal 
interference with 
usual social & 
functional activities 
Muscle pain 
causing inability 
to perform usual 
social & 
functional 
activities 
Disabling muscle 
pain causing 
inability to 
perform basic 
self-care 
functions 
Osteonecrosis 
NUCs 
Asymptomatic with 
radiographic 
findings AND No 
operative 
intervention 
indicated 
Symptomatic 
bone pain with 
radiographic 
findings OR 
Operative 
intervention 
indicated 
Disabling bone 
pain with 
radiographic 
findings causing 
inability to 
perform basic 
self-care 
functions 
 
1 
SYSTEMIC 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Acute Systemic 
Allergic 
Reaction  
Localized 
urticaria 
(wheals) with 
no medical 
intervention 
indicated 
Localized urticaria 
with medical 
intervention 
indicated OR Mild 
angioedema with no 
medical intervention 
indicated 
Generalized 
urticaria OR 
Angioedema with 
medical 
intervention 
indicated OR 
Symptomatic 
mild 
bronchospasm 
Acute anaphylaxis 
OR Life-
threatening 
bronchospasm 
OR laryngeal 
edema 


 
63
SYSTEMIC 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Chills 
Symptoms 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities 
Symptoms causing 
greater than 
minimal 
interference with 
usual social & 
functional activities  
Symptoms 
causing inability 
to perform usual 
social & 
functional 
activities 
NA 
Fatigue 
 
Malaise 
Symptoms 
causing no or 
minimal 
interference 
with usual 
social & 
functional 
activities 
Symptoms causing 
greater than 
minimal 
interference with 
usual social & 
functional activities 
Symptoms 
causing inability 
to perform usual 
social & 
functional 
activities 
Incapacitating 
fatigue/malaise 
symptoms 
causing inability 
to perform basic 
self-care 
functions 
Fever 
(nonaxillary) 
37.7C to 
38.6C 
 
99.8F to 
101.5F 
38.7C to 39.3C 
 
101.6F to 102.8F 
39.4C to 40.5C 
 
102.9F to 
104.9F 
 40.5C  
 
 104.9F 
Pain- Indicate 
Body Site 
 
See also 
Injection Site 
Pain, 
Headache, 
Arthralgia, and 
Myalgia 
Pain causing 
no or minimal 
interference 
with usual 
social & 
functional 
activities 
Pain causing greater 
than minimal 
interference with 
usual social & 
functional activities 
Pain causing 
inability to 
perform usual 
social & 
functional 
activities 
Disabling pain 
causing inability 
to perform basic 
self-care 
functions OR 
Hospitalization 
(other than ER 
visit) indicated 


 
64
SYSTEMIC 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Unintentional 
Weight Loss 
NA 
5% to 9% loss in 
body weight from 
baseline 
10% to 19% loss 
in body weight 
from baseline 
 20% loss in 
body weight from 
baseline OR  
Aggressive 
intervention 
indicated [eg, 
tube feeding or 
total parenteral 
nutrition] 
 
1 
INJECTION SITE REACTION 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Injection Site 
Pain (pain 
without 
touching) 
 
Or Tenderness 
(pain when area 
is touched)  
Pain/tenderness 
causing no or 
minimal 
limitation of use 
of limb 
Pain/tenderness 
limiting use of 
limb OR 
Pain/tenderness 
causing greater 
than minimal 
interference with 
usual social & 
functional 
activities 
Pain/tenderness 
causing inability 
to perform usual 
social & 
functional 
activities 
Pain/tenderness 
causing inability to 
perform basic self-
care function OR 
Hospitalization 
(other than ER 
visit) indicated for 
management of 
pain/tenderness 
Injection Site 
Reaction 
(Localized), 
 15 Years 
Erythema OR 
Induration 
of 5  5 cm to 
9  9 cm (or 
25−81  cm2) 
Erythema OR 
Induration OR 
Edema  9 cm any 
diameter (or  
81 cm2) 
Ulceration OR 
Secondary 
infection OR 
Phlebitis OR 
Sterile abscess 
OR Drainage 
Necrosis (involving 
dermis and deeper 
tissue) 
Pediatric 
 15 Years 
Erythema OR 
Induration OR 
Edema present 
but  2.5 cm 
diameter 
Erythema OR 
Induration OR 
Edema > 2.5 cm 
diameter but 
< 50% surface 
area of the 
Erythema OR 
Induration OR 
Edema involving 
 50% surface 
area of the 
extremity 
Necrosis (involving 
dermis and deeper 
tissue) 


 
65
extremity 
segment (eg, 
upper arm/thigh) 
segment (eg, 
upper 
arm/thigh) OR 
Ulceration OR 
Secondary 
infection OR 
Phlebitis OR 
Sterile abscess 
OR Drainage 
Pruritis 
Associated with 
Injection  
 
See also Skin: 
Pruritis 
(itching—no 
skin lesions) 
Itching localized 
to injection site 
AND Relieved 
spontaneously 
or with  48 h 
treatment  
Itching beyond 
the injection site 
but not 
generalized OR 
Itching localized 
to injection site 
requiring  48 h 
treatment  
Generalized 
itching causing 
inability to 
perform usual 
social & 
functional 
activities 
NA 
 
1 
ENDOCRINE/METABOLIC 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Lipodystrophy 
(eg, back of neck, 
breasts, 
abdomen) 
Detectable by 
study 
participant or 
caregiver (for 
young 
children and 
disabled 
adults) 
Detectable on 
physical exam by 
health care provider 
Disfiguring OR 
Obvious changes 
on casual visual 
inspection 
NA 
Diabetes 
Mellitus 
NA 
New onset without 
need to initiate 
medication OR 
Modification of 
current meds to 
regain glucose 
control 
New onset with 
initiation of 
indicated med 
OR Diabetes 
uncontrolled 
despite 
treatment 
modification 
Life-threatening 
consequences 
(eg, ketoacidosis, 
hyperosmolar 
non-ketotic 
coma)  


 
66
ENDOCRINE/METABOLIC 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Gynecomastia 
Detectable by 
study 
participant or 
caregiver (for 
young 
children and 
disabled 
adults) 
Detectable on 
physical exam by 
health care provider 
Disfiguring OR 
Obvious on 
casual visual 
inspection 
NA 
Hyperthyroidism  Asymptomatic 
Symptomatic 
causing greater than 
minimal 
interference with 
usual social & 
functional activities 
OR Thyroid 
suppression 
therapy indicated 
Symptoms 
causing inability 
to perform usual 
social & 
functional 
activities OR 
Uncontrolled 
despite 
treatment 
modification  
Life-threatening 
consequences 
(eg, thyroid 
storm) 
Hypothyroidism 
Asymptomatic 
Symptomatic 
causing greater than 
minimal 
interference with 
usual social & 
functional activities 
OR Thyroid 
replacement 
therapy indicated 
Symptoms 
causing inability 
to perform usual 
social & 
functional 
activities OR 
Uncontrolled 
despite 
treatment 
modification  
Life-threatening 
consequences 
(eg, myxedema 
coma) 


 
67
ENDOCRINE/METABOLIC 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Lipoatrophy (eg, 
fat loss from the 
face, extremities, 
buttocks) 
Detectable by 
study 
participant or 
caregiver (for 
young 
children and 
disabled 
adults) 
Detectable on 
physical exam by 
health care provider 
Disfiguring OR 
Obvious on 
casual visual 
inspection 
NA 
 
1 
GENITOURINUCsRY 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Intermenstrual 
Bleeding (IMB) 
Spotting observed 
by participant OR 
Minimal blood 
observed during 
clinical or 
colposcopic exam 
Intermenstrual 
bleeding not 
greater in 
duration or 
amount than 
usual menstrual 
cycle 
Intermenstrual 
bleeding greater in 
duration or 
amount than usual 
menstrual cycle 
Hemorrhage 
with life-
threatening 
hypotension OR 
Operative 
intervention 
indicated 
Urinary Tract 
obstruction 
(eg, stone) 
NA 
Signs or 
symptoms of 
urinary tract 
obstruction 
without 
hydronephrosis 
or renal 
dysfunction 
Signs or symptoms 
of urinary tract 
obstruction with 
hydronephrosis or 
renal dysfunction 
Obstruction 
causing life-
threatening 
consequences 
 
2 
INFECTION 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 


 
68
Infection (any 
other than 
HIV infection) 
Localized, no 
systemic antiμbial 
treatment 
indicated AND 
Symptoms causing 
no or minimal 
interference with 
usual social & 
functional 
activities 
Systemic 
antiμbial 
treatment 
indicated OR 
Symptoms 
causing greater 
than minimal 
interference with 
usual social & 
functional 
activities 
Systemic antiμbial 
treatment 
indicated AND 
Symptoms causing 
inability to 
perform usual 
social & functional 
activities OR 
Operative 
intervention (other 
than simple 
incision and 
drainage) indicated 
Life-threatening 
consequences 
(eg, septic 
shock) 
Basic Self-care Functions: Activities such as bathing, dressing, toileting, transfer/movement, 
1 
continence, and feeding. 
2 
Usual Social & Functional Activities: Adaptive tasks and desirable activities, such as going to work, 
3 
shopping, cooking, use of transportation, pursuing a hobby, etc 
4 
 
5 
 
6 
 
7 
 
8