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Impact of HCV core gene quasispecies on hepatocellular carcinoma risk among HALT-C trial patients

Mutations at positions 70 and/or 91 in the core protein of genotype-1b, hepatitis C virus (HCV) are associated with hepatocellular carcinoma (HCC) risk in Asian patients. To evaluate this in a US population, the relationship between the percentage of 70 and/or 91 mutant HCV quasispecies in baseline serum samples of chronic HCV patients from the HALT-C trial and the incidence of HCC was determined by deep sequencing. Quasispecies percentage cut-points, ≥42% of non-arginine at 70 (non-R70) or ≥98.5% of non-leucine at 91 (non-L91) had optimal sensitivity at discerning higher or lower HCC risk. In baseline samples, 88.5% of chronic HCV patients who later developed HCC and 68.8% of matched HCC-free control patients had ≥42% non-R70 quasispecies (P = 0.06). Furthermore, 30.8% of patients who developed HCC and 54.7% of matched HCC-free patients had quasispecies with ≥98.5% non-L91 (P = 0.06). By Kaplan-Meier analysis, HCC incidence was higher, but not statistically significant, among patients with quasispecies ≥42% non-R70 (P = 0.08), while HCC incidence was significantly reduced among patients with quasispecies ≥98.5% non-L91 (P = 0.01). In a Cox regression model, non-R70 ≥42% was associated with increased HCC risk. This study of US patients indicates the potential utility of HCV quasispecies analysis as a non-invasive biomarker of HCC risk.

The purpose of this study is to conduct a randomized, controlled trial to determine if long-term interferon therapy can reasonably reduce the risk of histologic progression to cirrhosis, decompensated liver disease and/or hepatocellular carcinoma in patients with chronic hepatitis C and advanced fibrosis or cirrhosis who failed to respond to previous interferon therapy.
A.2. Overview: Up to 1800 patients who meet the inclusion and exclusion criteria (Sections E and F) will be entered into a Lead-in Phase. They will be treated with a combination of peginterferon alfa-2a and ribavirin for a period of 24 weeks. Patients who have no detectable HCV RNA at week 20 will continue on combination treatment until week 48.
Patients who do not clear virus will be randomized 50:50 at week 24 to receive either peginterferon alfa-2a alone or no further therapy for the next three and a half years.
Nine hundred patients will be randomized and both randomized groups will be monitored quarterly during these 42 months. Biopsies for these patients will be obtained at 24 and 48 months after the start of the Lead-in Phase.
In August, 2001, to increase enrollment two modifications were made to the study design: Those Week 20 Responder patients with virologic breakthrough or relapse documented by detection of HCV RNA at weeks 36, 48, 60 or 72, and those patients who are adequately treated with pegylated interferon and ribavirin combination therapy outside of the HALT-C Lead-in (Express Group) will be considered for randomization into the maintenance phase. These patients will be followed for an additional 48 months and their biopsies performed 18 months and 42 months after randomization.
All treatment in the randomized phase will end in January 2007. A small number of patients may not have completed 48 months at that time. Every patient will stop treatment at that time point, regardless of what month of randomization they are in. These visits will primarily be to identify outcome events, and to provide information to patients concerning the current status of the trial. Some questionnaires, blood tests, and an ultrasonogram will be performed. This visit is not to take the place of patients establishing a relationship with a primary care or liver specialist physician once the Month 54 visit is completed.
Those patients who are currently being followed will be offered visits at 6 month intervals through October 2009. Depending on when the patient was randomized this visit will be a Month 54, Month

B. BACKGROUND
B.1. Natural history of chronic hepatitis C: Chronic hepatitis C, an illness caused by the hepatitis C virus (HCV), affects 4 million patients in the USA, and results in 10,000 deaths annually (1,2). Hepatitis C is the most common cause of liver transplant (3,4) and is a major predisposing factor to the development of hepatocellular carcinoma (HCC) (3,(4)(5)(6)(7)(8)(9) in the United States.
Additionally, hepatitis C produces debilitating fatigue in 12% of patients and a variety of extra-hepatic manifestations in 1% (chronic renal failure, hypothyroidism, etc.) (10). Recent evidence is consistent with a large population of undocumented hepatitis C patients that will yield triple the number of cases annually of hepatocellular carcinoma and decompensated liver disease in the next 20 years (1). The natural history of chronic HCV infection has not yet been fully defined (11). Current data suggest that the process runs an indolent course during the first two decades after initial infection, accounting for little morbidity and mortality. Serious sequelae are more likely to emerge as the disease process enters the third and fourth decades after infection. Three approaches have been used: the prospective study, the retrospective study, and the combined retrospective-prospective, otherwise referred to as non-concurrent prospective study. The prospective study would constitute the ideal approach but, as already noted, it suffers from the fact that the onset of the acute illness is rarely recognized and that the follow-up period needed is markedly protracted, lasting three or more decades, a difficult commitment for an individual investigator.
Currently, much of the reported data on the natural history of hepatitis C accrue from retrospective studies of persons with already established chronic liver disease, excluding from analysis the group that does not reach clinical awareness (12). Five prospective studies of transfusion-associated non-A, non-B hepatitis have been reported from the United States and Europe (9,(13)(14)(15)(16), the duration of evaluation ranging between 8 and 14 years. Clinical symptoms of varying type and severity were identified in 4% to 13% of patients, liver biopsies revealed the presence of cirrhosis in 16% to 24%, Hepatocellular carcinoma was reported in two of the studies (0.7% and 1.3%, respectively), and death attributed to liver disease was reported in between 1.6% and 6.0% over this period, averaging about 3%.
Although none of these five studies included a non-infected control group, and periods of follow-up were somewhat brief for the usually protracted course of chronic hepatitis C, the studies yielded important information. They demonstrated that over periods of about a decade to a decade and a half from acute onset, morbidity and mortality from liver disease could be clearly demonstrated, although at a relatively modest frequency.

Clinical Events of Chronic HCV
Version Date 07/21/2006 CONFIDENTIAL The majority of the clinical consequences of chronic liver disease (CLD) are related to the effect of progressive hepatic fibrosis in: 1) producing portal hypertension and 2) progressive decline in the functioning hepatic mass. Portal hypertension is usually present by the early cirrhotic stage and the amount of functioning hepatic tissue (functional mass) progressively decreases as the disease progresses from early to advanced cirrhosis (17,18). The interaction of these two factors produces most of the clinical outcomes of chronic liver disease. Thus, hypersplenism (19), marked collateral formation (with esophageal varices) and hepatic congestion related to portal hypertension characterize the cirrhotic patient (12)(13)(14)(15)(20)(21)(22)(23). Progressive CLD finally results in the major clinical events of variceal bleeding and ascites formation (12,15,20,21). A minimum portal pressure gradient  12 mm/Hg is required to initiate these clinical events, but the average portal pressure gradient at onset of these problems is greater than this (17 3 mm/Hg). (24)(25)(26). Although most cirrotic patients have varices, fewer than 30% of patients have variceal bleeding. The size and a p p e a r a n c e o f v a r i c e s c o r r e l a t e s wi t h t h e t e n d e n c y f o r b l e e d i n g ( 2 7 ) . A p a t i e n t ' s tolerance of a bleeding episode is related to the hepatic reserve as assessed by Ch i l d ' s c l a s s .
Ascites is a major clinical process that occurs spontaneously at the moderately advanced cirrhotic stage at a point when the functional mass is approximately half of normal (28-30). The 5 year survival after the onset of ascites is 20% (31)(32)(33). Further progression of liver disease leads to refractory ascites, hepatic encephalopathy, spontaneous bacterial peritonitis and renal dysfunction (34)(35)(36)(37)(38)39). The one-year survival is less than 50% in association with these clinical problems.
HCC is the cause of death in 10-20% of cirrhotics with chronic HCV and nearly all of these patients are at the cirrhotic stage (4)(5)(6)(7)(8). Iron overload, co-infection with other viruses, co-existent liver diseases and genetic factors may increase the likelihood of HCC (4)(5)(6)8). Symptomatic tumors are generally large and lead to an average survival of 3-4 months. There is no effective therapy for large tumors. Screening for HCC with alpha-fetoprotein (AFP) and ultrasound increases the detection of small tumors (40) that may be treated with surgery or alcohol injection (41,42).

Prognostic Models
The natural history and the prognostic factors in hepatic cirrhosis have been extensively studied but several aspects remain unclear and prognostic factors have not been validated fully (20,32,33,(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58). The level of hepatic fibrosis, Child-Turcotte-Pugh score, blood tests and/or the presence of clinical problems are traditional ways to assess prognosis. Most histologic scoring systems have cirrhosis as a single stage and the mean 10 year survival in patients with compensated cirrhosis is 50% (31,32,59). Clinical problems (particularly ascites) indicate patients with a poor prognosis. In the studies discussed above, those patients with compensated cirrhosis who developed ascites had a 2 year survival of 50% and 5 year survival of 20% (31)(32)(33). Various scores and models combining biochemical and histologic data have been proposed (45,46). One of these, the Child-Turcotte-Pugh score, has been shown to be a simple and valid index for assessing the severity of disease and prognosis in patients with cirrhosis (47)(48)(49).
Version Date 07/21/2006 CONFIDENTIAL Measurement of the functional hepatic mass could be useful in determining prognosis. Quantitative tests of liver function, such as the aminopyrine breath test, indocyanine clearance, as well as radiographic studies such as MRI, ultrasound, and CT scan have been evaluated in detection of cirrhosis and prognosis (30,(60)(61)(62)(63)(64)(65). Studies have demonstrated that the perfused hepatic mass (PHM) by quantitative liver spleen scan faithfully provides information regarding reserve hepatic function in patients with chronic liver disease (17,18,29,66). Quantitative expression of sulfur colloid distribution between liver, spleen and bone marrow expressed as PHM correlates with the severity of chronic liver disease by histologic assessment (17,29), severity of cirrhosis at peritonoscopy (66), and the functional hepatic mass of the explanted liver at transplant (66). The rate of decline in these functional indices may provide important insight into the natural history of HCV.

Factors Affecting Natural History
The major determinant of progressive liver disease in patients with chronic hepatitis C is hepatic inflammation which stimulates the progressive buildup of fibrosis (9,10,(12)(13)(14)(20)(21)(22)45,(67)(68)(69). This process generally takes 20-40 years from initiation of infection to death from cirrhosis and can be markedly accelerated by alcohol (3,(13)(14)(15)22,23). Other factors that are associated with a more rapid rate of progression are a high grade of inflammation, longer duration of disease and age (20). Male sex, iron overload and hepatic fat may be other risk factors for more rapid progression of fibrosis. The rate of fibrosis has been estimated in a selected population of patients with hepatitis C to be .133 METAVIR fibrosis units per year (20,70).
Viral factors that might determine outcome include the dose at the time of infection, genotype, and the presence of quasi-species (HCV genetic diversity). The effect of dose in initiating progressive disease is unknown (71,72).
Most studies have shown that genotype 1 provokes a more severe disease than does genotype 2 (73,74) and that 1b is more harmful than 1a. (53). The impact of HCV quasi-species was studied by comparing its presence among those with acute resolving hepatitis with those who progress to chronic hepatitis (75,76).
Host-related factors that might play a role include age at the time of infection, race, and gender. Genetic predilection warrants further study. With respect to age, the rate of disease progression seems to increase the older the age at the time of infection (20).
Extraneous factors that may conceivably promote progression include super-infection with other viruses (77)(78)(79)(80)(81)(82), smoking (83), exposure to defined or undefined environmental contaminants, or concomitant or pre-existing chronic alcoholism. Studies using univariate and multivariate regression analyses have suggested that chronic hepatitis C is worsened by infection with hepatitis B virus (84); this increases the risk of carcinogenesis as well (83,85).
An important co-factor in progression of chronic hepatitis C to cirrhosis and HCC is that of chronic alcoholism, even if it had existed only in the past. The relationship between HCV infection and alcoholic liver disease was first noted in early Version Date 07/21/2006 CONFIDENTIAL epidemiological surveys (86,87). It is now apparent that alcoholism may promote HCV replication (88,89). Numerous surveys using regression analysis target alcohol as a major factor in promoting progression of liver disease in persons with chronic HCV infection (90)(91)(92)(93)(94).
B.2. Response to interferon or interferon-ribavirin therapy: Virologic cure has been the major goal of treatment strategies. However, decrease in progression of fibrosis and attenuation of serious clinical events are secondary goals. Evidence supports the value of interferon in both aspects.

Virologic Cure
Interferon is the only medication acting either alone or in combination with other drugs that can cure a patient of chronic hepatitis C (95-97) through both anti-viral and immunologic mechanisms (98,99). The four types of interferons that have been evaluated in a large number of patients with chronic HCV infection have comparable safety and efficacy in the treatment of chronic HCV infection (97, [100][101][102][103]. However, less than half of the patients with chronic hepatitis C treated with interferon-alfa monotherapy develop a negative HCV RNA during treatment. Nearly all patients who respond to interferon do so within the first 3-4 months (104). No more than 20% have a sustained benefit from the initial course of interferon-alfa treatment as indicated by persistently normal SGOT/SGPT and approximately 11% of these will have virologic cure (105).
More potent anti-hepatitis C therapies have been developed. Combination therapy with interferon and ribavirin is more effective than interferon alone in inducing virologic, biochemical, and histologic improvement in both naïve and relapsed patients with chronic hepatitis C (106)(107)(108)(109)(110)(111). A sustained virological response of 38% and a sustained biochemical response of 36% has been demonstrated when this combination was used in naive patients (106). The response rate was better for non-1 genotype than genotype 1 (66% vs 28%) (106) and for low viral titer, (<2M) as opposed to high viral titers (43% vs 36%) (106). Combination therapy is well tolerated: side effects are similar to interferon alone, with the additional risk of hemolytic anemia (111). Twenty-four weeks of treatment with Intron-A plus ribavirin in patients who relapse following initial therapy with Intron-A yield a 48% sustained response. Similarly, consensus interferon (CIFN) in a dose of 15  g TIW for 48 weeks demonstrated a 13% sustained response rate in previous non-responders and a 58% response rate in previous relapsers (100,112,113,(114)(115)(116). However, despite improved therapy, more than 50% of all patients treated fail to clear HCV permanently.

Prevention of Progression and Adverse Clinical Outcomes
Although the major goal of therapy has been virologic cure, this is achieved in  40% of patients. However, patients treated with mono-therapy over the last 12 years appear to obtain secondary clinical benefits even without sustained virologic response.
B.3. Histologic improvement during treatment with interferon: Improvements in liver histology have been observed in numerous studies in which preand post-interferon treatment liver biopsies have been performed. Such improvements have not only been observed in patients who achieved a sustained virologic response, but in non-responding patients, who remain viremic, as well. In general, a histologic response to interferon therapy occurs in approximately 80% of patients who have achieved a biochemical and/or virologic response to interferon therapy and in about 40% of interferon non-responders (145)(146)(147)(148)(149)(150)(151)(152). Following a relatively brief, 6-12 month course of interferon, these histopathologic changes appeared to be confined to improvements in hepatic inflammation, with little change in hepatic fibrosis. However, since it is believed that hepatic inflammation drives progression to fibrosis (153)(154)(155)(156)(157), these observations suggest that long term interferon therapy may slow progression to more extensive fibrosis or cirrhosis.
A recent study has evaluated the hypothesis that long term interferon therapy, in those patients who remain virologic non-responders, may indeed prevent histologic progression (158). In this study patients underwent repeat liver biopsy following an initial 6-month course of interferon therapy. Those patients who exhibited a histologic response were randomly selected to either discontinue treatment and be followed prospectively over an additional two years, or to remain in interferon treatment at a dose of 3 mu TIW. After 2 years of continuous interferon therapy hepatic inflammation remained significantly less than at the pre-treatment baseline and hepatic fibrosis declined. In contrast, patients who discontinued interferon therapy had an increase in hepatic inflammation back to the pre-treatment baseline and an increase in hepatic fibrosis was observed after 2 years follow-up. A larger controlled randomized trial is now needed to confirm these preliminary observations.

B.4. Effect of interferon therapy on development of hepatocellular carcinoma:
A provocative report by Nishiguchi et al (159) suggested that treatment of cirrhotic patients with interferon reduced the incidence of HCC over a mean of 4.4 years from 38% in untreated controls to 4% in treated patients. What was most surprising about this report were the facts that a) such a profound impact over many years resulted from such a limited, six-month course of interferon therapy and b) the beneficial effect on incidence of HCC was not limited to interferon responders but occurred as well in patients who had not achieved biochemical or virologic responses. Schalm et al (160) summarized three studies on the impact of interferon therapy on the occurrence of HCC in cirrhotic patients with hepatitis C (159,(161)(162). Taken together, these three studies included 272 untreated patients, 371 nonresponders, and 60 sustained responders after interferon treatment, and the frequency of HCC in these three groups Version Date 07/21/2006 CONFIDENTIAL was 15%, 4%, and 0%, respectively, suggesting an apparent beneficial effect of interferon treatment in preventing HCC in cirrhotic patients with hepatitis C. Studies with similar results continue to appear in the literature. (163) At first glance, these data appear to provide strong support for the hypothesis that interferon therapy in cirrhotic patients with hepatitis C prevents HCC. Multivariate analysis, however, revealed that clinical differences at the time of entry, not interferon therapy, correlated with the incidence of HCC (160). In fact, progression from cirrhosis to HCC may be associated with such factors as genotype 1b (164-165) male gender, and age >60 (164), supporting the hypothesis that host and virus variables may play a more important role in the development of HCC than antiviral therapy. A similar conclusion was reached by Bruno et al (164) in a retrospective analysis of the incidence of HCC among 163 cirrhotic patients with hepatitis C. The apparent reduction of HCC in interferon-treated patients represented a treatment bias towards patients with less advanced disease.
Although the treatment bias in these studies does not support a role for interferon in preventing HCC, they do not prove that treatment will fail to be effective. The hypothesis remains viable that reduction of liver injury by antiviral therapy could slow the progression of fibrosis to cirrhosis, reduce the rate of new fibrosis in cirrhotics, and, as a consequence, reduce the likelihood of decompensation and even delay the onset of HCC. The only way to test this hypothesis adequately is in a prospective controlled trial. Given that a 1-4% annual incidence of HCC has been observed in cirrhotic patients with chronic hepatitis C (164-167), a prospective trial of sufficient study size and duration would have the power to identify an interferon-associated reduction in HCC.

B.5. Pegylated interferon (peginterferon):
A candidate drug to be used for long-term antiviral therapy must have an acceptable safety/tolerability profile. Considerable experience and safety data are available for interferon, and many patients have been treated for many years without an appreciable increase in adverse events over time. Ribavirin is now included routinely in combination therapy for chronic hepatitis C, but its safety profile when taken over several years has not been established. Because ribavirin causes hemolysis in a patient population with cirrhosis, leading to anemia, the risks of using the drug will be increased. Therefore, the use of combination therapy, especially for the four-year duration of this trial, is not planned.
Although standard preparations of interferon are available as candidate treatments for this trial, we propose the use of peginterferon, a long-acting preparation of interferonalfa conjugated to polyethylene glycol (PEG). Preliminary observations with peginterferon suggest that it is absorbed at rates slower than that of interferon-alfa, with a delayed Tmax and a prolonged concentration peak, achieved primarily as a result of the much longer (median seven-fold longer) elimination time of peginterferon. In addition, drug concentrations remain more stable and sustained over time than they do with more frequent administration of shorter-acting preparations with their resultant peaks and troughs. Trials of peginterferon given by subcutaneous injection once a Version Date 07/21/2006 CONFIDENTIAL week have shown levels of biochemical and virologic response comparable to those achieved with standard interferon-alfa given three times a week. Moreover, a recent trial of peginterferon among previously untreated patients with chronic hepatitis C suggested that the long-acting interferon may be more effective than standard interferon. Among patients treated with peginterferon, the sustained response rate measured six months after completion of therapy was 36% (168), which is comparable to the efficacy reported for combination interferon-ribavirin therapy (169)(170). By relying on therapy that can be administered once a week, we expect a higher patient acceptability, and therefore a longer enrollment and better compliance.
Data available indicate that adverse events associated with peginterferon are comparable to those observed in patients treated with standard interferon-alfa, and in multiple-rising-dose studies, fewer patients taking peginterferon reported adverse effects compared to those taking standard interferon-alfa. Recent experience in dosefinding studies suggest an increase in thrombocytopenia and neutropenia among peginterferon-treated patients compared to interferon-alfa-treated patients, although seldom leading to dose modification or discontinuation. Therefore, more vigilant platelet-count and WBC monitoring will be necessary in a trial of peginterferon, especially in cirrhotic patients.

B.6. Summary and Study Rationale:
Chronic, low-grade inflammation and liver injury due to hepatitis C infection leads to fibrosis and the development of cirrhosis in a significant portion of infected patients.
Although viral eradication has been the primary goal of treatment of hepatitis C thus far, patients who are non-responders constitute 50% of all those with hepatitis C. The evidence presented here suggests that interferons may slow or arrest progression of injury-related fibrosis, even in non-responder patients who fail to clear the virus. Regardless of whether these potential benefits are related to viral replication or to a direct anti-fibrotic mechanism, this potential benefit of interferon therapy needs further confirmation.
The aim of the present study is to determine whether prolonged interferon-based therapy can be achieved and maintained in a reasonable number of subjects over several years' time, and whether the anticipated benefits are worth the risk and expense involved. The study population will be limited to those at highest risk of progression to cirrhosis or its complications, namely, patients with established fibrosis or cirrhosis on biopsy at study entry. Since all patients enrolled will have had different previous treatments, the present study design provides that all patients entering the long-term portion of the study have begun at a common starting point and have had a second chance at reaching a sustained virologic response. In the initial or lead-in course of therapy all patients will be treated with peginterferon plus ribavirin for a period of 24 weeks, with virologic assessment at 20 weeks to refute their nonresponder status. Thus, patients entering the long-term study will know that they have been given the very latest regimen in an attempt to achieve virologic clearance and, having failed, now can be certain that they are non-responders to conventional therapy It is anticipated that approximately 20% will have no detectable virus in serum at the 20 week point, and that this group may continue treatment and receive a full course of Since patients with both fibrosis and cirrhosis will be included, therapeutic endpoints will be of two types: histologic progression of disease as well as development of evidence of cirrhotic decompensation. Ancillary studies will help define the role of interferon in slowing the evolution of portal hypertension and the role of fat change in disease progression. The extent of side effects of interferon as well as a detailed assessment of quality of life will be obtained for all study participants. Further studies will be aimed at increasing our understanding of the complex interactions of virus and host in this intriguing disease.
The purpose of the continued observation in this long-term follow-up phase of the HALT-C Trial is to determine the outcome of patients enrolled in this study over a longer time interval.

C. HYPOTHESES:
C.1. In patients with chronic hepatitis C who failed to respond to previous interferon therapy, long term treatment with interferon can safely prevent progression of advanced fibrosis to cirrhosis.

E. INCLUSION CRITERIA
Patients can be screened for the HALT-C T r i a l a s e i t h e r " L e a d -i n " o r " E x p r e s s " patients. Lead-in patients are treated for 24 weeks with peginterferon alfa-2a and ribavirin as part of the HALT-C Trial. Patients who have been treated with peginterferon and ribavirin outside of the HALT-C Trial can bypass the Lead-in Phase a n d b e r a n d o mi z e d a s " E x p r e s s " p a t i e n t s .
Sections E1, E2, E5, E8, and E9 are for all patients. Sections E3, E4, and E6 are for " L e a d -i n " p a t i e n t s a n d E 7 i s f o r " E x p r e s s " p a t i e n t s .  E.7.c. A pre-treatment liver biopsy which is available and has been performed within 18 months of randomization or a liver biopsy which is performed at least 8 weeks after the end of treatment and no more than 24 weeks prior to randomization. Slides from the biopsy must demonstrate at least Ishak stage 3 fibrosis as judged by the clinical center pathologist and then must be read centrally before randomization. E. 7. d. Patients with a history of Ishak stage 3 or higher fibrosis, who subsequently have fibrosis scored as Ishak 2 can also be enrolled as Express patients. Both biopsies must be read by the local HALT-C pathologist using the Ishak fibrosis scoring system. The most recent Ishak fibrosis score will be used for determination of the study outcome. 3. Liver histology, in the opinion of the study pathologist, that is consistent with either primary biliary cirrhosis or sclerosing cholangitis. F.1.e. Alpha-1-antitrypsin deficiency as defined by both of the following criteria: 1. A serum value for alpha-1-antitrypsin less than normal.
2. Liver histology which, in the opinion of the study pathologist, is consistent with alpha-1-antitrypsin deficiency.
F.1.f. Hemochromatosis or secondary iron overload as defined by 1 and 2 below: 1. An elevated value for serum ferritin or an iron saturation (serum iron/IBC x 100%) of greater than 50% and 2. Presence of 3+ or 4+ stainable iron on liver biopsy according to the study pathologist or a history of previous phlebotomy for iron overload.
All patients meeting the above criteria must undergo HFE genetic testing. Patients with an HFE genetic test demonstrating homozygosity for C282Y or compound heterozygosity, i.e. C282Y +/-and H63D -/+ are not eligible. F.24. Patients with a history of any one of the following: Suicide attempt or hospitalization for depression within the past 5 years.
2. The following patients must be assessed and followed (if recommended) by a psychiatrist or other mental health professional: a. Patients who have had a suicide attempt and/or hospitalization for depression more that 5 years ago. b. Patients who have had a severe or poorly-controlled psychiatric disorder (e.g., depression, schizophrenia, bipolar illness, obsessive-compulsive disorder, severe anxiety, personality disorder) more than 6 months ago but less than 5 years ago.

G. SCREENING OF PATIENTS
G.1. Identification of potential patients.
G.1.a. The medical records of patients with chronic hepatitis C who have been previously treated with interferon or interferon/ribavirin combination therapy, and who are being followed or have been recently referred to a HALT-C Trial Treatment Center will be reviewed. G.1.e. Recent legislation has required that the NIH ensure that federally-funded clinical trials include sufficient numbers of women and designated minority groups to determine whether the intervention affects women or members of minority groups differently from other subjects. The goal for this trial is to recruit at least 20% from minority groups and 30% women.
G.1.f. Screening for the HALT-C Trial is expected to last for 2 years.
G.1.g. Confidentiality of subject data will be maintained throughout the trial. No subject identifiers will be used and all data entered into the trial database will be under code.
The HALT-C Trial has received a Certificate of Confidentiality from the Department of Health and Human Services (DHHS). This certificate protects the investigators from being forced to release any research data with patient identifiers, even under court order or subpoena, without written consent from a patient. This includes information collected on genetic testing and HIV status, excluding required notification of health authorities for HIV positive test results. This protection, however, does not prohibit the G.1.h. The Principal Investigator or Co-Investigator, or approved designee will obtain informed consent from patients after full review of the pros and cons of the study. The possible benefits and complications of participation will be explained in detail. Every effort will be made to ensure that the patient fully comprehends the nature of the study and the details of his or her participation. A copy of the consent form will be provided to the subject.

G.2. Screening of patients.
G.2.a. The specific aims and general conduct of the protocol will be reviewed with each potential patient.
G.2.b. If the patient wishes to be screened for possible inclusion in this study, he or she must then sign the screening consent form.

G.3. Screening visit #1:
The patient will then undergo the first of two screening visits that will include the following: G.3.d. Eligible subjects with a history of severe or dose-limiting neuropsychiatric toxicity during prior interferon treatment will be referred to a consulting psychiatrist/psychologist. In addition to clarifying possible psychiatric disorders, the consultant will help determine current suitability for this study and the need for prophylactic therapy such as anti-depressants or counseling.

G.4. Screening visit #2:
All patients who meet the study criteria for the first screening examination will then proceed to the second screening visit and undergo the following testing: G.4.a. Laboratory blood tests to include:

H. LEAD-IN PHASE:
All patients who meet and completely fulfill study criteria at both screening visits are eligible to enter the Lead-in Phase of this trial except eligible Express patients who will enter the Randomized Phase of this trial. H.1.b. Baseline data will be obtained as shown in Table 1.
H.1.c. Instructions and study medications will be distributed to patients.
H.1.d. All patients who have neutrophils >1,500/mm 3 and platelet count >75,000/mm 3 will initially be treated for 24 weeks with peginterferon alfa-2a180  g once weekly plus ribavirin 1000-1200 mg (prescribed according to weight <75kg, >75kg) daily in two divided doses. Patients previously intolerant to ribavirin may receive peginterferon alfa-2a alone, at the discretion of the site investigator.
H.1.e. Patients who are admitted to the study with platelet count from 50,000/mm 3 up to 75,000/mm 3 and/or neutrophils between 1,000/mm 3 to 1,500/mm 3 , can be enrolled but the following special precautions will apply: Safety measures for patients with platelet count under 75,000/mm 3 and/or neutrophils under 1,500/mm 3

The ribavirin dosage may b e u n c h a n g e d o r l o we r e d a t t h e P I ' s d i s c r e t i o n .
Patients will start at a reduced dose of 90  g of peginterferon alfa-2a once weekly.
Version Date 07/21/2006 CONFIDENTIAL 2. Patients will be monitored more closely with the addition of a CBC with differential at Week 1 and Week 6.
3. Patients will be asked to hold weekly dose of peginterferon alfa-2a until the results of the CBC with differential are assessed by the PI.
4. The first 25 patients entering the Lead-in Phase with a lowered platelet and/or neutrophil count will have ongoing monitoring by the DSMB for the first 8 weeks of treatment.

5.
A separate dose reduction scheme will be followed for these patients (see Appendix D, Table c).
6. Dose adjustment upwards of peginterferon alfa 2-a should be made at the discretion of the PI after 8 weeks in the Lead-in.
H.1.f. G-CSF or GM-CSF may not be used in the Lead-in Phase.

H.2. Weeks 2-20.
During this lead-in treatment phase all patients will be seen and examined at regular intervals, and will undergo various laboratory studies as defined in Table I. I.3.b. Blood will be drawn and questionnaires will be completed at each visit according to schedule in Table 3.  J.1.d. Prior to randomization, patients will be stratified on the basis of the presence or absence of cirrhosis, as determined by central reading of baseline (Screening) biopsy. Breakthrough/Relpase patients will be stratified on the basis of the central reading of the most recent liver biopsy. Patients will not be excluded if the central reading of their biopsy is less than fibrosis stage 3 J.1.e. All eligible patients will be randomly assigned to one of two groups as follows:

I. CONTINUED FOLLOW-UP OF WEEK 20 RESPONDERS
1. To continue treatment with peginterferon alfa-2a alone at a dose of 90  g administered once weekly for an additional 42 months.
If a patient entering the Randomized Phase has been on 45 g peginterferon alfa-2a, a dose increase back to 90 g may be made at the Principal All patients, in both arms of the study, will be seen at regular intervals, examined and undergo various laboratory studies as defined in Table 2. Results of these tests and procedures may be shared with the patient.
In the event that a patient who is randomized cannot/will not complete all of the scheduled study visits, the patient will be offered HCV-RNA testing, an ultrasound, a liver biopsy and endoscopy at their last study visit, if deemed appropriate by the PI, in addition to all regularly scheduled tests and procedures. Every effort will be made to continue collecting any data possible on all patients who have been randomized.

J.3. Liver biopsy:
All patients will undergo repeat liver biopsy at study visit month 24 and study visit month 48 regardless of treatment group.
Only percutaneous biopsies will be performed. Patients with a prothrombin time 1.5 INR or platelet count 50,000 will not be biopsied.

J.4. Post-treatment follow-up:
Patients in both groups will be followed, but not treated, for an additional 6 months after study visit Month 48 ( L.3. Randomized patients who stop receiving peginterferon alfa-2a will continue to be followed at regular visit intervals for the duration of the trial (if possible). Patients who discontinue treatment during the Lead-in Phase will not be followed beyond 24 weeks except for follow-up on all unresolved adverse events.

M. DOSE MODIFICATION:
M.1. Peginterferon alfa-2a dosing: M.1.a. Factors that will lead to a reduction in the dose of peginterferon alfa-2a include: 1. Disabling symptoms, which, in the opinion of the investigator, are related to peginterferon alfa-2a treatment and prevent the patient from performing his/her occupation or daily tasks.

A rash consistent with allergic reaction or vasculitis.
3. Reductions in the platelet count according to the guidelines in Appendix D4 (page 67).

A reduction in neutrophil count according to the guidelines in Appendix D4
(page 67).
5. Any adverse reaction, which, in the opinion of the investigator, places the patient at increased risk.
M.1.b. The dose of peginterferon alfa-2a may be reduced as follows: There are 3 prescribed levels for dose reduction: During the randomized phase 90 g may be reduced to 45  g for cytopenias or other side effects. Peginterferon alfa-2a can be increased to 90 g a t t h e i n v e s t i g a t o r ' s discretion.  M.2.a. The dose of ribavirin will be reduced during the Lead-in Phase as follows:

M. 1 . c . On c e a p a t i e n t ' s d o s e h a s b e e n d e c r e a s e d , t h e i n v e s t i g a t o r ma yattempt
If either of the following is confirmed: (1) a patient without significant cardiovascular disease experiences a fall in hemoglobin to <10 g/dL and >8.5 g/dL or (2) a patient with stable cardiovascular disease experiences a fall in hemoglobin by >2 g/dL during any 4 weeks of treatment, the ribavirin dose should be reduced to 600 mg per day (200 mg in the morning and 400 mg in the evening). Further reductions may be considered.
Patients who have more than a 3 g/dL decrease from baseline in their hemoglobin concentration should have consideration given to an appropriate work-up for anemia, including reticulocyte count, search for sources of bleeding, etc., especially if a further drop occurs following some weeks of apparently stable hemoglobin levels on ribavirin.
M.2.b. If a patient cannot tolerate ribavirin, then he or she can be treated with peginterferon alfa-2a alone and randomized, providing eligibility criteria are met. Ribavirin should be discontinued under the following circumstances: 1. If a patient without significant cardiovascular disease experiences a fall in hemoglobin confirmed to be less than 8.5 g/dL.
2. If a patient with stable cardiovascular disease maintains a hemoglobin value <12 g/dL despite 4 weeks on a reduced dose [21].
Version Date 07/21/2006 CONFIDENTIAL In the event of ribavirin being discontinued, it can be reintroduced at a daily dose of 6 0 0 mg a n d

i n c r e a s e d t h e r e a f t e r a t t h e i n v e s t i g a t o r ' s d i s c r e t i o n . I n t h e e v e n t o f r i b a v i r i n b e i n g r e d u c e d , i t c a n b e i n c r e a s e d a t t h e i n v e s t i g a t o r ' s d i s c r e t i o n . I t i s n o t
considered necessary to tailor the reduced dosing according to the 75 kg cut-off level.

M.3. Pregnancy:
M.3.a. If a patient becomes pregnant during the Lead-in Phase, treatment will be stopped and she will not be eligible for the Randomized Phase of the trial.
M.3.b. If a patient becomes pregnant during the Randomized Phase, treatment will be discontinued for the duration of the pregnancy. Treatment may be resumed three months post-partum if the patient is not breast feeding.
M.3.c. I f a ma l e p a t i e n t ' s p a r t n e r ( s ) b e c o me s p r e g n a n t d u r i n g t h e L e a d -in Phase, ribavirin will be stopped, but not peginterferon alfa-2a. The male patient and his pregnant partner will be advised to use a barrier method of contraception for the remainder of the pregnancy, and post-p a r t u m i f t h e ma l e p a t i e n t ' s p a r t n e r i s b r e a s t feeding.

N. ADVERSE EVENTS
N.1. Definition N. 1 . a . A n a d v e r s e e v e n t i s a n y a d v e r s e c h a n g e f r o m t h e p a t i e n t ' s b a s e l i n e ( p r etreatment) condition, including intercurrent illness which occurs during the course of the trial, after the consent form has been signed, whether the event is considered related to treatment or not.
N.1.b. A serious adverse event is an untoward medical occurrence that results in any of the following: 1. Death 2. Is life threatening (risk of death at the time of the event). 3. Requires in-patient hospitalization or prolongation of existing 4. hospitalization 5. Results in persistent or significant disability/incapacity 6. Congenital abnormality or birth defect Important medical events that do not result in one of the events listed above may be considered a serious adverse event when, based upon appropriate medical judgment, they may jeopardize the patient and may require medical or surgical intervention to prevent one of the outcomes listed above.
N.1.c. A serious adverse event which is unexpected and is drug related (even remotely) will require expedited reporting (see N.3.a.).
N.1.d. The following events are trial outcomes and will not be considered to be serious adverse events: 1. Development of HCC or presumed HCC 2. A CTP score of 7 or higher at 2 consecutive study visits N.2.c. Patients will be followed for all ongoing unresolved adverse events until they are either resolved, or in the opinion of the Principal Investigator, the patient is medically stable.
N.3. Reporting procedures N.3.a. All serious adverse events, for patients in both the treated and the control group will be reported to the DCC and to Roche within 24 hours by telephone. The Serious Adverse Event form will be faxed to the DCC and Roche within 24-48 hours. This reporting includes serious adverse events that occur from the time the patient has signed the Screening or Trial informed consent to study visit Month 54 (6 month post Trial follow-up). Serious adverse events on patients who are in the control arm of the Randomized Phase and have stopped treatment for 12 weeks or patients who have not yet received treatment need to be reported only to the DCC. Roche will review all serious adverse events and will notify the DCC if an event is considered to be unexpected. The DCC will distribute the expedited report to NIDDK, the FDA, the DSMB, and clinical centers.
Status reports on serious adverse events will be generated by the DCC and sent to the DSMB every month and will include the relationship of the adverse event to trial medications, the severity of the event and if the event is resolved or ongoing. Adverse events will be reported annually to the FDA, as required.
N.3.b. All deaths, in both the treated and in the control group will be reported to the DCC within 24 hours by telephone. The Death Report will be faxed to the DCC within 24 hours. This reporting begins at the time the patient has signed the Trial informed consent up to the last scheduled patient visit (through October 2009). The report will include the relationship of the death to trial medications. A Clinical Outcome form will also be completed and sent to DCC for distribution and review by the Outcome Review Board (ORB). Deaths will be reported immediately to NIDDK and the DSMB. A death Version Date 07/21/2006 CONFIDENTIAL will be reported in an expedited report only if it is unexpected and drug related. A death must also be reported in accordance with local law and regulations.

O. SAMPLE SIZE
O.1. Estimates of rates for the primary outcome variable O.1.a. The primary outcome variable for the trial will be the progression of liver disease as judged by either: 1. An increase in the Ishak fibrosis score of 2 points or more at the study visit month 24 or study visit month 48 biopsies, or 2. The development of hepatic decompensation (as defined in K.1.), the development of HCC, or death from any cause. O.2. Sample size estimate O.2.a. The primary variable for this trial is the percent of patients whose liver disease has progressed (as defined in K.1.) at the time of the biopsy at study visit month 48. The sample size estimate is based on a chi-square test (171).
The null hypothesis is that the percent of patients with progression of liver disease will be the same in the peginterferon alfa-2a treated and control arms. The alternative hypothesis is that there will be at least a 50% reduction in the annual rate of progression in the peginterferon alfa-2a arm when compared to the control arm.
O.2.c. The sample size is based on a two-sided alpha (type I error) of 5% and a power of 90% (type II error).
O.2.e. Noncompliance rates of 3%, 6%, and 9% have been assumed for the peginterferon alfa-2a arm in years 2, 3, and 4. These patients will continue to be followed and will experience progression at the rate of the control arm.
O.2.f. Similarly, it is assumed that 5%, 10% and 15% of the control arm will elect to be treated and will receive peginterferon alfa-2a in years 2, 3 and 4. These patients will continue to be followed and will experience progression at the rate of the peginterferon alfa-2a arm.
O.2.g. Under these assumptions, 18.7% of the control arm and 10.6% of the peginterferon alfa-2a arm will have had progression of their liver disease 3.5 years after randomization. A sample size of 810 patients (405 per arm) will have 90% power to detect this difference. Nine hundred patients will be randomized for this trial, allowing for a loss to follow-up of 90 patients.

. Power Analyses
The following table shows the power for annual rates in the control arm of 5% and 6% and several noncompliance rates. It also shows the effect of assuming that the prerandomization treatment protects the control arm for 0 months or 3 months.

P. DATA MANAGEMENT AND ANALYSIS
P.1. Data Management P.1.a. The DCC will provide a web-based data entry system. This system will be an a d a p t a t i o n o f NE RI ' s A d v a n c e d Da t a E n t r y a n d P r o t o c o l Ma n a g e me n t S y s t e m (ADEPT). Clinical Center staff will use this system for data entry of study forms. Information entered into the data entry system will be by patient study ID; names will not be linked with patient data in the database. Clinical centers will maintain records linking the patient name with the ID assigned for this trial in locked files. The data entry system will include context specific help, automatic skip patterns, range checks, and intra-and inter-form checks of the data as it is being entered. P.1.b. The system will produce visit schedules to assist the Clinical Center staff in the scheduling of appointments and visit control sheets that will list all of the forms and procedures for a scheduled visit. The system will also be able to produce lists of data as needed.
P.1.c. The data sets created by ADEPT will be converted to SAS data sets at the DCC. Additional data editing will be performed at the DCC using SAS. Outliers will be identified using predetermined limits and graphical methods. These edits will also compare patient data across forms and over time. Edit messages will be sent to the Clinical Centers for clarification as needed. P.2.b. The analyses of the baseline data will include Clinical Center comparisons, comparisons of patients who are randomized and those who are not, and comparison of the two randomized treatment groups. An analysis of missing data will also be prepared.
P.2.c. Baseline characteristics that form continuous variables will be compared using analysis of variance or t-tests. Data will be transformed (e.g. logarithms) prior to analysis if transformation is needed to meet the assumptions of normality and homogeneity of variances that underlie these methods. Non-parametric methods, such as the Wilcoxon test, will be used if data fail to meet these assumptions even after transformation (172).
P.2.d. Characteristics that form ordinal categorical variables, (e.g., Child-Turcotte-Pugh score, biopsy severity scores) may be compared using analysis of variance or ttests if the number of categories is large enough for the data to approximately fit the assumption of normality. Otherwise non-parametric methods or methods for categorical data will be used. Categorical variables (e.g., presence or absence of cirrhosis) will be analyzed using chi-square tests (173,174). P.2.e. It is expected, due to randomization, that the patients in the treatment arm will, on average, be similar with respect to variables that might influence outcome. However, for imbalances that persist despite randomization, co-variates or strata will be included in the analysis of the primary and secondary endpoints. P.3. Analyses of Lead-in Phase P.3.a. The primary outcome for the Lead-in Phase is the disappearance of HCV RNA. Logistic regression will be used to determine the characteristics that are predictive of a response to peginterferon alfa-2a and ribavirin. P.4. Analyses of the Primary Outcome P.4.a. All randomized patients will be included in the analysis of the primary outcome. This includes patients who are later found to be ineligible and patients who do not receive the assigned treatment. P.4.b. The primary outcome of this trial is progression of liver disease as defined in Section K.
Version Date 07/21/2006 CONFIDENTIAL P.4.c. The analysis of this outcome is complicated because this is a composite outcome that consists of both the results of biopsies obtained at two-year intervals and the occurrence of events that may occur at any time during the follow-up period. In addition, the biopsy outcome, the development of cirrhosis, can occur only in patients in stratum 1 (pre-cirrhotic). Patients in stratum 2 will be much more likely to develop clinical events than patients in stratum 1.
P.4.d. If missing data are minimal, then the percent of patients having an outcome at the end of four years can be compared using logistic regression. This method will have less power than methods based on survival analysis. Several methods have been proposed for the analysis of interval-censored survival data (175)(176)(177)(178). The method to be used will be described in detail in the Manual of Operations (MOO). P.4.e. Hepatic events will be evaluated using life table methods and proportional hazards regression (45). The primary analysis will be life table analyses of time to first event. Life table estimates of time to hepatic event will be calculated for the two treatment groups with clinical center and baseline presence or absence of cirrhosis as strata. The log rank test will be used to test the difference between the treatment groups.
P.4.f. Analyses will be conducted for all hepatic events and the subset of death, HCC, or UNOS status 2b as defined in 1999.
P.5. Interim analyses P.5.a. An interim analysis will be performed once 50% of the study visit Month 24 biopsies have been evaluated by the Central Pathology Committee. We recommend i mp l e me n t a t i o n o f a n e a r l y s t o p p i n g r u l e b a s e d o n a n O' B r i e n -Fleming group sequential plan (179). This will be implemented using the alpha spending approach of Lan and DeMets (180-181).
P.5.b. Once the early stopping rule is established, NERI will produce interim analyses for the DSMB that will include: 1. Standard recruitment graphs comparing actual to goal over time and projected completion point.
2. Detailed tables and figures of recruitment experience at each CC (Number, by month or quarter by CC, as well as gender, age and minority crosstabulations by CC).
3. Tables comparing baseline characteristics, cumulatively, at each interim analysis by (blindly labeled) treatment groups. These tables will be presented in aggregate as well as by CC.
4. Protocol and eligibility violations in trial patients will be reported since the last interim analysis and cumulatively, by CC with full documentation. P.5.c. It is anticipated that the proposed stopping rule will have asymmetric boundaries to reflect the overriding concern with minimizing the possible deleterious Each of these components are discussed below. Figure 2 summarizes the main lines of communication between them.

Q.1.a. NIDDK
The Project Officer for the HALT-C Trial will be responsible for the close coordination of all aspects of the trial. All technical direction for the trial resides with the Project Officer who will serve as executive secretary of the Data and Safety Monitoring Board (DSMB) and will serve as a liaison between the DCC and the DSMB. The Project Officer will assist in quality control, interim data and safety monitoring, final data analysis and interpretation, preparation of publications, and coordination and performance monitoring. A representative of NIDDK will serve on each committee.

Q.1.b. Clinical Centers (CC)
Each clinical center will need to recruit as many as 180 patients and randomize and follow approximately 90 patients. Clinical Centers will be responsible for collecting trial data, recording information on the trial forms, sending these data to the DCC, and collecting and shipping specimens to the Repository. Key Clinical Center responsibilities include: The DCC shall have primary responsibility for the following tasks:  Preparation of the trial protocol, forms, data management system, and a management plan  Monitoring and support of enrollment and follow-up  Compilation and preparation of the data, assuring data quality, and data analysis  Administrative support of the work of the Project Officer and the trial subcommittees  Participation in manuscripts and presentations  Developing and monitoring all Quality Assurance/Quality Control procedures Q.1.d. Virology Laboratory (VL) This central laboratory will perform key assays for the trial. Key responsibilities include:  Participation in protocol development  Leadership in development of the specimen processing and analysis protocols and production of the laboratory  Completion of all assays in a timely manner  Implementation of adequate quality assurance in assay performance  Participation in manuscripts and presentations Q.1.e. Central Repository (CR) SeraCare Bio Services, formerly BBI-Biotech Research Laboratories, Inc, will serve as the Central Repository for the trial. This repository will be responsible for receipt, storage and distribution of serum, plasma, peripheral blood mononuclear cells (PBMC) and frozen liver tissue.
Specimens will be sent to the repository for indefinite storage. In the initial treatment phase, blood and tissue will be collected and white blood cells will be used for the generation of cell lines as well as for the extraction of DNA. These materials will be stored for genetic testing. Genetic tests will be confined to testing for genes relating to viral hepatitis and other liver diseases. It is possible that as new genes are identified that may be related to immune response to chronic viral infections such as hepatitis or o t h e r l i v e r d i s e a s e s o r l i v e r c a n c e r , p a t i e n t ' s DNA ma t e r i a l wi l l b e u s e d f o r s u c h t e s t i n g in order to improve our knowledge of the factors that influence disease progression in patients with hepatitis C.
Blood, cell lines, and tissue will be stored under a unique identifier, which can be traced back to the patient. If there is a medical reason to seek specific information from the patient in the future, patients will be notified and if appropriate, genetic counseling will be offered.   The primary purpose of this Board will be to monitor and provide independent ethical oversight for the trial. The DSMB will provide advice to NIDDK, as needed. This board will review the protocol developed during Phase 1, plans for recruitment and follow-up, and any other questions pertinent to the ethical conduct of the trial. It will review protocol changes and might suggest changes as needed. During Phase 2, it will monitor the data at regular intervals to determine whether significant benefit or harm has been demonstrated in either treatment group or whether there is other compelling need to stop the trial.
If the DSMB finds the treatment to be effective all patients enrolled into HALT-C will be notified. However, funds are not currently available to provide treatment to all study participants. Every effort will be made to assist all patients in obtaining treatment with peginterferon alfa-2a.
During the extension, a DSMB will continue to monitor the study.
The Principal Investigator of the DCC, the Project Officer, and at least one scientific advisor from NIDDK may participate as ex-officio, not-voting members of this board.

Q.1.j. Outcome Review Board (ORB)
An Outcome Review Board will be appointed to review all outcome events (Section K). Members of the Board will include Principal Investigators from the Clinical Centers. Clinical Centers will submit to the DCC the appropriate forms and source documents. Copies of these documents will be sent to two members of the ORB, and to a third if there is not consensus. Outcomes will be evaluated by the ORB based on predetermined criteria for each outcome variable. ORB members will not know the treatment status of patients.

Q.2. Changes to the Protocol
T h e S t e e r i n g Co mmi t t e e , NI DDK , c l i n i c a l c e n t e r I RB ' s a n d t h e DS MB mu s t a p p r o v e a l l changes to the protocol.

Q.3. Publication Policies
A Publications Committee will review all publications. No trial data may be presented or published without prior approval of the Publications Committee. Detailed publication policies will be included in the Manual of Operations.

Q.4. Ancillary Studies:
The Ancillary Studies Committee recommends and the Steering Committee approves all ancillary studies in the HALT-C Trial. The ancillary study policy and detailed protocols are in the HALT-C Manual of Operations.      1-3). Therefore, a heightened awareness of depression and its potential impact on patient safety and compliance is necessary when prescribing interferon.

Detection of Depression
In the HALT-C Trial, the CIDI will be administered pretreatment and the Beck Depression Index-II (BDI-II) will be administered every 3 months during treatment. In order to provide rapid and reliable psychiatric services for enrolled patients, a collaborative psychiatrist/ psychologist should be identified in each center for urgent and elective referrals.

CIDI
The CIDI-Auto 2.1 is the computerized version of the Composite International Diagnostic Interview (CIDI) developed by the World Health Organization (4). The CIDI is a comprehensive, fully standardized interview that can be used to assess mental disorders and provide diagnoses according to the definitions and criteria of the ICD-10 and DSM-IV (See Attachment 1). The CIDI can be administered by trained study personnel, does not require outside informants or medical records, and can be completed in 20-40 minutes.
Beck Depression Index-II (BDI-II) The BDI-II is a 21 item, self-administered survey used to screen for and monitor depression that takes 5-10 minutes to complete (5). The BDI-II has been shown to provide valid and reliable information in follow-up studies of patients with either psychiatric illness or medical illness (6,7). Although no arbitrary scores are available that can be used on all patients to classify the severity of depression, specific interpretation guidelines are available (8). The BDI-II was developed for the assessment of symptoms corresponding to DSM-IV criteria for diagnosing depressive disorders in 1996. The BDI-II has been extensively tested and validated and is felt to be an improvement over the previous versions of the Beck(9).
Version Date 07/21/2006 CONFIDENTIAL 4. The BDI-II is scored by summing the ratings for 21 items. Each item is rated on a 4 point scale ranging from 0-3.

5.
Subjects with abnormal BDI-II scores (range: 11 to 63 with higher scores indicative of more severe symptoms) should be assessed and managed as follows: BDI-II score Clinical Picture 0 -10 none to minimal 11 -14 Mild depression 15 -19 Mod depression 20 -28 Severe depression >29 Critical 6. Practitioners should keep in mind that all self-report inventories are subject to response bias. That is, some individuals may endorse more symptoms then they actually have and thus produce spuriously high scores while others might deny symptoms and receive spuriously low scores. In addition, the practitioner is cautioned that the BDI-II may simply reflect the degree of depression, not the diagnosis of depression. Determination of the severity of depression and the establishment of a diagnosis of depression require examination by a clinician (physician or psychologist/ psychiatrist).
7. Because the BDI-II total score provides only an estimate of the overall severity of depression, it is important to be attentive to specific items regarding suicidal ideation. Patients admitting to suicide ideation (Item 9) and hopelessness (Item 2) with a rating of 2 or 3 should be closely scrutinized for suicide potential.
8. Any patient who develops recurrent suicidal ideation, a suicide plan and/ or makes a suicide attempt should have IFN immediately discontinued and be referred to a psychiatrist for further management.
9. Suggested guidelines for interferon dose reduction:  Unpleasant and/or disabling side effects might prompt dose reduction, but this is not required.  A BDI score of 20-28 or a score of 15-19 that has doubled since the previous measurement might prompt further attention from the PI, but is not a requirement for dose reduction.  A score of >29 or higher should prompt attention from the PI and dose reduction or discontinuation should be considered.
10. Suggested guideline for withholding/discontinuing interferon:  Persistent suicide ideation/suicide plan/suicide attempt should lead to discontinuation of interferon.