Natural history and disease progression in Chinese chronic hepatitis B patients in immune-tolerant phase

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

In view of the findings that high hepatitis B virus (HBV) deoxyribonucleic acid (DNA) is associated with increased risk of chronic hepatitis B (CHB)–related complications, disease progression in CHB patients in the immune-tolerant phase is uncertain. We evaluated disease progression in 57 immune-tolerant CHB patients with high HBV DNA. Each subject underwent an initial liver biopsy. In those who remained in the immune-tolerant phase, a follow-up liver biopsy was performed after 5 years of follow-up. Patients who developed elevated serum alanine aminotransferase (ALT) levels were discontinued from the study after a follow-up liver biopsy. Disease progression was defined as a 1-point increase in fibrosis stage. Initial liver biopsies showed the median fibrosis stage of the study patients was 1 (range 0–1). By the end of follow-up, 9 of the 57 patients (15.8%) had developed elevated serum ALT. In those who remained in the immune-tolerant phase, follow-up fibrosis stage was comparable with the initial fibrosis stage (P = 0.58). However, disease progression was greater in patients who developed elevated serum ALT when compared with those who remained in the immune-tolerant phase (5 of 9 vs. 3 of 48, respectively, P = 0.001). The median rate of fibrosis progression of patients who remained in the immune-tolerant phase was lower than that of patients with high serum ALT (0 U/year [range −0.40–0.20 U/year] versus 0.21 U/year [range 0–1.11 U/year], respectively, P = 0.04). Conclusion: CHB patients in the immune-tolerant phase have mild disease. In those who remained in the immune-tolerant phase in the present study, disease progression was minimal. However, immune-tolerant patients who progressed to the immune clearance phase often faced disease progression. (HEPATOLOGY 2007.)

More than 350 million people worldwide have chronic hepatitis B (CHB) infection.1 Even with the advent of an effective hepatitis B vaccine, more than 50 million new cases of HBV infection occurs annually.2 In HBV-endemic areas, most individuals are infected with HBV in the perinatal period or during early childhood. CHB patients are at increased risk of developing liver cirrhosis and hepatocellular carcinoma (HCC).3

Traditionally, serum alanine aminotransferase (ALT) level and HBV serology analysis have been widely used for the assessment of patients with CHB infection. The annual incidence of cirrhosis has been estimated to be 2%–6% for hepatitis B e antigen (HBeAg)–positive CHB patients and 8%–10% for HBeAg-negative CHB patients.4, 5 The higher incidence of cirrhosis among HBeAg-negative patients is related to older age and more advanced liver disease at presentation.2, 4, 5 More advanced fibrosis stage at presentation correlates with risk of cirrhosis, and those with milder forms of hepatitis have a longer time to cirrhosis.6

HBeAg positivity has also been found to be associated with higher hepatic inflammation and an increased risk of HCC. Although seroconversion of HBeAg to hepatitis B e antibody (anti-HBe) often leads to hepatic inflammation subsiding and an improved prognosis, the identification of precore/core promoter mutations and recognition of HBeAg-negative CHB disease explain the disease progression noted after HBeAg seroconversion.7–10

Recent studies have demonstrated a strong link between viral replication, liver injury, and progression to cirrhosis with increased risk of HCC in CHB-infected patients.11–13 Serum HBV DNA level has been found to be directly correlated with the development of HCC and cirrhosis independent of serum ALT level, HBV genotype, and HBeAg status.11–13 This has led to promotion of the use of serum HBV DNA concentration as an indicator for treatment regardless of serum ALT level, and even patients with normal serum ALT levels are to be treated with antiviral therapy with the primary aim of suppressing HBV DNA,14, 15

However, the natural course of CHB infection among those infected at an early age comprises 4 phases: the immune-tolerant, immune clearance, inactive carrier and reactivation phases. The immune-tolerant phase is characterized by HBeAg positivity, normal serum ALT level, and minimal or no inflammation on liver biopsy, but high serum HBV DNA level.16 Whether there is long-term benefit in treating these immune-tolerant CHB patients cannot be ascertained without a better understanding of the natural history of CHB in patients in the immune-tolerant phase.14, 15 In this study, we evaluated disease progression in Chinese immune-tolerant patients over a 5-year period. Hepatic fibrosis score was used as the primary marker of disease progression.

Abbreviations

ALT, alanine aminotransferase; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen.

Patients and Methods

Patients.

Between January 1997 and December 1998, 57 consecutive Chinese CHB-infected adult patients in the immune-tolerant phase who had undergone a liver biopsy for assessment of their CHB infection at the Nethersole Hospital, Hong Kong, China, were recruited into this study. These 57 patients fulfilled the following criteria: (1) HBsAg positive for at least 6 months, (2) HBeAg positive for at least 6 months, (3) serum ALT levels within normal range (7–53 U/L for men and 7–31 U/L for women) on 3 consecutive readings 6 months apart before the initial liver biopsy, (4) HBV DNA more than 107 copies/mL at the time of initial liver biopsy, (5) treatment naive before the initial liver biopsy, and (6) alcohol intensity of less than 10 g/day as defined previously.17 Patients were excluded from the study if they had: (1) previous therapy with nucleoside/nucleotide analogues or immunomodulators; (2) coinfection with hepatitis C virus (HCV), human immunodeficiency virus (HIV), or hepatitis D virus; (3) fibrosis stage greater than 1 on initial liver biopsy; or (4) HCC.

Follow-up and HBV Virological Studies.

After the initial liver biopsy, the patients were prospectively followed up in the outpatient clinic every 6 months for 5 years. A follow-up liver biopsy was performed after 5 years for assessment of progression of the disease in those who remained in the immune-tolerant phase. Patients whose serum ALT had become elevated received the follow-up liver biopsy earlier, after which they were discontinued from the study. They were also followed up for 5 years.

Serum was tested for HBsAg, HBeAg, and anti-HBe with the enzyme-linked immunosorbent assay (ELISA; Abbott Laboratories, Chicago, IL) on every follow-up visit. Anti-HCV, hepatitis D, and HIV virus were tested for with a commercially available ELISA (Abbott GmbH Diagnostika, Wiesbaden-Delkenheim, Germany).

Serum HBV DNA was quantified by real-time polymerase chain reaction (PCR) assay on stored serum samples as described (linear range of 102 to 109 copies/mL).18, 19 Serum HBV DNA was measured at the time of initial liver biopsy and on every follow-up visit. HBV genotype was determined as described.20

Assessment of Liver Biopsy.

All liver biopsy specimens were assessed by a pathologist blinded to the chronological sequence of the liver biopsies and biochemistry and virological studies, who used the modified histology activity index (HAI) score to grade inflammation and necrosis and the Ishak fibrosis score to stage fibrosis.21, 22

Definitions of End Points.

Patients in the immune-tolerant phase of CHB infection were defined as those with a serum ALT level measured within normal range on 3 consecutive readings 6 months apart and with serum HBV DNA concentration of more than 107 copies/mL. HBeAg seroconversion was defined as loss of HBeAg accompanied by the development of anti-HBe on 2 consecutive readings 3 months apart. Elevated serum ALT level was defined as elevation of serum ALT above the upper limit of normal on 3 consecutive readings 6 months apart.

Disease progression was defined as at least a 1-point increase in fibrosis score in ranked assessment of fibrosis. Fibrosis reduction was defined as at least a 1-point decrease in fibrosis score. The rate of fibrosis progression was calculated as the difference in fibrosis score between the initial and follow-up liver biopsies divided by the duration of time between the biopsies and expressed as fibrosis units/year.17, 23

The study was approved by the local ethics committee.

Statistical Analysis.

All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 12.5 for Windows; SPSS Inc., Chicago, IL). The Mann-Whitney U test was used for comparisons of 2 continuous variables, and the chi square with Yates correction for continuity or the Fisher exact test was used for comparisons of 2 categorical variables. Kaplan-Meier survival analysis was used to calculate the percentage of patients in the immune-tolerant phase at the end of follow-up (5 years). The primary end point of this study was disease progression on follow-up liver biopsy. A secondary analysis to determine the sensitivity of employing serum ALT level below the recent revised cutoff upper limit of 30 U/L for men and 19 U/L for women as a predictor of patients without disease progression was also performed.24 All continuous variables are expressed as medians with ranges. All statistical analyses were performed on an intention-to-treat basis. A P value < 0.05 (2-tailed) was considered statistically significant.

Results

The baseline demographics of the 57 patients, ascertained at the time of the initial liver biopsy, are shown in Table 1. Median fibrosis stage of the cohort was 1 (range 0–1). Serum ALT level at the time of initial liver biopsy was not associated with fibrosis stage (r = 0.21, P = 0.52).

Table 1. Baseline Demographics of Immune-Tolerant Patients at Time of Initial Liver Biopsy
CharacteristicValue
  1. Abbreviations: ALT, alanine aminotransferase; HAI, histologic activity index; HBeAg, hepatitis B e antigen; anti-HBe- hepatitis B e antibody; HBV, hepatitis B virus.

Number of patientsn = 57
Sex (M:F)34:23
Age (years)31 (18–41)
ALT (U/L)30 (4–42)
HBeAg 
Positive57
Negative0
Anti-HBe 
Positive0
Negative57
HBV DNA (log10 copies/mL)9.81 (7.12–>10.00)
HBV genotype
B32
C25
Median modified HAI score4 (1––8)
Fibrosis stage 
F019
F138
Median fibrosis stage1 (0–1)
Number of portal tracts12 (9–14)

Follow-Up.

By the end of follow-up, 9 of the 57 patients (15.8%) had developed elevated serum ALT levels (Fig. 1). The baseline characteristics of patients whose serum ALT levels had become elevated and of those who remained in the immune-tolerant phase are shown in Table 2.

Figure 1.

Kaplan-Meier curve of patients who remained in the immune-tolerant phase after 5 years of follow-up.

Table 2. Baseline Demographics of Patients Who Remained in Immune-Tolerant Phase and of Those Who Had Developed Elevated Serum ALT on Follow-Up
CharacteristicPatients Who Remained Immune Tolerant (n = 48)Patients Who Developed Elevated Serum ALT (n = 9)P Value
  1. Abbreviations: ALT, alanine aminotransferase; HAI, histologic activity index; HBV, hepatitis B virus.

Sex (M:F)28:206:30.84
Age (years)31 (18–41)31 (24–36)0.90
ALT (U/L)14 (4–22)38 (20–42)0.03
HBV DNA (log10 copies/mL)9.81 (9.00–>10.00)9.92 (7.12–>10.00)0.24
HBV genotype   
 B2660.72
 C223 
Median modified HAI score on initial liver biopsy3 (1–5)6 (4–8)0.03
Median fibrosis stage on initial liver biopsy1 (0–1)1 (0–1)0.62
Median modified HAI score on follow-up liver biopsy3 (1–6)10 (8–18)0.002
Median fibrosis stage on follow-up liver biopsy1 (0–2)1 (1–3)0.57
Disease progression on follow-up liver biopsy350.001
Increase of 2 points in modified HAI on follow-up liver biopsy430.07
Rate of fibrosis progression (U/year)0 (−0.40–0.20)0.21 (0–1.11)0.04

Patients who remained in the immune-tolerant phase had a lower baseline serum ALT level (P = 0.03) and lower baseline modified HAI score (P = 0.03) compared with those of those who had developed elevated serum ALT levels on follow-up (Table 2).

The 9 patients with elevated serum ALT levels were discontinued from the study after a follow-up liver biopsy. All 9 had a follow-up liver biopsy after elevation in serum ALT was detected on 3 occasions 6 months apart (18 months after the first elevated serum ALT was detected). The median time between the initial and follow-up liver biopsies was 29.5 months (range 21.7–60.0 months). Five of the 9 patients (55.6%) whose serum ALT had become elevated on follow-up had disease progression, whereas 3 of the 9 patients (33.3%) had a 2-point increase in modified HAI score on follow-up liver biopsy. They were begun on anti-HBV therapy, as their follow-up liver biopsy only showed evidence of HBV disease. None of the 9 patients who developed elevated serum ALT levels had fibrosis reduction.

None of the 9 patients who developed elevated serum ALT levels showed HBeAg seroconversion by the time they were discontinued from the study.

The clinical characteristics of these 9 patients are shown in Table 3. There was no significant difference between those with and without HBeAg seroconversion in baseline characteristics (age, sex, serum ALT, modified HAI score, fibrosis stage, and HBV DNA) at the time of initial liver biopsy (all P = NS).

Table 3. Characteristics of the 9 Patients Who Developed Elevated Serum ALT Levels
PatientSerum ALT (U/L)HBV DNA (log10copies/mL)HBeAgAntiviral TherapyMonths to HBeAg Seroconversion After Follow-up Liver BiopsyHBeAg SeroconversionAntiviral TherapyHBV DNA (log10copies/mL)Serum ALT (U/L)
At Follow-up Liver BiopsyAt Completion of Study
  1. Abbreviations: LAM, lamivudine 100 mg daily; PEG, pegylated interferon alpha-2a 180 μg subcutaneous weekly for 48 weeks; ADV, adefovir dipivoxil; ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen.

1978.41+LAMNoYes<2.0013
22197.54+LAMNoYes<2.0027
32337.99+PEG2.3YesNo3.4631
4936.56+LAMNoYes3.1232
51097.32+ADVNoYes4.9819
64036.89+ADV27.8YesYes<2.0011
72118.52+LAMNoYes3.5442
81246.12+LAMNoYes<2.0034
92787.11+ADV37.9YesYes3.1227

Disease Progression in Patients Who Remained in Immune-Tolerant Phase.

All 48 patients who remained in the immune-tolerant phase had a follow-up liver biopsy after 5 years. Their fibrosis stage on the initial and follow-up liver biopsies is shown in Table 4. Fibrosis stage of the 48 patients on the follow-up liver biopsy did not differ significantly with that on the initial liver biopsy (P = 0.58; Table 4).

Table 4. Stage of Fibrosis on Initial and Follow-Up Liver Biopsies of the 48 Patients Who Remained in Immune-Tolerant Phase at End of 5-Year Follow-Up
Stage of fibrosisInitial liver biopsyFollow-up liver biopsyP value
F015160.58
F13331 
F201 

On follow-up liver biopsy, 4 of the 33 patients (12.1%) with stage 1 fibrosis on initial liver biopsy had fibrosis reduction, whereas only 3 of the 48 patients (6.3%) had disease progression. The fibrosis stage of the remaining 41 of these 48 patients (85.4%) was unchanged on follow-up liver biopsy.

None of the 48 patients HBeAg-seroconverted during the 5-year study period.

Modified HAI Score on Follow-up Liver Biopsy in Patients Who Remained in the Immune-Tolerant Phase.

Of the 48 patients who were still in the immune-tolerant phase at the end of follow-up, the modified HAI score on follow-up liver biopsy of 6 patients (12.5%) showed a 2-point reduction, and that of 4 patients (8.3%) showed a 2-point increase.

There was no significant change in the median modified HAI score on follow-up liver biopsy of patients who remained in the immune-tolerant phase compared with that at the initial liver biopsy: 3 (range 1-6) versus 3 (1–5), respectively (P = 0.34).

HBV DNA and Serum ALT Levels in Those Who Remained in the Immune-Tolerant Phase.

In patients who remained in the immune-tolerant phase, there was no significant change in median initial and follow-up serum HBV DNA and serum ALT levels: serum HBV DNA, 9.74 log10 copies/mL (range 8.35 to >10.00 log10 copies/mL) versus 9.81 log10 copies/mL (range 9.20 to >10.00 log10 copies/mL), P = 0.20; serum ALT, 17 U/L (range 6–24 U/L) versus 14 U/L (range 4–23 U/L), P = 0.47.

Serial median HBV DNA values of the 48 patients who remained in the immune-tolerant phase are shown in Fig. 2.

Figure 2.

Median serum HBV DNA of the 48 patients who remained in the immune-tolerant phase from the time of initial liver biopsy to completion of the study.

Disease Progression and 2-Point Increase in Modified HAI Score in Those with Elevated Serum ALT Levels.

Patients who developed elevated serum ALT levels were more likely to have disease progression on follow-up liver biopsy than were those who remained in the immune-tolerant phase (5 of 9 [55.6%] versus 3 of 48 [6.3%], respectively, P = 0.001; Table 2).

Furthermore, patients with elevated serum ALT showed a trend of being more likely to have a 2-point increase in modified HAI score on follow-up liver biopsy than did patients who remained in the immune-tolerant phase (3 of 9 [33.3%] versus 4 of 48 [8.3%], respectively, P = 0.07; Table 2).

Patients who developed elevated serum ALT levels also showed a trend toward having a significant increase in modified HAI score on follow-up liver biopsy compared with that on initial liver biopsy (median 10 [range 8–18] versus 6 [range 4–8], respectively, P = 0.06; Table 2), although there was no change in fibrosis stage (P = 0.62).

Rate of Fibrosis Progression.

Patients who remained in the immune-tolerant phase had a lower median rate of fibrosis progression than the 9 patients who developed elevated serum ALT levels on follow-up (0 U/year [range −0.40–0.20 U/year] versus 0.21 U/year [range 0–1.11 U/year], respectively, P = 0.04; Table 2).

Serum ALT Level at Time of Follow-Up Liver Biopsy Was Associated with Increased Rate of Fibrosis Progression.

Serum ALT level at the time of follow-up liver biopsy correlated with a higher rate of fibrosis progression (r = 0.83, P < 0.001).

Validation of Use of the Recently Revised Upper-Limit Cutoff of 30 U/L for Men and 19 U/L for Women for Predicting Patients Without Disease Progression.

Fifty-one of the 57 patients (89.5%) recruited into this study had serum ALT levels below the recently revised upper cutoff for normal-range ALT level (30 U/L for men and 19 U/L for women).24 Using the revised serum ALT cutoff limits, 3 of the 51 patients (5.9%) would be predicted to develop elevated serum ALT levels compared with a predicted 9 of 57 patients (15.8%) if the existing normal range were used (P = 0.10).

The sensitivity of the revised cutoff serum ALT level for predicting patients who would remain in the immune-tolerant phase was 94.1%, whereas the sensitivity of the existing normal range was 84.2%.

Among patients whose serum ALT was below the revised serum ALT upper cutoff levels, disease progression was also higher in patients who developed elevated serum ALT than in those who remained in the immune-tolerant phase (2 of 3 vs. 3 of 48, respectively, P = 0.02).

If the revised serum ALT cutoff values were employed, 5 of the 51 patients (9.8%) were predicted to have disease progression by the end of the study compared with the 8 of 57 patients (14.0%) predicted to have disease progression using the existing serum ALT normal range (P = 0.50).

The sensitivity of the revised serum ALT cutoff values for predicting patients without disease progression was 90.2%, whereas the sensitivity of the existing serum ALT normal range values for predicting patients without disease progression was 86.0%.

Discussion

With the discovery and understanding of HBeAg-negative disease, the natural course of CHB infection acquired perinatally is believed to have 4 phases, rather than the 3 phases as originally described in the 1980s.16 However, not all CHB patients go through every one of the 4 phases. The first immune-tolerant phase may last for 1–4 decades. During this phase, the rate of spontaneous or treatment-induced HBeAg seroconversion is less than 5% per year. Patients in the immune-tolerant phase are considered at low risk of progression to cirrhosis or HCC based on serial monitoring of virological, clinical, and ultrasonographic assessments.16, 25 However, histologic documentation has not been done.25

Recently, high serum HBV DNA has been reported to be an independent risk factor for progression of CHB disease, independent of genotype, serum ALT level, and HBeAg status. This prompted the need for a detailed study to better delineate the course of disease progression among patients in the immune-tolerant phase, who typically have high serum HBV DNA.

Although measuring serum ALT level remains the most common and convenient way to reflect the liver inflammation of CHB patients, the correlation is not ideal.26–28 Furthermore, serum ALT level is not a reliable marker of fibrosis stage. Similar to what was found in chronic hepatitis C patients with persistently normal serum ALT, in the present study we were unable to demonstrate a correlation between serum ALT level at the time of initial liver biopsy with fibrosis stage.29, 30 This may be related to the small sample size of the study and fluctuating nature of serum ALT level.

Thus, a normal serum ALT level alone does not indicate inactive or lack of active liver pathology. As shown in the present study, if the recently revised cutoff for serum ALT is used, the sensitivity of predicting those who would remain without disease progression or those who would progress to the immune-tolerant phase would be higher than the sensitivity of predicting using the existing normal range for serum ALT. This supports the idea that the upper limit of normal of serum ALT should be lowered and has clinical relevance, as illustrated in this cohort of immune-tolerant patients.

However, patients in the immune-tolerant phase with normal ALT have low modified HAI and fibrosis scores on liver biopsy, as revealed in this study. The median modified HAI score was only 4, and the median fibrosis stage was only 1 in this cohort of 57 patients.

This study has shown that CHB patients who remain in the immune-tolerant phase with persistently normal serum ALT are much less likely to have disease progression. Serum ALT level at the time of follow-up liver biopsy correlated positively with increased rate of fibrosis progression, indicating that the rate of fibrosis progression increased once these patients progressed into the immune clearance phase.

One interesting observation from this study is that although our cohort consisted of immune-tolerant patients with persistently normal serum ALT levels, those with serum ALT levels near the upper limit of normal are more likely to progress to the immune clearance phase. Furthermore, those patients who progressed to the immune clearance phase also had a higher modified HAI score on baseline liver biopsy compared with those remaining in immune-tolerant phase. The absolute level, apart from being in the normal range, is important to know for identifying patients with more active liver disease and those who are likely to progress to the immune clearance phase.

A few important clinical recommendations can be derived from this study. Firstly, although a high serum HBV DNA may be associated with a lifelong higher chance of developing HCC or cirrhosis, the risk assessment among Asians CHB patients in the immune-tolerant phase needs to be better defined. This prospective longitudinal study showed that although immune-tolerant patients have high HBV DNA, they have low disease activity and minimal or no disease progression over 5 years. Starting these patients on antiviral therapy such as nucleoside/nucleotide analogues in an attempt to suppress viral replication will not only bring little benefit to the patient but will encourage the development of viral resistance, which increases with prolonged therapy—70% for 5 years of lamivudine therapy and 29% for 5 years of adefovir dipivoxil therapy.3, 24, 31 In addition, the emergence of drug resistance may result in accelerated disease progression, as previously reported in patients with cirrhosis.32 Therapy with immunomodulators such as conventional or pegylated interferons may not be effective, as low response rate is associated with normal serum ALT levels.3, 24, 31, 33, 34 Second, although antiviral therapy is not recommended in immune-tolerant patients, they should still be monitored closely for progression to the immune clearance phase. Once this occurs, antiviral therapy may be considered more diligently after 6-12 months if HBeAg seroconversion does not occur. This is because disease progression can occur in the immune clearance phase.

This study had a few limitations. First, among the 3 patients who progressed to the immune clearance phase, a bias may have resulted from performing follow-up liver biopsies before their participation in the study was ended rather than at the end of the 5-year follow-up period. Progression of fibrosis may not be linear, especially during flares of disease, giving rise to incorrect analysis. Similarly, calculation of the rate of fibrosis progression assumes that fibrosis progression is linear between the 2 time points over 5 years.17, 23 Thus, the invented fractions also lack meaning.35 Although histological scoring systems are still considered the best gold standard, they are unreliable, especially when used to quantify fibrosis progression with potential sampling error.36–38 To overcome these shortcomings, imaging systems such as the Fibroscan, a noninvasive and reproducible method for measuring liver stiffness, are being explored as an alternative tool for disease assessment.39, 40 Sufficient data for Fibroscan validation are needed for its clinical application to be recommended. Second, because we do not have any data on the patients' ALT levels before they were referred to our center, we have no way of determining whether they had normal serum ALT levels historically or were in transient although prolonged biochemical remission at the time of presentation. Any prior ALT elevation was not known. The median age of our cohort was somewhat high at 31 years, and some patients may have been in the late immune-tolerant or early immune clearance phase. Prior to recruitment, 6 monthly assessments of serum ALT level may have missed serum ALT elevation other than a “drift” toward the upper end of normal. Third, as only a small number of patients progressed to the immune clearance phase, we are unable to identify predictive factors for HBeAg seroconversion, and the small number may also have introduced a type 2 error.

In conclusion, this prospective longitudinal study showed slow disease progression in CHB-infected patients in the immune-tolerant phase despite high serum HBV DNA. They do not require antiviral therapy but should be monitored for transition to the immune clearance phase, when antiviral therapy is more likely to result in long-term clinical benefit. Antiviral therapy should be considered 6–12 months after progression to the immune clearance phase if HBeAg seroconversion does not occur.

Acknowledgements

A. S. Lok for her wise words, Y. Y. Lau for her help, and A. Wong.

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