BMJ  2005;330:932 (23 April), doi:10.1136/bmj.38391.663287.E0 (published 3 March 2005)

Paper

Incidence and risk factors for non-alcoholic steatohepatitis: prospective study of 5408 women enrolled in Italian tamoxifen chemoprevention trial

Abstract

Objective To assess the incidence, cofactors, and excess risk of development of non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis, attributable to tamoxifen in women.

Design Prospective, randomised, double blind, placebo controlled trial.

Setting and participants 5408 healthy women who had had hysterectomies recruited into the Italian tamoxifen chemoprevention trial from 58 centres in Italy.

Intervention Women were randomly assigned to receive tamoxifen (20 mg daily) or placebo for five years.

Main outcome measure Development of non-alcoholic fatty liver disease in all women with normal baseline liver function who showed at least two elevations of alanine aminotransferase ( 1.5 times upper limit of normal) over a six month period.

Results During follow up, 64 women met the predefined criteria: 12 tested positive for hepatitis C virus, and the remaining 52 were suspected of having developed non-alcoholic fatty liver disease (34 tamoxifen, 18 placebo)—hazard ratio = 2.0 (95% confidence interval 1.1 to 3.5; P = 0.04). In all 52 women ultrasonography confirmed the presence of fatty liver. Other factors associated with the development of non-alcoholic fatty liver diseaseincluded overweight (2.4, 1.2 to 4.8), obesity (3.6, 1.7 to 7.6), hypercholesterolaemia (3.4, 1.4 to 7.8), and arterial hypertension (2.0, 1.0 to 3.8). Twenty women had liver biopsies: 15 were diagnosed as having mild to moderate steatohepatitis (12 tamoxifen, 3 placebo), and five had fatty liver alone (1 tamoxifen, 4 placebo). No clinical, biochemical, ultrasonic, or histological signs suggestive of progression to cirrhosis were observed after a median follow up of 8.7 years.

Conclusions Tamoxifen was associated with higher risk of development of non-alcoholic steatohepatitis only in overweight and obese women with features of metabolic syndrome, but the disease, in both the tamoxifen and the placebo group, after 10 years of follow-up seems to be indolent.

Introduction

The efficacy of tamoxifen in preventing breast cancer in women at high risk has been recently recognised. However, it is associated with an increased risk of endometrial cancer and other adverse reactions,¹ including the development of non-alcoholic fatty liver and non-alcoholic steatohepatitis.^2-4 We aimed to evaluate the incidence of, potential risk factors for, and excess risk attributable to tamoxifen in non-alcoholic fatty liver diseases in a large cohort of women who had had hysterectomies originally enrolled in the Italian tamoxifen chemoprevention randomised trial.

Methods

The Italian tamoxifen chemoprevention trial included healthy women aged 35 to 70 who had had a total hysterectomy.^{5 6} The study started in October 1992, and recruitment ended in 1997. Women were randomly assigned in a double blind manner to receive tamoxifen (20 mg daily) or placebo for five years. History taking, physical examination, and blood testing were done at baseline. Follow up included a physical examination every six months and blood testing and mammography every 12 months.

Design of the study on incidence of non-alcoholic fatty liver disease
We suspected development of non-alcoholic fatty liver disease in all women with normal baseline transaminase concentrations and no history of liver enzyme alterations who developed chronic unexplained hypertransaminasaemia defined as multiple elevations of alanine aminotransferase 1.5 times the normal upper limit over a six month period. We chose this arbitrary criterion to justify the morbidity and to limit the number of false positive liver biopsies in women at normal risk.⁷ For each woman identified, we collected detailed information on previous liver function tests, hepatitis B or C infection, autoantibodies indicative of autoimmune hepatitis, iron metabolism, alcohol intake, and use of hepatotoxic drugs. Women with unexplained hypertransaminasaemia had an ultrasound examination of the liver and were offered liver biopsy. Both procedures were done by operators blind to the treatment allocation. We invited women with suspected non-alcoholic fatty liver disease to a repeat physical examination, blood tests, ultrasonography, and repeat biopsy in March 2004.

Statistical analysis
We assessed statistical differences in the frequency of the outcomes among women in the different study groups and the cumulative incidence of events reported during follow-up, and we compared the frequency of events between groups. We assessed the independent effect of multiple factors on the development of events during the trial by using Cox proportional hazards regression.

Results

From 1992 to 1997, 5408 healthy women who had had hysterectomies (median age 51 years) were randomised to receive tamoxifen (n = 2708) or placebo (n = 2700) for five years (table 1).⁵ Fifty two women fulfilled our criteria for non-alcoholic fatty liver disease and were invited to have a liver biopsy for final diagnosis. In all cases ultrasonic findings were consistent with the presence of fatty liver.

View this table: [in this window] [in a new window]   Table 1 Distribution of women in Italian tamoxifen chemoprevention trial according to alanine aminotransferase concentrations and hepatitis B or C virus profile

 

Among women with normal alanine aminotransferase at baseline, development of suspected nonalcoholic fatty liver disease was associated with baseline hypercholesterolaemia (> 6.5 mmol/l) (P = 0.005) and hypertension (P = 0.15).The mean weight of these 52 women was 71.5 kg at randomisation and 74.4 kg at the first elevation of alanine aminotransferase (P < 0.0001).

Liver biopsies
Twenty women (13 tamoxifen, 7 placebo) had liver biopsy: 15 had steatohepatitis (12 tamoxifen, 3 placebo), and 5 had fatty liver (1 tamoxifen, 4 placebo). The positive predictive value for non-alcoholic steatohepatitis was 92% in the tamoxifen group and 43% in the placebo group. The mean weight of the 15 women with steatohepatitis was 70.2 kg at randomisation and 73.5 kg at the time of first elevation of alanine aminotransferase (P = 0.015), whereas the mean weight of the five women with fatty liver remained stable (69.2 kg at randomisation, 69.8 kg at first alanine aminotransferase elevation; P = 0.68). The women who refused biopsy did not differ in any of the considered characteristics from those who agreed to have the procedure.

Cumulative incidence of suspected non-alcoholic fatty liver disease
Suspicion of non-alcoholic fatty liver disease was more common in the tamoxifen group (n = 34) than in the placebo group (n = 18) (log rank P = 0.017). This excess was, however, limited to the first two years of intervention (tamoxifen 21, placebo 7), and no excess was apparent thereafter (tamoxifen 13, placebo 11). The mean interval between randomisation and first elevation of alanine aminotransferase was 23 (range 6-50) months, similar in both study groups.

Compared with the placebo group, the cumulative incidence of suspected non-alcoholic fatty liver disease after five years in the tamoxifen group was particularly high (3.8%, 95% confidence interval 1.6% to 6.0%) among obese women (body mass index > 30), moderately high (2.4%, 1.2% to 3.5%) among overweight women (body mass index 25-30), but similar (0.7%, 0.2% to 1.2%) in women of normal weight (body mass index < 25) (see bmj.com).

The incidence of suspected non-alcoholic fatty liver disease in women with normal alanine aminotransferase at baseline reached 0.37/100 women/year in the tamoxifen group compared with 0.19/100 in the placebo group. On the basis of the liver biopsy findings, we can reasonably assume that all women in the tamoxifen group and half of the women in the placebo group had steatohepatitis. Therefore, the rate of steatohepatitis would be approximately 0.4/100 women/year in the tamoxifen group compared with 0.1/100 in the placebo group, with an excess rate of steatohepatitis attributed to tamoxifen of 0.3/100 women/year. Similarly, among obese women the rate of steatohepatitis during the first two years of intervention would be 1.13/100 women/year in the tamoxifen group compared with 0.07/100 in the placebo group.

Prognostic factors for tamoxifen associated suspected non-alcoholic fatty liver disease
Overall, tamoxifen was associated with an increased risk of developing suspected non-alcoholic fatty liver disease, but the association was restricted to overweight women. Other predictors of non-alcoholic fatty liver disease included overweight, obesity, severe baseline hypercholesterolaemia (> 7.8 mmol/l), and hypertension (table 2). Among overweight and obese women, tamoxifen and hypertension were independent significant predictors for the development of suspected non-alcoholic fatty liver disease. Among women in the placebo group, diabetes and severe hypercholesterolaemia (> 7.8 mmol/l) were associated with an excess risk of developing suspected non-alcoholic fatty liver disease, but among women in the tamoxifen group, only overweight and obesity were associated with an excess risk.

View this table: [in this window] [in a new window]   Table 2 Prognostic factors for development of suspected non-alcoholic fatty liver disease in women with normal baseline transaminase concentrations in Italian tamoxifen chemoprevention trial. Values are hazard ratios (95% confidence intervals) obtained from Cox regression model

 

Follow-up
Women with suspected non-alcoholic fatty liver disease were blindly invited to suspend treatment. During follow-up (median 6.9 years, range 2.7-9.9), 22 women had persistently normalised liver enzymes, 14 had fluctuations of transaminases, and 16 maintained high liver enzymes; we found no difference between the tamoxifen and placebo groups. For the 20 women who had biopsies, the mean follow-up time was 8.7 (range 4.9-10.7) years. In this group, 11 women had features of the metabolic syndrome, two had an abnormal oral glucose tolerance test, and 14 had a homoeostasis model assessment test result that suggested insulin resistance; no difference existed between study groups or histological diagnosis of fatty liver or steatohepatitis. No drug treatment for insulin resistance was initiated. Three of the women who had persistently abnormal alanine aminotransferase had a second liver biopsy after 5-6.5 years which showed no histological changes. In March 2004 none of the 52 women had signs of cirrhosis.

Discussion

This study showed that treatment with 20 mg of tamoxifen daily for up to five years was associated with an excess risk of developing non-alcoholic steatohepatitis in overweight and obese, but otherwise healthy, women who had had hysterectomies. Our arbitrary cut-off point of 1.5 times the normal upper limit for alanine aminotransferase with multiple elevations over a six month period was chosen because the main objective of our study was to recognise more severe disease and not fatty liver in itself, and in the absence of defined standard, we adopted these criteria to ethically justify offering liver biopsy.

Cofactors associated with the development of tamoxifen related steatohepatitis
Although tamoxifen might induce hepatic fat content,⁸ the mechanism by which it can lead to fibrosis has not yet been identified. In our study, body mass index was strongly associated with the development of suspected non-alcoholic fatty liver disease. We can speculate that, despite normal liver enzymes at enrolment, obese women had an underlying fatty liver.⁹ In this context, our results support the "multiple hit" hypothesis, suggesting that fatty liver is vulnerable to oxidants, with progression to steatohepatitis occurring when a second agent (such as tamoxifen) generates liver cell death, inflammation, and activation of stellate cells with production of fibrosis.

In contrast with previous case reports,^{3 4} the women in our trial developed only mild to moderate steatohepatitis, and we found no cirrhosis. This discrepancy can be explained by our exclusion of women with a history of elevated liver enzymes or elevated baseline alanine aminotransferase, which could be attributed to undiagnosed non-alcoholic fatty liver disease.¹⁰

Natural history of non-alcoholic fatty liver disease
We have estimated for the first time the incidence of development of persistent mild elevations of alanine aminotransferase suggestive of non-alcoholic fatty liver disease in a cohort of low risk women receiving placebo. Our results could be representative of the general Italian female population.

Several retrospective studies have suggested an association between non-alcoholic steatohepatitis and severe liver disease, including hepatocellular carcinoma.¹¹ In our study, none of the 15 women with histologically confirmed steatohepatitis has developed clinical, biochemical, or ultrasonic features suggestive of cirrhosis. This suggests that liver fibrosis, when recognised early, remains stable over the years and that progression to cirrhosis is rare, in agreement with other studies.^12-14

Conclusion
This study indicates that 20 mg of tamoxifen daily is associated with a higher risk of developing nonalcoholic steatohepatitis in healthy women who had had hysterectomies. The risk is low, restricted to women with body mass index 25, associated with obesity and features of the metabolic syndrome, and limited to the first two years of treatment. The identification of unexplained abnormal alanine aminotransferase ( 1.5 times the normal upper limit) in women receiving tamoxifen accurately predicts the presence of fibrosis at histology, reducing the need for liver biopsy. In the placebo group, the annual incidence of persistent mild elevation of alanine aminotransferase associated with ultrasonic and histological features of non-alcoholic fatty liver disease was 0.19/100 women/year. Mild to moderate non-alcoholic steatohepatitis, when recognised at the onset, seems to be indolent in the long term.

What is already known on this topic Non-alcoholic fatty liver disease is very common, although the annual incidence is unknown; major risk factors are obesity, hyperlipidaemia, and diabetes Tamoxifen has been occasionally associated with the development of non-alcoholic steatohepatitis, characterised by necro-inflammation and fibrosis in addition to fatty liver What this study adds In women on placebo, the incidence of persistently abnormal alanine aminotransferase suggestive of non-alcoholic fatty liver disease is low (0.19/100 women/year) Tamoxifen is associated with a twofold increased risk of developing non-alcoholic steatohepatitis, but only in overweight women During tamoxifen treatment, unexplained multiple elevations of alanine aminotransferase 1.5 times the upper normal limit are associated with steatohepatitis, making histological examination unnecessary

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Details of authors' affiliations are in the full version of the paper on bmj.com Other members of the study group are listed on bmj.com

This is the abridged version of an article that was posted on bmj.com on 21 March 2005: http://bmj.com/cgi/doi/10.1136/bmj.38391.663287.E0

We thank the Italian women who participated in this study.

Contributors: See bmj.com

Funding: This study was supported in part by grants from the Italian National Research Council, the Italian Association for Cancer Research, the American-Italian Cancer Foundation, and the Italian League Against Cancer.

Competing interests: None declared.

Ethical approval: The trial was approved by the ethical committee of the National Health ministry.

References

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(Accepted 28 January 2005)

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