Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora

AJ, Adams JM, Sullivan MP. Acute hypoxaemia stimulates atrial natriuretic factor in vivo. Am J Physiol 1988; 255: H295-300. 13 Lang CC, Coutie WJ, Struthers AD, et al. Elevated levels of brain natriuretic peptide in acute hypoxaemic chronic obstructive pulmonary disease. Clin Sci 1992; 83: 529-33. 14 Hosoda K, Nakao K, Mukoyama M, et al. Expression of brain natriuretic peptide gene in human heart: production in the ventricle.

12 Baertschi

Hypertension 1991; 17: 1152-56. JG, McAlpine H, Kennedy N, Struthers AD. Plasma brain

15 Motwani

natriuretic peptide as an indicator for angiotensin-converting-enzyme inhibition after myocardial infarction. Lancet 1993; 341: 1109-13. 16 Nicholson S, Richards M, Espiner E, et al. Atrial and brain natriuretic peptide response to exercise in patients with ischaemic heart disease. Clin Exp Pharmacol Physiol 1993; 20: 535-40. 17 Holmes SJ, Espiner EA, Richards AM, et al. Renal, endocrine and hemodynamic effects of human brain natriuretic peptide in normal man. J Clin Endocrinol Metab 1993; 76: 91-96. 18 Borson DB. Roles of neutral endopeptidase in airways. AmJ Physiol 1991; 260: L212-25.

Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora

Summary

Introduction

Previous studies with scanning electron microscopy (SEM) have suggested that pigment gallstones contain bacteria. We set out to culture these bacteria and to study their membrane characteristics. We studied gallstones from 54 patients (36 men, 18 women; mean age 55·4 years) admitted consecutively to two hospitals for cholecystectomy. SEM detected bacteria in all of 14 brown pigment stones, 2 of 14 black pigment stones, and in the pigmented centres of 9 of 19 mixed cholesterol stones; no bacteria were detected in 14 pure cholesterol stones or within the cholesterol portions of mixed stones. We were able to culture bacteria from all gallstones with bacteria seen on SEM and for which sufficient material was available (n=16). 20 bacterial species were recovered from these stones. Gallstones containing bacteria were associated with clinical sepsis and cholangitis. All bacteria obtained from gallstones agglutinated human O P1 erythrocytes, which reflects the presence of P1-specific fimbriae. 5 strains were positive for Forssman-antigen-specific fimbriae. None showed evidence of mannose-specific fimbriae. All of the organisms bound antiGal, a ubiquitous naturally occurring IgG specific for &agr;-galactosyl residues. The presence of P1 fimbriae and &agr;-galactosyi residues and the absence of mannose-specific fimbriae distinguish these organisms from gut flora. We postulate that possession of these unusual properties may enhance the ability of bacteria to colonise the biliary tree and initiate pigment gallstone formation.

classified according to composition as cholesterol stones. Previous studies have pigment or suggested that bacteria may have an important role in the formation of pigment gallstones.1,2 Maki et aP showed that bacteria secrete the enzyme P-glucuronidase; they postulated that this enzyme can deconjugate bilirubin in bile and that the unconjugated bilirubin then precipitates with calcium to form calcium bilirubinate particles, the main component of pigment stones. We have found that patients with pigment gallstone disease have more severe clinical manifestations than patients with cholesterol stones4 and that the presence of bacteria within the stones as seen on scanning electron microscopy (SEM) correlates with more severe disease.5 In this study, we set out to culture bacteria from pigment and mixed cholesterol/pigment stones. On the organisms recovered we sought fimbriae specific for PI (Galcxl,4Gal), mannose, or Forssman antigen and galactose !Xl,3 galactose (Galoci ,3Gal) structures on the outer membrane. Binding of bacteria to anti-Gal, a well-characterised human antibody specific for Galcxl,3Gal,6,7 is associated with resistance of selected Enterobacteriaceae to complement-mediated killing in human serum.8 In addition, we sought antibodies with the same specificities as anti-Gal in human bile.9

Lancet 1994; 343: 444-48

Departments of Surgery and Laboratory Medicine, and Center for Immunochemistry, University of California, San Francisco, USA (L A Wetter MD, R M Hamadeh, Prof J McL Griffiss MD, A Oesterle BA, B Aagaard MD, Prof LW Way MD) Correspondence to: Prof L W Way, Surgical Service, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA

444

Gallstones

are

Patients and methods We studied 54 patients (36 men, 18 women; aged 17-84 [mean 55,4] years) consecutively admitted for cholecystectomy to the Veterans Affairs Medical Center or our university centre in San Francisco. The indications for surgery were gallstone disease with acute or chronic cholecystitis or cholangitis or cholecystectomy incidental to insertion of a hepatic artery catheter to treat metastatic liver disease. Sepsis was diagnosed when three of the following criteria were satisfied: temperature above 38-5°C, systolic blood pressure below 100 mm Hg, white-blood-cell count above 10 x 10/L, rigors, end-organ failure, or emergency surgery for acute biliary disease. Patients with sepsis, hyperbilirubinaemia, and obstructing common-duct stones were judged to have cholangitis. Samples taken at the time of surgery included, whenever possible, gallstones, bile, blood, and a sample of gallbladder wall. We cultured gallstones, bile, and blood from each patient. Detailed characterisation of bacteria was limited to organisms cultured from gallstones. All gallstones were photographed and visually classified as cholesterol, mixed cholesterol, brown pigment, or black pigment stones.

Cholesterol

gallstones characteristically

are

small

*3 SEM-positive stones could not be cultured because of insufficient matenal. All culturepositive stones were SEM positive. Table 1: Association of gallstone characteristics with sepsis

Figure: SEM appearance of cholesterol (A, cholesterol (C, D), and brown pigment (E,

B), mixed F) stones

No bacteria are visible in external or internal areas of cholesterol stones (A, B) or in external areas of mixed cholesterol gallstones (C). Bactena are seen in pigmented centres of mixed cholesterol gallstones and throughout brown pigment stones (D-F).

Mixed cholesterol stones have a faceted hard outer cholesterol shell with a pigmented core. Brown pigment stones are amorphous, friable, and range in colour from light to dark brown. Black stones are small, hard, irregular, and coal-black. We have shown previously4 that gross characteristics can accurately distinguish between cholesterol and

mulberry-shaped yellow

We used haemagglutination assays to test for fimbriae specific for PI (Galal,4Gal), mannose, and Forssman antigen. Bacteria were cultured overnight on Meuller-Hinton agar and resuspended in phosphate-buffered saline to 2 x 109 colony-forming units per mL. 20 uL volumes of 3 % suspensions of fresh human erythrocytes of blood group 0 PI, guineapig erythrocytes, or sheep erythrocytes were mixed with serial two-fold dilutions of the bacterial suspension on microtitre plates. Mannose was added to each well (final concentration 0-5%). For guineapig erythrocytes, duplicate reaction mixtures without mannose were included. The plates were incubated at 4°C and the haemagglutination titre was taken as the highest dilution of bacteria that gave a positive result. We used our previously reported method to purify anti-Gal from pooled normal human serum.8 Lysates of gallstone-associated gram-negative organisms were prepared (some treated with proteinase K)," dissolved in 2% sodium dodecyl sulphate, and subjected to electrophoresis at 200 V/100 mA for 1 h." The separated samples were imaged by silver staining. 13 Western blots of the gels14 were incubated for 30 min with 1 % casein to block non-specific binding and then placed in 1 ug/mL anti-Gal. The transblots were then washed and binding to anti-Gal was detected with goat antibody to human IgG conjugated with alkaline

phosphatase. Results

stones.

pigment stones. Fresh stones were processed for SEM under sterile conditions. We first washed the stones for 24 h in three sequential baths of saline. One or two stones from each patient were set aside for culture. The remaining stones were fractured and fixed for 3-7 days in 5% gluteraldehyde with 0-1mol/L cacodylate pH 7-0; they were then soaked in 01 mol/L cacodylate buffer for 24 h (two changes), then dehydrated through ascending concentrations of ethanol. Stones were dried to the critical point (Polaron CPD apparatus, Watford, UK), mounted on aluminium, and sputtercoated with platinum to 25-35 - nm thickness (Polaron). The exterior and interior of each stone were examined under SEM at 15 kV on an ISIDS-130 scanning electron microscope (International Scientific Instruments, Santa Clara, California). For bacterial culture gallstones were crushed then incubated in flasks with Mueller-Hinton broth at 37°C for 24 h. After centrifugation the pellet was cultured on Mueller-Hinton agar plates at 37°C for 12-24 h. Bile was cultured on Mueller-Hinton agar plates as part of our standard microbiological practice. Blood cultures (aerobes and anaerobes) were done for patients with fever. All bacteria recovered were identified by standard methods in the microbiology laboratory at the Veterans Affairs Medical Center, San Francisco. Stool samples from 10 healthy volunteers were cultured on Mueller-Hinton agar plates, and the organisms that grew were identified and saved. We used a dot-blot assayl° to detect specific antibacterial antibodies in the bile and serum of patients whose gallstones contained bacteria. Organisms from the gallstone were grown on agar, bile or serum was added, and binding detected by means of an alkaline-phosphate-conjugated goat antibody to human IgG, IgA, and IgM.

15 patients had acute cholecystitis, 28 chronic cholecystitis, 10 cholangitis, and 2 intrahepatic cholelithiasis, and 3 were undergoing cholecystectomy as part of management of metastatic liver disease. 13 (24%) patients had cholesterol gallstones, 19 (35%) mixed stones, 8 ( 15 °,-o ) brown pigment stones, and 14 (26%) black pigment stones. SEM of cholesterol gallstones showed the typical cholesterol formations, whereas brown pigment gallstones showed amorphous material (calciium bilirubinate) closely associated with bacteria (figure). The black stones appeared on SEM as amorphous material, in most cases without bacteria. No bacteria could be identified in cholesterol gallstones by SEM or cultures (figure, table 1). All 6 patients with sepsis had stones positive for bacteria on SEM and 5 had culture-positive stones (there was insufficient material for stone culture in the remaining patient). No patient with cholesterol stones had sepsis or cholangitis. Among the 6 patients with sepsis, the site of stone recovery was the common bileduct in 2, the intrahepatic ducts in 1, and the gallbladder in 3. 4 patients, all with positive stone cultures and bacteria on SEM, had cholangitis. All patients with brown pigment stones had bacteria on SEM, and all those cultured (6) had positive cultures. 8 of 18 patients with mixed cholesterol gallstones and 2 of 14 patients with black pigment stones had positive cultures (table 1). The organisms isolated in various cultures are listed in table 2. E coli was isolated less commonly than would be expected from previous reports of biliary infections. For all but 2 patients (A and 0), stone and bile cultures grew different organisms, and only 1 of 2 patients with positive blood cultures had the same organism cultured from his blood and gallstone. 445

ND= not done. *Not A, B. C, D, F, or G.

Table 2: Results of gallstone, bile, and blood cultures

All the gallstone-associated bacterial isolates tested showed haemagglutination with human blood-group-0 Pl erythrocytes, which indicates the presence of Pl-specific fimbriae (table 3). The highest titres were found for pseudomonal and enterococcal isolates. In control experiments, organisms cultured from normal stool samples did not agglutinate human O-Pl erythrocytes; these bacteria were therefore without P1 fimbriae.

Haemagglutination of guineapig erythrocytes could not be inhibited by mannose in any isolate, which indicates the absence of mannose-specific fimbriae. 5 organisms haemaggluinated sheep erythrocytes, which suggests the presence of Forssman antigen (GaINAccxl,3GalNAc)specific fimbriae. All 3 enterococcal isolates from gallstones strongly haemagglutinated sheep erythrocytes. The other 2 organisms giving haemagglutination with sheep erythrocytes had lower titres. All patients with culture-positive stones had antibodies against their infecting bacteria in bile and serum. Anti-Gal bound to all 20 gallstone-associated bacteria in a whole-organism dot-blot assay. By means of western blotting, we examined the binding of anti-Gal to the lipopolysaccharides of the 17 gram-negative gallstone bacteria. All these bacteria bound anti-Gal as shown by proteinase-K-resistant, regularly spaced, ladder-like binding. Anti-Gal bound to the core oligosaccharide and lower molecular weight lipopolysaccharide in most cases, with the exception of isolates 2, 4, and 13 in which there was binding to higher molecular weight lipopolysaccharides. Anti-Gal also bound to proteinase-K-sensitive components on some

of the isolates.

Discussion The biliary tree is normally sterile, but when gallstones are present, bacteria can be cultured from bile or gallbladder wall.15-17 One prospective study found that bile infection preceded pigment stone formation rather than following the biliary obstruction caused by the stones.18 How pigment gallstones form and the role of bacteria in this process is still largely unknown. Our finding that bacteria form an intimate part of pigment stones1.2 supports Maki and colleagues’ hypothesis3 on the formation of calcium bilirubinate stones. In this series, all patients with brown pigment gallstones had bacteria cultured and seen on SEM. We were also able to culture bacteria from 44% of mixed gallstones; SEM showed that they were located in the pigmented centre of the stones. No bacteria were cultured from or seen on SEM in pure cholesterol stones without pigment centres. There was complete agreement between the presence or absence of bacteria seen by SEM and the isolation of bacteria from stones. Positive stone cultures were associated with more

(sepsis and cholangitis). The species of bacteria and frequency of positive cultures differed between gallstones and bile. How these organisms colonise and survive within the biliary tree is unknown. They must overcome the bacteriostatic effects of bile and remain viable, presumably for years, within the substance of the stone. This ability must require special attributes. The lack of concordance between the species of bacteria cultured from a gallstone and those isolated from the bile or blood of the same patient is not therefore surprising. The

severe

NR = non-reactive, RBC = red blood cells.

Table 3:

446

Haemagglutination by gallstone-associated bacteria

illness

infectious process that initiated stone formation antedated the clinical events that caused their removal, probably by many years. In other words, different species are involved in stone formation and the subsequent septic consequences of the stones. In addition, bloodstream isolates would have to resist serum lysis, whereas stone isolates would not. The first step in any infectious process is adherence of the microorganism to a host tissue.19 Adherence of bacteria to biliary epithelium would allow them to avoid being washed out of the biliary tree. Adherence is mediated by specific adhesins on the bacterial surface and can be monitored in vitro by haemagglutination.20 One group of adhesins, the

organism’s fimbriae, have been extensively studied .20-26 Fimbriae can be grouped according to their receptor specificity. Only 10-20% of Enterobacteriaceae isolated from stools of healthy people have PI fimbriae.26 Pfimbriated E coli, however, are the predominant cause of upper urinary tract infections in women.21-23 By contrast, type-I fimbriae, binding of which can be inhibited by mannose, are widespread among E coli isolated from different sources.19,20 S-fimbriae recognise sialic acid and are associated with neonatal meningitis.25 The organisms we isolated from gallstones all expressed PI fimbriae. It has been postulated that PI fimbriae enhance the ability of bacteria to adhere to the uroepithelium.19-26 Biliary epithelium may have similar glycoconjugates to uroepithelium, in which case expression of PI fimbriae might promote adherence and colonisation by selected bacteria. However, since E coli represented only 15% of gallstone isolates, the requirements for biliary colonisation must involve attributes other than PI fimbriae. All enterococci cultured from stones had fimbriae specific for P1and Forssman antigen. The fimbriae of these enteric streptococci have not been reported previously. Enterococci commonly cause urinary tract infections, and the expression of Pl fimbriae by enterococci may explain this propensity as it does for uropathogenic E coli. None of the gallstone-associated bacteria had the mannose-specific fimbriae that are ubiquitous among normal stool flora. The presence of PIfimbriae, and of Forssman-antigen-specific fimbriae for some, and the absence of mannose-specific fimbriae distinguish gallstone-associated bacteria from other enteric flora. Haemagglutination assays, however, are a crude way to assign the specificity of an adhesin. Understanding how bacterial pili or fimbriae and host susceptibility factors contribute to biliary tract colonisation and subsequent disease will require identification of the biliary epithelial carbohydrate receptor repertoire of patients with gallstones. We would then be able to match the carbohydrate specificity of bacterial fimbriae to host biliary epithelial surface glycoconjugates. Some work has been done to examine carbohydrates on biliary epithelium with monoclonal antibodies against structurally defined

carbohydrate antigensY Another characteristic of gallstone-associated bacteria is their ability to bind serum anti-Gal, a ubiquitous, polyclonal, naturally occurring human antibody with specificity for Galcxl,3Gal. We also found anti-Gal in human bile. Binding of anti-Gal to the lipopolysaccharides of certain Enterobacteriaceae makes them resistant to the lytic effects of serum. All the gallstone-associated bacteria we studied bound anti-Gal to their lipopolysaccharide, but only 2 patients had bacteraemia, and only 1 of these had the same species isolated from stone and blood. Bacteria could invade the biliary tract by several routes.28 They could ascend from the duodenum, be filtered from portal or systemic blood into bile canaliculi, or spread from mesenteric lymph channels from the gut. The organism’s adhesin repertoire may affect its ability to attach and persist at any one of these sites. In addition, binding to serum anti-Gal may be one mechanism by which gallstoneassociated bacteria escape the complement-mediated

bacteriolytic

effect of portal-venous or systemic blood.29 The roles of serum and secretory anti-Gal in the pathogenesis of infectious stones, however, remain to be defined. We are now studying the characteristics of bacteria

from bile, gallbladder wall, and blood, and comparing them with features of bacteria obtained from gallstones. Whether they express similar fimbriae or bind to anti-Gal in a similar fashion is not yet known. grown

We thank Mr Donald Pardoe and Ms Ilma Rodriguez for expert technical assistance. This study was supported in part by the Department of Veterans Affairs and Public Health Service Grant AI 21171 (JMcLG) from the National Institutes of Health.

References 1

2 3

4

Stewart L, Smith AL, Pellegrini CA, Motson RW, Way LW. Pigment gallstones form as a composite of bacterial microcolonies and pigment solids. Ann Surg 1987; 206: 242-49. Cetta F. The role of bacteria in pigment gallstones disease. Ann Surg 1991; 213: 315-26. Maki T. Pathogenesis of calcium bilirubinate gallstones: role of E coli, &bgr;-glucuronidase and coagulation by inorganic ions, polyelectrolytes and agitation. Ann Surg 1966; 154: 90-100. Bernhoft RA, Pellegrini CA, Motson RW, Way LW. Composition and morphologic and clinical features of common duct stones. Am J Surg

1984; 148: 77-85. 5

6

Smith AL, Stewart L, Fine R, Pellegrini CA, Way LW. Gallstone disease: the clinical manifestations of infectious stones. Arch Surg 1989; 124: 629-33. Galili U, Rachmilewitz EA, Peleg A, Flechner I. A unique natural human IgG antibody with anti-&agr;galactosyl specificity. J Exp Med

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160: 1519-31.

Galili U, Macher BA, Buehler J, Shohet SB. Human natural anti&agr;galactosyl IgG: the specific recognition of &agr; 1,3-linked galactose residues. J Exp Med 1985; 162: 573-82. 8 Hamadeh RM, Jarvis G, Galili U, Mandrell R, Griffiss JM. Bindingsite specific regulation by the human natural anti-Gal IgG of alternative complement pathway activation on bacterial surfaces. J Clin Invest 1992; 89: 1223-35. 9 Hamadeh RM, Galili U, Zhou P, Chiu T, Griffiss JM. Secretory IgA anti-Gal in man. Clin Res 1991; 39: 439A. 10 Kim JJ, Mandrell RE, Griffiss JM. Neisseria lactamica and Neisseria meningitidis share lipoopigosaccharide epitopes, but lack common capsular and class 1,2 and 3 protein epitopes. Infect Immun 1989; 57: 7

602-08. 11 Hitchcock

PJ, Brown TM. Morphological heterogeneity among

Salmonella

12 13

lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 1983; 154: 269-77. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-85. Tsai CM, Frasch CE. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 1982; 119:

115-19. 14 Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified

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nitrocellulose and radiographic detection of antibody and radioiodinated protein A. Anal Biochem 1981; 112: 195-203. Skar V, Skar AG, Stromme JH. Beta-glucuronidase activity related to bacterial growth in common bile duct bile in gallstone patients. Scand J Gastroenterol 1988; 23: 83-90. Donovan JM, Carey MC. Pathogenesis and therapy of gallstone disease: physical-chemical basis of gallstone formation. Gastroenterol Clin North Am 1991; 20: 47-66. Akiyoshi T, Nakayama F. Bile acid composition in brown pigment stones. Dig Dis Sci 1990; 35: 27-32. Cetta FM. Bile infection documented as initial event in the pathogenesis of brown pigment biliary stones. Hepatology 1986; 6: 482-89. Duguid JP, Old DC. Adhesive properties of Enterobacteriaceae. In: Beachey EH, ed. Bacterial adherence, receptors and recognition. Series B, vol 6. London: Chapman and Hall, 1980: 185-217. Evans DJ, Evans DG, duPont HL. Hemagglutination patterns of enterotoxigenic and enteropathogenic Escherichia coli determined with human, bovine, chicken and guinea pig erythrocytes in the presence and absence of mannose. Infect Immun 1979; 23: 336-46. Kallenius G, Mollby R, Svenson SB, et al. The pk antigen as a receptor for the haemagglutinin of pyelonephritic Escherichia coli. FEMS Microbiol Lett 1980; 7: 297-302. O’Hanley P, Low D, Romero I, et al. Gal-gal binding and hemolysin phenotypes and genotypes associated with uropathogenic Escherichia coli. N Engl J Med 1985; 313: 414-20. Vaisanen V, Tallgren LG, Makela PH, et al. Mannose-resistant haemagglutination and P antigen recognition are characteristic of

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Escherichia coli causing primary pyelonephritis. Lancet 1981; ii: 1366-69. 24 Korhonen TK, Valtonen MV, Parkkinen J, et al. Serotype, hemolysin production, and receptor recognition of Escherichia coli stains associated with neonatal sepsis and meningitis. Infect Immun 1985; 48 486-91. 25 Old DC. Inhibition of the interaction between fimbrial hemagglutinin and erythrocytes by D-mannose and other carbohydrates. J Gen Microbiol 1972; 71: 149-57. 26 Green CP, Thomas VL. Hemagglutination of human type O erythrocytes, hemolysin production and serogrouping of Escherichia

Risk of endometrial

cancer

coli isolates from patients with acute pyelonephritis cystitis, and asymptomatic bacteriuria. Infect Immun 1981; 31: 309-15. 27 Okada Y, Tsuji T. Immunohistochemical application of monoclonal antibodies to reveal the structure and localization of carbohydrate antigens: N-acetyl-lactosamine related carbohydrates antigens in human biliary epithelial cells. J Immunol Method 1988; 112: 243-49. 28 Sung JY, Costerton JW, Shaffer EA. Defense system in the biliary tract against bacterial infection. Dig Dis Sci 1992; 37: 689-96. 29 Hamadeh RM, Mandrell RE, Griffiss JM. Immunophysical characterization of human isolates of Serratia marcescens. J Clin Microbiol 1990; 28: 20-26.

after tamoxifen treatment of breast

cancer

Summary

Introduction

Since large trials have been set up to assess whether tamoxifen decreases the risk of breast cancer in healthy women, it has become important to investigate the drug’s potential adverse effects, including occurrence of endometrial cancer. We undertook a case-control study in the Netherlands to assess the effect of tamoxifen on the risk of endometrial cancer after breast cancer. Through the population-based Netherlands Cancer Registry and two older, hospital-based, registries, we identified 98 patients who had endometrial cancer diagnosed at least 3 months after a diagnosis of primary breast cancer. Detailed information about treatment was obtained for all these patients, and for 285 controls, who were matched to the cases for age, year of breast cancer diagnosis, and survival time with intact uterus. Tamoxifen had been used by 24% of patients with subsequent endometrial cancer and 20% of controls (relative risk 1·3 [95% Cl 0·7-2·4]). Women who had used tamoxifen for more than 2 years had a 2 3 (0 9-5 9) times greater risk of endometrial cancer than never users. There was a significant trend of increasing risk of endometrial cancer with duration of tamoxifen use (p=0 049), and also with cumulative dose (p=0·046). The duration-response trends were similar with daily doses of 40 mg or 30 mg and less. These findings support the hypothesis that tamoxifen use increases the risk of endometrial cancer. This oestrogenic effect on the endometrium was not related to the dose intensity. Physicians should be aware of the higher risk of endometrial cancer in tamoxifen users.

Since its introduction in the early 1970s, tamoxifen has been widely and effectively used to treat advanced breast cancer. Adjuvant tamoxifen therapy in early-stage breast cancer became common in the 1980s, and has been convincingly shown to improve disease-free survival as well as overall survival for women older than 50 years.’ Several large trials have suggested, furthermore, that tamoxifen reduces the risk of cancer in the contralateral breast." Based on these findings, chemoprevention trials have been set up in the USA and Europe to find out whether tamoxifen decreases the risk of breast cancer developing in healthy women judged to be at high risk of the disease.5.6 Our knowledge of the long-term effects of tamoxifen is still limited. Tamoxifen’s mixed oestrogen-agonist and oestrogen-antagonist properties have led to concern that the drug may increase the risk of endometrial cancer. The Stockholm trial4 found that women receiving 40 mg tamoxifen daily for at least 2 years had a more than six-fold excess risk of endometrial cancer in comparison with untreated controls. No significant increase in risk has been reported in other major adjuvant trialsalthough a non-significant excess of endometrial cancer after tamoxifen treatment was noted in an evaluation of Danish trials and in an unpublished Southwest Oncology Group Trial.8-10 The 10-year cumulative risk of endometrial cancer in postmenopausal women is estimated to be 0 3% or less. Thus, a follow-up study with sufficient power to settle the question of whether there is an excess risk of endometrial cancer after tamoxifen treatment would require long-term follow-up of many thousands of breast cancer patients. We therefore used a case-control design to investigate, in a nationwide study, whether tamoxifen, at different doses and for different durations, increases the risk of endometrial cancer.

Lancet 1994; 343: 448-52

Department of Epidemiology, Netherlands Cancer Institute, (F E van Leeuwen MSPH, A W van den Belt-Dusebout); Comprehensive Cancer Centers—Amsterdam (F E van Leeuwen, J Benraadt MD, F W Diepenhorst, H van Tinteren MSc), South Netherlands (J W W Coebergh MD), East Netherlands (L A L M Kiemeney PhD), Middle Netherlands (C H F Gimbrère MD), Northern Netherlands (R Otter MD), Limburg (L J Schouten MD), Rotterdam (R A M Damhuis MD), and Stedendriehoek Twente (J Benraadt); and Dr Daniel den Hoed Cancer Center, Rotterdam (M Bontenbal MD), Netherlands Correspondence to: Ms Flora E van Leeuwen, Department of Epidemiology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam

1066 CX, Amsterdam, Netherlands

448

Patients and methods Since 1989, the Netherlands has had a population-based, nationwide cancer registry served by nine regional cancer registries." Most registries have been operating since 1986, the Eindhoven Cancer Registry since 1975,12 and the Middle Netherlands Breast Cancer Registry since 1973.13 Patients with endometrial cancer after breast cancer were identified from eight of the nine regional cancer registries; other cases (most with endometrial cancer diagnosed before 1986) were identified from the hospital tumour registries of two major cancer treatment