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Screening for Liver Fibrosis by Using a Noninvasive Biomarker in Patients With Diabetes

      Background & Aims: Patients with diabetes are at risk for nonalcoholic fatty liver disease leading to advanced fibrosis, cirrhosis, and liver cancer. We examined the efficacy of a screening strategy with a noninvasive fibrosis biomarker (FibroTest) in patients with diabetes. Methods: We prospectively studied 1131 consecutive patients without a history of liver disease seen for diabetes. The biomarker data were obtained, and patients with presumed advanced fibrosis were reinvestigated by a hepatologist using elastography and, if necessary, ultrasonography, endoscopy, or liver biopsy. Results: The biomarker predicted advanced fibrosis in 63 of 1131 (5.6%) patients. A total of 45 patients was reinvestigated, and advanced fibrosis was confirmed in 32 patients, a 2.8% (32/1131) prevalence of confirmed advanced fibrosis, 5 cases of cirrhosis, and 4 cases of hepatocellular carcinoma. In the population with type 2 diabetes who were 45 years or older, the prevalence of confirmed advanced fibrosis was 4.3% (30/696), and hepatocellular carcinoma was 5.7 of 1000 (4/696). Conclusions: The fibrosis biomarker might be used for the detection of advanced fibrosis in patients with type 2 diabetes.

      Abbreviations used in this paper:

      CI (confidence interval), FT (FibroTest), GGT (γ-glutamyltranspeptidase), HCC (hepatocellular carcinoma), NAFLD (nonalcoholic fatty liver disease), OR (odds ratio), PPV (positive predictive value)
      See Huwart L et al on page 32 for companion article in the July 2008 issue of Gastroenterology.
      Patients with diabetes are at risk for nonalcoholic fatty liver disease (NAFLD) leading to advanced fibrosis, cirrhosis, and liver cancer, with an increase in liver-related mortality.
      • Ekstedt M.
      • Franzen L.E.
      • Mathiesen U.L.
      • et al.
      Long-term follow-up of patients with NAFLD and elevated liver enzymes.
      • El-Serag H.B.
      • Hampel H.
      • Javadi F.
      The association between diabetes and hepatocellular carcinoma: a systematic review of epidemiologic evidence.
      The aim of the study was to use a noninvasive biomarker (FibroTest [FT]; Biopredictive, Paris, France; FibroSURE in the USA, Labcorp, Burlington, NC)
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      • Morra R.
      • Munteanu M.
      • Imbert-Bismut F.
      • et al.
      FibroMAX: towards a new universal biomarker of liver disease?.
      • Ratziu V.
      • Massard J.
      • Charlotte F.
      • et al.
      Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease.
      to identify patients with fibrosis among patients with diabetes. FT has been extensively validated with similar diagnostic value in patients with chronic hepatitis C, chronic hepatitis B, alcoholic liver disease, and NAFLD.
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      • Morra R.
      • Munteanu M.
      • Imbert-Bismut F.
      • et al.
      FibroMAX: towards a new universal biomarker of liver disease?.
      • Ratziu V.
      • Massard J.
      • Charlotte F.
      • et al.
      Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease.
      In a retrospective study in hyperlipidemic patients, the prevalence of presumed fibrosis by using FT was 6% in patients with type 2 diabetes.
      • Ratziu V.
      • Giral P.
      • Munteanu M.
      • et al.
      Screening for liver disease using non-invasive biomarkers (FibroTest-SteatoTest-NashTest-FibroSURE) in patients with hyperlipidaemia.

      Methods

      Consecutive patients with confirmed diabetes were eligible for inclusion. All procedures were followed in accordance with the current revised guidelines of the Declaration of Helsinki, and all reinvestigated participants gave informed consent.
      Patients with high-risk profiles of false-negatives/positives for FT were excluded by using security algorithms.
      • Imbert-Bismut F.
      • Messous D.
      • Thibault V.
      • et al.
      Intralaboratory analytical variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in healthy blood donors.
      Patients with presumed fibrosis were reinvestigated by a single hepatologist including a liver elastography (Fibroscan, Echosens, Paris) and, if necessary, ultrasonography, esophageal endoscopy, or liver biopsy.
      The analysis was performed in 2 populations, patients without any previous history of liver disease (the naïve population) and patients with a history of liver disease (non-naïve population), used only for the FT validation. A control group was prospectively made up of blood donors to estimate the specificity of FT.
      FT includes α2-macroglobulin, apolipoprotein A1, haptoglobin, total bilirubin, and γ-glutamyltranspeptidase (GGT), adjusted for age and gender. FT scores range from zero to 1.00.
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      • Morra R.
      • Munteanu M.
      • Imbert-Bismut F.
      • et al.
      FibroMAX: towards a new universal biomarker of liver disease?.
      Fibrosis (many septa, numerous septa, or cirrhosis) was predicted when FT was greater than 0.48, as in all chronic liver diseases, and defined a priori.
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      • Morra R.
      • Munteanu M.
      • Imbert-Bismut F.
      • et al.
      FibroMAX: towards a new universal biomarker of liver disease?.
      • Imbert-Bismut F.
      • Messous D.
      • Thibault V.
      • et al.
      Intralaboratory analytical variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in healthy blood donors.
      The biomarker components were analyzed according to published recommendations.
      • Gomez-Dominguez E.
      • Mendoza J.
      • Rubio S.
      • et al.
      Transient elastography: a valid alternative to biopsy in patients with chronic liver disease.
      Liver stiffness is expressed in kilopascals (kPa). The technique was performed by a trained hepatologist who was blinded and according to the manufacturer's recommendations. When less than 10 successful acquisitions were possible or with a less than 60% success rate or with interquartile range greater than 30% of the mean stiffness, the technique was considered noninterpretable.
      • Gomez-Dominguez E.
      • Mendoza J.
      • Rubio S.
      • et al.
      Transient elastography: a valid alternative to biopsy in patients with chronic liver disease.
      Advanced fibrosis was presumed when FT was greater than 0.48 and confirmed if elastography was above 6 kPa (a sensitivity analysis was performed with a higher cutoff of 7.0 kPa), if ultrasonography demonstrated signs of cirrhosis (dysmorphia, splenomegaly, patent umbilical vein), there were endoscopic signs of portal hypertension (esophageal varices, gastropathy), or fibrosis was demonstrated on liver biopsy.
      The Fisher exact, Mann-Whitney, Bonferroni, and Tukey-Kramer tests and logistic regression were used. Number Cruncher Statistical Systems 2007 software (NCSS, Kaysville, UT) was used.

      Results

      One thousand two hundred sixty-one patients were eligible; 74 were excluded because of high-risk profiles of false-positives or false-negatives, duplicate submissions, or unconfirmed diabetes. Among the 1187 included patients, 1131 had no history of liver disease, and 56 had a history of liver disease; 925 blood donors were included (Table 1).
      Table 1Characteristics of Included Patients
      CharacteristicsIncluded diabetics, naïve populationIncluded diabetics, non-naïve populationIncluded controls
      Number of patients113156925
      Age at serum, y53.8 (14.8)58.3 (10.4)35.7 (12.3)
      Male602 (53%)35 (63%)457 (49%)
      BMI, kg/m228.2 (6.1)28.3 (5.8)23.4 (3.2)
      Daily alcohol ≥30 g/day48 (4%)1 (2%)6/923 (0.6%)
      Liver fibrosis predicted by FT
       No advanced fibrosis1068 (95%)21 (38%)925 (100%)
        F0: no fibrosis799 (71%)6 (11%)856 (92%)
        F0–F1104 (9%)5 (9%)37 (4%)
        F1: fibrosis without septa45 (4%)2 (4%)16 (2%)
        F1–F2120 (11%)8 (14%)16 (2%)
       Advanced fibrosis
      Advanced fibrosis was presumed when FibroTest was greater than 0.48.
      63 (5.6%)35 (62%)0 (0%)
        F2: few septa36 (3.2%)5 (9%)0 (0%)
        F3: many septa19 (1.7%)9 (16%)0 (0%)
        F4: cirrhosis8 (0.7%)21 (37%)0 (0%)
      Markers (normal range)
       AST, IU/L (17–27 female, 20–32 male)26 (12)54 (37)24 (6)
       ALT, IU/L (11–26 female, 16–35 male)29 (22)64 (59)23 (13)
       Total bilirubin, mol/L (1–21)8.8 (4.58)15.4 (16.5)8.2 (4.2)
       GGT, U/L (7–32 female, 11–49 male)41 (56)159 (172)21 (15)
       Alpha2-macroglobulin, g/L (female 1.6–4.0, male 1.4–3.3)1.8 (0.7)2.7 (0.9)1.7 (0.5)
       Apolipoprotein A1, g/L (1.2–1.7)1.6 (0.3)1.4 (0.3)1.6 (0.3)
       Haptoglobin, g/L (0.35–2.00)1.4 (0.6)1.0 (0.7)1.0 (0.4)
       Glucose (mmol/L)9.6 (4.0)8.6 (3.0)5.4 (2.4)
       Cholesterol (mmol/L)4.9 (1.1)4.6 (1.0)4.9 (1.0)
       Triglycerides (mmol/L)1.5 (1.7)1.9 (1.9)1.1 (0.7)
      FibroTest0.18 (0.15)0.58 (0.28)0.10 (0.07)
      SteatoTest0.13 (0.12)0.39 (0.27)0.17 (0.16)
      Data are mean (standard deviation) or proportion.
      a Advanced fibrosis was presumed when FibroTest was greater than 0.48.
      Among the 1131 patients in the naïve population, 63 (5.6%; 95% confidence interval [CI], 4.3%–7.1%) had an FT with presumed fibrosis (Table 2).
      Table 2Characteristics of 63 Diabetic Patients With FT >0.48 (Presumed Advanced Fibrosis) in the Population Without a History of Liver Disease (Naïve Patients)
      All presumed advanced fibrosisReinvestigatedNot reinvestigated
      CharacteristicsAdvanced fibrosis confirmedAdvanced fibrosis not confirmed
      Number of patients63321318
      Prevalence of advanced fibrosis63/1131 (5.6%)32/1131 (2.8%)(32 + 13)/1131 (4.0%)
      Estimated prevalence assuming that the prevalence of advanced fibrosis would be the same in the population of patients not reinvestigated.
      Cause of liver disease
       NAFLD50221216
       Chronic hepatitis C2200
       Alcoholic liver disease7511
       Primary biliary cirrhosis1100
       Autoimmune hepatitis2101
       Neuroendocrine metastasis1100
      Liver complications
       HCC4400
       Cholangiocarcinoma1100
       Portal hypertension2200
      Stage advanced fibrosis predicted
      Advanced fibrosis was presumed when FT was greater than 0.48.
       Few septa3617811
       Many septa191045
       Cirrhosis8512
      Mode of confirmation
       Elastography4030120
       Biopsy2200
       Endoscopy3210
       Ultrasonography9540
      a Estimated prevalence assuming that the prevalence of advanced fibrosis would be the same in the population of patients not reinvestigated.
      b Advanced fibrosis was presumed when FT was greater than 0.48.
      Forty-five patients were reinvestigated, and fibrosis was confirmed in 32 (71%) by elastography, biopsy, or endoscopy. Five patients had cirrhosis. Five patients had a primary liver cancer: 4 hepatocellular carcinoma (HCC) and 1 cholangiocarcinoma. Two patients had large varices. There was no clinically obvious cirrhosis, and standard liver tests have been interpreted as NAFLD profile; none of them had an APRI index above 2.0.
      Elastography was not interpretable in 6 cases out of 40 measurements (15%). In intention to diagnose, the prevalence of fibrosis screened was 2.8% (95% CI, 1.9%–4.0%), and the positive predictive value (PPV) was 50.8% (95% CI, 37.9%–63.6%). In per protocol analysis, assuming that 71% of the noninvestigated patients would have confirmed fibrosis, the prevalence of fibrosis screened would be 4.0% (95% CI, 2.9%–5.3%), and the PPV would be 68.3% (95% CI, 55.3%–79.4%). A sensitivity analysis taking 7 kPa as the optimal cutoff would have obtained a prevalence of 2.5% in intention to diagnose and 3.6% in per protocol analysis.
      Among the 56 patients of the non-naïve population, 35 (63%) had an FT with presumed fibrosis; 32 patients were reinvestigated, and the presumed fibrosis was confirmed in all 32 (100%).
      No presumed fibrosis was identified in blood donors.
      Multivariate analysis identified 3 major risk factors of confirmed fibrosis: age (odds ratio [OR], 1.06; 95% CI, 1.02–1.10; P = .001), male gender (OR, 4.2; 95% CI, 1.6–11.1; P = .004), and type 2 diabetes (OR, 5.8; 95% CI, 0.8–45.0; P = .08).
      Among patients of the naïve population with type 2 diabetes and aged 45 years or older, the prevalence of confirmed fibrosis was 4.3% (95% CI, 2.9%–6.1%) (30/696), presumed fibrosis was 7.5% (95% CI, 5.6%–9.7%) (52/696), and HCC was 5.7 of 1000 (95% CI, 1.7/1000–14.6/1000 (4/696).
      Among the 30 confirmed cases of fibrosis in the naïve population, 8 had GGT lower than 50 IU/mL (27% false-negatives), and 182 of 1070 in the presumed nonfibrosis group had greater than 50 IU/mL (17% false-positives); 20 patients with confirmed fibrosis had ALT lower than 50 IU/mL (67% false-negatives), and 87/1070 in the presumed nonfibrosis group had greater than 50 IU/mL (8% false-positives).

      Discussion

      This prospective study identifies patients with fibrosis and primary liver cancers in a large number of diabetic patients.

       Limitations of the Study

      The major shortcomings of this study are the failure to use liver biopsy to confirm positive test results and the failure to evaluate fibrosis in patients who tested negatively by FT.
      Several points support our methodology. First, most of the patients with chronic liver disease refused liver biopsy.
      • Castera L.
      • Denis J.
      • Babany G.
      • et al.
      Evolving practices of non-invasive markers of liver fibrosis in patients with chronic hepatitis C in France: time for new guidelines?.
      Second, liver biopsy is an imperfect gold standard with a high sampling error.
      • Ratziu V.
      • Charlotte F.
      • Heurtier A.
      • et al.
      Sampling variability of liver biopsy in nonalcoholic fatty liver disease.
      Around 50% of discordances between FT and biopsy can be attributable to biopsy failure.
      • Imbert-Bismut F.
      • Messous D.
      • Thibault V.
      • et al.
      Intralaboratory analytical variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in healthy blood donors.
      • Poynard T.
      • Munteanu M.
      • Imbert-Bismut F.
      • et al.
      Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C.
      Third, even if all the misclassified subjects were attributable to FT, the negative predictive value of FT at the 0.48 threshold is still high; among patients with NAFLD, we previously observed a 0.88 negative predictive value.
      • Ratziu V.
      • Massard J.
      • Charlotte F.
      • et al.
      Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease.
      We acknowledge that an evaluation with elastography of the patients with FT below the 0.48 threshold would have improved the study. We also acknowledge that the control group was not useful for estimating false-negatives of FT. However, the risk of false-negatives seems low, because several comparisons, independent from FT inventors, have observed that FT is the most sensitive fibrosis biomarker.
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      Fourth, the PPV of FT in the present study was similar to that previously observed with biopsy.
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      • Ratziu V.
      • Massard J.
      • Charlotte F.
      • et al.
      Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease.
      The PPV of FT was 71% among the naïve patients and 100% among non-naïve patients. The high specificity of FT was also confirmed without FT greater than 0.48 among blood donors. Fifth, elastography can be considered as an acceptable validation method in a screening strategy. Health authorities in France have approved elastography in patients with chronic hepatitis C.
      La Haute Autorité de Santé (HAS) in France
      The HAS recommendations for the management of the chronic hepatitis C using non-invasive biomarkers.
      Several published results validated elastography in NAFLD, despite a lower reproducibility in patients with steatosis, increased body mass index, and lower stage of fibrosis.
      • Foucher J.
      • Castera L.
      • Bernard P.H.
      • et al.
      Prevalence and factors associated with failure of liver stiffness measurement using FibroScan in a prospective study of 2114 examinations.
      FT has limitations. Less than 5% of FT are not interpretable, mainly related to Gilbert's syndrome, hemolysis, and acute sepsis,
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      • Morra R.
      • Munteanu M.
      • Imbert-Bismut F.
      • et al.
      FibroMAX: towards a new universal biomarker of liver disease?.
      • Imbert-Bismut F.
      • Messous D.
      • Thibault V.
      • et al.
      Intralaboratory analytical variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in healthy blood donors.
      which is lower than elastography, which is not interpretable in 15% of cases.
      The population studied was a population of diabetic patients seen in a tertiary care center. However, all the patients identified in the naïve population had no history and no clinical sign of chronic liver disease.

       Advantages of the Study

      FT has similar diagnostic value than biopsy in the most frequent chronic liver diseases.
      • Poynard T.
      • Morra R.
      • Halfon P.
      • et al.
      Meta-analyses of Fibrotest diagnostic value in chronic liver disease.
      In France FT is approved and used by 81% of hepatologists.
      • Ratziu V.
      • Charlotte F.
      • Heurtier A.
      • et al.
      Sampling variability of liver biopsy in nonalcoholic fatty liver disease.
      La Haute Autorité de Santé (HAS) in France
      The HAS recommendations for the management of the chronic hepatitis C using non-invasive biomarkers.
      Most previous small studies focused on nonalcoholic steatohepatitis, with prevalence of fibrosis at biopsy ranging from 10%–57%.
      • Younossi Z.M.
      • Gramlich T.
      • Matteoni C.A.
      • et al.
      Nonalcoholic fatty liver disease in patients with type 2 diabetes.
      With FT we observed 5.6% cases of presumed fibrosis, including 2.8% cases of confirmed fibrosis.
      We observed a very high prevalence of HCC in the naïve population, 3.5 of 1000, in accordance with published meta-analysis.
      • El-Serag H.B.
      • Hampel H.
      • Javadi F.
      The association between diabetes and hepatocellular carcinoma: a systematic review of epidemiologic evidence.
      A cholangiocarcinoma was also identified, an association previously suspected.
      • Shaib Y.H.
      • El-Serag H.B.
      • Davila J.A.
      • et al.
      Risk factors of intrahepatic cholangiocarcinoma in the United States: a case-control study.
      Indirectly, FT seems effective in the detection of HCC because the majority occurs in patients with cirrhosis.
      We confirmed that the rates of false-negatives and false-positives of transaminases and GGT were too high for screening purposes.
      • Ratziu V.
      • Massard J.
      • Charlotte F.
      • et al.
      Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease.
      • Ratziu V.
      • Giral P.
      • Munteanu M.
      • et al.
      Screening for liver disease using non-invasive biomarkers (FibroTest-SteatoTest-NashTest-FibroSURE) in patients with hyperlipidaemia.
      We also observed for NAFLD score
      • Angulo P.
      • Hui J.M.
      • Marchesini G.
      • et al.
      The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD.
      a low sensitivity for confirmed fibrosis: 12 (57%) false-negatives among 28 naïve patients and 18 false-negatives (80%) among 20 non-naïve patients, as already observed.
      • Munteanu M.
      • Charlotte F.
      • Ratziu V.
      • et al.
      Direct comparison of two non invasive biomarkers for the diagnosis of advanced fibrosis in patients with non alcoholic fatty liver disease (NAFLD): NAFLD score and FibroTest.
      A screening program should not be considered unless there is an effective therapeutic option. Two screened patients with cirrhosis and large varices were treated with beta-blockers to prevent bleeding; 1 patient with HCC has been successfully treated with radiofrequency and is waiting for a liver transplant. We recognize that for 3 patients with HCC the cancer was already not treatable by surgery. Patients for whom the screening permitted to diagnose hepatitis C, autoimmune hepatitis, and primary biliary cirrhosis received the appropriate treatments. Patients with fibrosis and NAFLD represented 25 of 30 patients with confirmed fibrosis, but new drugs are being investigated with promising results for those with NAFLD.

      Conclusion

      This study suggests that screening diabetic patients with biomarkers such as FT is effective. A high-risk population was identified, which was patients aged 45 years or older with type 2 diabetes. External validation studies are necessary to replicate these results and better estimate the cost-efficiency of such screening.
      We thank all the medical staff and nurses of the diabetology unit.

      Appendix. Members of the FibroSucre Group

      Service de Diabetologie-Metabolisme: Sophie Jacqueminet, Agnes Hartemann-Heurtier, Andre Grimaldi, Claude Sachon, Sandrine Jeanne; Service d'Hépatologie: Pascal Lebray, Patrick Ingilitz, Mercedes de Torres, Brigitte Hautecoeur, Luminita Bonihay, Joseph Moussalli, Julien Massard, Dominique Thabut, Yves Benhamou, Vlad Ratziu, Thierry Poynard; Unité de Transfusion: Laure Devers, Anne Mercadier; Biopredictive: Rachel Morra, Mona Munteanu, Fabienne Drane; Service de Biochimie: Djamila Messous, Francoise Imbert-Bismut; Laboratoire de biochimie métabolique: Maguy Bernard, Dominique Bonnefont-Rousselot.

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