Most hepatocellular carcinoma (HCC) arises on a background

Does Nonalcoholic Fatty Liver Disease Predispose Patients to Hepatocellular Carcinoma in the Absence of Cirrhosis? Grace Guzman, MD; Elizabeth M. Brunt, MD; Lydia M. Petrovic, MD; Gregorio Chejfec, MD; Thomas J. Layden, MD; Scott J. Cotler, MD Context. Hepatocellular carcinoma (HCC) is recognized as a complication of cirrhosis related to nonalcoholic fatty liver disease (NAFLD). Diabetes and the metabolic syndrome are also associated with HCC. However, it is not clear whether NAFLD predisposes patients to HCC in the absence of cirrhosis. Objective. To seek evidence that HCC can develop in NAFLD unaccompanied by cirrhosis. Design. Retrospective case study was performed on cases from 2004 to 2007 at the University of Illinois at Chicago Medical Center, using the key words hepatocellular carcinoma, liver explant, and liver resection. The diagnosis of HCC was identified and confirmed by hematoxylin-eosin stained slides in 50 cases. Cause of liver disease was determined by review of liver histology, clinical history, and laboratory data. Results. Three patients presented with advanced HCC with features of metabolic syndrome, including an elevated body mass index. Each patient had bland steatosis on liver biopsy, without fibrosis or cirrhosis. None of the 3 patients had evidence of any cause for liver disease other than NAFLD. Conclusions. The cases presented here suggest that NAFLD may predispose patients to HCC in the absence of cirrhosis. Further studies are needed to confirm this potentially important observation. (Arch Pathol Lab Med. 2008;132:1761 1766) Most hepatocellular carcinoma (HCC) arises on a background of cirrhosis, in which ongoing liver injury and regeneration predispose patients to neoplasia over time. 1 6 A notable exception is hepatitis B, in which HCC often occurs in the absence of cirrhosis. However, even in hepatitis B, HCC typically occurs with advanced fibrosis. There have also been sporadic reports 2,7,8 of HCC in noncirrhotic patients with hepatitis C and alcohol-related liver disease. More important, there are well-known cases 9 14 of HCC that arise in otherwise healthy liver. Whether these cases arise from preexisting adenoma is not known. The current series of HCC arising in nonalcoholic fatty liver disease (NAFLD) without fibrosis consists of cases that are clinically and pathologically distinct from the fibrolamellar type of HCC, which also develops in the absence of cirrhosis or fibrosis. Nonalcoholic fatty liver disease is now recognized as the Accepted for publication April 10, 2008. From the Departments of Pathology (Drs Guzman and Chejfec) and Medicine (Drs Layden and Cotler), University of Illinois, Chicago; the Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Mo (Dr Brunt); and the Department of Pathology, New York University Medical Center, New York (Dr Petrovic). Dr Petrovic is now with the Department of Pathology, Keck School of Medicine, Health Sciences Campus, University of Southern California, Los Angeles. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Grace Guzman, MD, Department of Pathology, University of Illinois at Chicago, 840 S Wood St, Chicago, IL 60614 (e-mail: graceguz@uic.edu). most common cause of liver disease in the United States. Patients with nonalcoholic steatohepatitis (NASH) may progress to advanced fibrosis and cirrhosis. Nonalcoholic fatty liver disease is considered the hepatic manifestation of the metabolic syndrome, a cluster of conditions that are related by obesity and insulin resistance. 9 Hepatocellular carcinoma has been recognized in cirrhosis related to NASH and to cryptogenic cirrhosis presumed to have arisen from NASH. 8,9,11,15,16 Moreover, diabetes, which is sometimes associated with NAFLD, has been identified by epidemiologic studies as a risk factor for HCC when present either alone, 17 19 or in combination with other causes of chronic liver disease, including viral hepatitis and alcohol. 15,20 It is not known whether cirrhosis is a necessary precondition for the development of HCC in patients with NAFLD. Herein, we report on 3 patients with features of obesity and metabolic syndrome without cirrhosis. These cases raise questions about the possible pathogenesis of HCC in NAFLD. Moreover, they raise a clinical concern regarding whether patients with NAFLD are possibly at an increased risk for HCC before the development of cirrhosis. MATERIALS AND METHODS Forty-nine cases with histologic evidence of HCC were identified through a search of data from 2004 to 2007 in the pathology computer system at the University of Illinois at Chicago Medical Center, using the key words hepatocellular carcinoma, liver explant, and liver resection. The diagnosis of HCC was confirmed by reviewing hematoxylin-eosin stained slides from all cases. Cause of liver disease was determined by review of liver histology, clinical history, and laboratory data. Arch Pathol Lab Med Vol 132, November 2008 HCC in NAFLD and the Metabolic Syndrome Guzman et al 1761

Table 1. Cause of Liver Disease in the 50 Patients Identified With Hepatocellular Carcinoma (HCC) Etiology HCC Without Cirrhosis HCC With Cirrhosis Nonalcoholic fatty liver disease 3 Nonalcoholic steatohepatitis 2 Cryptogenic liver disease 5 Hepatitis C 16 Hepatitis C and alcohol 7 Hepatitis B 7 Alcohol 7 Autoimmune hepatitis 3 Total 3 47 Two (4%) of the 49 cases had histologic evidence of HCC without cirrhosis. During the course of the retrospective review, an additional case of HCC arising in the absence of cirrhosis was identified in a needle biopsy of a patient from the hepatology practice. These 3 cases are the focus of this case series. Detailed historic and laboratory data were obtained for the cases of HCC without cirrhosis from the University of Illinois at Chicago electronic medical record. Type of HCC, grade, and stage were categorized following the guidelines of the World Health Organization and International Union Against Cancer. 21 The study protocol was approved by the institutional review board at the University of Illinois at Chicago. RESULTS Among the 50 patients identified with HCC, the mean age was 56 years ( 12 years); 17 (34%) of the patients were women. The mean body mass index (BMI) was 28 kg/m 2 ( 6 kg/m 2 ); 11 (22%) of the patients had diabetes. The causes of liver disease are listed in Table 1. Patients With HCC Without Cirrhosis The clinicopathologic features of the 3 patients with HCC in the absence of cirrhosis are summarized in Tables 2 and 3. They consisted of 2 women and 1 man, with a mean age of 57 years ( 13 years). All had features of the metabolic syndrome: all 3 were overweight (BMI 25 kg/ m 2 ) or obese (BMI 30 kg/m 2 ) and had hypertension and dyslipidemia. One patient was on treatment for diabetes, and another had hyperglycemia. The third patient (case 2) did not have evidence of diabetes during his hospital admission. Case 1. A 57-year-old white woman presented to our liver center with a recent diagnosis of HCC. She had a computed tomography (CT) scan for evaluation of back pain that showed 2 masses in the right lobe of the liver. She did not report a history of an exposure known to be associated with the development of HCC. Her medical history included type 2 diabetes and hypertension. She reported a weight gain of 100 lb during the past 5 years. She had no risk factors for viral hepatitis. She reported only occasional social use of alcohol. Her medications included only lovastatin and insulin. On physical examination, the patient was obese, with a BMI of 41 kg/m 2. She had no stigmata of chronic liver disease. She had mild right-upper-quadrant tenderness on deep palpation. There was no ascites or peripheral edema. Laboratory studies disclosed the following values: total Table 2. Clinical Features of Patients With Hepatocellular Carcinoma and Nonalcoholic Fatty Liver Disease Clinical Features Case 1 Case 2 Case 3 Age, y/sex/race 57/F/non-Hispanic white 70/M/non-Hispanic white 45/F/non-Hispanic white Body mass index, kg/m 2 41 30 29.6 Diabetes status Yes No Hyperglycemia Hypertension Yes Yes Yes Dyslipidemia Yes Yes Yes Alcohol use Occasional, social use No No Autoimmune serologies ANA* Negative Negative Not available Smooth muscle antibody Negative Negative Not available Hepatitis B and C serologies Negative Negative Negative -Fetoprotein, ng/ml 12 3 1533 * ANA indicates antinuclear antibody. Table 3. Histopathologic Features of Patients With Hepatocellular Carcinoma and Nonalcoholic Fatty Liver Disease (NAFLD) Histopathologic Features* Case 1 Case 2 Case 3 Pathologic specimen Core biopsies Wedge resection Right lobectomy Tumor status Multiple Multiple Multiple Range of tumor sizes, cm 0.5 9.6 0.5 10 0.3 6.8 Hepatocellular carcinoma grade 2 2 1 Underlying liver disease NAFLD NAFLD NAFLD Steatosis, % Mild, 5 33 Mild, 5 33 Moderate, 33 66 Lobular inflammation No No No Balloon hepatocytes No No No Fibrosis stage 0 0 0 Total NAS 39 1 1 2 Hepatitis B core-antigen immunostain Negative Negative Negative PAS/D stain Negative Negative Negative Iron stain Negative Negative Negative * NAS indicates NAFLD activity score; PAS/D, periodic acid Schiff/diastase. 1762 Arch Pathol Lab Med Vol 132, November 2008 HCC in NAFLD and the Metabolic Syndrome Guzman et al

bilirubin, 0.6 mg/dl; alanine transferase, 36 U/L; aspartate aminotransferase, 35 U/L; total protein, 4.8 g/dl; albumin, 2.8 g/dl; platelets, 268 000/ L; prothrombin time/international normalized ratio, 0.9; -fetoprotein, 12 ng/ml; total cholesterol, 157 mg/dl; triglycerides, 129 mg/dl; low-density lipoprotein, 129 mg/l; high-density lipoprotein, 49 mg/dl; and blood glucose, 285 mg/dl. Autoimmune (antinuclear antibody and smooth muscle antibody) and hepatitis B and hepatitis C serology findings were negative, and hepatitis C RNA was undetectable by polymerase chain reaction (Table 2). A triple-phase CT scan showed a 9.6-cm mass in segment 8 of the liver and several smaller lesions in segment 4a with arterial enhancement and portal venous washout. The large mass and the uninvolved liver were sampled by ultrasound-guided core biopsies. Pathologic Findings. The neoplastic liver was diagnostic of HCC with expanded trabecular cell plates, mild variation in nuclear size, clear cytoplasm, and interspersed unaccompanied arteries. The nonneoplastic liver showed a mild degree (20%) of zone 3, large-droplet steatosis, and ballooning, but no lobular inflammation. Trichrome staining showed an absence of fibrosis. There was no histopathologic evidence for viral hepatitis, 1 -antitrypsin deficiency, or hemochromatosis. Hepatitis B core antigen immunostain, periodic acid Schiff/diastase (PAS/D), and iron histochemistry findings were negative. There were no changes indicative of any chronic liver disease other than NAFLD (Table 3). Case 2. A 70-year-old white man, with no history of liver disease, presented to an outside institution with fever and right-upper-quadrant abdominal pain. A contrast-enhanced CT scan showed a 10 6-cm mass lesion in the right lobe of the liver. A CT-guided biopsy of the mass was diagnostic of HCC. The patient was referred to the Surgery department at the University of Illinois at Chicago for further intervention. He did not report a history of exposure associated with the development of HCC. He had no known risk factors for viral hepatitis and did not drink alcohol. His medical history was significant for obesity and dyslipidemia. He did not have evidence of diabetes. His medications were limited to diphenhydramine. On physical examination, the patient had a BMI of 30 kg/m 2. He did not have any stigmata of chronic liver disease. There was a tender, palpable mass in the right lobe of the liver. There was no ascites or peripheral edema. Laboratory studies showed the following values: total bilirubin, 0.7 mg/dl; alanine transferase, 131 U/L; aspartate aminotransferase, 123 U/L; total protein, 6.5 g/dl; albumin, 3.3 g/dl; platelets, 180 000/ L; and prothrombin time/international normalized ratio, 0.9. The -fetoprotein level was 3 ng/ml. Autoimmune (antinuclear antibody and smooth muscle antibody) and hepatitis B and C serology findings were negative (Table 2). An arteriogram showed a large, hypervascular lesion in the right lobe of the liver with satellite lesions consistent with HCC (Figure 1). A solitary hypervascular density was present within the left lobe of the liver. An exploratory laparotomy with intraoperative ultrasound showed a 10-cm hypoechoic, nonhomogeneous tumor within segment 8, with multiple satellite nodules. The liver surface was smooth and did not appear cirrhotic on visual inspection but did look fatty. A total of 4 masses were identified, ranging from 0.5 to 10 cm, in segments 8, Figure 1. An arteriogram showing a large hypervascular lesion in the right lobe of the liver with satellite lesions consistent with hepatocellular carcinoma (case 2). 6, 5, and 3. The presence of bilateral lobe involvement, multiple lesions, and a large total tumor volume precluded therapeutic liver resection. Liver-wedge resections of accessible masses were performed to confirm the diagnosis. The larger, nonresectable masses were subjected to radiofrequency ablation following an intraoperative histopathologic diagnosis of HCC. Pathologic Findings. Three portions of liver tissues varying from 1 to 3 cm, containing a total of 3 separate nodules, ranging from 0.5 to 0.8 cm at greatest dimension, were examined. The tumor nodules were grossly distinct from the surrounding noncirrhotic liver tissue. On microscopy, the tumors were HCC grade 2, with pseudoglandular and trabecular architectural features (Figure 2). The surrounding nonneoplastic liver tissue showed 33% centrilobular steatosis. There were no other features of active steatohepatitis. A trichrome stain was negative for zone 1, zone 3, or pericellular fibrosis. There was no evidence for viral hepatitis, 1 -antitrypsin deficiency, or iron-storage disease. Hepatitis B core antigen, PAS/ D, and iron histochemistry findings were negative. There were no changes indicative of any cause of liver disease other than NAFLD (Table 3). Case 3. A 45-year-old white woman was referred for management of a liver tumor diagnosed on CT scan at an outside institution. She did not report a history of an exposure known to be associated with the development of HCC. Her medical history included hypertension and dyslipidemia. She had no history of alcohol use or known risk factors for viral hepatitis. Her medications included diphenhydramine and an albuterol inhaler. On physical examination, the patient was overweight with a BMI of 29.6 kg/m 2. She had no stigmata of chronic liver disease. The liver-span findings were normal to percussion. There were no ascites or peripheral edema. Arch Pathol Lab Med Vol 132, November 2008 HCC in NAFLD and the Metabolic Syndrome Guzman et al 1763

Table 4. Reported Cases of Hepatocellular Carcinoma (HCC) Arising in Nonalcoholic Steatohepatitis With High-Grade Fibrosis Source, y HCC, No. (n 20) Stage* Hai et al, 26 2006 2 3 4 Hashimoto et al, 27 2005 10 4 Ikeda et al, 28 2006 1 4 Mori et al, 24 2004 1 4 Shimada et al, 13 2002 5 4 Yoshioka et al, 25 2004 1 4 * Stage indicates combined nonalcoholic steatohepatitis/fibrosis stage. Table 5. Reported Cases of Hepatocellular Carcinoma (HCC) Arising in Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH) With Low-Grade Fibrosis Source, y HCC, No. (n 21) Disease, Stage Brunt, 9 2004 2 NASH, 0 2 Bullock et al, 10 2004 2 NAFLD, 0 2 Cuadrado et al, 11 2005 2 NASH, 0 2 Komorizono et al, 12 2006 8 NASH, 1 Komorizono et al, 12 2006 2 NASH, 2 Shimada et al, 13 2002 1 NASH, 2 Zen et al, 14 2001 1 NASH, 2 Current study 3 NAFLD, 0 Figure 2. A trabecular and pseudoglandular configuration predominate in the hepatocellular carcinoma in case 2. A malignant hepatocyte at 6 o clock contains intracytoplasmic bile (hematoxylin-eosin, original magnification 40). Figure 3. Moderate degree of large droplet steatosis without steatohepatitis and without significant fibrosis is seen in case 3 (trichrome, original magnification 10). Serum chemistries showed the following values: total bilirubin, 0.3 mg/dl; alanine transferase, 20 U/L; aspartate aminotransferase, 20 U/L; total protein, 7.0 g/dl; albumin, 3.9 g/dl; and glucose, 183 mg/dl. The platelet count was 289 000/ L; and prothrombin time/international normalized ratio, 0.9. The serum -fetoprotein level was 1533 ng/ml. Autoimmune markers were not obtained. Hepatitis B and C serology findings were negative (Table 2). The abdominal CT scan showed a 7 6-cm arterially enhancing lesion with well-defined margins within the right lobe of liver as well as features suggestive of fatty infiltration of the liver. At laparotomy, the liver was fatty and without evidence of cirrhosis. A large mass in segment 6 was completely resected. Pathologic Findings. The right liver lobectomy was pale, yellow-tan, and had a greasy surface. The main tumor mass was 6.8 5 4.5 cm. There were 3 additional satellite lesions; the largest was 3 2.5 2.5 cm, and the remaining 2 lesions were 0.5 and 0.3 cm. The tumors were all well-differentiated HCC, grade 1, with a predominant pseudoglandular pattern. The nonneoplastic liver had 40% zone 3 large-droplet steatosis. There were no features of active steatohepatitis. There was no significant portal or zone 3 pericellular fibrosis on trichrome stain (Figure 3). There were no histopathologic features indicative of viral hepatitis, 1 -antitrypsin deficiency, or hemochromatosis. Hepatitis B core antigen, PAS/D, and iron histochemistry findings were negative. There was no evidence for any chronic liver disease other than NAFLD (Table 3). COMMENT Nonalcoholic fatty liver disease is the most common cause of liver disease in the United States, where the condition is thought to affect up to one third of the adult population. 22 It represents the hepatic manifestation of the metabolic syndrome, characterized by obesity, insulin resistance, dyslipidemia, and elevated blood pressure. 9 In recent reports, 13,23 28 HCC was identified in patients with cirrhosis and metabolic syndrome (Table 4). However, it is not clear whether cirrhosis is necessary for the development of HCC in patients with NAFLD. 29,30 Cases of HCC arising in NASH, without cirrhosis, raise the possibility that hepatocarcinogenesis can occur in patients with the metabolic syndrome in the absence of advanced liver disease (Table 5). 9 14 We describe 3 additional cases of HCC in patients with features of metabolic syndrome and histopathologic evidence of NAFLD, but without steatohepatitis, fibrosis, or cirrhosis. The patients had bland steatosis on liver biopsy, with NAFLD activity scores ranging from 1 to 2 out of a total score of 8. All had an increased BMI. The first and third patients had diabetes or hyperglycemia. None of the patients had evidence of a cause of liver disease other than 1764 Arch Pathol Lab Med Vol 132, November 2008 HCC in NAFLD and the Metabolic Syndrome Guzman et al

NAFLD by history, laboratory testing, or histopathologic assessment. Notably, all 3 patients had advanced HCC with multiple lesions at the time of presentation. These cases provide further evidence that HCC can develop in patients with NAFLD in the absence of both hepatic fibrosis and the vascular architectural remodeling of cirrhosis. A number of factors have been identified that might contribute to the pathogenesis of HCC in NAFLD. Diabetes was found to be a risk factor for HCC independent of age, sex, and race, 31 leading some investigators to postulate that chronic hyperinsulinemia and insulin-like growth factor 1 might be involved in carcinogenesis. 17 Ratziu et al 29 observed that cirrhosis developed later in obese patients with cryptogenic liver disease than in those with hepatitis C virus, whereas HCC occurred at a similar age, indicating that insulin resistance might promote carcinogenesis in the absence of cirrhosis. 29 In fact, experimental models of NASH in mice have demonstrated spontaneous development of HCC. 14 Oxidative stress is considered to be increased in NAFLD and causes hepatocyte injury while inhibiting the replication of mature hepatocytes. 32 Yang et al 33 35 showed that liver cell hyperplasia is present in the earliest stages of NAFLD in ob/ob mice, suggesting that metabolic abnormalities, rather than cirrhosis, may initiate the hepatic hyperplastic/neoplastic changes. Equivalent studies in humans, however, have not been done. Caldwell et al 30 hypothesized that oxidative stress in NAFLD might induce cancer-promoting mutations and that growth factors might drive proliferation of neoplastic cells. A combination of amplified cell proliferation and continuous oxidative damage contributed to the development of HCC in a mouse model for impaired antioxidant expression. 36 Although provocative conclusions have been drawn from animal studies regarding the role of oxidative stress in hepatocarcinogenesis, it remains to be proven whether similar mechanisms of carcinogenesis apply to humans with NAFLD. Recently, 37 a single gene was identified linking oxidative stress to NAFLD, NASH, and hepatic tumors. Liver-specific inactivation of Nrf1, a transcription factor essential for embryonic development, activates oxidative stress response genes and might stimulate development of HCC. Genomic and proteomic studies 38 showed differential expression of several genes and proteins in patients with the spectrum of NAFLD, notably the downregulation of Muclass glutathione S-transferases, the enzymes that play a role in the cellular defense against oxidative stress. These changes were observed in the early stages of NAFLD. Taken together, the available data suggest that processes involved in hepatocarcinogenesis may occur in NAFLD before the onset of cirrhosis. Finally, 2 important clinical features are raised by these cases. The first is all 3 cases had advanced HCC at the time of presentation. They all had more than one tumor, and 2 were unresectable. That fact and the fact that none of our cases or those previously reported in the literature 9 14 had a history of liver disease or cirrhosis and were, thus, not in an HCC-surveillance screening program, call attention to a clinical need to be attentive to patients with metabolic syndrome who have NAFLD for this putative complication. Some important limitations in this report should be noted. Liver resection or transplantation is performed in only a small percentage of cases of HCC. The development of HCC in the absence of cirrhosis is an uncommon event, and the data from this study should not be interpreted to suggest that 6% (3/50) of HCC cases arise in this way. Information regarding features of the metabolic syndrome, which is associated with NAFLD and HCC was limited because of the retrospective design. We cannot exclude the possibility that the cases of HCC occurred by chance in patients with NAFLD, given the high prevalence of NAFLD in the United States. Finally, although serologic testing for hepatitis B and C was negative, virologic testing was not performed except in case 1. However, the presence of occult viral hepatitis seems unlikely based on the histologic findings. In conclusion, the cases of HCC described here were notable not only for the absence of cirrhosis but also for the lack of histologically identifiable steatohepatitis, fibrosis, or vascular remodeling of cirrhosis and for advanced tumor stage at the time of presentation. These cases suggest that HCC can arise in patients with both NAFLD and features of metabolic syndrome, without evidence of cirrhosis. Further studies are needed to confirm this observation and to determine the prevalence and pathogenesis of such events. References 1. Suriawinata A, Xu R. An update on the molecular genetics of hepatocellular carcinoma. Semin Liver Dis. 2004;24(1):77 88. 2. Fattovich G, Stroffolini T, Zagni I, Donato F. 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