Steatohepatitic Hepatocellular Carcinoma, a Morphologic Indicator of Associated Metabolic Risk Factors

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1 Steatohepatitic Hepatocellular Carcinoma, a Morphologic Indicator of Associated Metabolic Risk Factors A Study From India Deepali Jain, MD, DNB; Nabeen C. Nayak, MD, FRCPath; Vinay Kumaran, MS, MCh; Sanjiv Saigal, MD, DM Context. The common risk factors for hepatocellular carcinoma (HCC) include persistent viral infection with either hepatitis B or C virus, alcohol abuse, hemochromatosis, and metabolic syndrome. Steatohepatitic (SH) HCC has been recently recognized as a special morphologic variant of HCC associated with metabolic risk factors. Objective. To assess the SH pattern in HCC cases of various etiologies in Indian patients and to further correlate this morphology with the presence of metabolic risk factors. Design. A total of 101 cases of HCC with various etiologies in explanted livers from adults were included in the study. Morphologic examination was performed to identify SH lesions within the tumor and in the nontumorous liver parenchyma. Correlation of nontumor and tumor SH morphology with clinically identifiable metabolic risk factors and with non-sh type of HCC was performed. Results. The SH variant of HCC was identified in 19 livers (18.8%). Most SH-HCC cases were associated with metabolic risk factors such as obesity, diabetes, hypertension, and hyperlipidemias. Comparison of SH-HCC with non SH-HCC was statistically significant in terms of presence of metabolic risk factors. Conclusions. Steatohepatitic morphology in HCC is frequent in nonalcoholic fatty liver disease associated cirrhosis (P ¼.009) and is significantly associated with metabolic risk factors (P ¼.03). By recognizing SH pattern, one may predict associated metabolic diseases and determine the prognosis both in pretransplant and posttransplant patients. (Arch Pathol Lab Med. 2013;137: ; doi: / arpa oa) Hepatocellular carcinoma (HCC) accounts for 85% to development of HCC in NAFLD, which include obesity, 90% of all primary liver cancers. 1 There is wide diabetes, and iron deposition. 7,8 Recently a new morphologic geographic variation of prevalence of HCC, with Asia and Africa having 40 times more cases than other parts of the variant of HCC named steatohepatitic hepatocellular carcinoma has been identified that relates to the presence of world. 2 However, the incidence of HCC has been rising metabolic syndrome. 9,10 The aim of the study is to recognize throughout the world, with particularly large increases seen in industrialized nations such as the United States and Denmark. 3 Nonalcoholic fatty liver disease (NAFLD) ranges steatohepatitic (SH) pattern in HCC cases of various etiologies in Indian patients and to further correlate this variant with the presence of metabolic risk factors. in severity from steatosis to steatohepatitis (NASH, nonalcoholic steatohepatitis) and finally to cirrhosis, which is MATERIALS AND METHODS known to be a risk factor for the development of HCC. 4 In Between January 2004 and January 2011, 450 adult patients Western and Asian countries, the prevalence of NAFLD in underwent living donor liver transplant for end-stage liver disease. the general population is increasing dramatically. 5 Genetic factors may in part contribute to the rise in NAFLD. Polymorphisms within various genes have been linked to For each patient a pre liver transplant diagnosis of the etiologic type of end-stage chronic liver disease was recorded, but the final diagnostic category of native liver disease was assigned only on the NAFLD in lean Indian men. 6 Emerging evidence has basis of a combination of pretransplant clinical data and established multiple independent risk factors for the morphologic findings in the explant liver. Among 450 patients, 101 underwent transplant for HCC secondary to chronic hepatitis C virus (HCV) infection (n ¼ 42), chronic hepatitis B virus (HBV) infection (n ¼ 32), alcoholic liver disease (ALD) (n ¼ 11), NAFLD (n Accepted for publication August 29, ¼ 12), and mixed (HBVþHCVþALD) etiology (n ¼ 4). Most (11 of From the Departments of Pathology (Drs Jain and Nayak) and Liver 12) NAFLD transplant cases were associated with the clinical Transplant (Dr Kumaran and Dr Saigal), Sir Ganga Ram Hospital, diagnosis of cryptogenic cirrhosis. The diagnosis of NAFLD was New Delhi, India. based on clinical history of nonalcoholism and established The authors have no relevant financial interest in the products or companies described in this article. histologic features such as steatosis, lobular inflammation, Mallory Reprints: Deepali Jain, MD, DNB, Department of Pathology, Sir hyaline, and hydropic change. Ganga Ram Hospital, Rajinder Nagar, New Delhi, India ( In all cases, information on pretransplant clinical and investiga- deepalijain76@gmail.com). tional data were analyzed. Basic demographics, body mass index, Arch Pathol Lab Med Vol 137, July 2013 Steatohepatitic Hepatocellular Carcinoma Jain et al 961

2 Clinical and Pathologic Differences Between 2 Groups of Steatohepatitic (SH) and Conventional Hepatocellular Carcinoma (HCC) Clinical Features SH-HCC (19) Conventional HCC (82) Total Cases (101) P Value Age, mean (range), y 54.8 (47 65) 51.2 (45 67) 52.3 (45 67)..99 Sex, M/F 17/2 74/8 91/10.99 Associated etiology HCV (9), NAFLD (6), HBV (2), HCV (35), HBV (26), ALD HCV (44), HBV (28), ALD.32 ALD (1), mixed a (1) (10), NAFLD (6), CC (2), mixed a (3) (11), NAFLD (12), CC (2), mixed a (4) Risk factors, b No. (%) 10/16 c (62.5) 22/64 c (34.3) 32/80 (40).03 NAFLD Tumor size, mean (range), cm d 3.38 (1 7) 3.17 (0.5 5) 3.58 (0.5 7)..99 No. of lesions, single/multiple 8/11 17/65 25/76.15 Differentiation, well/moderate/poor 6/13/0 20/55/7 26/68/7.74 Vessel invasion None None None Staging T1 T3a T1 T3a T1 T3a Abbreviations: ALD, alcoholic liver disease; CC, cryptogenic cirrhosis (no etiology could be determined even after clinical and histologic examination); HBV, hepatitis B virus; HCV, hepatitis C virus; NAFLD, nonalcoholic fatty liver disease; T, pathologic tumor stage. a Mixed etiology: HBVþHCVþALD. b Metabolic risk factors such as obesity (increase in body mass index), diabetes, hyperlipidemia, hypertension. c Risk factor information is available for 16 (SH-HCC) and 64 (conventional HCC) patients only. d The largest tumor size was taken in multifocal tumors for comparison. and presence of metabolic abnormalities, such as obesity, diabetes, hypertension, and hyperlipidemia, were particularly noted. Triglyceride levels of 150 mg/dl or higher and cholesterol levels of 250 mg/dl or higher were considered as hyperlipidemic. Body mass index (BMI) of more than 25 kg/m 2 was taken as increased BMI/ obesity. Patients carrying a previous clinical diagnosis of either hypertension or diabetes mellitus were identified as hypertensive and diabetic, respectively. Explant Liver Morphology Gross examination of the explant liver was done in respect of the size and location of the tumor. The number of lesions was recorded in cases of multicentric or multifocal tumors. Presence or absence of portal vein invasion was noted. Gross appearance of nontumorous background liver was documented. Histologic Analysis The analysis was performed on formalin-fixed, paraffin-embedded tissue sections stained with hematoxylin-eosin and Masson trichrome stains. A morphologic examination was performed to identify SH pattern within the tumor and in the nontumorous parenchyma. Correlation of SH morphology (tumor and nontumor) and non-sh type of HCC (conventional/standard HCC) with clinically identifiable metabolic risk factors was performed. In each case a minimum of 2 sections from the tumor were examined. More sections were studied when tumor was larger than 4 cm. The features of each tumor, including grade of differentiation and presence of vascular invasion, were noted. Presence of steatosis, fibrosis, inflammation, ballooning degeneration, and Mallory hyaline were recorded by 2 pathologists (D.J. and N.C.N.) without any knowledge of pretransplant clinical information for identifying SH variant of HCC. Hepatocellular carcinomas assigned as SH-HCC type showed steatohepatitic features in more than 5% of the tumor area 9 (including overall area in multiple HCCs), and displayed the combination of features described in steatohepatitis, including macrovesicular steatosis, ballooning degeneration, Mallory hyaline, fibrosis, and inflammation. The pattern of fibrosis was reported as trabecular (thick bundles of fibrous tissue within the tumor) or pericellular (thin strands of fibrosis). Inflammatory cells (neutrophils, mixed [neutrophils and lymphocytes]) were scored as þ (,2 foci of inflammatory cells under the 310 objective), þþ (2 to 5 foci), and þþþ (.5 foci). The degree of steatosis, presence of Mallory hyaline, and ballooning in the tumor were used to classify the cases as mild (5% 25%), moderate (25% 50%), or severe (more than 50%). Conventional and SH-HCC tumors were graded according to the Edmondson-Steiner classification into well-differentiated (grade I), moderately differentiated (grade II, III), and poorly differentiated (grade IV) types. Immunohistochemistry Glypican-3 immunostaining was performed to further confirm the diagnosis of HCC in SH variant. Immunohistochemical staining was carried out by using a mouse monoclonal antibody (B0134R, clone 1G12 isotype IgG1 human glypican-3, prediluted, BioMosaics Inc, Burlington, Vermont). Statistical Analysis For comparing categorical data, a Fisher exact test was used and for frequency distributions, a v 2 test was used. A difference was defined as significant at P.05. RESULTS Clinical features of all 101 patients with differences in SH and conventional HCC are summarized in the Table. Clinical Features and Metabolic Risk Factors The SH variant of HCC was identified in 19 livers (18.8%). In the SH-HCC group, there were 17 men and 2 women. Ages ranged from 47 to 65 years (mean, 54.8 years). Associated etiologies in these cases were HCV (9), NAFLD (6), HBV (2), ALD (1), and mixed (HBVþHCVþALD) infection (1). Age and sex were not found to be significantly different in the SH-HCC and the standard HCC groups. There was no difference in terms of size, location, number of lesions, overall tumor differentiation, and vascular invasion between SH and conventional HCC cases. Information about metabolic risk factors was available for 16 patients only. Of these, only 10 (62.5%) were associated with metabolic risk factors in the form of obesity, diabetes, hypertension, or hyperlipidemia. Four patients were obese, 4 were both obese and diabetic, and 2 were obese, hypertensive, and hyperlipidemic. Of these 10 patients, 4 were clinically and histologically diagnosed as having NAFLD, and 3 had associated HCV-related end-stage liver disease. The remaining 3 patients had underlying HBVrelated liver disease, ALD, and mixed etiology (HBVþHCVþALD). 962 Arch Pathol Lab Med Vol 137, July 2013 Steatohepatitic Hepatocellular Carcinoma Jain et al

3 Figure 1. A, Photomicrograph shows well-demarcated areas of steatohepatitic hepatocellular carcinoma (SH-HCC) (left side) in an otherwise welldifferentiated trabecular HCC (right side). B, The SH-HCC pattern merges into a more classical pseudoglandular region at lower left. C, Macrovesicular steatosis, inflammation, and ballooning are appreciated in SH-HCC area. D, Another case of SH-HCC illustrates prominent ballooning with conspicuous Mallory hyaline. E and F, Polymorphonuclear inflammatory infiltrate (E), and prominent Mallory hyaline and fibrosis (F) are seen in SH-HCC (hematoxylin-eosin, original magnifications 340 [A, B], 3200 [C, E, and F], 3400 [D]). In the conventional HCC group, 22 (22 of 64, 34.3%) patients had metabolic risk factors (Table). Fourteen patients were obese and 8 had both obesity and diabetes. Associated etiologies were as follows: HCV (9), NAFLD (6), HBV (4), ALD (2), and mixed HBVþHCVþALD infection (1). Comparison of SH-HCC with non SH-HCC was statistically significant (P ¼.03) in terms of presence of metabolic risk factors. Steatohepatitic morphology in HCC was frequent in NAFLD cirrhosis (P ¼.009) in comparison to non-nafld cases (Table). Gross Examination Grossly, tumors in both categories were either solitary or multiple. Size varied from 0.5 to 7 cm. Macroscopically, tumors were nodular and well demarcated from surrounding cirrhotic liver parenchyma. The color of the tumors was tan yellow to grey white. No vessel invasion was identified grossly. Histologic Analysis Most of the SH-HCC cases were moderately differentiated and showed trabecular and pseudoglandular patterns. Among the 19 SH-HCC cases, all showed macrovesicular steatosis, inflammation and ballooning degeneration, with Mallory hyaline in 16 of 19 cases (84.2%), and pericellular fibrosis in 18 of 19 cases (94.7%). Features of steatohepatitis were moderate to severe in intensity. All cases showed moderate (þþ) (10 of 19, 52.6%) to marked (þþþ)(9of19 cases, 47.3%) infiltration of the SH-HCC areas by neutrophils with admixed lymphocytes and plasma cells. Neutrophils were seen in the vicinity of ballooned hepatocytes. Lymphocytes and plasma cells were distributed through areas of tumor. Masson trichrome stain demonstrated characteristic pericellular fibrosis; associated trabecular thick bands of fibrosis were seen in some of these cases (Figures 1, A through F; 2, A through C). Glypican-3 immunostaining was performed in 18 cases. It showed strong cytoplasmic (11 of 13) and focal canalicular (2 of 13) positivity in 13 cases (Figure 2, D). Five cases showed negativity for this stain. Diagnosis of HCC was established in these 5 cases by recognizing paucireticulin pattern on reticulin staining and infiltrative margins of the tumor. Prevalence of Steatosis and NASH in the Adjacent Nonneoplastic Liver Background nontumorous liver was cirrhotic in all cases. Histologic analysis of the adjacent liver showed an increased prevalence of steatosis and steatohepatitis in explants of SH-HCC cases. Among these were 6 cases of NAFLD and 9 cases of HCV, which showed moderate steatohepatitis (Figure 3, A through C). Cases of HBV (2), ALD (1), and mixed etiology (1) also showed mild steatosis. Among the conventional HCC cases, metabolic risk factors were present in 22 cases. Of these 22 cases, background liver showed steatohepatitis in 3 NAFLD cases. Most cases of HCV (8 of 9) showed steatosis in addition to changes associated with viral hepatitis in the form of portalbased lymphoid aggregates and interface hepatitis (Figure 3, D). Three cases of HBV, with 1 case of mixed etiology and 1 other case of ALD, showed mild to moderate macrovesicular steatosis. One case each of HBV, HCV, and ALD and 3 cases of NAFLD did not show steatosis/steatohepatitis. The remaining cases of conventional HCC (60 of 82) showed variable features of end-stage liver disease with or without fatty change. Arch Pathol Lab Med Vol 137, July 2013 Steatohepatitic Hepatocellular Carcinoma Jain et al 963

4 Figure 2. A through C, Photomicrographs of steatohepatitic hepatocellular carcinoma (SH-HCC). Fibrosis is better appreciated in Masson trichrome stain. Trichrome stain shows characteristic pericellular fibrosis in (A) and thick fibrous bands in (B) and (C). D, Glypican-3 immunostain shows strong cytoplasmic positivity in SH-HCC (original magnifications 3400 [A, C, and D] and 3200 [B]). Of a total of 19 patients with SH-HCC, 16 were alive and disease-free at last follow-up of 24 to 72 months. Three patients died, with one death due to recurrence of tumor (HBV-associated HCC) after 30 months of diagnosis. Two other patients died (NAFLD-associated HCC) at the time of diagnosis after surgery owing to sepsis and multiorgan dysfunction. Follow-up information was available for 76 patients with conventional HCC. Of these, 16 patients died with 6 deaths due to recurrence of HCC. COMMENT Available evidence in the literature indicates that NAFLD is responsible for a large proportion of cases of cryptogenic cirrhosis and cryptogenic HCC. 11,12 Development of HCC in the setting of NAFLD/NASH supports the association of diabetes and obesity, as well as age, hepatic iron deposition, and advanced fibrosis, as a significant risk factor. 7,8,13 Since published literature on NAFLD in India is sparse, it is not clear whether these risk factors are similar in this part of the world. In general, differences in Indian patients include a lower frequency of metabolic syndrome. 14,15 Despite having a lower average BMI, Asians have a higher percentage of body fat compared to whites and African Americans. It has been suggested that the BMI limits for overweight and obesity should be lower for Asian Indians. 16,17 This explains why we have taken a BMI of 25 kg/m 2 or higher as a cutoff for obesity. Multiple case reports and reviews of HCC in the setting of NAFLD have been published. These suggest a prevalence of 4% to 27% of HCC after the development of cirrhosis, although the overall occurrence of HCC in the setting of NAFLD remains a rare complication. 5 Salomao et al 9 identified a distinctive histologic variant named steatohepatitic hepatocellular carcinoma in patients with HCV with associated NAFLD/NASH. They raised the question of altered biologic/morphologic characteristics of HCC in presence of risk factors for NAFLD and NASH. Recently, the same group of authors 10 has identified SH- HCC in 13.5% of HCCs with various etiologies in a span of 3.5 years. Steatohepatitic HCC is characterized by a constellation of SH features such as fatty change, ballooning degeneration, neutrophilic inflammation, perisinusoidal fibrosis, and Mallory bodies. Mallory bodies are known to occur in hepatocellular carcinoma and these were earlier considered as cellular markers for liver cell preneoplasia and 964 Arch Pathol Lab Med Vol 137, July 2013 Steatohepatitic Hepatocellular Carcinoma Jain et al

5 Figure 3. A through C, Case of nonalcoholic fatty liver disease (NAFLD) with steatohepatitic hepatocellular carcinoma (SH-HCC). Background liver shows macrovesicular steatosis with prominent ballooning and lobular inflammation. Mallory hyaline is seen in (C). D, A case of hepatitis C virus infection with conventional HCC. Photomicrograph shows cirrhosis with macrovesicular steatosis and portal-based chronic inflammation (hematoxylin-eosin, original magnifications 3100 [A and D], 3200 [B], and 3400 [C]). neoplastic change. 18,19 Nakanuma and Ohta 19 found a positive correlation between the presence of Mallory bodies in HCC tissue and the presence of Mallory bodies in the associated nonneoplastic hepatocytes in cryptogenic cirrhotic livers. It might be the process of steatohepatitis in HCC and nonneoplastic hepatocytes that has recently been identified by Salomao et al. 9,10 Overall, we found SH variant of HCC in approximately 19% of cases during a period of 7 years and in 50% of cases of NAFLD-HCC. The remaining 50% of SH-HCC cases were largely of HCV etiology. Hepatitis C virus infection itself induces insulin resistance and is associated with a 2- to 3-fold risk of developing type 2 diabetes 20 ; this may explain the higher association of metabolic risk factors and SH- HCC morphology in HCV cases. However, it is not known whether SH morphology in these patients is associated with metabolic syndrome or is due to viral steatosis related to HCV. Since HCV genotype 3 is predominant in India (genotyping not done in this study) and is associated with macrovesicular steatosis, 21 the SH morphology may possibly be due to the viral infection in some of these cases. This may explain the difference with the recent study by Salomao et al, 10 as these authors found a strong relationship between SH-HCC and background steatohepatitis; however, we found a significant association of SH-HCC with metabolic risk factors and NAFLD. Identifying SH pattern in HCC is important as it may indirectly hint toward metabolic dysfunction even if background liver overshadows underlying steatohepatitis due to end-stage liver disease. Steatohepatitis was seen only in 50% (3 of 6) of NAFLD cases It is known that steatosis, which is prominent and universal in the established stage of NAFLD/NASH, is inconspicuous or even absent in the advanced/cirrhotic stage of the disease This may be the reason for not finding features of fatty liver disease in half of the NAFLD cases. For patients with HCV, HBV, and ALD, steatosis was the only feature, which may represent a spectrum of fatty liver disease. Although it is difficult to determine the prognosis of SH- HCC owing to the small number of cases examined, it can be predicted that these tumors behave better, as only 1 patient died from tumor recurrence on follow-up, which also was associated with HBV. Certainly metabolic risk factors were present in a large number of cases (32 of 80, 40%) including SH and conventional HCC groups. This observation denotes that Arch Pathol Lab Med Vol 137, July 2013 Steatohepatitic Hepatocellular Carcinoma Jain et al 965

6 metabolic syndrome has now become an important risk factor directly in addition to playing an additive role in hepatic carcinogenesis. This is the first study of patients of Asian (Indian) origin that identifies SH-HCC originating in the setting of ALD, NAFLD, and viral hepatitis on both clinical and pathologic grounds. Further, correlation of SH-HCC with identifiable metabolic risk factors is discussed, which represents additional evidence to suggest a possible role of NAFLD in liver carcinogenesis. This study includes a cohort from an overall North Indian population and spans a 7-year period. However, it is limited by the selection of cases, as all of our patients had end-stage disease and needed a liver transplant. To know the exact prevalence of SH-HCC for all the recognized causes of liver disease, and its association with metabolic dysfunction, a further large-scale study of differently staged cases with inclusion of HCV genotyping may be required. CONCLUSION Nonalcoholic fatty liver disease has become an important cause of liver transplant in India. Prevalence of HCC in NAFLD is almost similar to that for ALD, signifying the important role of metabolic syndrome in liver carcinogenesis. Steatohepatitic morphology in HCC is frequent in NAFLD cirrhosis and has a significant association with metabolic risk factors. By recognizing SH pattern in HCC, one may predict associated metabolic syndrome and associated outcome of patients. Steatohepatitic pattern among HCCs may be considered as a prototype of metabolic dysfunction. References 1. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132(7): Bosch FX, Ribes J, Cléries R, Diaz M. Epidemiology of hepatocellular carcinoma. Clin Liver Dis. 2005;9(2): Jepsen P, Vilstrup H, Tarone RE, Friis S, Srensen HT. Incidence rates of hepatocellular carcinoma in the U.S. and Denmark: recent trends. Int J Cancer. 2007;121(7): Starley BQ, Calcagno CJ, Harrison SA. Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection. Hepatology. 2010;51(5): Siegel AB, Zhu AX. Metabolic syndrome and hepatocellular carcinoma: two growing epidemics with a potential link. Cancer. 2009:115(24): Petersen KF, Dufour S, Hariri A, et al. Apolipoprotein C3 gene variants in nonalcoholic fatty liver disease. N Engl J Med. 2010;362(12): Regimbeau JM, Colombat M, Mogol P, et al. Obesity and diabetes as a risk factor for hepatocellular carcinoma. Liver Transpl. 2004;10(2 suppl 1):S69 S Sorrentino P, D Angelo S, Ferbo U, Micheli P, Bracigliano A, Vecchione R. Liver iron excess in patients with hepatocellular carcinoma developed on nonalcoholic steato-hepatitis. J Hepatol. 2009;50(2): Salomao M, Yu WM, Brown RS Jr, Emond JC, Lefkowitch JH. 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End stage chronic liver disease yesterday, today and tomorrow. In: Michelli ML, ed. Liver Cirrhosis Causes, Diagnosis and Treatment. New York, NY: Nova Science Publishers; 2011: Arch Pathol Lab Med Vol 137, July 2013 Steatohepatitic Hepatocellular Carcinoma Jain et al