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1 346 Cldwell SH, et l.,, 2009; 8 (4): ORIGINAL ARTICLE October-December, Vol. 8 No.4, 2009: NASH nd cryptogenic cirrhosis: A histologicl nlysis Stephen H. Cldwell,* Vness D. Lee,* Dvid E. Kleiner,** Abdullh M.S. Al Osimi,* Curtis K. Argo,* Ptrick G. Northup,* Crl L. Berg* * Division of Gstroenterology nd Heptology, Digestive Helth Center. University of Virgini, Chrlottesville, Virgini. ** Lbortory of Pthology, Ntionl Cncer Institute, Bethesd, Mrylnd, USA. ABSTRACT Introduction. Epidemiologicl studies indicte tht nonlcoholic stetoheptitis (NASH) is common cuse of cirrhosis described s cryptogenic. To ddress this from histologicl perspective nd to exmine the significnce of residul histologicl findings s n indiction of prior NASH, we looked bck t biopsies in ptients who presented with cirrhosis without sufficient histologicl fetures to dignose NASH but who hd prior histologiclly confirmed non-cirrhotic NASH. Methods. Seven ptients were identified with biopsy pirs showing non-specific (cryptogenic) cirrhosis in the ltest specimen nd prior biopsy showing non-cirrhotic NASH. Using n expnded NASH-CRN system scored blindly by light microscopy, we compred the erly nd lte biopsies to ech other nd to cohort of 13 ptients with cirrhosis due to heptitis C without co-existing metbolic syndrome. Results. Mcrostetosis, lthough uniformly present in the noncirrhotic NASH specimens, declined in the lte stge cirrhotic NASH specimens nd ws not useful in the distinction of lte cirrhotic NASH from cirrhotic virl heptitis. However, the presence of bllooned cells, Mllory-Denk bodies, nd megmitochondri nd the bsence of poptotic bodies were significntly different in lte stge cirrhotic NASH compred to cirrhosis due to heptitis C. Conclusions. Histologiclly dvnced NASH presenting s non-specific or cryptogenic cirrhosis hs residul chnges which re consistent with prior stetoheptitis but which differ from cirrhosis due to heptitis C. These results provide histologicl support for the more estblished epidemiologicl ssocitions of NASH with cryptogenic cirrhosis nd for criteri used in severl proposed clssifictions of cryptogenic cirrhosis. Key words. Cryptogenic cirrhosis. Non-lcoholic stetoheptitis (NASH). Non-lcoholic ftty liver disese (NAFLD). Mcrostetosis. Acidophil bodies. INTRODUCTION Correspondence nd reprint request: Stephen H. Cldwell, M.D. GI/Heptology, Box , University of Virgini, Chrlottesville, VA, 22908, USA. Tel.: , fx: E-mil: shc5c@virgini.edu Finncil Support/Grnts: This reserch ws supported in prt by the Intrmurl Reserch Progrm of the NIH, Ntionl Cncer Institute. Mnuscript received: November 4, Mnuscript ccepted: November 11, A vriety of conditions cn cuse cirrhosis presenting in ptients without definitive histologicl, historicl or lbortory findings to indicte the underlying cuse. This is usully referred to s nonspecific or cryptogenic cirrhosis nd ccounts for pproximtely 8-9% of liver trnsplnttions in the US. 1 Non-lcoholic stetoheptitis (NASH) hs been implicted s common cuse of this condition. Seril biopsy studies hve shown tht bout 5-15% of ptients with non-cirrhotic NASH will develop cirrhosis within 5-15 yers lthough in most such cses the finl biopsy ws interpreted either simply s cirrhosis or s consistent with cirrhotic NASH rther thn s non-specific or cryptogenic cirrhosis. 2 Loss of stetosis s key indictor of ftty liver disese in lte stge NASH ws first described by Powell, et l. in We subsequently described strong epidemiologicl reltionship between NASH nd cryptogenic cirrhosis in This ssocition ws lter confirmed in number of epidemiologicl studies 5-7 nd in reports regrding the post-opertive clinicl course of ptients undergoing liver trnsplnttion for cryptogenic cirrhosis. 8,9 Although the epidemiologicl dt hs been strong in most reports ddressing this problem, very few biopsy pirs hve ctully been reported wherein ptient with non-specific or cryptogenic cirrhosis (without defi-

2 NASH nd Cryptogenic Cirrhosis., 2009; 8 (4): nitive histologicl or clinicl/lbortory prmeters of n underlying disese) hs n ntecedent biopsy demonstrting non-cirrhotic NASH. This issue hs prcticl implictions relted to ccurte dignosis of underlying disese nd in proposed clssifiction schemes for cryptogenic cirrhosis. 10,11 To ddress this further, we undertook systemtic look bck t prior histology in ptients presenting to us with histologicl cirrhosis identified s non-specific by biopsy but with prior histologiclly proven non-cirrhotic NASH. METHODS Seven ptients were identified who met the stringent study criteri including current biopsy dignosed s non-specific cirrhosis by histologicl criteri, prior biopsy nd comptible history confirming non-cirrhotic NASH in the pst nd bsence of other liver disese by conventionl testing to exclude virl, utoimmune or other liver diseses such s Wilson disese, hemochromtosis or lph-1-ntitrypsin deficiency. The medin time between the biopsy showing non-cirrhotic NASH nd tht showing non-specific cirrhosis ws 6 yers. Demogrphic dt of this group re shown in Tbles 1 nd 2. All but one of these ptients met ATP III criteri for metbolic syndrome. 12 Hemtoxylin nd eosin (H&E) nd Msson trichrome stined specimens from the lte nd erly stge specimens were ssessed blindly by n experienced pthologist (DK) using n expnded NASH-Clinicl Reserch Network (NASH-CRN) system which included 14 histologicl fetures scored semi-quntittively (Tble 3). 13 In order to scertin the reltive frequency of NASH ssocited histologicl prmeters in nother form of cirrhosis, the specimens from the NASH-nonspecific cirrhosis pirs were compred to cohort of 13 heptitis C ptients with cirrhosis undergoing liver trnsplnttion who were selected bsed on the bsence of metbolic syndrome. Demogrphic dt for these ptients is shown in Tble 1. These ptients tended to be slimmer (men BMI 25.8 ± 4), nd none hd hyperlipidemi lthough four did hve history of hypertension nd two of these ptients hd insulin dependnt dibetes. Four Tble 2. Comprtive demogrphics: Lte stge NASH (nonspecific cirrhosis) versus heptitis C-relted cirrhosis. Chrcteristic NASH (n = 7) HCV (n = 13) Gender (m/f) 1/6 9/4 BMI 40.8 ± ± 4 Ethnicity Cucsin 7 11 Blck 0 1 Other 0 1 Comorbidities Dibetes 5 2 * Hypertension 5 4 Hyperlipidemi 40 Previous liver trnsplnt 1 1 Drugs Metformin 3 0 Thizolidinedione 40 Insulin 2 2 Sttin 3 0 Ezetimibe 1 0 * Insulin dependnt dibetes. Tble 1. Subjects with non-specific cirrhosis nd prior non-cirrhotic NASH. Chrcteristic Vlue Mesure Age t 1 st NASH biopsy 47.9 ± 12 Men ± SD Yers to cirrhosis 6 Medin 1 st qurtile 5 yrs; 3 rd qurtile 8 yrs Weight t initil biopsy ± 24.4 kg Men ± SD Weight t cirrhosis biopsy ± 25.1 kg Men ± SD Chnge in weight kg Medin 1 st qurtile ; 3 rd qurtile BMI t initil biopsy 38.1 ± 4 Men ± SD BMI t cirrhosis biopsy 40.8 ± 5.10 Men ± SD Chnge in BMI Medin 1 st qurtile ; 3 rd qurtile

3 348 Cldwell SH, et l.,, 2009; 8 (4): Tble 3. Comprison of NASH-CRN histologicl fetures identified in NASH, NASH cirrhosis, nd heptitis C cirrhosis. Chrcteristic / N / Comprisons P Non-cirrhotic Non-specific Heptitis Group Group NASH Cirrhosis C Cirrhosis 1 vs. 2 2 vs. 3 N = 7 N = 7 N = 13 Mcrovesiculr stetosis Present (> 5%) Mcrovesiculr stetosis < 5% % % > 66% Stetosis distribution Zone Zone Azonl Pncinr Microvesiculr stetosis Present in contiguous ptches Portl Inflmmtion None Mild Greter thn mild Prenchyml Inflmmtion No foci < 2 foci per 200x field foci per 200x field > 4 foci per 200x field Microgrnulom (present) Lrge lipogrnulom (present) Bllooned Cells (present) Bllooned Cells (grde) None Few Mny Acidophil Bodies Pigmented mcrophges Megmitochondri Mllory-Denk Bodies Glycogented Nuclei Fibrosis Stge hd distnt history of moderte or less ethnol use but none within six months nd none were felt to hve lcohol-relted liver disese. Virl genotypes included genotype 1 (n 8), genotype 4 (n 2), indeterminte genotype (n 2) nd genotype 3 (n 1). One ptient in the NASH group nd one in the heptitis C group hd previously undergone liver trnsplnttion, one for cryptogenic cirrhosis in the former group nd one for heptitis C relted cirrhosis in the ltter group. Both of these ptients were included becuse they met our inclusion criteri nd

4 NASH nd Cryptogenic Cirrhosis., 2009; 8 (4): COLOR it ws felt tht the inclusion of these ptients brodened the findings. The Fisher s exct test nd signed rnk test performed with SAS version 9.1 (SAS Institute Inc., Cry, NC, USA) were used to compre the ctegoriclly scored histologicl fetures in the non-cirrhotic NASH ptients, their pired biopsy showing non-specific cirrhosis nd the cirrhotic heptitis C ptients. The protocol for this study ws pproved by the University of Virgini Institutionl Review Bord. RESULTS Among the ptients with histologicl NASH, ll were obese t the time of the first biopsy with Figure 1. Cellulr bllooning with Mllory-Denk bodies (rrows) in subject with non-cirrhotic NASH. (H&E, 400x). men BMI of 38.1 (rnge ) nd men ge of 47.9 yers (rnge 29 to 58 yrs) t the time of dignosis (Tble 1). These ptients progressed from NASH with fibrosis stge 2 (3 subjects) or 3 (4 subjects) to the ppernce of non-specific cirrhosis over medin of 6 yers (rnge 3 to 11 yers). All but one of the ptients gined weight in this intervl with medin chnge in weight nd BMI of kg nd respectively without evidence of scites. Compred to the heptitis C group (Tble 2), there ws significntly higher proportion of dibetes (p 0.02) nd hyperlipidemi (p 0.007) in the NASH group. Consistent with these conditions, the NASH ptients were more frequently treted with metformin (n 3), thizolidinediones (n 4), nd sttin-type medictions (n 3), p < 0.05 for ech gent. Tble 3 shows the comprisons of histologicl fetures between the prior NASH biopsy nd the subsequent non-specific cirrhosis specimens in the NASH group. There ws sttisticl trend towrds complete loss of mcrostetosis. When clssified s present or bsent, ll subjects hd mcrostetosis in the prior non-cirrhotic NASH specimen but 4 out of 7 subjects hd complete loss of mcrostetosis in the cirrhosis biopsy specimen (p 0.07). However, there ws no significnt difference in the presence of bllooning (Figures 1 nd 2) evident in 5 out of 7 ptients t both the NASH stge nd the non-specific cirrhosis stge or in the presence of microstetosis in the erly versus lte stge smples. Portl inflmmtion incresed s subjects progressed from NASH to cirrhosis (p 0.03) wheres prenchyml inflmmtion ws unchnged. Mllory-Denk bodies COLOR COLOR Figure 2. Bllooned heptocytes (center) within cirrhotic nodule in subject with non-specific cirrhosis but with ntecedent biopsy showing non-cirrhotic NASH. (H&E, 200x). Figure 3. Megmitochondri (rrows) within cirrhotic nodule in subject with non-specific cirrhosis nd with prior biopsy showing non-cirrhotic NASH. (H&E, 400x).

5 350 Cldwell SH, et l.,, 2009; 8 (4): Mcroveciculr Ft Bllooned Cells Mllory-Den Body Group Non-cirrhotic NASH Figure 4. Differences in mcrostetosis, cellulr bllooning nd Mllory-Denk body histologicl scores between the three groups including the erly non-cirrhotic NASH biopsy, the lter pired non-specific cirrhosis specimen nd the control group of heptitis C ptients who lcked metbolic syndrome. Indictes sttisticl trend (p = 0.07) in the loss of mcrovesiculr ft in the erly (non-cirrhotic NASH) versus lte (non-specific cirrhosis) NASH ptients. * Indictes sttisticlly significnt difference (p < 0.05) between the erly or lte NASH smples versus the heptitis C group. (Figures 1 nd 2), megmitochondri (Figure 3), glycogented nuclei, microgrnuloms, lipogrnuloms, nd pigmented mcrophges were noted eqully in both the non-cirrhotic NASH nd the lte stge cirrhosis specimens. Acidophil (poptotic) bodies were notbly bsent in both stges. Results compring the histologicl fetures of the NASH group to those from heptitis C cirrhosis re shown in Tble 3 nd Figure 4. In terms of mcrostetosis, lte stge NASH resembled cirrhosis due to virl HCV becuse mcrostetosis ws miniml in both cirrhotic groups including the single ptient (grde 1 stetosis) with genotype 3 heptitis C. Overll, there ws no sttisticlly significnt difference in the presence (p 0.29) or the grde of mcrostetosis (p 0.35) between the NASH-relted nonspecific cirrhosis group nd the heptitis C cirrhosis group. However, the presence nd the grde of bllooned cells (p 0.015, p 0.03 respectively), the presence of megmitochondri (p 0.03) nd the presence of Mllory-Denk bodies (p 0.02) were significntly greter in the NASH-relted cryptogenic cirrhosis group compred to heptitis C relted cirrhosis. There ws lso strong trend towrds significnce in the bsence of cidophil bodies in the NASH group. Acidophil bodies were identified in 6 * NASH: Nonspecific Cirrhosis * Heptitis C Cirrhosis out of 13 heptitis C cirrhosis specimens but were bsent in both the erly nd the lte stge NASH group (p 0.05). DISCUSSION Epidemiologic dt support tht progression of NASH is one of the more common cuses of cirrhosis chrcterized histologiclly s non-specific nd cliniclly s cryptogenic. Compred to the epidemiologicl literture, the histologicl literture is reltively less estblished in this regrd. Although progression of NASH to cirrhosis is very well known from seril biopsy studies, we re wre of only 21 histologiclly confirmed cses in published ppers which document progression of histologicl NASH to stge of nonspecific (without definitive fetures of NASH) cirrhosis by biopsy. 3,14-21 In the present study, we hve looked bck t prior histologicl specimens in ptients presenting to us with cirrhosis which histologiclly ws dignosed s non-specific nd compred these specimens to the prior NASH smples nd to cirrhosis relted to heptitis C. Strict entry criteri limited the focus of this study to these unique ptients presenting with histologiclly non-specific cirrhosis who lso hd prior biopsy (usully from n outside institution) consistent with non-cirrhotic NASH. Blinded ssessment of these biopsies nd comprison of these pirs with cirrhosis due to heptitis C in ptients without co-existing metbolic syndrome reveled tht lte stge NASH possesses some distinctive fetures consistent with ntecedent NASH including bllooned heptocytes, Mllory-Denk bodies, nd megmitochondri (Figures 1-3). These results support previously proposed clssifiction schemes for cryptogenic cirrhosis The most prominent histologicl feture which chnged in erly versus lte stge NASH ws the loss of mcrostetosis -usully considered n essentil but not sufficient indictor of NASH. Indeed, there ws no significnt difference in mcrostetosis between the lte stge NASH specimens nd the lte stge heptitis C specimens from ptients without metbolic syndrome-both of the cirrhotic groups hd reltively low mcrostetosis compred to the NASH smples (Figure 4). Counted s present or bsent, 4 of 7 NASH ptients completely lost evidence of this feture in the lte-stge, non-specific cirrhosis biopsy. Although unresolved, severl explntions for loss of stetosis hve been proposed including ltered heptic blood flow resulting in ltered insulin exposure nd bnorml glucose nd lipoprotein delivery due to sinusoidl injury Another less est-

6 NASH nd Cryptogenic Cirrhosis., 2009; 8 (4): blished explntion involves repopultion of the ntive liver with one derived from disese-resistnt stem cells (ovl cells) leding to fundmentl chnges in heptic ft metbolism nd diminished ft storing cpcity. In support of this mechnism, such cells constitute the mjority of cells in regenerting cirrhotic nodules in NASH-cirrhosis. 25 Cirrhosis-relted mlnutrition is nother possibility. However, weight gin ws seen in the NASH ptients in the intervl between the first nd second biopsy. Becuse none of these ptients hd over complictions of portl hypertension such s scites, the weight gin ws unlikely to be due to simple fluid retention. This suggests tht diminished mcrostetosis in dvncing NASH ws not simply due to systemic cirrhosis-relted mlnutrition. Acidophil bodies were distinctly less evident in both erly nd lte stge NASH compred to heptitis C cirrhosis specimens. This ws somewht prdoxicl given the prominent role of poptosis pthwy ctivtion in NASH pthogenesis. It suggests tht cell deth my proceed more by necrosis or necropoptosis but the issue remins to be resolved. We lso noted significntly incresed inflmmtion in the residul portl res s NASH progressed to non-specific cirrhosis. This is in keeping with more recent reports on portl involvement in NASH but contrsts to prior studies on this issue. 3,16,26,27 The reson for this discrepncy is uncler. There re severl inherent limittions in this work. As look bck study, it is retrospective by design s we worked bck from ptients presenting with non-specific cirrhosis defined histologiclly. Strict entry criteri lso required tht such ptients hve n ntecedent biopsy showing non-cirrhotic NASH thus introducing specific bis but which ctully constituted n im of the study to compre these well-defined biopsy pirs. However, this lso limited cse scertinment becuse most of the ntecedent biopsies were performed t outside institutions s long go s 11 yers thus limiting the size of the study to those in whom both specimens could be obtined for blinded review. Another limittion of our nlysis of this dt is the inherently ctegoricl nture of the NASH-CRN scoring system chrcteristic of ll such scoring systems. Becuse the primry im of this study ws to ssess residul fetures of NASH in non-specific cirrhosis in ptients with proven prior NASH, we elected to utilize system designed specificlly for NASH rther thn other systems such s Ishk often pplied to virl heptitis. Also, the inclusion of two ptients (one in the NASH group nd one in the heptitis C group) who hd prior liver trnsplnttions my hve dded degree of heterogeneity. However, both subjects met strict entry criteri nd were included s it ws felt to broden the study nd demonstrted the occurrence of post-trnsplnt NASH with progression to non-specific cirrhosis. In conclusion, our results provide further histologicl evidence to support the progression of NASH to lte stge in which there is loss of dignostic fetures of NASH leding to histologicl dignosis of non-specific cirrhosis nd clinicl dignosis of cryptogenic cirrhosis. These results confirm tht residul histologicl fetures of NASH remin even in these dvnced cses nd distinguish this form of cryptogenic cirrhosis from cirrhosis cused by dvnced heptitis C. In prticulr, heptocyte bllooning, Mllory-Denk body formtion nd megmitochondri nd pucity of cidophil bodies my id in the interprettion of these cses. Further studies re wrrnted to understnd the mechnisms involved with loss of stetosis s the disese progresses nd to develop other histologicl mrkers of prior lipid-induced oxidtive injury. ABBREVIATIONS NASH: Non-lcoholic stetoheptitis. NASH-CRN: NASH-Clinicl Reserch Network. NIDDK: Ntionl Institute of Dibetes nd Digestive nd Kidney Diseses. BMI: Body mss index. REFERENCES 1. Angulo P. Nonlcoholic ftty liver disese. N Engl J Med 2002; 346(16): Argo CK, Northup PG, Al-Osimi AM, Cldwell SH. Systemtic review of risk fctors for fibrosis progression in nonlcoholic stetoheptitis. J Heptol 2009; 51: Powell EE, Cooksley WG, Hnson R, Serll J, Hllidy JW, Powell LW. The nturl history of nonlcoholic stetoheptitis: follow-up study of forty-two ptients for up to 21 yers. Heptology 1990; 11: Cldwell SH. Oelsner DH. Iezzoni JC. et l. Cryptogenic cirrhosis: clinicl chrcteriztion nd risk fctors for underlying disese. Heptology 1999; 29: Mheshwri A. Thuluvth PJ. Cryptogenic cirrhosis nd NAFLD: Are they relted? Am J Gstroenterol 2006; 101: Poonwl A, Nir SP, Thuluvth PJ. Prevlence of obesity nd dibetes in ptients with cryptogenic cirrhosis: A cse-control study. Heptology 2000; 32(4 Pt 1): Browning JD, Kumr KS, Sboorin MH, Thiele DL. Ethnic differences in the prevlence of cryptogenic cirrhosis. Am J Gstroenterol 2004; 99:

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