A randomized trial of iron depletion in patients with nonalcoholic fatty liver disease and hyperferritinemia

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1 Online Submissions: doi:1.3748/wjg.v2.i11.32 World J Gstroenterol 214 Mrch 21; 2(11): ISSN (print) ISSN (online) 214 Bishideng Publishing Group Co., Limited. All rights reserved. A rndomized tril of iron depletion in ptients with nonlcoholic ftty liver disese nd hyperferritinemi BRIEF ARTICLE Luc Vlenti, Ann Ludovic Frcnzni, Pol Dongiovnni, Seren Rovid, Rffel Rmett, Erik Ftt, Edordo Alessndro Pulixi, Mrco Mggioni, Silvi Frgion Luc Vlenti, Ann Ludovic Frcnzni, Pol Dongiovnni, Seren Rovid, Rffel Rmett, Erik Ftt, Edordo Alessndro Pulixi, Silvi Frgion, Deprtment of Pthophysiology nd Trnsplnttion, Metbolic Liver Diseses Reserch Center, Università degli Studi di Milno, Fondzione IRCCS C' Grnd Ospedle Policlinico, vi F Sforz 35, 2122, Milno, Itly Mrco Mggioni, Pthology, Fondzione IRCCS C Grnd Ospedle Policlinico, 2122 Milno, Itly Author contributions: All Authors contributed to literture review nd writing of this pper. Supported by Associzione Mlttie Metboliche del Fegto ONLUS (Non-profit orgniztion for the Study nd Cre of Metbolic Liver Diseses), Centro Studi Mlttie Metboliche del Fegto, Università degli Studi di Milno Correspondence to: Luc Vlenti, MD, Deprtment of Pthophysiology nd Trnsplnttion, Metbolic Liver Diseses Reserch Center, Università degli Studi di Milno, Fondzione IRCCS C' Grnd Ospedle Policlinico, vi F Sforz 35, 2122, Milno, Itly. luc.vlenti@unimi.it Telephone: Fx: Received: September 16, 213 Revised: November 6, 213 Accepted: December 3, 213 Published online: Mrch 21, 214 Abstrct AIM: To compre iron depletion to lifestyle chnges lone in ptients with severe nonlcoholic ftty liver disese (NAFLD) nd hyperferritinemi, frequent feture ssocited with more severe liver dmge, despite t lest 6 mo of lifestyle chnges. METHODS: Eligible subjects hd to be yers old who underwent liver biopsy for ultrsonogrphiclly detected liver stetosis nd hyperferritinemi, ferritin levels 25 ng/ml, nd NAFLD ctivity score > 1. Iron depletion hd to be chieved by removing 35 cc of blood every 1-15 d ccording to bseline hemoglobin vlues nd venesection tolernce, until ferritin < 3 ng/ ml nd/or trnsferrin sturtion (TS) < 25%. Thirtyeight ptients were rndomized 1:1 to phlebotomy (n = 21) or lifestyle chnges lone (n = 17). The min outcome of the study ws improvement in liver dmge ccording to the NAFLD ctivity score t 2 yers, secondry outcomes were improvements in liver enzymes [lnine minotrnsferses (ALT), sprtte minotrnsferse (AST), nd gmm-glutmyl-trnsferses (GGT)]. RESULTS: Phlebotomy ws ssocited with normliztion of iron prmeters without dverse events. In the 21 ptients complint to the study protocol, the rte of histologicl improvement ws higher in iron depleted vs control subjects (8/12, 67% vs 2/9, 22%, p =.39). There ws better improvement in stetosis grde in iron depleted vs control ptients (p =.2). In ptients followed-up t two yers (n = 35), ALT, AST, nd GGT levels were lower in iron-depleted thn in control ptients (p <.5). The prevlence of subjects with improvement in histologicl dmge or, in the bsence of liver biopsy, ALT decrese 2% (ssocited with histologicl improvement in biopsied ptients) ws higher in the phlebotomy thn in the control rm (p =.22). The effect of iron depletion on liver dmge improvement s ssessed by histology or ALT decrese 2% ws independent of bseline AST/ALT rtio nd insulin resistnce (p =.1). CONCLUSION: Iron depletion by phlebotomy is likely ssocited with higher rte of improvement of histologicl liver dmge thn lifestyle chnges lone in ptients with NAFLD nd hyperferritinemi, nd with meliortion of liver enzymes. 214 Bishideng Publishing Group Co., Limited. All rights reserved. Key words: Nonlcoholic ftty liver disese; Iron depletion; Rndomized controlled tril; Ferritin 32 Mrch 21, 214 Volume 2 Issue 11

2 Core tip: We compred in rndomized controlled tril iron depletion by phlebotomy (n = 21) to stndrd therpy (n = 17) in ptients with severe nonlcoholic ftty liver disese nd hyperferritinemi despite t lest 6 mo of lifestyle chnges. Phlebotomy ws ssocited with normliztion of iron prmeters without dverse events. In the 21 ptients complint to the study protocol, the rte of histologicl improvement ws higher in iron depleted vs control subjects (p =.39). In ptients followed-up t two yers (n = 35), by the end of the study lnine minotrnsferses, sprtte minotrnsferse, nd gmm-glutmyl-trnsferses levels were lower in iron-depleted thn in control ptients (p <.5). Vlenti L, Frcnzni AL, Dongiovnni P, Rovid S, Rmett R, Ftt E, Pulixi EA, Mggioni M, Frgion S. A rndomized tril of iron depletion in ptients with nonlcoholic ftty liver disese nd hyperferritinemi. World J Gstroenterol 214; 2(11): Avilble from: URL: com/ /full/v2/i11/32.htm DOI: org/1.3748/wjg.v2.i11.32 INTRODUCTION Nonlcoholic ftty liver disese (NAFLD), the heptic expression of the metbolic syndrome [1], ffects 2%-3% of the Western popultion [2,3]. When ssocited with necroinflmmtion nd bllooning, defining nonlcoholic stetoheptitis (NASH), NAFLD my evolve to cirrhosis nd heptocrcinom [4]. Although lifestyle modifictions improve liver dmge [5], long-term complince is difficult to chieve [6], nd no phrmcologicl pproch is yet pproved for NASH. Hyperferritinemi with mild heptic iron ccumultion is observed in 2%-3% of ptients with NAFLD referred to tertiry centres [7-1], nd is commonly referred s dysmetbolic iron overlod syndrome [11]. Besides directly inducing liver dmge [11], excess iron is involved in the pthogenesis of metbolic syndrome by inducing dipose tissue insulin resistnce nd modifying the relese of dipokines [11-13]. Furthermore, hyperferritinemi nd incresed iron stores hve been ssocited with the severity of liver disese nd heptocellulr crcinom in ptients with NAFLD [1,14-17]. Therefore, previous studies hve evluted the therpeutic vlue of iron depletion in NAFLD. Phlebotomy meliorted insulin resistnce more thn lifestyle chnges lone in mtched cse-control study including 128 ptients [18], wheres in propensity score djusted multicenter study with prospective evlution of 198 ptients, it incresed the likelihood of lnine minotrnsferses (ALT) normliztion t the end of follow-up [19]. Furthermore, in phse Ⅱ tril in 31 ptients with NAFLD, phlebotomy ws ssocited with nd improvement in disese ctivity [2]. Aim of this study ws to compre the effect of iron depletion by phlebotomy to tht of mintennce of lifestyle chnges lone (i.e., the stndrd of cre) on histologicl liver dmge evluted t 24 mo in rndomized controlled phse Ⅲ tril in subjects with NAFLD nd hyperferritinemi (CliniclTrils.gov Identifier: NCT658164). MATERIALS AND METHODS Eligible subjects hd to be yers old who underwent liver biopsy between Jnury 1 st 27, nd December 31 st 21 for ultrsonogrphiclly detected liver stetosis nd hyperferritinemi despite t lest 6 mo of lifestyle modifictions, ferritin levels 25 ng/ml or histologicl evidence of incresed iron stores, nd NAFLD ctivity score (NAS) > 1 (henceforth defined s severe NAFLD ) t liver biopsy obtined within 6 mo before rndomiztion, s commonly ccepted in NAFLD trils [21]. Subjects hd to be willing to mintin diet nd exercise during the full course of the study, to give written informed consent, nd to comply with ll study requirements. We excluded ptients with decompensted liver disese, pregnnt or lctting femles, type 1 or secondry forms of dibetes, thyroid diseses (by TSH evlution), lcohol consumption > 2 g/d for femles nd > 3 g/d for mles, BMI 35 kg/m 2, other liver diseses, lph- 1-ntitrypsin deficiency (lph-1-ntitrypsin levels < 8 mg/dl or PiZ/PiZ or PiZ/PiS genotype), nd hereditry hemochromtosis (HFE C282Y/C282Y or C282Y/ H63D or heptic iron index 1.9), previous or ctive chronic HCV nd HBV heptitis (HBsAg, ABcAg, nd nti-hcv Ab were evluted in ll subjects), congestive hert filure nd ischemic hert disese, systolic dysfunction (ejection frction 45%), ECG bnormlities, mlignncy within the lst 5 yers, serum cretinine levels > 1.5 mg/dl mles, > 1.4 mg/dl femles, use of drugs known to induce NAFLD, nd bsl hemoglobin levels < 11 g/dl. Before rndomiztion, ll ptients underwent electrocrdiogrphic evlution, nd in the presence of hyperglycemi or hypertension lso echocrdiogrphy. Crriers of HFE gene muttion without hemochromtosis were enrolled, s HFE muttions re strong risk fctors for mildly incresed iron stores in NAFLD [17,22]. Ptients were rndomized 1:1 by rndom numbers genertion to lifestyle chnges lone or lifestyle chnges ssocited with iron depletion by phlebotomy. In the phlebotomy group, iron depletion hd to be chieved by removing 35 cc of blood every 1-15 d ccording to bseline hemoglobin (Hb) vlues nd venesection tolernce, until ferritin < 3 ng/ml nd/or trnsferrin sturtion (TS) < 25%. Weekly phlebotomies were llowed for crriers of the C282Y HFE muttion, smller phlebotomies (25 cc) for crriers of bet-thlssemi trit. Mintennce phlebotomies (s much s required) were then performed to keep iron stores depleted (trget: ferritin < 5 ng/ml nd < 25%, MCV < 85 fl). Iron removed to 33 Mrch 21, 214 Volume 2 Issue 11

3 128 ptients underwent liver biopsy for suspected NAFLD excluded becuse of norml ferritin nd iron stores 23 excluded becuse of mild liver disese 3 refused rndomiztion 38 ptients underwent rndomiztion: 21 iron depletion by phlebotomy 17 lifestyle chnges 3 lost to follow-up 2 developed serious dverse events (neoplsi) 14 refused control liver biopsy 19 ptients underwent follow-up liver biopsy Figure 1 Study flow chrt. NAFLD: Nonlcoholic ftty liver disese. depletion ws estimted s previously described [23]. Visits were scheduled t week: -2, bseline (dy 1), nd t months 6, 12, 18, 24, for physicl exmintion, evlution of vitl signs nd nthropometric mesures (weight, bdominl circumference), lbortory evlution, including complete blood count, liver enzymes, iron prmeters, C rective protein, glucose, insulin, serum lipids. Heptic ultrsonogrphy ws performed t bseline, yer 1, yer 2. Adverse event were reported t ech visit, nd when notified by the ptients. Chnges in dibetes mediction dosge or strt of new therpy (metformin) hve been llowed for HbA1C vlues < 6% or 7%. The primry outcome of the study ws improvement of histologicl dmge relted to NAFLD, s defined by ny improvement of NAS (bsed on the severity of stetosis grded -3, lobulr necroinflmmtion grded -3, nd heptocellulr bllooning grded -2) without worsening of liver fibrosis, or n improvement of liver fibrosis without worsening of NAS [21]. Liver biopsies hve been reviewed in blinded fshion by n expert pthologist (Mggioni M). Heptic iron concentrtion ws quntified by tomic bsorption spectrometry [24], nd scored ccording to Deugnier et l [25]. Secondry outcomes included the evlution of the effect of phlebotomy on liver enzymes [ALT, sprtte minotrnsferreses (AST), nd gmmglutmyl-trnsferses (GGT) levels]. The protocol ws pproved by the Ethicl Committee of the Fondzione IRCCS C Grnd Ospedle Policlinico Milno, nd conforms to the principles of the Declrtion of Helsinki. Informed written consent hs been recorded for ech subject prticipting in the study. The study flow chrt is presented in Figure 1. The clinicl fetures of enrolled ptients re shown in Tble 1. Sttisticl nlysis The smple size ws bsed on the estimte tht 2% of the ptients would spontneously improve liver histology in the lifestyle chnges group. To detect 45% response rte mong the tretment ptients, using n lph level of.5 with 8% power, we estimted we needed to include 62 ptients per group. However, the study ws closed before the completion of enrolment becuse of the difficulties in ccepting 24 mo control liver biopsies. Therefore, we report the results obtined in ptients enrolled in the proposing center (Fondzione IRCCS C Grnd Milno) during the predefined time period (27-21). Dt re expression s men ± SD, medin (interqurtile rnge), nd frequency (%) ccording to distribution. Study outcomes were compred between the two tretment rms by χ 2 test, Student t test or Wilcoxon, s required ccording to dt distribution. Improvement in histologicl indices of liver dmge ctivity were compred by Wilcoxon signed-rnk test. Logistic regression nlysis considering the tretment rms nd mjor clinicl confounders ws conducted to djust the effect of tretment on histologicl improvement nd ALT normliztion for clinicl fctors (included vribles re reported in the results section). P vlues were considered significnt when.5 (two-tiled). RESULTS Study cohort We enrolled 38 subjects: 21 were rndomized to phlebotomy nd 17 to the lifestyle chnges rm (Figure 1). Ptients were mostly middle-ged overweight men with centrl distribution of diposity, moderte to severe ste- 34 Mrch 21, 214 Volume 2 Issue 11

4 Tble 1 Bseline fetures of 38 rndomized subjects with nonlcoholic ftty liver disese nd hyperferritinemi/ incresed iron stores ccording to tretment lloction Bseline fetures Lifestyle chnges lone Phlebotomy (n = 21) P vlue (n = 17) Age (yr) 53 ± ± 8.45 Gender (femle) 3 (18) 1 (5).31 Wist circumference, cm 13 ± ± 1.7 BMI (kg/m 2 ) 28.2 ± ± HOMA-IR 4.1 ± ± HDL chol (mg/dl) 47 ± ± 8.88 Triglycerides (mg/dl) 141 ± ± Type 2 dibetes 1 (6) 4 (19).23 Ferritin (ng/ml) 642 (55-97) 71 ( ).4 TS (%) 4 ± ± HIC (μg/1 mg dry tissue) 234 (98-358) 33 (238-57).23 TIS 6 (4-12) 9 (6-11).19 ALT (UI/mL) 39 ± ± AST (UI/mL) 3 ± ± 14.7 GGT (UI/mL) 4 (24-56) 28 (21-41).36 US Stetosis grde > 1 17 (1) 17 (81).11 NAS 3 11 (65) 13 (63) 1. Fibrosis stge (18) 7 (33).46 HFE genotype C282Y+ or H63D +/+ 2 (11) 2 (1).82 Bet-thlssemi trit 4 (23) 4 (19) 1. Dt re expressed s bsolute numbers (percentge), men ± SD or medin (interqurtile rnge). ALT: Alnine minotrnsferses; AST: Asprtte minotrnsferses; BMI: Body mss index; Chol: Cholesterol; GGT: Gmm-glutmyl-trnsferses; HDL: High density lipoprotein; HFE: Hemochromtosis gene; HIC: Heptic iron concentrtion; HOMA-IR: Homeostsis model ssessment insulin resistnce index; NAS: Nonlcoholic ftty liver disese ctivity score; TIS: Tissue iron score; TS: Trnsferrin sturtion; US: Ultrsonogrphic. tosis nd insulin resistnce (Tble 1). At bseline, bout two thirds of ptients hd NASH, nd roughly qurter moderte to severe fibrosis (stge 2-4; Tble 1). There were no significnt differences in bsl demogrphic, clinicl, histologicl nd genetic fetures between the two tretment rms (Tble 1). The mjority of ptients (22/38, 58%) hd lredy lost > 5% of body weight fter clinicl presenttion, while the other were not ble to modify body weight despite t lest 6 mo of lifestyle counseling. All ptients hd hyperferritinemi t bseline. Four ptients (1%) hd heptic iron concentrtion within the upper reference rnge (15 μg-1 mg), but ll were positive for stnible iron t liver biopsy. Effect of tretment on iron metbolism nd body mss In ptients rndomized to iron depletion, phlebotomy chieved reduction in biochemicl iron prmeters (Figure 2A, B, p <.1). Ferritin levels dropped progressively towrds the tretment trget, wheres TS ws lredy reduced to low to norml levels t 6 mo nd remined stble throughout the study period. In phlebotomized ptients, iron depletion ws reched fter medin of 16 phlebotomies, interqurtile rnge (IQR) 12-18, corresponding to medin of 4.3 g of iron, IQR In ptients with follow-up liver biopsies, tissue iron score ws significntly decresed in phlebotomized ptients (medin -9, IQR -9/-1, p =.18), but not in controls (medin -2, IQR -5/, p >.5). No ptient reported dverse events nor developed nemi (Hb levels during the study re shown in Figure 2C), or ltertions in renl function tests (not shown). No significnt loss in body weigth ws chieved nd mintined during the study in either rms (p >.7), nd there ws no significnt difference in body mss between tretment groups t the end of the study (27.2 ± 3.9 kg/m 2 in the phlebotomy rm vs 27.6 ± 5.2 kg/m 2 in the control rm, p =.8). Effect of tretment on histologicl dmge Follow-up liver biopsy ws performed in 19/38 ptients (5%). Two ptients developed severe dverse events (neoplstic diseses, ll in the control group), precluding follow-up liver biopsy. Therefore, 21 ptients were evluted in per protocol nlysis. Of the remining ptients, three were lost to follow-up, nd 14 refused control liver biopsy (Figure 1). Resons most commonly reported for retiring consent from control liver biopsy were complete normliztion of iron indices nd liver function tests in the phlebotomy group, nd the possibility to undergo phlebotomy for persistently bnorml iron indices in the control group. Histologicl improvement of liver dmge in n intention to tret nlysis considering ll rndomized ptients ws non-significntly higher in iron depleted vs control ptients (Figure 3A, p =.67), but the difference ws sttisticlly significnt in the per protocol nlysis (Figure 3B, p =.39). Vritions in histologicl indices of liver dmge ctivity in individul ptients subdivided ccording to tretment rm re presented in Figure 4. Ptients rndomized to iron depletion hd significntly better improvement in stetosis grde to tht observed in the control group (p =.2), wheres differences in the improvement of necroinflmmtion nd bllooning were not sttisticlly significnt. Effect of tretment on liver enzymes The time course of liver enzymes during the study period is shown in Figure 5. At lter time points, ALT, AST, nd GGT levels were significntly lower in phlebotomized ptients thn in controls (p <.5). At receiver-operting chrcteristic curve nlysis conducted in the 19 ptients who underwent both bseline nd follow-up liver biopsy (not shown), decrese in ALT levels 2% from bseline to the end of the study represented the best cut-off mong the vilble biomrkers for predicting improvement of histologicl dmge (sensitivity = 1%, specificity = 78%, positive predictive vlue = 83%, negtive predictive vlue = 1%, positive likelihood rtio = 4.54, negtive likelihood rtio =.). The prevlence of subjects with improvement in histologicl dmge or, in the bsence of liver biopsy, ALT decrese 2% (overll liver dmge ssessment) ws 35 Mrch 21, 214 Volume 2 Issue 11

5 A 12 B 5 b b b b Ferritin ng/ml b b b Trnsferrin sturtion % C Phlebotomy Control Hemoglobin g/dl Figure 2 Effect of tretment on iron sttus nd erythropoiesis. Effect of tretment on the levels of ferritin (A), trnsferrin sturtion (B), nd hemoglobin (C). Phlebotomy: dshed lines nd blck squres; Lifestyle chnges lone: continuous lines nd empty squres. P <.5, b P <.1 between phlebotomy nd lifestyle chnges lone tretment rms. A 1 9 B 1 9 C 8 P =.67 8 P =.39 Histologicl improvement % Control Phlebotomy (n = 17) (n = 21) Histologicl improvement % Control Phlebotomy (n = 9) (n = 12) Liver dmge improvement % P =.22 Control Phlebotomy (n = 15) (n = 2) Figure 3 Effect of tretment on liver dmge. Prevlence of demonstrted histologicl improvement of liver dmge t 2 yers (ny improvement in NAS score without worsening of fibrosis stge; min outcome of the study) in ll ptients who underwent rndomiztion (pnel A; n = 38; intention to tret nlysis), nd in ptients complint to the study protocol [pnel B; n = 21 (19 biopsied nd 2 filures due to serious dverse events), per protocol nlysis]. The effect of tretment on overll liver dmge ssessment [improvement in liver histology or lnine minotrnsferses (ALT) decrese 2% t the end of the study (n = 35, three ptients could not be ssessed becuse lost to finl follow-up evlution); direct nd indirect nlysis of liver dmge evolution in ll ptients tht completed follow-up] is shown in pnel C. higher in the phlebotomy thn in the control rm (p =.22; Figure 3C). Independent predictors of liver dmge progression Independent predictors of liver dmge improvement (overll ssessment) by multivrite logistic regression nlysis considering vribles most strongly ssocited t univrite nlysis (p <.1) re shown in Tble 2. Iron depletion ws strongly ssocited with improvement in liver dmge independently of confounders. In ddition, higher bseline AST/ALT rtio ( vlidted non-invsive index of liver fibrosis) nd insulin resistnce (homeosttic metbolic ssessment insulin resistnce: HOMA- IR index) were negtive predictors of fvourble outcome. There ws no significnt influence of ge, gender, bseline iron prmeters nd heptic iron stores, nd 36 Mrch 21, 214 Volume 2 Issue 11

6 A 3 P =.2 B 3 C Control Phlebotomy Bseline Stetosis 24 mo Bseline 24 mo Lobulr necroinflmmtion Bseline Bllooning 24 mo Figure 4 Vritions in histologicl indices of liver dmge ctivity in individul ptients subdivided ccording to tretment rm. A: Stetosis grde (P =.2 t Wilcoxon signed-rnk test); B: Necroinflmmtion (P >.5); C: Bllooning (P >.5). A 6 B ALT IU/mL 4 3 AST IU/mL C 1 Phlebotomy Control GGT IU/mL Figure 5 Effect of tretment on liver enzymes. Time course of liver enzymes: lnine minotrnsferses (ALT, A), sprtte minotrnsferses (AST, B), nd gmm-glutmyl-trnsferses (GGT, C) during the study. Phlebotomy: Dshed lines nd blck squres; Lifestyle chnges lone: Continuous lines nd empty squres. P <.5 between phlebotomy nd lifestyle chnges lone tretment rms. the genetic bckground on liver dmge improvement (not shown). The outcome ws worse in ptients with higher bseline bdominl circumference, but the effect ws not independent of insulin resistnce (not shown). There ws no significnt interction between tretment rm nd either AST/ALT rtio or HOMA-IR on the improvement of liver dmge. DISCUSSION In this pper, we present the results of phse Ⅲ rndomized controlled tril of iron depletion by phlebotomy on histologicl liver dmge in ptients with severe NAFLD nd hyperferritinemi, commonly observed clinicl syndrome ssocited with substntil risk of progressive liver disese [1,11,14-17]. Difficulties in recruiting ptients due to the requirement of follow-up liver biopsy, nd to the low rte of complince to this procedure, reduced the sttisticl power of the study. Notwithstnding, results, tht for the first time re derived from rndomized tril with histologicl outcome, re consistent with improvement 37 Mrch 21, 214 Volume 2 Issue 11

7 Tble 2 Independent predictors of improvement of liver dmge evluted by multivrite logistic regression nlysis t the end of the tril in 35 ptients with nonlcoholic ftty liver disese nd hyperferritinemi/incresed iron stores (P <.1 for model) Independent predictors 1 OR 95%CI P vlue Iron depletion by phlebotomy AST/ALT rtio <.1 HOMA-IR index Liver dmge included direct or indirect evlution of liver dmge in ll ptients tht completed follow-up. AST/ALT: Alnine minotrnsferses/ sprtte minotrnsferses; HOMA-IR: Homeostsis model ssessment insulin resistnce index. of histologicl dmge in ptients who underwent iron depletion, lso in line with previous results bsed on the ssessment of non-invsive mrkers of liver dmge nd on the results of uncontrolled studies [18-2]. Indeed, despite iron depletion ws not ssocited with significnt improvement of liver dmge t the intention to tret nlysis, there ws higher rte of histologicl improvement of NAFLD ctivity score in ptients rndomized to iron depletion who ttined to the clinicl protocol. The difference in the improvement rte (roughly 65% vs 25% in the control group) ws mintined when liver dmge improvement ws estimted by non-invsive predictor of liver dmge evolution, i.e., serum ALT decrese 2% compred to bseline, which showed high ccurcy in the prediction of histologicl improvement in ptients with follow-up liver biopsy. The limited rte of improvement in the control group is consistent with the selection of ptients tht were lredy resistnt to lifestyle chnges t bseline [6], nd by the persistence of ltered iron metbolism during the study. Strengths of the study include the high comprbility of clinicl fetures in the two tretment rms t bseline, nd the fct tht ll the secondry outcomes relted to improvement of liver enzymes (which could be evluted in the mjority on rndomized ptients) were met. Finlly, t multivrite logistic regression nlysis the fvourble effect of iron depletion on liver dmge improvement ws independent of the other identified predictors, including the AST/ALT rtio, reflecting bseline severity of liver dmge, nd of insulin resistnce. Notbly, phlebotomy normlized iron prmeters nd heptic iron stores without dverse effects, wheres two severe dverse events were observed in the control group, consisting in dignosis of neoplstic diseses fter 1 yer of follow-up. The difference is likely due to chnce, even if incresed iron stores hve been linked with cncer risk [26], nd iron depletion my reduce crcinogenesis [27]. Limittions relted to the limited power of the present study must be weighted ginst the lck of vilble phrmcologicl tretments for progressive liver disese in NAFLD nd NASH, which is t lest prtly relted to the difficulty of conducting trils requiring short-term followup liver biopsies [28]. The difficulties in mintining protocol complince were mplified in the tretment rm by the positive effect of phlebotomy on biochemicl indices, nd in the controls by the possibility to switch to ctive tretment (which cn be prescribed even in the bsence of evidence of efficcy on liver dmge progression) without the need of control biopsy. We believe tht these difficulties re inherent to the lck of possibility to perform phlebotomy tril in blinded fshion, nd to the vilbility of ctive tretment outside the reserch setting. Finlly, we think tht the choice of considering ny improvement of NAFLD ctivity score without worsening fibrosis s primry outcome (insted of higher cutoffs), nd liver enzymes s secondry outcomes ws pproprite for this study. Indeed, the pthophysiology of liver dmge progression is different in ptients with incresed heptic iron stores thn in those with clssic NAFLD, in tht in the presence of heptocellulr iron liver dmge is ssocited with lesser severity of stetosis nd is not invribly dependent on poptosis [11-13,29,3], but lso on other forms of cellulr dmge including ferroptosis [31]. Given the evidence tht, when ssocited with histopthologicl signs of liver dmge, even stetosis without dignosis of definite NASH frequently progress to fibrosis [32], we lso believe tht the inclusion in the tril of subjects without definite NASH, but with iron overlod coupled with severe stetosis or necroinflmmtion/heptocellulr dmge, ws pproprite. In conclusion, iron depletion by phlebotomy is probbly ssocited with higher rte of improvement of liver dmge improvement thn the mintennce of lifestyle chnges lone in ptients with severe NAFLD nd hyperferritinemi, who did not normlize liver enzymes fter t lest 6 mo of lifestyle counseling. In ddition, iron depletion meliortes liver enzymes, with possible impct on the nturl history of the disese. The present nd previous results [18-2] suggest tht, in the bsence of phrmcologicl tretments, lrger trils evluting phlebotomy tretment my be conducted in ptients with severe NAFLD nd hyperferritinemi resistnt to lifestyle chnges. ACKNOWLEDGMENTS We thnk ll the members of the Metbolic Liver Diseses Resech Center, Università degli Studi di Milno. COMMENTS Bckground Hyperferritinemi ssocited with mildly incresed heptic iron stores is frequently observed in nonlcoholic ftty liver disese (NAFLD), the heptic mnifesttion of the metbolic syndrome nd now leding cuse of liver disese, nd hs been ssocited with more dvnced liver dmge. Pthophysiologicl studies suggested tht iron stores my be involved in the pthogenesis of insulin resistnce nd liver disese progression, nd controlled studies indicted tht iron depletion by phlebotomy my decrese insulin resistnce nd liver enzymes in ptients with NAFLD. Reserch frontiers However, the effect of iron depletion on the progression of liver dmge s evluted by histologicl exmintion, the gold stndrd for ssessment, hs 38 Mrch 21, 214 Volume 2 Issue 11

8 not been previously tested in rndomized controlled tril in ptients with NAFLD. Innovtions nd brekthroughs In selected popultion of Itlin ptients with NAFLD nd hyperferritinemi which persisted despite t lest 6 mo of lifestyle counseling, who were found to hve mild heptic iron overlod, iron depletion by phlebotomy ws well tolerted nd induced more mrked meliortion of liver enzymes, non-invsive mrkers of liver dmge, thn lifestyle counseling lone. In ddition, in subgroup of ptients who underwent follow-up liver biopsy t 2 yers, iron depletion ws ssocited with higher probbility of improvement of the histologicl ctivity of NAFLD. Applictions These findings suggest tht iron depletion by phlebotomy should be further studied, s it represents s n ttrctive therpeutic pproch for ptients with NAFLD with persistent hyperferritinemi despite tril of lifestyle chnge/dditionl therpies or with incresed iron stores. 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9 24 Brry M, Sherlock S. Mesurement of liver-iron concentrtion in needle-biopsy specimens. Lncet 1971; 1: 1-13 [PMID: 4996] 25 Deugnier YM, Lorél O, Turlin B, Guyder D, Jounolle H, Moirnd R, Jcquelinet C, Brissot P. Liver pthology in genetic hemochromtosis: review of 135 homozygous cses nd their bioclinicl correltions. Gstroenterology 1992; 12: [PMID: ] 26 Frgion S, Vlenti L, Frcnzni AL. Hemochromtosis gene (HFE) muttions nd cncer risk: expnding the clinicl mnifesttions of hereditry iron overlod. Heptology 21; 51: [PMID: DOI: 1.12/hep.23541] 27 Zchrski LR, Chow BK, Howes PS, Shmyev G, Bron JA, Dlmn RL, Mlenk DJ, Ozki CK, Lvori PW. Decresed cncer risk fter iron reduction in ptients with peripherl rteril disese: results from rndomized tril. J Ntl Cncer Inst 28; 1: [PMID: DOI: 1.193/jnci/djn29] 28 Chlsni N, Younossi Z, Lvine JE, Diehl AM, Brunt EM, Cusi K, Chrlton M, Snyl AJ. The dignosis nd mngement of non-lcoholic ftty liver disese: prctice Guideline by the Americn Assocition for the Study of Liver Diseses, Americn College of Gstroenterology, nd the Americn Gstroenterologicl Assocition. Heptology 212; 55: [PMID: DOI: 1.12/hep.25762] 29 Mliken BD, Nelson JE, Klintworth HM, Beuchmp M, Yeh MM, Kowdley KV. Heptic reticuloendothelil system cell iron deposition is ssocited with incresed poptosis in nonlcoholic ftty liver disese. Heptology 213; 57: [PMID: DOI: 1.12/hep.26238] 3 Guldi R, Cslgrndi G, Montosi G, Ventur E, Pietrngelo A. Excess iron into heptocytes is required for ctivtion of collgen type I gene during experimentl siderosis. Gstroenterology 1994; 17: [PMID: ] 31 Dixon SJ, Lemberg KM, Lmprecht MR, Skout R, Zitsev EM, Gleson CE, Ptel DN, Buer AJ, Cntley AM, Yng WS, Morrison B, Stockwell BR. Ferroptosis: n irondependent form of nonpoptotic cell deth. Cell 212; 149: [PMID: DOI: 1.116/j.cell ] 32 Pis R, Chrlotte F, Fedchuk L, Bedoss P, Lebry P, Poynrd T, Rtziu V. A systemtic review of follow-up biopsies revels disese progression in ptients with non-lcoholic ftty liver. J Heptol 213; 59: [PMID: DOI: 1.116/j.jhep ] P- Reviewers: Buchler C, Czj MJ, Rostmi-Nejd M S- Editor: Zhi HH L- Editor: A E- Editor: M S 31 Mrch 21, 214 Volume 2 Issue 11

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