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Absence ofKLF6 gene mutation in 71 hepatocellular carcinomas

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Absence ofKLF6 gene mutation in 71 hepatocellular carcinomas
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  CORRESPONDENCE  Staging for Hepatocellular Carcinoma: Treatment Strategy Matters To the Editor:  We read with interest a paper by Kudo et al. published in a recentissue of H EPATOLOGY   that compared the predictive power between Japan Integrated Staging (JIS) system and Cancer of the Liver ItalianProgram (CLIP) system in a large patient cohort with hepatocellularcarcinoma (HCC). 1 The authors concluded that the JIS system isbetter and may be a preferred staging model. Because the CLIP systemhas been prospectively validated and proposed as the primary staging system, it would be interesting to examine how these commonly usedstagingsystemswerederivedandtoexplorethepotentiallimitationsof their conclusion.The vast majority (96%) of patients in the Kudo et al. study hadundergone radical treatment (resection or loco-regional therapy), sug-gesting that most of them belonged to an early or intermediate stage.This distinct feature made the JIS system, which is intrinsically similarto the Barcelona-ClI`nic Liver Cancer (BCLC) system proposed by theBarcelona group, 2 more favorable. A recent study showed that theBCLC system was the best one compared with the CLIP, Okuda, andother systems in a surgically oriented referral center. 3 It should benoted that the CLIP and Okuda systems were srcinally derived froma large, unselected population, and the majority of patients weretreated conservatively. Therefore, although the selected prognosticpredictorsarenotmutuallyexclusive,certainriskfactors(tumorsize  3 or 2 cm in the BCLC or tumor-node-metastasis [TNM]/JIS system)may only be meaningful in the population that is predominantly treated with radical therapy. This may explain why the JIS system isbetter, because the outcome is intimately associated with baseline de-mographics and subsequent treatment strategy. In keeping with thesefindings,anotherstudyinwhichmorethanhalf(52%)ofpatientsweretreated conservatively because of advanced stage demonstrated thatCLIPwasagoodpredictivemodel. 4 Moreover,inamulticentersurvey we have recently shown that the CLIP system was not superior to the JIS system in patients undergoing resection. 5 Therefore, it is not sur-prising that the JIS system can beat others in an appropriate study environment. Another important factor that may alter the predictive ability of a staging system is the distribution of patients. The proportion in eachcategory was considered fairly balanced for JIS scores 0 (12%), 1(31%),2(33%),3(17%),and4or5(7%),whereasthedistributioninthe CLIP model was rather skewed. 1 There will be a substantial degreeof swing in the survival curves if the denominator is too small or thepredictors are analyzed in an otherwise unbalanced population.Collectively, the JIS system contains treatment-derived parametersand is similar to the BCLC system that may work well in areas whereHCC is diagnosed at a relatively early stage, whereas the CLIP systemwouldonlyprevailwhenpatientspredominantlybelongedtoaninter-mediate or late stage, a condition in which aggressive therapy is lesslikely. It is necessary to consider all risk factors from early to advancedstages when building an ideal staging system for HCC.T EH -I  A   H UO  Y  I -H SIANG  H UANG National Yang-Ming University School of Medicine Division of Gastroenterology Department of Medicine Taipei Veterans General Hospital Taipei, Taiwan References 1. Kudo M, Chung H, Haji S, Osaki Y, Oka H, Seki T, et al. Validation of a new prognostic staging system for hepatocellular carcinoma: the JIS scorecompared with the CLIP score. H EPATOLOGY   2004;40:1396-1405.2. Llovet JM, Bru C, Bruix J. Prognosis of hepatocellular carcinoma: theBCLC staging classification. Semin Liver Dis 1999;19:329-338.3. Cillo U, Bassanello M, Vitale A, Grigoletto FA, Burra P, Fagiuoli S, et al.The critical issue of hepatocellular carcinoma prognostic classification:which is the best tool available? J Hepatol 2004;40:124-131.4. Levy I, Sherman M, the Liver Cancer Study Group of the University of Toronto. Staging of hepatocellular carcinoma: assessment of the CLIP,Okuda, and Child-Pugh staging systems in a cohort of 257 patients inToronto. Gut 2002;50:881-885.5. Huang YH, Chen CH, Chang TT, Chen SC, Wang SY, Lee HS, et al.Evaluation of predictive value of CLIP, Okuda, TNM and JIS staging systems for hepatocellular carcinoma patients undergoing surgical resec-tion. J Gastroenterol Hepatol 2005, in press. Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20583Conflict of interest: Nothing to report. Reply:  We appreciate the comments of Huo and Huang related to ourrecentpublication.HuoandHuangarenotinfullagreementwithourconclusions. First, Huo and Huang misunderstood that the majority (96%) of the patients in our study had undergone radical treatment(resection or loco-regional therapy), suggesting that most of them be-longed to an early or intermediate stage. However, the potentially curative(radical)treatmentssuchasresectionorpercutaneousablationwere performed only in 45% (2,023 of 4,525) of the patients. Trans-catheter arterial chemoembolization (TACE) was performed on 39%of the patients. Patients who received TACE, a noncurative modality,are categorized mostly into the advanced stage and partly into theintermediate stage. In that sense, Huo and Huang’s claim is not cor-rect.Second, it is incorrect to state that the JIS score is similar to theBCLC system. There is a difference between a prognostic staging sys-temandatreatmentallocationstagingsystem.TheCLIPscoreandthe JIS score were developed for the prediction of prognosis in patientswith HCC. In contrast, BCLC staging was developed for the adequateallocation of the HCC patients into given treatment modalities.Third,HuoandHuangstatethattheJISsystemisonlymeaningfulin the population that is predominantly treated with radical therapy. As already noted, 55% of our patient population was allocated to therelatively conservative treatment modalities such as TACE, chemoli-piodolization, or no treatment; therefore, their statement is again outof focus. Conversely, the JIS score works well in the stratification of HCC patients from early stage to advanced stage. The JIS system isuseful not only in selected patients but also the entire spectrum, fromearly to end-stage HCC patients. We agree with Huo and Huang that the JIS system works well inareas where HCC is diagnosed at a relatively early stage, whereas theCLIP system would only prevail when patients predominantly be-longedtoanintermediateorlatestage,aconditioninwhichaggressivetherapy is less likely. In that sense, tumor-node-metastasis (TNM)stage by the Liver Cancer Study Group of Japan 1-3 is fairly importantbecause the cutoff size of 2 cm for the nodule is crucial for curativetreatment. Microscopic portal venous invasion and intrahepatic me-tastasisarealreadynotedeveninthesmallHCCnoduleslessthan2cmin diameter in 27% and 10% of cases, respectively. 4  Actually, until 10years ago the CLIP and JIS scores had an identical prognostic signifi-canceamongJapanesepatients,butoverthepast10yearstheJISscoreis apparently superior to CLIP score (M. Kudo et al., unpublisheddata). 678  M  ASATOSHI  K  UDO Department of Gastroenterology and Hepatology Kinki University School of Medicine Osaka, Japan References 1. Kudo M, Chung H, Haji S, Osaki Y, Oka H, Seki T, et al. Validation of a new prognostic staging system for hepatocellular carcinoma: the JIS scorecompared with the CLIP score. H EPATOLOGY   2004;40:1396-1405.2. UenoS,TanabeG,NurukiK,HamanoueM,KomorizonoY,OketaniM,et al. Prognostic performance of the new classification of primary livercancer of Japan (4th edition) for patients with hepatocellular carcinoma: a validation analysis. Hepatol Res 2002;24:395-403.3. MakuuchiM,BelghitiJ,BelliG,FanST,LauJWY,RingeB,etal.IHPBA concordant classification of primary liver cancer: working group report. J Hepatobiliary Pancreat Surg 2003;10:26-30.4. Nakashima O, Sugihara S, Kage M, Kojiro M. Pathomorphologic charac-teristics of small hepatocellular carcinoma: a special reference to smallhepatocellular carcinoma with indistinct margins. H EPATOLOGY  1995;22:101-105. Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20625 Conflict of interest: Nothing to report. Host Response to Hepatitis C Virus To the Editor: IntheirrecentstudypublishedinH EPATOLOGY  ,Majoretal. 1 analyzeda panel of host responses to a hepatitis C virus (HCV) infection in chim-panzees.Theauthorsinvestigatedinnateimmuneresponsesbyexamining oligoadenylate synthetase expression as a surrogate for type I interferon(IFN)-regulatedprocesses,intrahepaticcytokineexpressionwithinterleu-kin10(IL-10)andIFN-   assurrogatesforT-lymphocyteornaturalkiller(NK)cellactivation,intrahepaticchemokineexpression,andthepresenceof CD3  lymphocytes within the liver. By closely monitoring the serumviral load and carrying out kinetic analyses, they were able to decipher 3phases of virus-host interactions: an acute phase of infection with unim-peded virus replication, an early post-acute phase with a reduced replica-tionrate,andathirdphasewhichwasmarkedbyadecreaseinviraltiters.Theauthorsobservedthedecelerationintheviralreplicationtobeassoci-atedwiththeonsetofatypeIIFNresponseandthedecliningvirustitertobecorrelatedwithincreasedIFN-   expressioninallanimals.Theparam-etersthattheyassumedtoberelevantforresolvingtheinfection,however,were (1) an earlier onset of the host immune response indicated by anincrease in ALT activity, and (2) an intrahepatic CD3e and macrophageinflammatory protein 1 alpha (MIP-1  ) messenger RNA induction. We find this comprehensive view on the host parameters to beparticularly noteworthy because it gives innate, humoral, and cellularimmune responses equal attention. Based on our experience, we seesome important parallels to the HCV infection in humans and to liverdisorders in general. As has been found to be the case in chimpanzees with steady-statelevels of viral titers, livers of chronic hepatitis C patients also containelevated levels of IFN-    transcripts. 2,3 Through combined immuno-histochemicalmeansand insitu hybridization,IFN-   messengerRNA was demonstrated to be localized in CD3  lymphocytes infiltrating portal tracts and hepatic lobules. 4 In contrast to the human HCV-infected liver, however, livers of persistently infected chimpanzees aregenerally poorly infiltrated. Active hepatitis in persistent infection israrely manifested; which, of note, is also the case with some humanHCV carriers. 5 This poor infiltration is reflected in lower intrahepaticCD3e  messenger RNA levels in persistently infected animals com-pared with those animals that eliminated the virus. As suggested by Major et al., the source(s) of IFN-    transcripts should thus be differ-ent. In the former group of animals it should be the NK cells, and inthe latter group of animals it should be either the infiltrating T lym-phocytes or both the NK cells and T lymphocytes. This assumptionreflects a recent observation made on an animal model of acute toxicliver injury, in which IFN-    expression was found to occur in twodifferent instances: (1) in the acute injury phase before the start of cellular infiltration, and (2) in the post-acute injury phase with prom-inent inflammation. 6 In human chronic HCV carriers, the clearance of HCV RNA canalso occur during a superinfection with the hepatitis B virus (HBV). 7,8  We were able to monitor serological parameters for one such patient,who successfully resolved both infections. 7  With the detectability of hepatitis B surface antigen (HBsAg), serum alanine aminotransferase(ALT) activity rose. In accordance with what has been observed inchimpanzees, we also noted an increase in the IFN-    expression. Thisincrease coincided with both the temporal increase in serum ALT andaspartateaminotransferase(AST)activitiesandthelossofHCVRNA.In contrast, disappearance of HBsAg took place later with the onset of an anti-HBsAg antibody response.S  ABINE  M IHM , P H .D. Department of Gastroenterology Georg-August-Universita¨t Go¨ttingen, Germany  References 1. Major ME, Dahari H, Mihalik K, Puig M, Rice CM, Neumann AU, et al.Hepatitis C virus kinetics and host responses associated with disease andoutcome of infection in chimpanzees. H EPATOLOGY   2004;39:1709-1720.2. Mihm S, Hutschenreiter A, Fayyazi A, Pingel S, Ramadori G. High in-flammatoryactivityisassociatedwithanincreasedamountofIFN-gamma transcripts in peripheral blood cells of patients with chronic hepatitis Cvirus infection. Med Microbiol Immunol (Berl) 1996;185:95-102.3. MihmS,SchweyerS,RamadoriG.ExpressionofthechemokineIP-10corre-lates with the accumulation of hepatic IFN-gamma and IL-18 mRNA inchronic hepatitis C but not in hepatitis B. J Med Virol 2003;70:562-570.4. Schweyer S, Mihm S, Radzun HJ, Hartmann H, Fayyazi A. Liver infiltrat-ing T lymphocytes express interferon gamma and inducible nitric oxidesynthase in chronic hepatitis C virus infection. Gut 2000;46:255-259.5. WieseM,BerrF,LafrenzM,PorstH,OesenU.Lowfrequencyofcirrhosisin a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study. H EPATOLOGY   2000;32:91-96.6. Batusic DS, Armbrust T, Saile B, Ramadori G. Induction of Mx-2 in ratliver by toxic injury. J Hepatol 2004;40:446-453.7. Wietzke P, Schott P, Braun F, Mihm S, Ramadori G. Clearance of HCV RNA in a chronic hepatitis C virus-infected patient during acute hepatitisB virus superinfection. Liver 1999;19:348-353.8. Sagnelli E, Coppola N, Messina V, Di Caprio D, Marrocco C, Marotta A,et al. HBV superinfection in hepatitis C virus chronic carriers, viral inter-action, and clinical course. H EPATOLOGY   2002;36:1285-1291. Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20586 Conflict of interest: Nothing to report. HEPATOLOGY, Vol. 41, No. 3, 2005 CORRESPONDENCE 679  Biliverdin, Immune-Mediated Liver Injury, and the Gigo Effect To the Editor: Once considered mere metabolic waste, the products of heme ca-tabolism are now increasingly viewed as molecules with a mission. 1 Bilirubin, potent interceptor of free radicals, hit the spotlight first, butthefocushasnowwidenedtoembracethetwoprimaryproductsoftheprocess,COandbiliverdin.Arecentexampleisthereportoftheeffectsof CO and biliverdin on immune-related liver injury. 2 However, thefindings of that paper and an earlier related paper 3 are seriously under-mined by a fundamental methodological flaw that may not be evidentto most readers and seems to have been overlooked or ignored by theauthors. The flaw concerns the nature and identity of the “biliverdin”used for the study and on which major conclusions of the paper ulti-mately depend. Described as srcinating from Sigma Chemical Co.,the“biliverdin”wasadministeredtomiceintraperitoneallyatadoseof 25 mg/kg dissolved in saline. The volumes injected and the concen-tration of pigment in the injectate were not given.To my knowledge, Sigma has never sold biliverdin. At one time itdid sell “biliverdin dihydrochloride,” which is not the same as the freeacidandwouldhavedifferentsolubilityproperties.Moreimportantly,the material that was sold was clearly labeled as “Approx. 80%.” Ma-terial of even that degree and uncertainty of purity is unsuitable andinadequateforreliablebiomedicalinvestigations,especiallythosedeal-ing with immune responses, cytokines, and enzyme induction. How-ever, it has been known for years that “Approx. 80%” is an optimisticestimate, some batches of the product containing far less than 80%. 4-6 Figure 1 shows thin-layer chromatograms of authentic biliverdin andof two batches of Sigma “biliverdin dihydrochloride” (Catalog no.B-3753). The commercial product is clearly not homogenous andcontains a great deal of (greenish) material that is not biliverdin andmainly remains at the srcin. Analysis of the same two batches by high-pressureliquidchromatography(HPLC)indicatedthatonecon-tained only   38% biliverdin and the other  68%. In each case thebiliverdinwasamixtureofIII  ,IX   ,andXIII  isomers(whichdonotseparate on thin-layer chromatography), making the actual content of biliverdin IX   , the natural biosynthetic isomer, even less than 38%and 68% respectively.Sigma stopped making and selling “biliverdin dihydrochloride”about 6 years ago and no longer offer it in the Sigma-Aldrich onlinecatalog. Thus, the experiments described in the H EPATOLOGY   paper 2 are based not only on material of uncertain identity, but also on a product that is no longer available, making them independently un-verifiable.  Without reliable experimental evidence that the material used was authentic biliverdin of a high degree of purity, it is impossible to judge the scientific significance of the recent studies. 2,3  A similar caveat appliesto other papers in the literature that have unquestioningly used theSigma product or similar “  80% pure” material from other ven-dors. 7,8 That is not to say that biliverdin does not have the biologicaleffects suggested—it might have even stronger effects—only that itstrue biological activity cannot be divined from the experiments asdescribed.The biological activities of the impurities in Sigma “biliverdin di-hydrochloride”areunknown.Biliverdinispoorlysolubleinwater,andthereforepreparationofinjectatesbydissolvingthepigmentdirectlyinsaline, as described, 2,3 would be impracticable or impossible with purematerial unless a co-solvent or relatively vast volumes of saline wereused. When assessing the purity of bile pigments it is important to beaware that criteria such as HPLC, absorbance spectrophotometry, andmass spectrometry are inadequate and potentially misleading whenconsidered alone. For example, a single peak on HPLC does  not   nec-essarily signify a pure compound because impurities may be retainedon the column, the system may be inadequate, or a detector optimizedfor biliverdin may not “see” impurities. Absorption spectrophotome-try is also unreliable because bile pigments have broad absorbancebands and the spectra of impurities may resemble those of biliverdinitself. To establish purity, several methods need to be used and thematerialshould,atleast,givesatisfactoryC,H,Nanalysis.Fortunately,simple,inexpensivethin-layerchromatographyprocedurescanbeuse-ful informative indicators of purity (as shown in Fig. 1), if appropriatesystems are used.Investigators can hardly be expected to analyze all solvents andreagents, and for common materials this is generally unnecessary. Butbottle labels and suppliers’ descriptions and analyses are not infallibleand it is foolish (and irresponsible) not to confirm the identity andsuitability of key materials before their use in extensive investigations.Otherwise, as in the present instance 2 and earlier work on “biliver-din,” 3,7,8 much effort may be wasted to no useful scientific end, clut-tering the literature with questionable, possibly unreproducible,findings.InthecaseoftherecentH EPATOLOGY  papers, 2,3 studiesdonewith grossly impure material could be particularly misleading. It is notonly in the computer world that the Gigo effect 9 applies. Note added in proof.  ICN Pharmaceuticals, Inc., no longer exists.The “biliverdin dihydrochloride” that they once sold (Cat. no.194886) was listed in their catalog, like the srcinal Sigma product, as  80%pure.MPBiomedicals(Irvine,CA),whichnowsellstheformerICN Biomedical product line, also describes their material (2004 cat-alog)as  80%pure.However,asamplepurchasedinDecember2004was found to be much purer and chromatographically similar to biliv-erdin hydrochloride from Frontier Scientific (Logan, UT). ICN/MPdo not make biliverdin, but resell material purchased from other sup-pliers. It is unlikely that what is sold really is a   di  hydrochloride, asdescribed. Biliverdin hydrochloride made in-house by Frontier Scien-tific is likely to be pure enough for most biomedical investigations. A  NTONY   F. M C D ONAGH Division of Gastroenterology Department of Medicine University of California San FranciscoSan Francisco, CA References 1. Sedlak TW, Snyder SH. Bilirubin benefits: cellular protection by a biliver-din reductase antioxidant cycle. Pediatrics 2004;113:1776-1782.2. Sass G, Seyfried S, Soares MC, Yamashita K, Kaczmarek E, Neuhuber WL,et al. Cooperative effect of biliverdin and carbon monoxide on survival of mice in immune-mediated liver injury. H EPATOLOGY   2004;40:1128-1135. Fig. 1. Thin-layer chromatogram of two different batches of Sigma “biliverdin dihydro-chloride” (lanes a and c) and of authentic biliverdin IX    (lane b). Pigments were applied assolutions in MeOH (1 mg/mL); the adsorbent was Silica gel G; and the irrigation solvent chloroform:MeOH:acetic acid (90:10:0.1 vol/vol). The plate was scanned on an HP color scanner and the contrast enhanced after scanning to improve black-and-white reproduction. The plate was also overloaded to improve reproduction, accounting for the “rocketing” of thebiliverdin bands. The horizontal line marks the application srcin. 680 CORRESPONDENCE HEPATOLOGY, March 2005  3. Sass G, Soares MC, Yamashita K, Seyfried S, Zimmermann WH, Eschen-hagen T, et al. Heme oxygenase-1 and its reaction product, carbon monox-ide, prevent inflammation-related apoptotic liver damage in mice.H EPATOLOGY   2003;38:909-918.4. McDonaghAF.BilePigments:biladienesand5,15-bilatrienes.In:DolphinD,ed. The Porphyrins. Volume 6. New York: Academic Press, 1979:293-491.5. McDonagh AF, Palma LA. Preparation and properties of crystalline biliver-din IX alpha. Simple methods for preparing isomerically homogeneousbiliverdin and [ 14 C[biliverdin by using 2,3-dichloro-5,6-dicyanobenzoqui-none. Biochem J 1980;189:193-208.6. McPhee F, Caldera PS, Bemis GW, McDonagh AF, Kuntz ID, Craik CS.Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viralmaturation and infectivity in vitro. Biochem J 1996;320:681-686.7. Chiu H, Brittingham JA, Laskin DL. Differential induction of heme oxy-genase-1 in macrophages and hepatocytes during acetaminophen-inducedhepatotoxicity in the rat: effects of hemin and biliverdin. Toxicol ApplPharmacol 2002;181:106-115.8. Fondevila C, Katori M, Lassman C, Carmody I, Busuttil RW, Bach FH, etal. Biliverdin protects rat livers from ischemia/reperfusion injury. Trans-plant Proc 2003;35:1798-1799.9. Oxford English Dictionary, on-line edition. Available at: http://dictionary.oed.com/cgi/entry/00293026?single  1&query_type  word&queryword  Gigo&first  1&max_to_show   10.(Alternatively,see:http://www.bennetyee.org/http_webster.cgi?isindex   gigo&method  exact). Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20587 Conflict of interest: Nothing to report. Reply: For both studies published in H EPATOLOGY  , 1,2 we used biliverdinIX dihydrochloride (Formula C 33 H 34 N 4 O 6  2HCl) from ICN Bio-medicals Inc., not, as it was indicated, from Sigma Chemicals. Weapologize for this mistake, which we overlooked in Materials andMethods, and thank H EPATOLOGY   for giving us the opportunity tocorrect this. ICN Biomedicals Inc. now sells this product under thename of MP Biomedicals, LLC (Ohio, USA; available under Cat. no.194886). This substance from ICN was commonly used when inves-tigating effects of HO-1 products  in vivo  and exerted biological andprotective effects not only in our hands. 3,4  According to its data sheet,itpassedtheidentitytestandwasabout95%pure.Weactuallydidnotperform thin-layer chromatography of any batch of the biliverdin IX dihydrochloride used in our experiments but instead relied on thedescription of ICN regarding identity and purity. Biliverdin IX dihy-drochloride was dissolved in saline, kept in the dark at anytime, andapplied to mice at 25 mg/kg in a volume of 200   L intraperitoneally. As controls for the activity of biliverdin IX dihydrochloride we usedheat- and light-inactivated aliquots in parallel groups of mice to ruleout, for example, lipopolysaccharide effects or effects of other un-knowncontaminants.Wedidnotaddanyingredients,whichwerenotindicated in the manuscripts, to increase solubility. In our hands,solubility was good enough to inject mice using a 0.4-mm diameterneedle. The experiments described in our manuscripts were repeatedseveral times and gave consistent results.Regarding the controls used, we feel that the readers of H EPATOL - OGY   do not have to fear that we or others presented a Gigo effect by usingbiliverdinIXdihydrochloride.Furthermore,assuggestedbyMc-Donagh, effects of biliverdin  in vivo  might be even stronger thanobserved so far.G ISA   T IEGS G  ABRIELE  S  ASS University of ErlangenDepartment of Pharmacology and Toxicology Erlangen, Germany  References 1. Sass G, Soares MC, Yamashita K, Seyfried S, Zimmermann WH, Eschen-hagen T, et al. Heme oxygenase-1 and its reaction product, carbon mon-oxide, prevent inflammation-related apoptotic liver damage in mice.H EPATOLOGY   2003;38:909-918.2. Sass G, Seyfried S, Parreira Soares M, Yamashita K, Kaczmarek E, Neu-huber WL, et al. Cooperative effect of biliverdin and carbon monoxide onsurvival of mice in immune-mediated liver injury. H EPATOLOGY   2004;40:1128-1135.3. FondevilaC,KatoriM,LassmanC,CarmodyI,BusuttilRW,BachFH,etal. Biliverdin protects rat livers from ischemia/reperfusion injury. TransplProc 2003;35:1798-1799.4. Yamashita K, McDaid J, Ollinger R, Tsui TY, Berberat PO, Usheva A, etal. Biliverdin, a natural product of heme catabolism, induces tolerance tocardiac allografts. FASEB J 2004;18:765-767. Epub 2004 Feb 20. Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20624 Conflict of interest: Nothing to report.  Absence of   KLF6  Gene Mutation in 71 Hepatocellular Carcinomas To the Editor: KLF6   is a gene encoding the Kruppel-like factor 6, a transcriptionfactor involved in the regulation of cell proliferation and differentia-tion. Mutations of   KLF6   have been reported in prostate cancer, 1,2 inastrocyticgliomas 3 andincoloncancer. 4 Inarecentstudy,Kremer-Talet al. 5 reported somatic mutations of   KLF6   in 6 of 41 hepatocellularcarcinomas (HCCs). We have screened for  KLF6   mutations in a largerseries of HCCs to search for genotype-phenotype correlations. Aseriesof71HCCswascollectedaftersurgicalresectioninFrance.The sex ratio (M/F) was 4:1 and the mean age of the patients was 61years. Risk factors for HCC of hepatitis B virus, hepatitis C virus,alcohol abuse and heamochromatosis occurred in 25%, 17%, 31%,and 4% of the tumors, respectively. All DNAs were extracted fromfrozentissuesectionsafterapathologicalcontrol.Directsequencingof exon2of  KLF6  wasperformedbecausealmostallmutationspreviously found in tumors, including HCC, were clustered in this exon. Se-quences were systematically determined on both DNA strands using a Big Dye terminator chemistry kit (Applied Biosystems, Foster City,CA). The sensitivity of our screening method has been previously validated in the same series of tumor DNA samples by the identifica-tion of   -catenin ,  TP53 , and  axin1  gene mutations in 34%, 21%, and6% of the cases, respectively. 6 In our series, we observed neither somatic nor germline mutationsin  KLF6  . Known silent nucleotide polymorphisms were observed intwocases(R201RandS277S).Thelackof  KLF6  mutationobservedinthe 71 HCCs analyzed was significatively discordant with the 15% of mutations reported by analyzing 41 samples, Fisher exact test  P    .001. 5 This discrepancy is not likely to be due to a failure in ourscreening process since we found the usual frequency of    -catenin , TP53 , and  axin1  mutation in the same series of DNA when comparedwith similar published HCC series. 7  A similar discrepancy in the fre- HEPATOLOGY, Vol. 41, No. 3, 2005 CORRESPONDENCE 681  quency of   KLF6   mutation has also been observed in prostate cancerand astrocytic gliomas: the high frequencies of   KLF6   mutations ini-tially reported in 55% of prostate cancer 1 and in 9% of astrocyticgliomas 3 havenotbeenconfirmedin5subsequentstudieswhichfound0%-7% of mutations in prostate cancers 2,8,9 and no mutation in astro-cytomas and glioblastomas. 10-12 The reasons for these discrepancies in KLF6   mutational frequency remain to be elucidated. One possiblereason for the differences could be that the srcinal study was carriedout using DNA extracted from paraffin-embedded tissue samples, a procedure that has been previously shown to carry a risk of detecting false-positive nucleotide mutations. 13 It is also possible that the differ-ences may be due to differences in tumor sampling, including thegeographical srcin of patients, variation in risk factors and/or in tu-morgradesincetheseinformationwerenotprovidedbyKremer-Taletal. 5 Finally,theabsenceof  KLF6  mutationinourstudysuggestsatbesta minor role for mutations in this gene in hepatocarcinogenesis.S  ANDRINE  B OYAULT 1  A  URÉLIE  H ÉRAULT 1 C HARLES  B  ALABAUD 2  J ESSICA   Z UCMAN -R  OSSI 1 1 Inserm U434, Paris, France   2  Inserm E362, Bordeaux, France  References 1. Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, et al.KLF6, a candidate tumor suppressor gene mutated in prostate cancer.Science 2001;294:2563-2566.2. Chen C, Hyytinen ER, Sun X, Helin HJ, Koivisto PA, Frierson HF Jr, etal. Deletion, mutation, and loss of expression of KLF6 in human prostatecancer. Am J Pathol 2003;162:1349-1354.3. JengYM,HsuHC.KLF6,aputativetumorsuppressorgene,ismutatedinastrocytic gliomas. Int J Cancer 2003;105:625-629.4. Reeves HL, Narla G, Ogunbiyi O, Haq AI, Katz A, Benzeno S, et al.Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inac-tivated in colorectal cancer. Gastroenterology 2004;126:1090-1103.5. Kremer-Tal S, Reeves HL, Narla G, Thung SN, Schwartz M, Difeo A, etal. Frequent inactivation of the tumor suppressor Kruppel-like factor 6(KLF6) in hepatocellular carcinoma. H EPATOLOGY   2004;40:1047-1052.6. Laurent-Puig P, Legoix P, Bluteau O, Belghiti J, Franco D, Binot F, et al.Genetic alterations associated with hepatocellular carcinomas define distinctpathways of hepatocarcinogenesis. Gastroenterology 2001;120:1763-1773.7. Buendia MA. Genetics of hepatocellular carcinoma. Semin Cancer Biol2000;10:185-200.8. Koivisto PA, Hyytinen ER, Matikainen M, Tammela TL, Ikonen T,Schleutker J. Kruppel-like factor 6 germ-line mutations are infrequent inFinnish hereditary prostate cancer. J Urol 2004;172:506-507.9. MuhlbauerKR,GroneHJ,ErnstT,GroneE,TschadaR,HergenhahnM,et al. Analysis of human prostate cancers and cell lines for mutations in theTP53 and KLF6 tumour suppressor genes. Br J Cancer 2003;89:687-690.10. Koivisto PA, Zhang X, Sallinen SL, Sallinen P, Helin HJ, Dong JT, et al. Absence of KLF6 gene mutations in human astrocytic tumors and celllines. Int J Cancer 2004;111:642-643.11. KohlerB,WolterM,BlaschkeB,ReifenbergerG.Absenceofmutationsinthe putative tumor suppressor gene KLF6 in glioblastomas and meningi-omas. Int J Cancer 2004;111:644-645.12. Montanini L, Bissola L, Finocchiaro G. KLF6 is not the major target of chromosome10plossesinglioblastomas.IntJCancer2004;111:640-641.13. Williams C, Ponten F, Moberg C, Soderkvist P, Uhlen M, Ponten J, et al. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol 1999;155:1467-1471. Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20588 Conflict of interest: Nothing to report. Reply:  We appreciate the interest of Drs. Boyault and colleagues in thepotential role of KLF6 in hepatocellular cancer (HCC). As we havepreviously outlined, 1 somatic mutation detection in cancer can behighly variable owing to differences in methodology and sample sets.Indeed, reported mutational frequencies of even well-characterizedtumor suppressor genes—for example, p53—can range from 13%-67% in HCC. 2 Similarly, in prostate cancer, whereas one study failedto identify KLF6 mutations, 3 a second one apart from our own 4 iden-tified loss of heterozygosity (LOH) in approximately 30% of samplesand somatic mutations in 15%. 5 In contrast to the negative findings suggested by Drs. Boyault andcolleagues,arecentpublication 6 hasidentifiedKLF6LOHandmutationin HCC at levels almost identical to those we srcinally reported. 7  WewouldalsoemphasizethatLOHoftheKLF6locusispresentmuchmorecommonly than mutation in HCC. 6,7 This result parallels the publishedfindingsinothercancers,includingprostate, 4,5 colorectal, 8 andnon–smallcell lung cancer. 9 This feature was not examined by Drs. Boyault andcolleagueseventhoughitmayleadtoagrowthadvantagethroughhaplo-insufficiency.Interestingly,wehavealsodefinedanumberoftumorsthatdemonstrateonlysignificantdegreesofLOH,withoutsomaticmutation.These include ovarian cancer, head and neck squamous cell carcinoma,smallcelllungcancer,andglioblastoma,aswellasprostatecancercelllines(manuscripts in preparation), suggesting that the presence of KLF6 mu-tations is by no means ubiquitous in human cancer. We agree that se-quencing artifacts secondary to tissue preservation are an importantconcern, which is why we have always used similarly prepared and storedpaired normal tissue specimens as controls. 4,7,8 Emerging KLF6 studies are now characterizing functions and fea-tures consistent with its role as a tumor suppressor, including interac-tions with cell cycle regulators, 10 suppression of tumsrcenicity   invivo , 11 induction of proto-oncogene degradation, 12 regulation of apo-ptosis, 9 and promoter methylation and/or downregulation in can-cer, 9,13 withreducedgeneexpressionlevelspredictingdiseaseoutcomein both pulmonary adenocarcinoma  14 and prostate cancer. 15 Takentogether,webelievethesefindingshighlighttheimportanceof continuedindependentstudiesusingmultiplesamplesetstofirmlyclarify the role and mutation frequency of KLF6, or any candidate tumor sup-pressor, in both HCC and other human cancers. We welcome interest inthis topic and look forward to its continued exploration.S IGAL  K  REMER  -T  AL , M.D. 1 H ELEN  R  EEVES , M.D., P H .D. 2 G OUTHAM  N  ARLA  , B.S. 1  J OHN  M  ARTIGNETTI , M.D., P H .D. 1 S COTT  F RIEDMAN , M.D. 1 1  Mount Sinai School of Medicine, New York, NY   2  University of Newcastle upon Tyne, United Kingdom References 1. Narla G, Friedman SL, Martignetti JA. Kruppel cripples prostate cancer:KLF6 progress and prospects. Am J Pathol 2003;162:1047-1052.2. Buendia MA. Genetics of hepatocellular carcinoma. Semin Cancer Biol2000;10:185-200.3. MuhlbauerKR,GroneHJ,ErnstT,GroneE,TschadaR,HergenhahnM,et al. Analysis of human prostate cancers and cell lines for mutations in theTP53 and KLF6 tumour suppressor genes. Br J Cancer 2003;89:687-690.4. Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, et al.KLF6, a candidate tumor suppressor gene mutated in prostate cancer.Science 2001;294:2563-2566.5. Chen C, Hyytinen ER, Sun X, Helin HJ, Koivisto PA, Frierson HF Jr, etal. Deletion, mutation, and loss of expression of KLF6 in human prostatecancer. Am J Pathol 2003;162:1349-1354.6. Wang SP, Chen XP, Qiu FZ. A candidate tumor suppressor gene mutatedin primary hepatocellular carcinoma: Kruppel-like factor 6. Zhonghua  Wai Ke Za Zhi 2004;42:1258-1261.682 CORRESPONDENCE HEPATOLOGY, March 2005
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