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{=0A= document.writeln('

3D"Click

');=0A= }=0A= =0A= function get_text_link(url, linktext)=0A= {=0A= document.writeln('' + = linktext + '');=0A= }=0A= =0A= function get_htmlftext_link(a, b) {=0A= document.writeln("" + = b + ", ");=0A= }=0A= =0A= ------=_NextPart_000_0000_01C98BBF.C033BB80 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://download.interscience.wiley.com/freeflow/js/personalisation.js /*=0A= * Project : Interscience Redesign=0A= * File : personalisation.js=0A= * Creation Date : 25th March 2003=0A= * Author : Jonathan Sugar=0A= *=0A= * Various functions to deliver personalisation functionality.=0A= */=0A= =0A= // Pre-declare userName so it's available when we need to test if the = page=0A= // assembler ran OK=0A= var userName =3D null;=0A= var shcrtCookie =3D null;=0A= =0A= // Browser detect globals:=0A= var uaStr =3D navigator.userAgent.toLowerCase();=0A= var uaVer =3D parseInt(navigator.appVersion);=0A= // for detecting netscape 4=0A= var netscape =3D (uaStr.indexOf("mozilla") > -1);=0A= var netscape4 =3D (netscape && uaVer < 5);=0A= =0A= // Factory Methods=0A= =0A= // Simple wraper object to encapsulate the data for a co-branding item.=0A= function coBrandingItem(image, imageAltText, href)=0A= {=0A= //alert("Making new coBrandingItem:\nImage: " + image + = "\nimageAltText" + imageAltText + "\nhref: " + href);=0A= this.image =3D image;=0A= this.imageAltText =3D imageAltText;=0A= this.href =3D href;=0A= }=0A= =0A= /*=0A= * Insert the 'Customer co-branding' section.=0A= * -----------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= *=0A= * 1. Insert customer co branding. Loop through 'customerCoBranding' array=0A= * inserting XHTML where needed.=0A= *=0A= * NOTE: the array contains ONLY the resource location (the location of a = gif=0A= * on the file system), the required XHTML is then added where needed. = This way=0A= * we maintain the seperation between presentation and content.=0A= */=0A= function insertCustomerCoBranding(maxBranding)=0A= {=0A= if( customerCoBranding[0].image !=3D "none")=0A= {=0A= document.writeln('

');=0A= document.writeln('

');=0A= document.writeln('

' + imageAltText + '
' + imageAltText + = '
More Providers

');=0A= document.writeln('

');=0A= document.writeln('

3D""

');=0A= }=0A= }=0A= =0A= /*=0A= * Insert the 'My Area' section.=0A= * -----------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= * Updated: Ben Marvell, April 2006=0A= *=0A= * My profile link absolute as this file isnt only run from w3 anymore.=0A= */=0A= function insertMyArea(userdata)=0A= {=0A= // just in case the page assembler isn't running.=0A= =0A= =0A= if (userName !=3D null)=0A= {=0A= document.writeln("

" + = userName.substring(0,33) + "..., access your
" + userName + = ", access your

My Profile"); =0A= } =0A= document.writeln(getLoginOutLink());=0A= //CR 3652 getAthenStatus() as added=0A= document.writeln(getAthenStatus());=0A= document.writeln("

");=0A= } else {=0A= if(typeof(environment)!=3D'undefined' && = environment=3D=3D'PRODUCTION') {=0A= document.writeln("

Log Out");=0A= document.writeln("

"); =0A= }=0A= }=0A= if (menuloadedflag){=0A= wisMenu();=0A= }=0A= }=0A= =0A= /*=0A= * Insert the Customer Admin 'Login/Logout' link=0A= * -----------------------------=0A= *=0A= * Author: Bobby Jack, December 2004.=0A= *=0A= */=0A= function insertAdminLink(page)=0A= {=0A= val =3D getCookie("AID");=0A= =0A= if (val && val !=3D "-1")=0A= {=0A= document.writeln('

Logout');=0A= }=0A= else=0A= {=0A= document.writeln('

');=0A= }=0A= }=0A= =0A= /*=0A= * Insert the username section.=0A= * -----------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= *=0A= * 1. Insert the username.=0A= */=0A= function insertUserName()=0A= {=0A= document.write(userName);=0A= }=0A= =0A= /*=0A= * Return the appropriate link to the login/logout cgi=0A= * ---------------------------------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= * Updated: Ben Marvell, April 2006=0A= * Had to change the links absolute as this file isnt only run from w3 = anymore.=0A= */=0A= function getLoginOutLink()=0A= {=0A= if(userName !=3D "")=0A= {=0A= return "

";=0A= }else=0A= {=0A= return "

";=0A= }=0A= }=0A= =0A= /*=0A= * Return the appropriate link to the shopping cart content page=0A= * ---------------------------------------------------=0A= *=0A= * Author: Zhiming & Warrell June 2007=0A= */=0A= function getShCrtLink(logged)=0A= {=0A= if(logged =3D=3D true )=0A= return "

";=0A= else=0A= return "

";=0A= }=0A= =0A= function toggleShCrtLink()=0A= {=0A= shcrtCookie =3D readCookie("SHCRT");=0A= if(shcrtCookie !=3D null)=0A= {=0A= if(document.all)=0A= {=0A= return true;=0A= }=0A= else=0A= {=0A= document.getElementById("shcrt_id").style.display =3D "block";=0A= }=0A= }=0A= else=0A= {=0A= if(document.all)=0A= {=0A= return true;=0A= }=0A= else=0A= {=0A= document.getElementById("shcrt_id").style.display =3D "none";=0A= }=0A= }=0A= }=0A= =0A= =0A= =0A= function readCookie(name)=0A= {=0A= var nameEQ =3D name + "=3D";=0A= var ca =3D document.cookie.split(';');=0A= for(var i=3D0; iYou are logged in = to Athens';=0A= }=0A= else=0A= {=0A= return "

";=0A= }=0A= =0A= } ------=_NextPart_000_0000_01C98BBF.C033BB80 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://download.interscience.wiley.com/freeflow/js/site.js /************************************************************************= ***************************=0A= FILE: site.js=0A= AUTHOR: Jonathan Sugar, Chris Neatby-Smith and Digital Pulp. July = 2003.=0A= =0A= DESCRIPTION: Javascript relating to site wide functionality across =0A= InterScience.=0A= =0A= INCLUDES FUNCTIONS=0A= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=0A= =0A= Search Functions=0A= ----------------=0A= =0A= selectAndSubmit() - for deciding where to submit a query based on the = state of the form.=0A= processallContentSearch() - for validating form input for an all = content search.=0A= processTitleSearch() - for validating form input for a Title search.=0A= checkCitationSearch() - for validating the citation search form.=0A= selectAll() - check all the checkboxes in a form.=0A= do_popup() - stasa Acronym Finder window popup function.=0A= =0A= =0A= Other Functions=0A= ---------------=0A= =0A= fixInlineLists() - fix netscape 4.x bug where

tags with images = display both an image and=0A= the default bullet. =0A= =0A= = *************************************************************************= **************************/=0A= =0A= var http =3D false;=0A= if(navigator.appName =3D=3D "Microsoft Internet Explorer") {=0A= http =3D new ActiveXObject("Microsoft.XMLHTTP");=0A= } else {=0A= http =3D new XMLHttpRequest();=0A= } =0A= =0A= // Global Variables=0A= var debug =3D false;=0A= var first =3D true;=0A= =0A= // Browser detect globals:=0A= var uaStr =3D navigator.userAgent.toLowerCase();=0A= var uaVer =3D parseInt(navigator.appVersion);=0A= // for detecting netscape 4=0A= var netscape =3D (uaStr.indexOf("mozilla") > -1);=0A= var netscape4 =3D (netscape && uaVer < 5);=0A= =0A= /*=0A= * Used to switch search mode between APS and TitleFinder on quicksearch = boxes=0A= * = -------------------------------------------------------------------------= --=0A= *=0A= * 1. Decides which search is required based on the value of the radio = buttons=0A= * in 'document.formSelector.whichForm'.=0A= *=0A= * 2. Calls individual functions to process the forms and submit them.=0A= *=0A= */=0A= function selectAndSubmit()=0A= {=0A= var formSelection;=0A= =0A= // find out which form we need to process.=0A= var selector =3D document.formSelector.whichForm;=0A= for (var i =3D 0; i < selector.length; i++){=0A= if (selector[i].checked){=0A= formSelection =3D selector[i].value;=0A= }=0A= }=0A= =0A= // Call the relevent function to process the required form.=0A= if(formSelection =3D=3D "allContentSearch"){=0A= processallContentSearch(document.allContentSearchForm, = document.formSelector)=0A= }=0A= else if(formSelection =3D=3D "searchTitles"){=0A= processTitleSearch(document.searchTitles, document.formSelector)=0A= }=0A= else{=0A= return false;=0A= }=0A= =0A= //@@@DM It was anoying me this error =0A= return false;=0A= }=0A= =0A= /*=0A= *Process the ADS search form=0A= *=0A= */=0A= function trim(str)=0A= {=0A= return str.replace(/(^\s*)|(\s*$)/g,"");=0A= }=0A= =0A= function ADSquery(t) {=0A= var keyword =3D trim(t.getElementsByTagName("input")[0].value);=0A= if (keyword!=3D"") {=0A= = window.open("","popup","toolbar=3Dyes,location=3Dyes,status=3Dyes,menubar= =3Dno,scrollbars=3Dyes,resizable=3Dyes,width=3D800,height=3D500");=0A= t.target=3D"popup";=0A= t.qsearch.value=3Dkeyword;=0A= return true;=0A= = //processADSSearch(document.ADSSearchForm,document.ADSQueryForm,keyword);=0A= } else {=0A= alert("Please input a keyword for ADS searching before clicking go");=0A= return false;=0A= }=0A= }=0A= /*=0A= function processADSSearch(hiddenForm ,queryForm , str) {=0A= var terms =3D str;=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= formToSubmit.qsearch.value =3D terms;=0A= = window.open("","popup","toolbar=3Dyes,location=3Dyes,status=3Dyes,menubar= =3Dno,scrollbars=3Dyes,resizable=3Dyes,width=3D800,height=3D500");=0A= formToSubmit.action =3D = "http://adsabs.harvard.edu/cgi-bin/nph-basic_connect"=0A= formToSubmit.target =3D "popup";=0A= formToSubmit.submit();=0A= }=0A= */=0A= =0A= /*=0A= * Process the allContentSearch form=0A= * ---------------------------------=0A= * 1. Perform form validation, checking for empty fields.=0A= * Give the user a relevent message if required.=0A= *=0A= * 2. Process/Assign the appropriate form elements and submit the form.=0A= */=0A= function processallContentSearch(hiddenForm, queryForm)=0A= {=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= =0A= // Do some basic form validation=0A= if(formToProcess.terms.value !=3D "")=0A= {=0A= formToSubmit.WISsearch1.value =3D formToProcess.terms.value;=0A= formToSubmit.submit(); =0A= }=0A= else=0A= {=0A= alert("You must enter a search term in the text box before you click = go.");=0A= }=0A= }=0A= =0A= /*=0A= * Process the Title Search form=0A= * -----------------------------=0A= * 1. Perform form validation, checking for empty fields.=0A= * Give the user a relevent message if required.=0A= *=0A= * 2. Process/Assign the appropriate form elements and submit the form.=0A= */=0A= =0A= function processTitleSearch(hiddenForm, queryForm)=0A= {=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= =0A= // Do some basic form validation=0A= if(formToProcess.terms.value !=3D "")=0A= {=0A= formToSubmit.query.value =3D formToProcess.terms.value;=0A= formToSubmit.submit();=0A= }=0A= else=0A= {=0A= alert("You must enter a search term in the text box before you click = go.");=0A= }=0A= }=0A= =0A= function doCitationSearch()=0A= {=0A= var form =3D document.citsearchform;=0A= if(checkCitationSearch())=0A= {=0A= form.action =3D "/search/allsearch";=0A= form.submit();=0A= }=0A= }=0A= =0A= // Preprocesses the arguments sent to CitationSearch and checks validity = of user inputs.=0A= function checkCitationSearch()=0A= {=0A= var supplementFound =3D 0; //increment if found;=0A= var hyphenFound =3D 0; //increment if found;=0A= var fm =3D document.citsearchform;=0A= var errmsg =3D "Please use the valid digits '0' to '9'";=0A= // remove all spaces=0A= fm.volume.value =3D fm.volume.value.replace(/ /g, "");=0A= fm.issue.value =3D fm.issue.value.replace(/ /g, "");=0A= fm.pages.value =3D fm.pages.value.replace(/ /g, "");=0A= =0A= if (fm.issn.value.length > 0 && fm.volume.value.length > 0 && = fm.pages.value.length > 0)=0A= {=0A= // check page range=0A= var pageRange =3D fm.pages.value;=0A= var volume =3D fm.volume.value;=0A= var issue =3D fm.issue.value;=0A= var continueFlag =3D 0;=0A= //check the volume=0A= // if (isNaN(volume))=0A= // {=0A= // // volume failed checking=0A= // alert("Volume is invalid. "+errmsg+".");=0A= // return false;=0A= // }=0A= //check the issue=0A= if ((issue.length !=3D 0) && isNaN(issue))=0A= {=0A= // issue failed checking=0A= alert("Issue is invalid. "+errmsg+" or leave blank.");=0A= return false;=0A= }=0A= //check the page range=0A= if (isNaN(pageRange))=0A= {=0A= var isValid =3D false;=0A= // check for hyphen=0A= var hyphenIndex =3D pageRange.indexOf("-");=0A= if ((hyphenIndex !=3D -1) && (hyphenIndex =3D=3D = pageRange.lastIndexOf("-")))=0A= {=0A= // we have only one hyphen, so extract each number=0A= var firstNum =3D pageRange.substring(0, hyphenIndex);=0A= var secondNum =3D pageRange.substring(hyphenIndex + 1);=0A= // check for S=0A= var firstS =3D firstNum.indexOf("S");=0A= var secondS =3D secondNum.indexOf("S");=0A= if (firstS =3D=3D secondS)=0A= {=0A= if ((firstS =3D=3D -1) && isNum(firstNum) && isNum(secondNum) && = (firstNum.length > 0) && (secondNum.length > 0))=0A= isValid =3D true; // both inputs are digits=0A= else if (firstS =3D=3D 0)=0A= {=0A= var firstNumNoS =3D firstNum.substring(1);=0A= var secondNumNoS =3D secondNum.substring(1);=0A= if (isNum(firstNumNoS) && isNum(secondNumNoS) && = (firstNumNoS.length > 0) && (secondNumNoS.length > 0))=0A= isValid =3D true; // both inputs are Sdigits=0A= }=0A= =0A= }=0A= }=0A= if (isValid)=0A= {=0A= return true;=0A= }=0A= else=0A= {=0A= alert("The page range is invalid. "+errmsg+" and a hyphen '-' if = required.");=0A= return false;=0A= }=0A= }=0A= return true;=0A= }=0A= else=0A= {=0A= alert("Volume and pages are required fields. Please make sure data has = been entered for both fields.");=0A= return false;=0A= }=0A= }=0A= =0A= // returns true if a is an Integer.=0A= function isNum(a)=0A= { return !(isNaN(a)); }=0A= =0A= function fixInlineLists() {=0A= if (!document.getElementById) return;=0A= var allLIs =3D document.getElementsByTagName('li');=0A= var bulletNode =3D document.createElement('img');=0A= bulletNode.src = =3D"http://download.interscience.wiley.com/images/icon.bullet.gif";=0A= =0A= for (var i=3D0; iAbstract | References';=0A= }=0A= //alert("URL: " + loc + "\nStart Index: " + startIndex + "\nEnd Index: = " + endIndex + "\nPage: " + page + "\nLink Text: " + links);=0A= return links;=0A= }=0A= =0A= =0A= function selectAll(fm)=0A= {=0A= //var fm =3D document.viewSelectedForm;=0A= =0A= for (var i =3D 0; i < fm.elements.length; i++)=0A= {=0A= if (fm.elements[i].type =3D=3D 'checkbox' /*&& fm.elements[i].name = =3D=3D 'ID'*/)=0A= {=0A= fm.elements[i].checked =3D true;=0A= }=0A= }=0A= }=0A= =0A= =0A= function do_popup(file)=0A= {=0A= var w =3D window.open(file, 'popWin', = 'width=3D350,height=3D500,scrollbars=3Dyes');=0A= =0A= }=0A= =0A= =0A= function mailTool(itemID, useurl, skin)=0A= {=0A= //var url =3D "/cgi-bin/mailform?item=3D"+itemID;=0A= var url =3D "/cgi-bin/mailform?item=3D" + itemID + "&skin=3D" + skin;=0A= var parameters =3D = 'status=3Dno,scrollbars=3Dno,resizable=3Dyes,width=3D400,height=3D475';=0A= =0A= if (useurl !=3D "")=0A= {=0A= url +=3D "&useurl=3D"+useurl;=0A= }=0A= =0A= if ((isNaN(parseInt(itemID))=3D=3Dtrue)||(arguments.length >=3D 4 && = arguments[3] =3D=3D 'cluster'))=0A= {=0A= url +=3D '&type=3Dcluster';=0A= }=0A= =0A= //check whether the last 2 arguments is used for urllink.=0A= // urllink is needed to open up mail form for mrw pages on tomcat = port=0A= if (arguments.length>=3D5 && = arguments[arguments.length-2]=3D=3D'urllink' && = arguments[arguments.length-1]!=3D'')=0A= {=0A= url =3D arguments[arguments.length-1] + url;=0A= }=0A= =0A= if (arguments.length>=3D5 && arguments[3]=3D=3D'currentlink' && = arguments[4]!=3D'')=0A= {=0A= url =3D url + '¤tLink=3D' + arguments[4];=0A= =0A= //processing specific cases=0A= if (arguments[4]=3D=3D'jcatalogjsphdr')=0A= {=0A= //get the issn and pass it the next stage for processing=0A= var currloc =3D location.href;=0A= var issnloc =3D currloc.indexOf("&issn=3D");=0A= if (issnloc !=3D -1)=0A= {=0A= url =3D url + '¤tISSN=3D' + = currloc.substring(issnloc+6);=0A= }=0A= }=0A= // issn is needed for cocoon rendered mrw to form the final url.=0A= else if ( (arguments[4]=3D=3D'cocoonmrwhome') || = (arguments[4]=3D=3D'cocoonmrwarticle') )=0A= {=0A= if (arguments.length>=3D7 && arguments[5]=3D=3D'issn' && = arguments[6]!=3D'')=0A= {=0A= url =3D url + '¤tISSN=3D' + arguments[6];=0A= }=0A= //getting the parameters of the currently shown mrw from the = url=0A= //the method used here is a little crude, it extracts the = values from the current url of the page the user is in =0A= //to pass it to the background program to reconstruct the = requred url=0A= if (arguments[4]=3D=3D'cocoonmrwhome')=0A= {=0A= var currloc =3D location.href;=0A= var mrwParamArr =3D currloc.split("/");=0A= if (mrwParamArr.length >=3D 7)=0A= {=0A= url =3D url + '&mrwPath=3D' + mrwParamArr[5];=0A= var mrwFileParam =3D mrwParamArr[6];=0A= var seploc =3D mrwFileParam.indexOf("#");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwSection=3D' + = mrwFileParam.substring(seploc+1);=0A= mrwFileParam =3D = mrwFileParam.substring(0,seploc);=0A= }=0A= seploc =3D mrwFileParam.indexOf("?");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwUrlParam=3D' + = mrwFileParam.substring(seploc+1);=0A= url =3D url + '&mrwDocType=3D' + = mrwFileParam.substring(0,seploc);=0A= }=0A= else=0A= {=0A= url =3D url + '&mrwDocType=3D' + mrwFileParam;=0A= }=0A= }=0A= =0A= }=0A= else if (arguments[4]=3D=3D'cocoonmrwarticle')=0A= {=0A= var currloc =3D location.href;=0A= var mrwParamArr =3D currloc.split("/");=0A= if (mrwParamArr.length >=3D 7)=0A= {=0A= url =3D url + '&mrwPath=3D' + mrwParamArr[5];=0A= url =3D url + '&mrwAid=3D' + mrwParamArr[7];=0A= url =3D url + '&mrwVersion=3D' + mrwParamArr[8];=0A= var mrwFileParam =3D mrwParamArr[9];=0A= var seploc =3D mrwFileParam.indexOf("#");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwSection=3D' + = mrwFileParam.substring(seploc+1);=0A= url =3D url + '&mrwDocType=3D' + = mrwFileParam.substring(0,seploc);=0A= }=0A= else=0A= {=0A= url =3D url + '&mrwDocType=3D' + mrwFileParam;=0A= }=0A= }=0A= }=0A= }=0A= }=0A= =0A= //checking if a html file exists in the url=0A= var tmpfname =3D location.href;=0A= var loc =3D tmpfname.indexOf("?");=0A= if (loc !=3D -1)=0A= {=0A= tmpfname =3D tmpfname.substring(0,loc);=0A= }=0A= loc =3D tmpfname.indexOf(".html");=0A= if (loc !=3D -1)=0A= {=0A= loc =3D tmpfname.lastIndexOf("/");=0A= if (loc !=3D -1)=0A= {=0A= tmpfname =3D tmpfname.substring(loc+1);=0A= url =3D url + '¤tFile=3D' + tmpfname;=0A= }=0A= }=0A= =0A= if (arguments.length>=3D7 && arguments[5]=3D=3D'currentyear' && = arguments[6]!=3D'')=0A= {=0A= url =3D url + '¤tYear=3D' + arguments[6];=0A= }=0A= =0A= eval("wileyMailTool =3D = window.open('"+url+"','MailTool','"+parameters+"')");=0A= =0A= // If object exists ensure has focus=0A= if (eval("wileyMailTool") && window.focus)=0A= eval("wileyMailTool").focus();=0A= }=0A= =0A= =0A= /*=0A= * 1. Find the links we need to alter.=0A= * 2. For each link apply the new styles we need.=0A= */=0A= function fixAbsRefRendering()=0A= { =0A= var table =3D document.getElementById('prerendered');=0A= var anchors =3D table.getElementsByTagName('a');=0A= for (var i =3D 1; i < anchors.length; i++)=0A= {=0A= if(anchors[i].href !=3D"") =0A= { =0A= applyAbsRefStyleCorrection(anchors[i]);=0A= =0A= }=0A= }=0A= }=0A= =0A= function applyAbsRefStyleCorrection(currentLink)=0A= {=0A= currentLink.style["color"] =3D "#369";=0A= currentLink.style["fontWeight"] =3D "normal";=0A= currentLink.style["marginLeft"] =3D "3px";=0A= currentLink.style["marginRight"] =3D "5px";=0A= }=0A= =0A= function populatePage(formName)=0A= {=0A= if (formName.Page.value =3D=3D '')=0A= {=0A= formName.Page.value =3D document.location;=0A= }=0A= }=0A= =0A= function equalizeColumnWidth(numFeatures)=0A= {=0A= var numberOfFeatures =3D numFeatures; =0A= // Percentage width of table to use for each column.=0A= var percentPerFeature =3D Math.floor(100 / numberOfFeatures); =0A= // Number of columns in total. Content cols + seperators. =0A= =0A= document.writeln("");=0A= for(var i =3D 0; i < numberOfFeatures; i++)=0A= { =0A= document.writeln(' 3D"blank"

');=0A= }=0A= document.writeln("");=0A= }=0A= =0A= function old_equalizeColumnWidth(numFeatures)=0A= {=0A= var numberOfFeatures =3D numFeatures; =0A= // Percentage width of table to use for each column.=0A= var percentPerFeature =3D Math.floor(100 / numberOfFeatures); =0A= // Number of columns in total. Content cols + seperators. =0A= =0A= if(debug)=0A= {=0A= alert("Number of feature boxes: " + numberOfFeatures + "\nPercent per = feature: " + percentPerFeature);=0A= }=0A= =0A= document.writeln("");=0A= for(var i =3D 0; i < numberOfFeatures; i++)=0A= { =0A= if (netscape4 =3D=3D false)=0A= {=0A= document.writeln(' 3D"blank"

'); =0A= }=0A= }=0A= document.writeln("");=0A= }=0A= =0A= function writeFBoxCell()=0A= {=0A= if(first)=0A= {=0A= document.write('');=0A= }else=0A= {=0A= document.write('');=0A= }=0A= numFeatures ++;=0A= }=0A= =0A= =0A= //=0A= // escape apostrophe characters within a string=0A= //=0A= function encode(str)=0A= {=0A= out =3D '';=0A= =0A= for (a =3D 0; a < str.length; a++)=0A= {=0A= c =3D str.charAt(a);=0A= =0A= if (c =3D=3D "'")=0A= {=0A= out +=3D '\\';=0A= }=0A= =0A= out +=3D c;=0A= }=0A= =0A= return out;=0A= }=0A= =0A= =0A= //=0A= //=0A= //=0A= function escapeEntities(str)=0A= {=0A= while (str.indexOf("'") > -1)=0A= {=0A= pos =3D str.indexOf("'");=0A= str =3D str.substr(0, pos) + '\\\'' + str.substr(pos + 6);=0A= }=0A= =0A= return str;=0A= }=0A= =0A= =0A= // new popup function less intrusive just uses the DOM to pickup links = with an exwin class.=0A= // currently implemented for Figure popups on D&T homepage.=0A= =0A= function exLinks()=0A= {=0A= if (document.getElementById)=0A= {=0A= var aObject =3D document.getElementsByTagName("a");=0A= for (var x=3D0; x" + = liels[j].innerHTML + "

You do not have = anything saved in this menu.

";=0A= else=0A= tmpinnerhtml +=3D "

" = + liels[j].innerHTML + "

"; =0A= } =0A= } else {=0A= //setup var to see if more is required=0A= var more =3D (subnodeslength < lilength) ? true : = false; =0A= var flag =3D true;=0A= for (var j=3D0; j" + liels[j].innerHTML + = "";=0A= } else if (j > lilength-3){=0A= if (j =3D=3D lilength-2){=0A= var url =3D liels[j].innerHTML.split('"')[1];=0A= tmpinnerhtml +=3D "

" + liels[j].innerHTML + "

";=0A= } else {=0A= tmpinnerhtml +=3D "

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"; =0A= } =0A= } =0A= }=0A= // set last two menu items to have no rollover and = a border at the top =0A= }=0A= menu.childNodes[x].childNodes[y].innerHTML =3D = tmpinnerhtml;=0A= // set default menu elements with diff style=0A= if (flag){=0A= if (more){=0A= liels[liels.length-3].style.borderTop =3D "1px solid = #DDDDDD";=0A= liels[liels.length-3].className =3D "noroll";=0A= liels[liels.length-2].style.borderTop =3D "1px solid = #DDDDDD";=0A= liels[liels.length-2].className =3D "noroll";=0A= liels[liels.length-1].className =3D "noroll";=0A= more =3D false;=0A= } else {=0A= liels[liels.length-2].style.borderTop =3D "1px solid = #DDDDDD";=0A= liels[liels.length-2].className =3D "noroll";=0A= liels[liels.length-1].className =3D "noroll";=0A= } =0A= flag =3D false; =0A= } =0A= =0A= // set sub menu title for the msnu=0A= liels[0].className =3D "hoverTitle";=0A= // apply rollover functions to hide/show sub menus=0A= if (classname =3D=3D "menuDOM"){=0A= menu.childNodes[x].onmouseover =3D function(){=0A= // safari doesnt handle auto in absoulte = positioning correctly so this code makes the menus display correctly.=0A= if (navigator.userAgent.indexOf("Safari") !=3D -1){=0A= if(!window.devicePixelRatio) {=0A= this.getElementsByTagName("UL")[0].style.marginTop =3D "20px";=0A= this.getElementsByTagName("UL")[0].style.marginLeft =3D "0";=0A= }=0A= }=0A= this.className =3D"menuhover domArrowOver";=0A= if(document.all) = this.getElementsByTagName("iframe")[0].style.height=3Dthis.getElementsByT= agName("ul")[0].offsetHeight;=0A= }=0A= menu.childNodes[x].onmouseout=3Dfunction() {=0A= this.className =3D "domArrowOut";=0A= }=0A= }=0A= =0A= if(document.all) {=0A= var newIframe =3D document.createElement("iframe");=0A= newIframe.style.filter =3D = "progid:DXImageTransform.Microsoft.Alpha(style=3D0,opacity=3D0)";=0A= menu.childNodes[x].appendChild(newIframe);=0A= }=0A= =0A= } =0A= }=0A= // deletes the sub menus on the IEMAC as they have a = background bug, will still remain on NS6=0A= if ((navigator.userAgent.indexOf("Mac") !=3D -1) && = (navigator.userAgent.indexOf("MSIE") !=3D -1)){=0A= menu.childNodes[x].innerHTML =3D = menu.childNodes[x].innerHTML.split(""=0A= var menu =3D document.all["menu"].children[0];=0A= menu.style.marginBottom =3D "-3px";=0A= for (var x=3D1; 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Neuropathology

ISSN: 1440-1789 (Online)

ISSN: 0919-6544 (Print)

Volume 9999, Issue 9999, 2009.

Pages: ???=96???

Top of Page
Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
References

Tables: 1, 2, 3

List of=20 Images

Abstract=20 Only

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3D"About

Online ISSN: = 1096-9896 Print ISSN: 0022-3417
The Journal of = Pathology
Volume 214, Issue 3, 2008. Pages: 328-336

Copyright =C2=A9 2007 Pathological Society of Great = Britain and Ireland. Published by John Wiley & Sons, Ltd.

'); } =0A= function copyright(hwnd) {var title =3D pagetitle(); = hwnd.document.write(title.bold() + '
Copyright © 2008 Japanese = Society of Neuropathology

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'); } =0A= =0A= =0A= function caption_f1(hwnd) = {hwnd.document.write('Fig. 1 Representative = results of methylation-specific PCR assays. Lanes U and M indicate the = presence of unmethylated and methylated DNA, respectively. The negative = control for methylation was normal lymphocyte DNA and normal lymphocyte = DNA treated with SssI methylase was the positive control. Water was the = negative control for the PCR. The lanes marked 1 show the results from a = patient without methylation and those in lanes 2 the results from a = patient with methylation.'); }=0A= function display_f1(size) { var i =3D new Array(); i[0] =3D 'nf1'; i[1] = =3D 'nf1'; var hwnd =3D newfigwin(); dochead(hwnd, ''); copyright(hwnd); = otherlinks(hwnd, 'f1', size); imagelink(hwnd, i[size]); = caption_f1(hwnd); doctail(hwnd); hwnd.document.close(); hwnd.focus(); }=0A= function caption_f2(hwnd) = {hwnd.document.write('Fig. 2 Overall survival = expressed in Kaplan–Meier curves, showing statistically = insignificant differences between the methylation and unmethylated = groups (log rank test, P =3D 0.516). Number of patients = at risk is shown at the bottom.'); }=0A= function display_f2(size) { var i =3D new Array(); i[0] =3D 'nf2'; i[1] = =3D 'nf2'; var hwnd =3D newfigwin(); dochead(hwnd, ''); copyright(hwnd); = otherlinks(hwnd, 'f2', size); imagelink(hwnd, i[size]); = caption_f2(hwnd); doctail(hwnd); hwnd.document.close(); hwnd.focus(); }=0A= =0A= =0A= function imagelist() {var hwnd =3D newfigwin(); dochead(hwnd, 'List of = Images - '); copyright(hwnd); =0A= hwnd.document.write('');=0A= =0A= =0A= hwnd.document.write('');=0A= hwnd.document.write('');hwnd.document.write('

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3D""=20

Original Article

Methylation status of the MGMT gene = promoter=20 fails to predict the clinical outcome of glioblastoma patients = treated=20 with ACNU plus cisplatin

Chul-Kee Park,=20 ^1,2 Sung-Hye Park,=20 ^3,6 Se-Hoon Lee, = ⁴ Chae-Yong Kim,=20 ¹ Dong-Wan Kim,=20 ⁴ Sun Ha Paek, = ^1,6 Dong Gyu Kim,=20 ¹ Dae Seog Heo,=20 ^2,4 Il Han Kim=20 ^2,5 and Hee-Won Jung = ¹

Departments of ¹Neurosurgery, ³ Pathology, = ⁴ Internal Medicine = and=20 = ⁵ Radiation = Oncology,=20 and ⁶ Neuroscience = Research=20 Institute, Seoul National University College of Medicine, Seoul = National=20 University, and ² = Cancer Research=20 Institute, Seoul National University Hospital, Seoul, Korea =

Correspondence to Sung-Hye Park, MD,=20 PhD, Department of Pathology, Seoul National University College of = Medicine, 101 Daehangno, Jongno-gu, Seoul, 110-744, Korea. Email: = shparknp@snu.ac.kr=20

Copyright =A9 2008 Japanese Society of = Neuropathology

KEYWORDS

glioblastoma =95 = O6-methylguanine-DNA-methyltransferase=20 =95 survival

ABSTRACT

3DAbstract=20

=

=

=

=

=

=

We analyzed the methylation status of the=20 O6-methylguanine-DNA methyltransferase (MGMT) promoter using a=20 methylation-specific polymerase chain reaction (MSP) in = glioblastoma=20 patients treated with=20 = 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea = (ACNU) plus cisplatin followed by radiation therapy. Forty-eight = patients=20 with interpretable MSP results were included in this study. The = MGMT=20 promoter was methylated in 26 patients (54.2%, methylated group) = and=20 unmethylated in 22 patients (45.8%, unmethylated group). = Comparison of=20 clinical outcomes between the two groups revealed that the = methylation=20 status of the MGMT gene promoter was not a prognostic factor for = overall=20 survival (P =3D 0.516) or a = predictive factor=20 for radiological response to ACNU plus cisplatin treatment (P =3D 0.529). The most noteworthy = explanation for=20 the result is that the synergistic antitumor effects of ACNU and = cisplatin=20 resulting from inactivation of MGMT by cisplatin in MGMT active = tumors=20 offset the drug resistance.

Received 7 October 2008 and accepted 13 November=20 2008.

DIGITAL OBJECT IDENTIFIER (DOI)

10.1111/j.1440-1789.2008.00998.x About=20 DOI

=20

=20

=20

INTRODUCTION

=

=

=

=

=

=

O6-methylguanine-DNA methyltransferase (MGMT) is = an enzyme=20 in the DNA repair process that specifically removes cytotoxic=20 O6-alkylguanine adducts, thus mediating resistance to alkylating = agents.¹=20 This DNA repair enzyme plays a role in maintaining the integrity = of the=20 DNA in normal cells but also protects tumor cells against = alkylating and=20 methylating chemotherapeutic agents, resulting in drug = resistance.²=20 There have been several studies suggesting the clinical value of = MGMT=20 evaluation in predicting benefit from alkylating agents in = malignant=20 glioma patients treated with nitrosourea as well as with = temozolomide.³⁼⁹⁶⁷=20 However, there is also evidence denying a connection between the = MGMT=20 methylation status of gliomas and the clinical outcome when = treated with=20 nitrosourea.⁸⁼⁹⁶¹²=20 There is also in vitro evidence indicating that the enhancement of = the=20 radiation response by temozolomide is independent of the = epigenetically=20 silenced MGMT gene.¹³

Previous MGMT analysis studies were performed = using a=20 variety of different techniques, and the best technique for = analyzing MGMT=20 status is a matter of debate. However, it is generally accepted = that a=20 methylation-specific polymerase chain reaction (MSP) evaluating = the=20 methylation status of the MGMT promoter is the best way to predict = the=20 MGMT expression of the tumor in a manner that also correlates with = clinical outcome.¹⁴

Since nitrosoureas were introduced in the early = 1970s, a=20 large number of clinical trials have evaluated their role in = glioblastoma=20 and other malignant gliomas, and they have been used as a mainstay = agent=20 for the treatment of malignant gliomas.^15,16=20 We previously reported the effectiveness and feasibility of = preradiation=20 chemotherapy with=20 = 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea = (ACNU) plus cisplatin in glioblastomas in the setting of a single = arm=20 phase II clinical trial.¹⁶=20 Based on this evidence, we have managed newly diagnosed = glioblastoma=20 patients using preradiation chemotherapy with an ACNU plus = cisplatin=20 protocol. Our hypothesis is that the methylation status of the = MGMT=20 promoter is associated with prognosis in glioblastoma patients = treated=20 uniformly with surgery, ACNU plus cisplatin chemotherapy and = radiation=20 therapy. We analyzed the methylation status of the MGMT promoter = in tumor=20 cells using MSP and determined its relationship with radiological = response=20 after ACNU plus cisplatin treatment and overall = survival.

=20

=20

MATERIALS AND METHODS

=

=

=

=

=

=20

=20

Patients and management

Between January 2000 and December 2003, 77 newly = diagnosed=20 glioblastoma patients were treated in our institute with = preradiation=20 chemotherapy with ACNU plus cisplatin after surgery, followed by = radiation=20 therapy. Among them, proper tissue samples for MGMT promoter MSP = analysis=20 were available from 50 patients, who were included in this study.=20 Interpretable results from the MGMT-promoter MSP were obtained in = 48 of=20 these 50 patients, and these results were analyzed. Patient data = were=20 based on information contained in hospital charts and radiological = studies=20 and were collected using a case record form approved by the = institutional=20 review board.

The study population consisted of 31 men and 17 = women=20 ranging in age from 20 to 69 years (mean, 49 years). All patients=20 underwent surgical removal or biopsy sampling of their tumor. The=20 histological diagnosis of glioblastoma was confirmed according to = World=20 Health Organization criteria.¹⁷=20 The preradiation chemotherapy of ACNU=20 (40 mg/m²/day) and cisplatin=20 (40 mg/m²/day) by continuous = intravenous=20 infusion for 72 h started 2=963 weeks after surgery and was = repeated=20 after 6 weeks. Two cycles were administered unless the patient = showed=20 progressive disease, unacceptable toxicity or refused further = treatment.=20 Before the second cycle of treatment, neurological examinations = and MRI=20 were performed. Patients who showed a radiological response or = stable=20 state without neurological deterioration were eligible for a = second cycle=20 of chemotherapy, and patients with tumor progression were referred = for=20 radiation therapy. In addition to tumor response, the treatment = schedule=20 was readjusted according to bone marrow, renal and hepatic = function, as=20 described previously.¹⁶=20 Radiation therapy was initiated 6 weeks after the last cycle of=20 chemotherapy, with a total dose of 60 Gy, 1.8 Gy per = fraction=20 with five fractions per week. The target volume included the=20 post-chemotherapy tumor volume and surrounding edema with a margin = of=20 3 cm, as defined by contrast-enhanced MRI taken 5 weeks after = the=20 last cycle of chemotherapy. Thirty-five patients completed the two = cycles=20 of ACNU plus cisplatin, and 13 patients underwent one cycle before = radiation therapy. The survival time was defined as the time from = the date=20 of the surgery to the date of death. Post-surgical performance = status,=20 expressed using the Karnofsky performance scale, and the extent of = resection, classified as complete and incomplete, were analyzed as = possible compounding prognostic factors.

=20

=20

Tumor samples and MSP

The methylation status of the MGMT promoter was = confirmed by=20 MSP. After microscopic examination of HE-stained slides, we = delineated the=20 most representative areas. Tissue was dissected from paraffin = blocks and=20 put into polyethylene microtubes. After deparaffinization with = xylene and=20 alcohol, the blocks were dissolved in a lysis buffer solution = containing=20 proteinase K. For the MSP, purified DNA was modified by sodium = bisulfite=20 treatment using an EZ DNA methylation-Gold Kit=99 (Catalog No. = D5005; Zymo=20 Research, Orange, CA, US). The primer sequences for the MGMT were = as=20 follows: methylated forward: 5' TTT CGA CGT TCG TAG GTT TTC GC 3', = methylated reverse: 5' GCA CTC TTC CGA AAA CGA AAC G 3', = unmethylated=20 forward: 5' TTT GTG TTT TGA TGT TTG TAG GTT TTT GT 3', = unmethylated=20 reverse: 5' AAC TCC ACA CTC TTC CAA AAA CAA AAC A 3'. The = annealing=20 temperature was 64=B0C. The PCR products obtained were = electrophoresed in 2%=20 agarose gels and visualized under ultraviolet illumination after = staining=20 with ethidium bromide. CpGenome universal unmethylated (Catalog = No. S7822;=20 Chemicon, Temecula, CA, US) and methylated DNA sets (Catalog No. S = 7821;=20 Chemicon) were used as negative and positive controls = respectively.

For the evaluation of the assay results, the = products from=20 the controls were examined first. The MGMT promoter fragments in = the=20 controls should be observed at 80 and 92 base pairs in the = methylated=20 DNA=96methylated primer and unmethylated DNA=96unmethylated primer = combinations respectively. The methylated DNA=96unmethylated = primer and=20 unmethylated DNA=96methylated primer controls should not show any = bands. If=20 the control results were acceptable, patient samples were = evaluated for=20 the presence of amplification with the methylated and unmethylated = primers. The results were interpreted as positive if MGMT promoter = methylation was detected as a fragment of 80 base pairs observed = on the=20 gel, and negative if MGMT promoter methylation was not detected = with the=20 methylated primers.

=20

=20

Radiological response analysis

Radiological responses were assessed by comparing = patients'=20 post-surgical MRI taken within 48 h of the operation with the = preradiation-therapy MRI obtained 5 weeks after the last cycle of = ACNU=20 plus cisplatin treatment. The volume of enhanced tumor was = measured=20 3-dimensionally in gadolinium-enhanced T1-weighted images using a=20 volumetric program (Osiris^=AE v4.0, = Digital Imaging=20 Unit, Informatics Center, University Hospital of Geneva, Geneva,=20 Switzerland). The error associated with repeat measurements of the = volume=20 of the same MRI was within =B1 2%. Response criteria proposed = for=20 3-dimensional volume changes in solid tumors were used.¹⁸=20 A complete response (CR) was defined as total disappearance of all = enhancing tumor, a partial response (PR) was defined as a 65% or = more=20 reduction in volume, stable disease (SD) was defined as no change = or=20 < 65% reduction or < 40% increase, and progressive = disease=20 (PD) was defined as ≥ 40% increase in volume or the = appearance of new=20 lesions.

=20

=20

Statistical analysis

Overall survival curves were estimated by the = Kaplan=96Meier=20 technique and compared using the two-sided log-rank test. The Cox=20 proportional-hazards model was fitted to assess the prognostic = value of=20 the methylation status of the MGMT promoter and potential = prognostic=20 factors such as age, performance status, and the extent of = resection. For=20 analyzing group differences and responses according to the = methylation=20 status of the MGMT promoter, the chi-square test and Student's = t-test were used for parametric comparisons. = Statistical=20 significance was accepted at probability values of < 0.05. = These=20 statistical analyses were performed with the aid of SPSS software = (version=20 10.5; SPSS, Inc., Chicago, IL, US).

=20

=20

RESULTS

=

=

=

=

=20

=20

Methylation status of the MGMT = promoter

Representative results of MGMT-promoter MSP assays = are shown=20 in Figure 1.=20 Of the 48 patients whose samples were analyzed, the MGMT promoter = was=20 methylated in 26 (54.2%, methylated group) and unmethylated in 22 = (45.8%,=20 unmethylated group). The clinical characteristics of the = methylated and=20 unmethylated groups are summarized in Table 1.=20 There were no significant differences in the distribution of age, = extent=20 of resection and performance status between the two groups.

=20

= Fig. 1 Representative results=20 of methylation-specific PCR assays. Lanes U and M indicate = the=20 presence of unmethylated and methylated DNA, respectively. = The=20 negative control for methylation was normal lymphocyte DNA = and=20 normal lymphocyte DNA treated with SssI methylase was the = positive=20 control. Water was the negative control for the PCR. The = lanes=20 marked 1 show the results from a patient without methylation = and=20 those in lanes 2 the results from a patient with = methylation.=20

[Normal View=20 ]

=20

Table 1 Comparison of = clinical=20 characteristics of the patients in the methylated MGMT promoter = group=20 (n =3D 26) and unmethylated MGMT = promoter=20 group (n =3D 22) =

	MGMT = promoter=20 status	P=20 value

Methylated (n =3D 26)	Unmethylated (N =3D 22)

Mean age	49.7 =B1 12.4 years	48.5 =B1 13.2 years	0.757
Extent of = resection
Complete = resection	9 (34.6%)	7 (31.8%)	0.838

Incomplete = resection	17 (65.4%)	15 (68.2%)
Functional = status
KPS 80 or = more	22 (84.6%)	17 (77.3%)	0.516

KPS less than = 80	4 (15.4%)	5 (22.7%)

MGMT, O6-methylguanine-DNA = methyltransferase;=20 KPS, Karnofsky performance = scale

=20

=20

Methylation status of the MGMT = promoter and=20 overall survival

Overall survival curves are presented in Figure 2.=20 The Kaplan=96Meier survival curves showed that the methylated = group did not=20 have a longer survival time than the unmethylated group did (P =3D 0.516). No difference was seen in = the median=20 survival time between the methylation (17.0 months, 95% CI: = 12.0=9621.9) and=20 unmethylated (17.0 months, 95% CI: 10.1=9623.9) groups. Estimated = survival=20 rates at 1 and 2 years in the methylation and unmethylated groups = were=20 similar (76.9% vs 72.7% and 30.8% vs 31.8% respectively). Multivariate analysis = using a Cox=20 proportional-hazards model including possible prognostic factors = such as=20 age, extent of resection, performance status as well as the = methylation=20 status of the MGMT promoter also did not show any significant=20 relationships with overall survival (Table 2).

=20

= Fig. 2 Overall = survival=20 expressed in Kaplan=96Meier curves, showing statistically=20 insignificant differences between the methylation and = unmethylated=20 groups (log rank test, P =3D 0.516).=20 Number of patients at risk is shown at the bottom.=20

[Normal View=20 ]

=20

Table 2 Results=20 of the Cox proportional-hazard analysis of overall survival =

Variable	P value	Hazard ratio	95% CI

Age	0.100	1.025	0.995=961.055
Complete resection	0.509	1.266	0.628=962.552
KPS 80 or more	0.400	0.719	0.333=961.551
MGMT promoter = methylation	0.518	0.813	0.434=961.523

MGMT,=20 O6-methylguanine-DNA methyltransferase; KPS, Karnofsky = performance scale

=20

=20

Methylation status of the MGMT = promoter and=20 radiological response

Table 3=20 shows the tumor response rate results and the tumor control rate = for the=20 methylation and unmethylated groups as determined from MRI taken = before=20 and after ACNU plus cisplatin treatment. Although small = superiorities in=20 response rates (50% vs 41%) and control = rates (81%=20 vs. 68%) were observed in the methylated = group, their=20 differences were statistically insignificant (P =3D 0.529 and P =3D 0.316, respectively).

=20

Table 3 Tumor=20 response rates and control rates after ACNU plus cisplatin=20 treatment

	MGMT = promoter=20 status	P-value

Methylated (n =3D 26)	Unmethylated (N =3D 22)

Response
CR + PR	13 (50%)	9 (41%)	0.529

SD + PD	13 (50%)	13 (59%)
Control
CR + PR + SD	21 (81%)	15 (68%)	0.316

PD	5 (19%)	7 (32%)

MGMT,=20 O6-methylguanine-DNA methyltransferase; CR, complete = response;=20 PR, partial response; SD, stable disease; PD, = progressive=20 disease

=20

=20

DISCUSSION

=

=

=

Many attempts to correlate the MGMT activity of = tumor cells=20 with clinical outcome in patients with high-grade gliomas treated = with=20 nitrosourea-based chemotherapy have been made previously, but the = results=20 have been controversial. Nakasu et al. = reported=20 the results of immunohistochemical staining of neoplastic cells = from 51=20 patients with high-grade gliomas who were treated with radiation = therapy=20 followed by ACNU chemotherapy, and showed a significant benefit in = median=20 survival in the MGMT-negative group (23 months) over the = MGMT-positive=20 group (14 months).⁵=20 Tanaka et al. found a reverse = correlation=20 between MGMT mRNA copy number as measured by real-time RT=96PCR = and the=20 response rate after ACNU treatment in patients with high-grade = glioma.⁶=20 Esteller et al., using an MSP method, = reported=20 that the methylation of the MGMT promoter in gliomas was a useful=20 predictor of the responsiveness of the tumors and overall survival = in=20 patients with high-grade glioma treated with=20 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).³=20 In contrast, there are many reports denying that MGMT has any = prognostic=20 value in cases of high-grade gliomas. Kamiryo et al. analyzed the methylation status of = the MGMT=20 promoter by MSP in 116 high-grade gliomas in patients treated with = radiation therapy and ACNU-based chemotherapy.⁹=20 They found that it had significant predictive value for overall = survival=20 and progression-free survival in anaplastic astrocytoma patients, = but no=20 relationship was observed in glioblastoma patients.⁹=20 Belanich et al. reported that no = correlations=20 were found between low MGMT expression in tumor cells, determined = by=20 immunofluorescence assay, and longer overall survival or = progression-free=20 survival in 167 primary brain tumor patients, including 99 = patients with=20 glioblastomas treated with radiation therapy and BCNU.⁷=20 Jaeckle et al. also did not find any=20 statistically significant correlation between low MGMT expression = in tumor=20 cells, as assessed by immunofluorescence assay, and longer = survival in 64=20 patients with high-grade glioma treated with radiation therapy and = BCNU.¹⁹=20 Blanc et al. studied methylation of = the MGMT=20 promoter in 44 glioblastomas using an MSP method and found that = there was=20 no relationship between the methylation status of the MGMT = promoter and=20 overall survival and response to BCNU.⁸=20 Brell et al. observed a correlation = between MGMT=20 protein expression and survival in glioblastoma patients, but they = could=20 not find any correlation between the methylation status of the = MGMT=20 promoter and survival period.²⁰=20 Rodriguez et al. evaluated MGMT status = in a=20 group of 50 glioblastoma patients using both immunohistochemistry = and MSP,=20 but neither approach showed a predictive value for overall = survival.¹²

The controversial results regarding the prognostic = significance of MGMT protein expression and MGMT promoter = methylation in=20 high-grade glioma patients may be explained by several reasons. = First, the=20 various methods used for the measurement of MGMT activity in tumor = cells=20 might have led to inconsistent results among studies. There are = reports=20 showing an inconsistent correlation between aberrant MGMT promoter = methylation and loss of MGMT protein expression.^12,20,21=20 In addition, discrepancies in results between assays on frozen and = paraffin samples have been observed.²²=20 Immunohistochemical staining has merits in that it is a method for = measurement of functional proteins at the endpoint of regulation, = as well=20 as being fairly easy technically and generally available. = Nevertheless, it=20 has shortcomings in that confused results are produced if MGMT = positive=20 inflammatory cells have infiltrated the tumor, and it cannot = predict the=20 MGMT induction by radiation therapy or chemotherapy.^5,23=20 Methylation-specific PCR of MGMT has advantage that it detects the = functional status of gene expression, even if alkyltransferase is = absent,=20 and it has been clearly advocated as the mainstay of clinical = evaluation=20 of MGMT status in glioblastomas.^2,24=20 However, MSP also has shortcomings in that it is a highly = sensitive=20 qualitative technique, and a positive result is observed even if = cells=20 that carry MGMT promoter hypermethylation represent only a minor = portion=20 of the tumor.^20,25=20 This means that it cannot fully reflect the heterogeneity in MGMT = status=20 of the tumor, which can frequently be seen in high-grade = gliomas.^8,23,26=20 A major problem is that the discordant results for = immunohistochemical=20 staining, enzyme activity assays and MSP prevent the use of these = assays=20 interchangeably.²⁶=20 Second, as observed in the report by Kamiryo et al., there might be pitfalls in analyzing = glioblastomas and other high-grade gliomas together as a single = entity.⁹=20 The discrepancy in determining the prognostic value of MGMT status = between=20 glioblastoma and other high-grade gliomas may have originated from = the=20 degree of tumor cellularity, as considerable contamination of the = MGMT=20 status results from non-tumor cells might occur if the tumor = cellularity=20 is low. Third, many patients in previously reported studies = received=20 nitrosourea after radiation therapy, and it was difficult to = establish=20 whether MGMT expression was a predictive factor in nitrosourea = treatment=20 or merely a prognostic factor.²⁷=20 Careful interpretation of study results is needed to assess the = predictive=20 value of MGMT expression for alkylating agent treatment, and large = intergroup prospective studies using standardized techniques are = required=20 for proper validation.

Few studies have evaluated the impact of MGMT on = the=20 radiological response to chemotherapy.¹⁴=20 In this study, the methylation status of the MGMT gene promoter = was not a=20 prognostic factor for overall survival of glioblastoma patients = treated=20 with ACNU plus cisplatin followed by radiation therapy or a = predictive=20 factor for radiological response to ACNU plus cisplatin treatment. = It is=20 true that this study is not free from weak points such as = retrospective=20 analysis, a single institution series and the above-mentioned = pitfalls in=20 MSP. However, the homogeneous group of glioblastoma patients and = the=20 neoadjuvant application of ACNU plus cisplatin before radiation = therapy in=20 this study population provided more reliable assessment of the = influence=20 of MGMT activity on nitrosourea treatment.

Hypotheses explaining the lack of difference in = clinical=20 outcomes between the methylation and unmethylated groups shown in = this=20 study can be proposed. One is that there may be an unknown = nitrosourea=20 resistance mechanism in addition to MGMT.⁹=20 The other is that the dose schedule for ACNU administration in = this series=20 resulted in sufficient MGMT depletion to overcome drug resistance = in the=20 unmethylated group. Another is that regulation of MGMT expression = is a=20 rather complex phenomenon, and the simple expectation of abnormal=20 methylation of the promoter being a determinant factor may not be = true,=20 and effects on other associated genes or post-translation = modification of=20 MGMT may have distorted the results.^{20,25,28=9630}=20 However, the most likely explanation for the result of this study = is the=20 effect of the combination of cisplatin with ACNU. Previous studies = have=20 indicated that cisplatin can inactivate or attenuate MGMT in tumor = cells=20 and MGMT inactivators can enhance the toxicity of cisplatin.^31=9633=20 Therefore cisplatin plus ACNU might have improved antitumor = activity, even=20 in the unmethylated group, so that their MGMT-related drug = resistance is=20 offset. The excellent median overall survival of 17.0 months and = the=20 response rate of 41% observed in the unmethylated group support = such=20 reasoning. This possible synergistic effect of cisplatin and = alkylating=20 agents extends the strategies for glioblastoma treatment and is = currently=20 being evaluated.^34=9638

The methylation status of the MGMT promoter failed = to=20 predict the clinical outcome of glioblastoma patients treated with = ACNU=20 plus cisplatin. Among the plausible explanations for this result, = the=20 synergistic antitumor effects of ACNU and cisplatin resulting from = inactivation of MGMT by cisplatin in MGMT active tumor is = noteworthy.=20 However, controversies regarding the predictive value of MGMT in=20 high-grade glioma treatment, including the correct method for = MGMT-status=20 evaluation, remain to be solved in the future.

=20

=20

=20

ACKNOWLEDGMENT

=

=

This study was supported by a grant of the Seoul = National=20 University Hospital research fund (No. 04-2008-095).

=20

=20

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3D""=20

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