tumor markers: discovery to practice

The Tumor Markers: Discovery toPractice conference was held in Santa Barbara (CA, USA) between28 February and 4 March 2003 andspecialized in new tumor markers thatare currently in R&D or clinicalvalidation studies, and newtechnologies for cancer diagnostictests. The conference was organized byThe University of Texas MD AndersonCenter (http://www.mdanderson.org).

Basic conceptsThe conference chairman, Herbert A.Fritsche (Texas MD Anderson CancerCenter), overviewed recent advancesin biotechnology that fueled thediscovery process and generated many new potential tumor markers.The progress was mainly from thecompletion of human genomesequencing and applications of geneexpression profiling with DNAmicroarrays [1]. Two-dimensional (2D)gel electrophoresis coupled with MS,multi-dimensional HPLC, LC–MS–MS,surface enhanced laser desorption-ionization time-of-flight MS (SELDI-TOF MS) [2], helped to analyze thepotential markers at the protein level.Tissue microarray applications alsointroduced a multiplex approach tovalidate gene-expression profiles at theprotein level in situ [3]. New areas fortumor marker investigations includemethylated DNA gene promoterregions and alternatively splicedmRNA. A newly developing field oftumor markers is the ‘circulatingepithelial cell’. There is muchcontroversy in this area related to thedetection methods used for circulatingcells, the malignant nature of thecirculating epithelial cell, and its

metastatic potential. Highly sensitiveRT-PCR analysis of these cells couldprovide new insights into their use ascancer diagnostics.

Sudhir Srivastava from the NationalCancer Institute (NIH; http://www.nci.nih.gov) discussed the importanceof discovering new tumor markers andearly detection by understanding thevariations and complex situations incancer proteomics [4]. Proteinexpression and function are subject to modulation through transcription,as well as post-transcriptional andtranslational events. More than oneRNA can result from one gene througha process of differential splicing.Additionally, there are >200 post-translational modifications thatproteins could undergo that affectfunction, protein–protein interaction,stability, targeting and half-life, allcontributing to potentially largenumbers of protein products from one gene. At the protein level, distinct changes occur during thetransformation of a normal cell into aneoplastic cell that range from alteredexpression, differential proteinmodification, changes in specificactivities and aberrant localization, allof which affect cell function. Srivastavaintroduced the US National CancerInstitute’s Early Detection ResearchNetwork consortium, in whichgenomics and proteomics approachesare being employed for the rapiddiscovery and evaluation of cancerbiomarkers for early detection and risk assessment.

Genomics and proteomicsThe conference provided a portfolio ofpresentations and discussions on the

progress in genomics and proteomicsthat is bringing tumor marker discoveryto a higher level [5,6]. DiaDexus’(http://www.diadexus.com)genomics–proteomics strategies, usingbioinformatics to curate and prioritize30,000 differentially regulatedsequences and screening them viahybridizing with panels of RNA derivedfrom human tumor and normal tissues,were shared by Jackie Papkoff. Thedifferential expression profiling wasconfirmed by quantitative PCR and MS analysis on the samples of panelsof tumor and matched normaladjacent tissues, as well as a set ofnormal tissues.

Stephen Naylor (Beyond Genomics;http://www.beyondgenomics.com)also discussed approaches of usingcombined genomics and proteomicsmethods to quantify and identifydifferentially expressed proteins as afunction of disease or treatment ofdisease. The essence of what isrequired to perform differentialproteomics was described, bydiscussing the formidable challengesin wide dynamic ranges of proteinconcentrations in sample preparationand analytical limitations in detectionsensitivity.

Brigitta Brinkman from ExonhitTherapeutics (http://www.exonhit.com)reported the development of aninnovative approach to qualitativegenome analysis that enables theidentification of the changes that takeplace during RNA processing. Themethodology, designated DATAS(Differential Analysis of Transcripts withAlternative Splicing), provides insightinto the alternative RNA processingevents that occur as part of disease

DDT Vol. 8, No. 10 May 2003 updateconference

1359-6446/02/$ – see front matter ©2003 Elsevier Science Ltd. All rights reserved. PII: S1359-6446(03)02682-5 441www.drugdiscoverytoday.com

Tumor markers: discovery to practiceWei-Wei Zhang, GenWay Biotech, 10130 Sorrento Valley Road, Suite C, San Diego, CA 92121, USA; tel: +1 858 458 0866 ext.101, fax: +1 858 458 0833, e-mail: wzhang@genwaybio.com

progression or following exposure tocertain drugs. Susan Contrell(Epigenomic; http://www.epigenomics.com) reported on the discovery ofcancer markers with analysis ofmethylated DNA in tissue and serumsamples from cancer patients. Onemarker, Calcitonin, has previously beenshown to be aberrantly methylated incancer, particularly leukemia.Methylation-sensitive microarrays were used to identify new markers,which were validated by real-time PCR analysis.

Phillip Bernard from HuntsmanCancer Institute (http://www.huntsmancancer.org) discussed alarge-scale analysis of clinical sampleson different types of solid tumorsusing quantitative real-time PCR forexpression profiling and for detectinggene amplifications and detection. Themelting curve analysis immediatelyfollowing PCR to identify smallmutations, down to single basechanges, was also reported.

MS and 2D gel approachesSome results of clinical sample analysisusing SELDI-TOF MS were presentedby Daniel Chan (Johns HopkinsMedical Institutions; http://www.hopkinsmedicine.org), showing severalcases of cancer biomarker discoveryfrom serum or plasma of ovarian, breastand prostate cancers. The strength ofthe method was characterized byhigh-throughput and ease of samplepreparation, and the limitation of themethod was characterized by a largenumber of variables (the mass peaks).

Karrin Rodland (Pacific NorthwestNational Laboratory; http://www.pnl.gov) also presented novel MS approaches to characterize theproteome by using multi-prongedmethodology, based upon acombination of instrumentation,separation technologies and uniqueaffinity reagents. By combining strongcation exchange of tryptic peptides

from albumin- and immunoglobulin-depleted serum with reverse-phase LCand ion-trap MS, >490 low-abundanceproteins in human plasma sample were identified.

David Beer (University of MichiganComprehensive Cancer Center;http://www.cancer.med.umich.edu)reported a case of 2D-classifier for Stage I lung adenocarcinoma. A total of 682 individual protein spots werequantified in 90 lung adenocarcinomasusing quantitative 2D-gel analysis. Of 46 survival-associated proteins, 33 were identified using matrix-assistedlaser desorption ionization time-of-flight(MALDI-TOF) MS or nanoflow capillaryLC coupled with electrospray tandemspectrometry. The expression of 12 candidate proteins was confirmed as tumor-derived withimmunohistochemical analysis andtissue arrays using tumors from this study.

Tissue bankingMichael Misialek (Impath PredictiveOncology; http://www.impath.com)addressed the importance of creating acancer biorepository and tissue bank,which are becoming a platform fornovel biomarker discovery. A large-scaleoperation is in process with a plannedcollection of 50,000 patient cases withcomplete demographics, clinical dataand 3-year outcome analyses. Thesamples will be ethically obtainedafter informed consent and formalInstitutional Review Board review. Theestablishment of the tissue bank is acrucial resource for tumor markerdiscovery and validation in suchdownstream applications asimmunohistochemistry, tissue andDNA microarrays, flow cytometry,image analysis, serum analysis,molecular genetics and cytogenetics,northern and Southern hybridization,PCR, fluorescence in situ hybridization(FISH), western blotting, drugresistance assays, cell culture and,

most importantly, for futuretechnological advances.

New tumor markersEleftherios Diamandis (Mount SinaiHospital; http://www.mtsinai.on.ca)addressed a comprehensive study ofkallikreins as tumor markers. Humankallikreins are a family of secreted serineproteases, which was recently identifiedencoded by a cluster of genes onhuman chromosome 19q13.4 [7].Among these genes is prostate-specificantigen (PSA), the premier biomarkerfor the diagnosis and monitoring ofprostatic carcinoma. It was found thatmany kallikrein genes were over- orunderexpressed at the mRNA level, invarious malignancies, including thosethat are endocrine-related, such asovarian, breast and prostate cancer.More specifically, elevations of humankallikreins 5, 6, 7, 8, 10 and 11 in theserum of patients with ovariancarcinoma were observed. Humankallikreins 4, 5, 10, 11 and 15 werealso found to be elevated in breast and prostate cancers.

John Robertson (Nottingham CityHospital; http://www.ncht.org.uk)presented the discovery of auto-antibodies as tumor markers. RichardBabaian and Barton Grossman (TexasMD Anderson Cancer Center) discussedprostate cancer markers by applyingnew assays to measure truncated andcleaved isoforms of free PSA, and gavean update on bladder cancer markerstudies, respectively.

Ingegerd Hellstrom from the PacificNorthwest Research Institute(http://www.pnri.org) further reportedtwo new assays for diagnosis ofovarian carcinoma. One of the assaysapplied a combination of ELISA formeasuring both CA125 and solubleMPF-related molecules [8]. The otherwas HE4 protein-based ELISA, whichwas shown to have an advantage over the CA125 assay. DanielO’Shannessy (Fujirebio Diagnostics;

update conference DDT Vol. 8, No. 10 May 2003

442 www.drugdiscoverytoday.com

http://www.fujirebiodiagnostics.com)reported on diagnostic potential ofsurviving – an inhibitor of apoptosis,and Raymond Houghton from CorixaCorporation (http://www.corixa.com)discussed real-time RT-PCR assays forthe detection of disseminated tumorcells in breast and prostate cancer.

Circulating cellsBlood-borne distant metastasis is theleading cause of cancer-related deaths.New initiatives and approaches havebeen developed to look into the onsetof this fundamental process in cancerpatients, using ultrasensitiveimmunocytochemical and molecularassays that are able to detect evensingle metastatic cells. Stephan Braun(Leopold-Franzens-Universitaet,Innsbruk; http://www.uibk.ac.at)discussed the clinical impact of occultmetastatic cells in breast cancer. A noveldevice, Rare Cell Detection (RCD)system, which enables the counting oftumor cells in the blood using aninexpensive disposable that prepares arelatively large sample of whole bloodfor automated microscopic examination

in a single step, was presented byHerbert Bresler (Battelle HealthcareInstitute; http://www.battelle.com).

Paul Ts’o (Cell Works;http://www.cell-works.com) alsopresented the BloodBiopsy™ as auniversal test system for thequantitative measurement andcharacterization of circulating cancercells for various types of cancers. TheBloodBiopsy™ is a procedure forisolation and immunochemical-stainingto detect rare cells in blood. Tim Allen-Mersh from Imperial CollegeLondon (http://www.ic.ac.uk) reportedon the detection of circulating tumorcells at 24 hours after primary resectionto predict colorectal cancer recurrence.

ConclusionsThe message from the conference isclear: the field of tumor markers fromdiscovery to practice is quicklyadvancing, thanks to recentdevelopments in new technologies and approaches. It is hoped that byapplying and further developing themultiplex and high-throughputmethodologies of genomics and

proteomics, more tumor markers will bediscovered, validated and put to use inmeeting the urgent clinical needs ofcancer patients.

References1 van’t Veer, L.J. et al. (2002) Gene expression

profiling predicts clinical outcome of breastcancer. Nature 415, 530–536

2 Issaq, H.J. et al. (2002) The SELDI-TOF MSapproach to proteomics: protein profilingand biomarker identification. Biochem.Biophys. Res. Comm. 292, 587–592

3 Kononen J. et al. (1998) Tissue microarraysfor high- throughput molecular profiling oftumor specimens. Nature Med. 4, 844–847

4 Srinivas, P.R. et al. (2001) Proteomics inearly detection of cancer. Clin. Chem. 47,1901–1911

5 Shalhoub, P. et al. (2001) Proteomic-basedapproach for the identification of tumormarkers associated with hepatocellularcarcinoma. Dis. Markers 17, 217–223

6 Jones, M.B. et al. (2002) Proteomic analysisand identification of new biomarkers andtherapeutic targets for invasive ovariancancer. Proteomics 2, 76–84

7 Yousef, G.M. and Diamandis, E.P. (2001) The new human tissue kallikrein gene family:structure, function and association to disease.Endocr. Rev. 22:148–204

8 Scholler, N. et al. (1999) Soluble member(s) ofthe mesothelin/megakaryocyte potentiatingfactor family are detectable in sera frompatients with ovarian carcinoma. Proc. Natl.Acad. Sci. U. S. A. 96, 11531–11536

DDT Vol. 8, No. 10 May 2003 updatebook

1359-6446/03/$ – see front matter ©2003 Elsevier Science Ltd. All rights reserved. PII: S1359-6446(03)02694-1 443www.drugdiscoverytoday.com

Textbook ofDrug Designand Discovery

Edited by Povl Krogsgaard-Larsen, TommyLiljefors and Ulf Madsen, Taylor and Francis2002, 572 pages in paperback, £32.00, ISBN 0-4152-8288-8

The editors of this textbook include this line in the preface to the book.‘In order to attract the attention of

intelligent students, the creative andfascinating nature of drug design mustbe the underlying theme of basic andadvanced student courses in medicinalchemistry.’

This textbook is comprised of17 chapters and 572 pages. The text is reasonably easy to read and thechapter, section and subsectionheadings are clear. This is the thirdedition of this textbook.

The Textbook of Drug Design andDiscovery gives an excellentintroduction into the required basics inmany important areas in this field. Withfew exceptions, each chapter provides

a good introductory section and morein-depth discussions of each topic.Many studies from industry arepresented to exemplify or supportvarious important points and topics.The collection of these chaptershighlights the complexities anddifficulties inherent in this field.

Experts in the field author eachchapter, highlighting to readers thatdifferent perspectives and expertise willbe encountered and that they shouldoften be embraced, rather thanignored. With this type of organization,the editors must bring together thewriting styles, ensure a consistent