Editorial

Krishnarao AppasaniGeneExpression Systems, Inc.,PO Box 540170, Waltham, MA 02454-0170, USATel.: +1 781 891 8181Fax: +1 781 891 8234DrAppasani@expressgenes.com

© Future Drugs Ltd. All rights reserved. ISSN 1473-7159 1

From bioarrays to diagnostics: a systemomics approachBillion dollar technique draws a crowd to New York: optimism abounds for developing diagnosticsExpert Rev. Mol. Diagn. 4(1), 1–5 (2004)

Several meetings have been held in the micro-arrays field, however, none of them detailedthe potential applications for developingmolecular diagnostics. With that focus inmind, GeneExpression Systems (MA, USA)identified a market niche and organized itsfirst international conference on the theme ofBioArrays 2003 – New York Meeting on Biochipsin Diagnostics, Industrial Genomics & Proteomics:Systemomics™ Approaches, held in New YorkCity (NY, USA) on October 1–2, 2003. Theone-track, two-day event was very wellattended with many scien-tists from several coun-tries, including Taiwan,Russia, Greece and theUK. The meeting wasseparated into nine scien-tific sessions and onetechnology workshop,and ended with a panel discussion. There were28 speakers invited from elite schools, such asHarvard, Yale, Rockefeller, Cornell, Columbia,New York University, Memorial Sloan-KetteringCancer Center, Mount Sinai School of Medi-cine, University of Michigan and the NationalCancer Institute. Executives from bioarraydeveloping companies including Affymetrix,Amersham, Protometrix, MetriGenix, LynxTherapeutics, Psychiatric Genomics, VentanaMedical Systems and Schleicher & SchuellBioscience presented details of recent productlines. The meeting was well balanced bybringing speakers from large pharmaceuticalcompanies, such as Pfizer, Abbott, Johnson &Johnson, AstraZeneca and Millennium, whoexploit the bioarray technology platformsfor the development of gene-based drugsand molecular diagnostics. There were

approximately 15 poster abstracts and tenvendors presented their products. The associ-ated exhibits demonstrated Go-To-Market andBench-To-Marketplace strategies, providing sci-entists with cutting-edge tools, reagents andinstruments for the biochips field.

In his welcoming address to a packed room,the chairman of the meeting pointed out thatalthough disease prevention is an importantaspect of a patient’s health, accurate diagnosisis the primary issue to be addressed. The chal-lenge for scientists in both academia and

industry is to identifythe whole complementof disease-associatedgenes and elucidate theirfunctions in gene expres-sion and regulation, andsubsequently developnew diagnostics. People

anticipate reduction of the entire drug devel-opment pipeline as bioarrays significantlyspeed up drug target validation. However, aswith any new technology in the life sciencesfield, it is not always apparent whether thispromise will be borne out by results. Is it hypeor reality?

Most are aware of the terms genome, pro-teome, transcriptome, glycome and RNome,however a new term, Systemomics™, theholistic study of expression profiling (gene,protein, lipid and drug), function, physiologicalcircuits and developmental networks in human(animal) body systems, has been introduced.As we advance in the post genomic era, with ahope to discover novel drug targets and thera-pies, validation is a pivotal step preceding clini-cal practice. Target identification and validationcan be achieved with the use of bioarrays in

‘...although disease prevention is an important

aspect of a patient’s health, accurate diagnosis is the

primary issue to be addressed.’

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Appasani

2 Expert Rev. Mol. Diagn. 4(1), (2004)

combination with high-throughput methods [1]. DNA arrays,protein arrays and glycoarrays, collectively referred to as bio-arrays, have a tremendous commercial promise to provide newdiagnostics and drugs.

There are two driving forces behind the upheaval of microarraysin the drug discovery field:

• Time: It takes 8–10 years and about a billion dollars todevelop a new drug and, most importantly, a very high failurerate occurs during the drug development life cycle [2]

• Costs: During the past 5 years, drug companies haveinvested a vast amount of money into developing newdrugs, however, this has not been reflected by the numberof products on the market [3]. Identification of druggabletargets is easier with the help of target discovery and validationapproaches, which derive from genomics and proteomics.This is the main driving force for the hype and urgency ofusing bioarrays in drug industry

Krishnarao Appasani (GeneExpression Systems) presentedfindings from research performed in collaboration with a groupfrom Harvard Medical School (MA,USA) on the expression profiling ofbreast cancer. Twelve marker genes wereidentified that were highly expressed inblood samples from breast cancerpatients. Using these breast cancer signa-tures, the disease was detected in one of amillion normal cells [4]. In addition, Appasani gave an overviewof the surge in publications related to bioarrays. Until today,there have been approximately 200 and 6000 papers publishedon protein and DNA arrays, respectively. The ultimate goal ofbioarrays or biochips is to get diagnostic kits into the market-place, which has been achieved recently by the introduction ofthe P450 Amplichip™ (Roche, Basel, Switzerland), whichmonitors a patient’s response to common drugs. Although thereare several players in the market, some companies have alreadyintroduced chips for research applications but not for clinicaldiagnostics, and these have to seek US Food and DrugAdministration (FDA) approval.

Faster, smaller and more is powerful. This mantra is appli-cable not only to microprocessors and the high-tech industry,but also to the diagnostic and biotechnology industries. Sci-entists in biology have taken advantage of the semiconductorindustry and the genetic information that deciphered throughthe human genome project, and spotted all possible humangenes on a glass surface. These are popularly known as DNAarrays or DNA chips. The same procedure can be used forproteins, using protein arrays or protein chips. In the biotechindustry, these are jointly referred to as bioarrays or biochips.Since the discovery of these chips a few years ago, this fieldhas opened up several avenues to scientists to dissect andunderstand the molecular pathogenesis of the biological cell.Specifically, revolutions in this field will change the futurediagnosis and treatment of disease over the next decade. Bioarraytechnology has potential applications in biology, medicine

and agriculture, and has drawn the attention of investors inWall Street and subsequently become labeled as the billiondollar technique.

Democratic Senator Hillary Rodham Clinton sent hergreeting letter to GeneExpression Systems and hailed theeffort made to bring together internationally reputed scientistsfrom academia, biotech and pharmaceutical industries toexplore the applications of biochips in the development ofdiagnostics to allow for the treatment of human diseases. Shealso pointed out that these efforts will enhance cutting-edgeand emerging technologies that will allow early diagnosis ofleukemia, prostate, breast, brain, liver, colon and ovarian cancers,as well as psychiatric diseases.

Bioarray platform technologiesMonica Mody (Affymetrix, CA, USA) presented GeneChip®

platform technology and its applications in clinical research.Her talk detailed the experimental stages of molecular profilingfrom discovery to preclinical trials and disease management.Disease signatures were identified from acute lymphoblastic

leukemia (ALL) patients who were treatedwith single agents (such as mercaptopurineand methotrexate) and combination ther-apy. Only 13% of the gene changes insingle-agent versus combination therapywere observed. In addition, resistance toimatinib mesylate (Gleevec®, Novartis,

Basel, Switzerland) was shown by gene expression profiles ofbone marrow samples of ALL patients. In a Phase II study, 17patients were analyzed pre treatment and six matched samplesfrom patients analyzed before and after treatment. The idea ofdeveloping a leukemia custom chip that consists of four panelsof gene sets – one panel (of 32 genes) identifies the disease stageclassification, another (of 25 gene sets) provides the status ofdisease prognosis, the third (of 96 genes) is for choosing atherapeutic approach, and the fourth (of 140 genes) includesappropriate array controls – was discussed, with the conclusionthat this type of custom chip is ideal for many diseases and willsoon emerge in the marketplace.

John Tonkinson (Schleicher & Schuell Bioscience, NH,USA) discussed practical proteomics and how to spot proteinsmore efficiently on a nitrocellulose surface for the developmentof microspot assays on glass slides (FASTQuant™). Correla-tion was demonstrated for several human cytokines for micro-spot versus traditional ELISA. Schleicher & Schuell Biosciencehas also developed a custom slide called ProVision™ to screencancer cell activity before and after drug treatment. JasonGonclaves (Iobion Informatics, LLC, Toronto, Canada) gave atutorial on PathwayAssist™, a bioinformatic tool to analyzemetabolic pathways from gene expression profile data. Hispeeling-the-onion approach gave a quick explanation to theattendees of how simple to use the PathwayAssist™ software isfor determining the direct interacting and binding molecules inbiological pathways. However, it did not address the issue ofhow to differentiate genes from different organisms.

‘People anticipate reduction of the entire drug

development pipeline as bioarrays significantly speed up drug target validation.’

From bioarrays to diagnostics

www.future-drugs.com 3

Diagnostics & drug developmentColorectal cancer is a deadly cancer that affects hundreds ofthousands of people across the globe. Mutations in transcriptionfactor p53 have been implicated in colorectal cancer. However,commercially available p53 antibodies fail to detect 20–25% offrame shift mutations. Francis Barany (Weill College of Medi-cine of Cornell University, NY, USA), the keynote speaker,cautioned that if the patient’s samples are involved, we need tohave more sensitive and 100% accurate methods. His group hasdevised a p53 chip (in collaboration with Arnold J Levine, Uni-versity of Medicine and Dentistry of New Jersey, USA) whichconsists of 125 genes that could detect only known mutationsbut miss unknown mutations. Therefore, Barany’s colleaguescombined universal DNA array analysis with multiplex PCR/ligase chain detection reaction using endonuclease V/ligase anddeveloped a mismatch scanning assay that could accuratelydiagnose colorectal cancer. His seminal presentation stressed tocombine two or three techniques and to investigate the changesin expression of 20–50 genes, or possibly100, for accurate clinical diagnosis.

Jeffrey Ross, a pathologist from Mil-lennium Pharmaceuticals (MA, USA),described a personalized medicineapproach and presented a case study onhow the pharmacogenomics strategy wasused to develop the protease inhibitordrug bortezomib (Velcade™) for thetreatment of late-stage multiple myeloma.This drug, in combination with cytotoxic agents, was alsotested for the treatment of ovarian and breast cancers. In fact,it is the first drug to be developed from the integration of thepharmacogenomics approach into the development of ananticancer agent.

Biomarkers 1: surgiomics & clinical genomicsDo we need a massive number of genes to diagnose a disease?According to Steven Gullans (Harvard Medical School), theanswer is that expression ratios of a few genes (4 or 5) are suf-ficient to differentiate mesothelioma from adenocarcinoma ofthe lung. He felt that the most important element is cross-platform validation prior to development of any set of diag-nostic markers. In contrast, Eric Eastman (MetriGenix, MD,USA) felt that five is too little to rely on for diagnosis. There-fore, his team came up with a focused lung cancer array panelof 20 selected genes for the diagnosis of lung cancer. Embry-onic stem-cell-enriched genes were identified using bothgenomic and proteomic approaches by a team led by TowiaLibermann (Beth Israel Deaconess Hospital, MA, USA).Using the Bayesian Analysis of Differential Gene Expression(BADGE) method, his team discovered molecular markers forlong/short disease-free survival and response to chemotherapyin ovarian cancer and renal carcinoma. In addition, massspectrometry and other proteomic technologies were adoptedto discover serum biomarkers in the muscular dystrophysyndrome patients.

Emerging technology & toxicogenomicsThomas Vasicek (Lynx Therapeutics, CA, USA) claims that themassively parallel signature sequencing method is an alternativeor at least a complimentary approach to the study of expressionprofiling. Using the Complete Accurate Permanent (CAP)tagging method, his group sequenced more than 64 millionclones. From another perspective, Jingyue Ju and colleaguesfrom the Columbia Genome Center (Columbia UniversityCollege of Physicians & Surgeons, NY, USA) developed massiveparallel fluorescence imaging chip technology, a novel molecularengineering approach for developing a new generation of DNAsequencing and digital gene expression. This novel method usesfour fluorophores or mass tags to label all four nucleotides via a2-nitrobenzyl linker. In addition, DNA arrays were successfullyused in predictive toxicology by Jeffrey Waring and his col-leagues from Abbott Laboratories (IL, USA) to identify acompendium of hepatotoxin liver signatures after testing theeffects of 1000 drugs.

Industrial genomicsThe industrial genomics session waschaired by Jose Walewski (Mount SinaiSchool of Medicine, NY, USA) andwelcomed John Burczak, an industrialspeaker form Amersham Biosciences(NJ, USA), to present the details of theCodeLink™ 3D Bioarray platform.CodeLink™ Bioarrays have 30mer

oligonucleotides on a 3D surface, which mimics solution-phase hybridization and provides high sensitivity comparedwith the Affymetrix GeneChip®. Although at present theseCodeLink™ Bioarrays have 20,000 elements, by next yearthey hope to come with an entire human genome array thatconsists of 40,000 genes. The application of CodeLink™ 3Dplatform technology was exploited by Jose Waleski’s groupin the study of human liver cancer and hepatitis infection.Heat map analysis was generated and identified 200 livergenes that distinguish normal liver from tumor tissue.Another version of 3D biochips called Immobilized Micro-Array of Gel Elements (IMAGE), which uses amino-modifiedprobes, was introduced by Victor Barsky from the RussianAcademy of Sciences (Moscow, Russia). These biochips wereadopted in the identification of Mycobacterium tuberculosisstrains and the detection of infectious agents causing anthraxand smallpox.

In his greetings letter to GeneExpression Systems, New YorkState Governor George Pataki wrote that:

“The Empire State is proud to host events that bringtogether people who represent the best in ingenuity and whoare at the forefront of developments and advancements thatimprove our lives. With vision and perseverance, the inspiringscientists continue to bring positive change to the worldwith revolutionary breakthroughs in the early diagnosis ofvarious diseases and man’s capacity to treat them.”

‘Bioarray technology has potential applications in biology, medicine and

agriculture, and has drawn the attention of investors in

Wall Street and subsequently become labeled as the billion

dollar technique.’

Appasani

4 Expert Rev. Mol. Diagn. 4(1), (2004)

High-throughput genomic methodologiesIf massive expression profiling yields a set of ten or 20 genes asdiagnostic markers for disease, their routine use in high-throughput technology has to be established for pathologicalpurposes. Such technologies were developed by several com-panies. Most importantly, Chris Boles (Matrix Technologies,NH, USA) and Ralph Martel (High Throughput Genomics,AZ, USA) described sensitive, quantitatively reproducible andhigh-throughput array platforms that could be used for routineexpression analysis of a set of genes in a 96-well format. In adownstream approach of characterizing the so-called markergenes identified from expression analysis, their tissue distribu-tion is pivotal prior to developing them as accurate and sensi-tive biomarkers. This type of functional validation can beachieved by immunohistochemistry and in situ hybridizationmethods. If one could work with several markers, high-throughput and automated procedures are needed, and theseare provided by the technology developed by Ventana MedicalSystems (AZ, USA).

Biomarkers 2: clinical proteomicsClinical proteomics is an emerging fieldthat describes the application of pro-teomic techniques for clinical use.Development of protein biomarkers iscomplicated because of the post-translational modifications ofproteins in the cell. The second-day keynote speaker, SamHanash (University of Michigan, Ann Arbor and Human Pro-teome Organization [HUPO]), provided a wish list stating thatyou need to obtain samples in a noninvasive manner (i.e.,blood, serum or saliva), with low cost and with 100% accuracy.Using techniques such as direct mass spectrometry, antibody-based protein arrays and separation-based quantitative pro-teomics, Hanash and colleagues dissected the proteome ofhuman lung and colon cancer patients and developed a panel ofprotein biomarkers. The use of either colon or lung chips willhelp in the identification of tumor antigens and kinase sub-strates, and in the study of drug binding and protein interac-tions. Under his leadership, with the help of a consortiumbetween industry and government, the HUPO has set up variousother organizations, such as HUPO Antibody Initiative, Euro-HUPO and Asia/Oceanic HUPO. In a complimentaryapproach, Thomas Conrads and his team from the NationalCancer Institute (MD, USA) provided a proteome snap-shotapproach for developing pathological signatures for the diagnosisof ovarian cancer from serum samples. So far, his team hasidentified a total of 1361 proteins and 2387 unique peptides aspart of the Human Serum Cellular Proteome Initiative Project.

Transcription profiling & oncogenomicsTo understand the molecular basis of acute leukemias and toidentify potential targets for directed therapies in the clinic,oncologists Stephen Nimer and Joseph Scandura (Memorial-Sloan Kettering Institute, NY, USA) have unraveled acuteleukemias by transcriptional profiling and identified novel

leukemia-associated oncogenes. They identified a novel onco-gene, p57kiP2, and further confirmed its role as a tumor sup-pressor using small interference RNA-mediated gene silencing.David Munroe and his team in their core facility at theNational Cancer Institute routinely analyze several hundreds ofbiopsy samples per day and integrate the bioinformatics datawith patient’s clinical data to maintain and develop systematicdisease management tools and reagents. This oncogenomicsapproach was well exploited by pharmaceutical giant Pfizer(CT, USA), and several kinase drug targets were identified inprostate and lung cancers.

Neurogenomics, proteomics & bioinformaticsIn order to understand the yeast proteome, Ghil Jona andMichael Snyder from Yale University (CT, USA) developedantibody and functional protein microarrays and characterizedapproximately 6282 coding proteins (a third of which werealready characterized) of the Saccharomyces pombe (yeast)

genome, consisting of 4824 genes andcoding for more than 10,000 proteins.They are in the process of developing theProteomeChip in collaboration with Pro-tometrix (CT, USA). DNA microarrays incombination with the laser-capture micro-dissection method were extensively

adopted by Michael Mallamaci and his team at AstraZeneca(DE, USA), who discovered new G-protein-coupled receptorsinvolved in signal transduction in the CNS. A similar approachwas used by Jeffrey Brockman and his colleagues (PsychiatricGenomics, MD, USA) and led to the identification of a set ofgene signatures for psychiatric diseases, such as schizophreniaand depression. In order to elucidate novel, custom-tailoredbioinformatics tools for the gene expression analysis, Johnson& Johnson (NJ, USA) developed an interesting scientificcollaboration with Javier Cabrera (Rutgers University, NJ, USA).

Business trends & economicsAppasani talked about the fact that while the study of all omics isimportant, their controlling economics is crucial. His commentfrom the business perspective is appropriate, valid and timely.The molecular diagnostics market in the USA alone is currentlyworth around US$890 million and the number is expected toincrease to US$2.4 billion by 2009. The reason we use bioarraysis for developing molecular fingerprints or barcodes to allow earlydiagnosis of disease. The market for DNA arrays was started ataround US$250 million in 2000 and is forecast to become abillion dollar market by the year 2005. On the other hand, theprotein arrays market is reaching US$400 million. Due to amarket surge for bioarrays, the demand is expected to grow to1 million chips per application by the year 2008 [5]. However,Appasani cautioned the audience that these numbers may not bereal, just forecast! The success of this whole diagnostics industrywill depend on how many low-density chips containing relevantsets of genes for a given condition or disease will becomeimportant in the future and how many will gain FDA approval.

‘The reason we use bioarrays is for developing molecular fingerprints or barcodes to allow early diagnosis of disease.’

From bioarrays to diagnostics

www.future-drugs.com 5

Outstanding accomplishmentsOn behalf of the scientific committee, Appasani honored FrancisBarany of the Weill College of Medicine of Cornell University,the keynote speaker on the first day, with the Technology Inno-vator Award for developing the Universal Array Readout Assayfor the accurate diagnosis of colorectal cancers. In addition, thescientific committee chose Thomas Conrads of the NationalInstitutes of Health as Best Speaker Presentation for developingnovel biomarkers for the detection of ovarian cancer. IndustryBest Presentation was awarded to John Burczak of AmershamBiosciences for developing the 3D CodeLink™ technologyplatform. Best Poster Award was given to Tae Seok Seo ofColumbia University for his work on novel imaging chiptechnology. Iobion Informatics won Best Company Award.

Panel discussionThe panel discussion, which took place at the end of the meeting,provided a unique opportunity for all attendees to share viewswith experts from venture capital, technology licensing andintellectual property firms, along with business and scientificjournalists. While all speakers addressed the critical issues in thedevelopment path of the bioarray technology, there were otherissues at play, including intellectual property rights and funding.Appasani ended the conference as a moderator with a paneldiscussion on these topics by an academic technology transferofficer, a venture capitalist and two lawyers specializing in intel-lectual property practice. Joseph Lawler, an oncologist by trainingfrom the New York venture capital firm JP Morgan says:

“Many investors are not as excited about platform technologies,rather they are interested in looking for a unique technologyplatform that can speed a drug’s progress to the clinic.”

Sam Hanash, an expert in the field of proteomics, argues thatwhile the hope of venture capital funding may be dim, there areplenty of avenues to obtain government funding. DuncanGreenhalgh, an attorney with the Boston-based law firm ofTesta, Hurwitz & Thibeault, said that patents can be grantedfor new uses for old compositions of matter. Kenneth Son-nefeld, an attorney with the New York firm of Morgan &Finnegan, suggested partnerships with bioarray content inven-tors avoids patent infringements. Nivea Almaula, a technologytransfer manager from Rockefeller University (NY, USA) feltthat early collaboration with academics by industry scientistswill get a competitive advantage.

Final thoughtsThe message from the meeting is that less is really more. Thefuture of diagnostic prognostication involves a small numberof genes; a small number but one where the quality counts.Current bioarray platforms allow the analysis of the function,expression and disease involvement of several thousands ofgenes. However, in reality, looking for a few good genes isenough for routine clinical diagnosis. In order to develop 100%accuracy of disease diagnosis, the combination of two or moremethods is pivotal.

Most attendees felt that BioArrays 2003 was a unique,coherent, well-organized, target meeting for learning cutting-edge technology and for meeting authorities in the bioarraysfield. I personally believe that bioarrays will revolutionize thefuture of molecular diagnostics, providing improved reagents andallowing a better understanding of the molecular pathogenesisof human diseases.

References

1 Appasani K. RNA interference: a technology platform for target validation, drug discovery and therapeutic development. Drug Discov. World Summer Issue, 61–68 (2003).

2 Dimasi JA, Hansen RW, Grabowski HG. The price of innovation: new estimates of drug development costs. J. Health Econ. 22, 51–185 (2003).

3 Dove A. Screening for content: the evolution of high-throughput. Nature Biotechnol. 21, 859–864 (2003).

4 Ford HL, Biswas DK, Martin KJ, Pardee AB. Discovery of expressed genes by differential display and their applications. In: Perspectives in Gene Expression. Appasani K (Ed.), Eaton Publication, MA, USA, 3–20 (2003).

5 Mounier E. Biochips and microfluidics: technologies and market trends. DrugPlus Int. July/August, 18–21 (2003).

Affiliation• Krishnarao Appasani, PhD, MBA

GeneExpression Systems, Inc.,PO Box 540170, Waltham,MA 02454-0170, USATel.: +1 781 891 8181Fax: +1 781 891 8234DrAppasani@expressgenes.com