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20032 Blackwell Publishing, Inc., 1075-122X/02/$15.00/0The Breast Journal, Volume 9, Suppl. 1, 2003 000–000

Address correspondence and reprint requests to: Joe W. Gray, PhD, UCSFCancer Center, 2340 Sutter St., N415, San Francisco, CA 94115, or email:jgray@cc.ucsf.edu.

Blackwell Science, LtdOxford, UKTBJThe Breast Journal1075-122X2003 Blackwell PublishingJanuary/February 20039suppl

Original Article

Translating Laboratory Knowledge

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Translating Laboratory Knowledge into Biological Therapy and

Genetic Analysis

Joe Gray, PhD

Professor of Laboratory Medicine and Radiation Oncology, UCSF Cancer Center, San Francisco, California

j

Abstract:

Genomic instability is one of the earliest fea-tures of cancer cell behavior and can lead to gene mutation,amplification, or deletion. Rarely one of these genomic eventscan give the cell a growth advantage or some other charac

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teristic that contributes to carcinogenesis and also leads toclonal expansion. Solid tumors contain numerous geneticabnormalities and these vary among individuals. New tech-niques from the laboratory allow unprecedented levels ofdetail in cancer genetic analysis of human tumors. One tech-nique called comparative genomic hybridization (CGH) canpinpoint areas of the genome that are amplified or deleted.These changes that occur at a high frequency are likely torepresent genes that are important in cancer developmentand progression. How can this be translated into new biologictherapy as well as a better understanding of factors thatpredict responses to standard chemotherapy to allow betterindividualized tailoring of treatment? Through the linkage ofCGH data on human tumors to their clinical outcomes, specificquestions can be asked about the relationship of specificgenes to clinical variables. For example, genes that aregained in patients who are resistant to anti-HER-2 antibody(Herceptin) might help select patients for such therapy oridentify genes that could be pharmacologically targeted toovercome Herceptin resistance. Prospects for better treatmentand recent advances in genomic research may lead to anincreased understanding of the basic mechanisms resulting ininitiation and progression of breast cancer.

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THE ROLE OF GENOMICS

Genomic instability plays a major role in the evolutionof cancers, which in the case of breast cancer seems tooccur during transition from hyperplasia to carcinoma insitu. Numerous genomic abnormalities have been foundthat differ dramatically between clinically similar tumors.However, the process of mapping out more than 30 regionsof recurrent abnormality, many of which are potentialtherapeutic targets or treatment-specific predictivemarkers, continues. Understanding the implications ofspecific genomic abnormalities in relation to prognosisand response to specific therapies remains an importantresearch priority. Specifically,• Genome instability arises during transition from hyper-

plasia to carcinoma in situ.• Genomic aberrations differ dramatically between clini-

cally similar tumors, but more than 30 regions of recur-rent abnormality have been defined. These occur inmany tumor types.

• Many of the recurrent aberrations influence growthfactor receptor tyrosine kinase (RTK) signaling,although response to targeted RTK inhibitors (suchas Herceptin) is not uniform, even in patients whosetumors exhibit amplification and overexpression ofthe RTK HER-2/

neu

(HER-2).Dr. Gray, along with his colleague Thea, Tisty, PhD,

also at the University of California, San Francisco (UCSF),suspect that the key to genomic instability and alterationsis controlled by the enzyme telomerase, which is inactiveduring hyperplasia and active during carcinoma in situ.

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2003 Blackwell Publishing, Inc., 1075-122X/03/$15.00/0The Breast Journal, Volume 9, Suppl. 1, 2003 S29–S31

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Telomerase restores telomeres, DNA base pair sequencesthat cap and protect the ends of individual chromosomesfrom degradation from repeated chromosome shorteningthat occurs with each cell division.

The DNA replication machinery can’t quite replicateall the way out to the end of each chromosome. In theabsence of this enzyme you lose a little telomere every timethe cell divides. The cell eventually runs out of telomereand enters a “telomeric crisis” in which the DNA repairmachinery sees these ends of chromosomes as broken andtries to join them together. This leads to chromosomefusion, chromosome breakage, and rearrangement asthese fused chromosomes try to divide during mitosis.Without telomerase, cells with these damaged chromo-somes die. Occasionally telomerase turns on again in sucha cell and starts “healing” the ends of chromosomes. Thisallows the abnormal cell to stabilize and further evolveinto a malignancy. There is a substantial increase ingenomic complexity from hyperplasia to ductal carci-noma in situ (DCIS), but not much from DCIS to aninvasive cancer.

The general concept is that genomic abnormalities suchas chromosome rearrangements, deletions, and amplifica-tions are a strong driving force that enable breast cancerdevelopment and progression. For example, some onco-genes, such as HER, are activated by amplification and thesubsequent overexpression of the protein that results froma high gene copy number. Others, like

ras

, can acquiremutations that result in an abnormally active function,a common abnormality in lung, colon, and pancreaticcancer. Also, genetic loss of tumor suppressor genes, whichencode proteins that negatively regulate cell growth andother key functions, can also contribute to malignantprogression. It is likely that specific genetic changes canpredict clinical behavior and response to both standardand newer targeted therapies. By identifying these abnor-malities and understanding the mechanisms by which theyoccur, the development of better predictive markers andtherapeutic targeting strategies will be facilitated.

Comparative genomic hybridization (CGH) is aninnovative laboratory tool that has been developed by Dr.Gray’s group. With this technique, tumor DNA is labeledwith a green fluorescent dye and normal breast (or othercontrol) cells are labeled with a red fluorescent dye. Thetwo are combined and allowed to hybridize to a normalset of chromosomes (taken from a normal cell in meta-phase, when the chromosomes condense and are identifi-able). Since extra labeled DNA copies will bind in anoverrepresented manner, the result is a chromosome thatis colored when analyzed with a fluorescence microscope.

Areas of the chromosome that are amplified in the tumorDNA are seen as green, whereas areas that are deleted areseen as red.

Says Dr. Gray, “We’ve been able to eliminate the chro-mosomes that we were hybridizing to and replace themwith these clones of DNA. This is interesting because eachone of these clones now can be specifically mapped onto thenormal, now nearly completed genomic sequence. And soif we see an abnormality that is involving these three clones,we can immediately go to the DNA sequence for thatregion and ask what are the genes that are located there.”

The newer technique is called “array CGH,” since smallDNA clones spanning shorter distances are arrayed on aglass slide instead of the whole chromosomes (1). A laserscanner and camera can quickly acquire the data, whichis then analyzed by computer and subjected to complexstatistical analyses. This provides a much higher resolu-tion and a better estimate of gene copy number, or genedeletion. An example of gene copy number plotted againstthe length of the chromosome is shown in Figure 1. Thetwo samples shown illustrate the genetic diversity seenamong patients. Areas that are commonly amplified anddeleted would be candidates for oncogenes or tumorsuppressor genes. Now that the human genome has beensequenced and expressed genes are highly annotated andshared on databases, this tool can be applied rapidly forthe discovery of genes associated with cancer. As we beginto assemble pathway maps as to how these genes and theirencoded proteins interact, we can begin to rationallyunderstand this vast amount of data and apply it in theclinic. By linking CGH information of patients’ tumorsto their clinical outcomes, one can gain insight into whichspecific genetic changes might best correlate with thepotential for clinical benefit. Furthermore, these genesmay represent targets against which new drugs can bedeveloped.

Genomic instability plays a major role in the evolutionof cancers. Most genetic alterations seem to occur dur-ing transition from hyperplasia to carcinoma in situ. Dr.Gray’s team has found numerous genomic abnormalitiesamong human breast cancers, and they differ dramaticallybetween clinically similar tumors. They have been able tomap out more than 30 regions of recurrent abnormalityand many of these are therapeutic targets or perhapspredictive markers. Many of these genes are involved inreceptor tyrosine kinase signaling pathways, and thusmay be useful in developing better tools to predict whowill and will not respond to these RTK inhibitors. A largecell line consortium has been formed to try to understandhow these abnormalities actually function, and many of

Translating Laboratory Knowledge •

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these recurrent abnormalities may be therapeutic andpreventive targets. Once candidate genes are identified,they will be tested on human tumors where therapy withHerceptin has been used and linked with clinical outcometo verify the relationship.

MOLECULAR TAILORING OF THERAPY

There are potential implications in predicting whetherpatients will respond to some of the new therapeuticagents that are being targeted against this particularpathway. One of these drugs is Herceptin, which binds tothe HER-2 proteins (receptors) on the tumor cell surface.While the mechanisms of Herceptin are not completelyunderstood, it appears that Herceptin modifies the func-tion of the HER-2 receptor (normally HER-2 binds therelated proteins HER-3 and HER-4 to activate a variety ofcellular pathways).

Herceptin targets tumors that overexpress HER-2:Why is it that only 20–30% of patients whose tumors areHER-2 positive actually respond to Herceptin as single-agent therapy? The hypothesis based on these data is thatthe abnormalities that are involving either parallel ordownstream genes actually modulate response.

The pathways mediated by HER and modulated byHerceptin are complex and involve numerous other pro-teins. Hence CGH abnormalities (or other markers at theRNA or protein levels) may distinguish further those

likely to respond, and these markers probably indicate thefunction of other proteins in the signaling pathway. It ispossible that a combination of markers will be the bestpredictive test that will allow choosing the optimal ther-apy of combination therapies for a patient and alsospare them the side effects from treatments that will notbe beneficial.

Says Dr. Gray, “It will be important in the phase I andearly phase II trials for the community to be collectingclinical material extensively on patients that have beentreated with these novel therapeutics. While they’re stillbeing administered as single agents, it’s good for us to havethese mechanisms in place to get objective measures ofresponse. I think that this is going to require some collab-oration with the pharmaceutical industry that is runningsome of these trials. But it is just absolutely critical to getthat clinical material archived and made available forthese kinds of correlative studies.”

Joe Gray, PhD, “Genomic Events in Breast Cancer:Prospects for Better Treatment.” Presented at the 26thAnnual Symposium of the American Society of BreastDisease, San Francisco, California, April 19–20, 2002.

REFERENCE

1. Pinkel D, Seagraves R, Sudar D,

et al.

High resolution ana-lysis of DNA copy number variation using comparative genomichybridization to microarrays.

Nat Genet

1998;20:207–11.

Figure 1. Genome diversity in human breastcancer.