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Emerging issues of the Emerging issues of the expression profiling expression profiling

technologies for the study of technologies for the study of gynecologic cancergynecologic cancer

American Journal of Obstetrics and Gynecology American Journal of Obstetrics and Gynecology (2005) 193, 908-18(2005) 193, 908-18

R4 R4 박영미박영미

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The technology of complementary DNA microarrays The technology of complementary DNA microarrays and its impact in cancer biologyand its impact in cancer biology

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The novel experimental approachesThe novel experimental approaches

: : the technology of complementary DNA orthe technology of complementary DNA or oligonucleotide microarraysoligonucleotide microarrays

The analysis of the levels of expression of thousands The analysis of the levels of expression of thousands of cellular genes of cellular genes

The establishment of distinct patterns in different kindThe establishment of distinct patterns in different kinds of tumorss of tumors

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GenomeGenome 생물이 살아가기 위해서 필요한 최소한의 유전자군을 가지고 생물이 살아가기 위해서 필요한 최소한의 유전자군을 가지고

있는 염색체의 있는 염색체의 1 1 세트세트 유전자유전자 (gene)(gene) 와 염색체와 염색체 (chromosome)(chromosome) 의 두 단어를 합성한 말 의 두 단어를 합성한 말 단상성단상성 (n)(n) 의 염색체의 염색체 , , 또는 거기에 포함되는 유전 정보 전체또는 거기에 포함되는 유전 정보 전체

complementary DNAcomplementary DNA mRNAmRNA 를 를 Reverse transcripaseReverse transcripase 란 효소를 사용하여 만든 란 효소를 사용하여 만든 mRNAmRNA

의 상보적인 의 상보적인 DNADNA 게놈에서 전사된 게놈에서 전사된 11 차 산물인 차 산물인 RNARNA 는 단일나선으로 불안정하며 는 단일나선으로 불안정하며

또한 수명이 짧기 때문에 연구자가 시험관내에서 취급하기가 또한 수명이 짧기 때문에 연구자가 시험관내에서 취급하기가 쉽지 않아쉽지 않아 , , 이러한 이러한 RNARNA 를 인위적으로 이중나선인 를 인위적으로 이중나선인 DNADNA 로 로 전환시켜 연구에 사용전환시켜 연구에 사용

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The differential gene expression assay between 2 tissue The differential gene expression assay between 2 tissue samples using the DNA microarray technologysamples using the DNA microarray technology

: normal or reference RNA vs cancer or test RNA : normal or reference RNA vs cancer or test RNA

Total cellular mRNA is reversed transcribed to cDNA in thTotal cellular mRNA is reversed transcribed to cDNA in the presence of 2 differentially fluor-labelled nucleotides (Ue presence of 2 differentially fluor-labelled nucleotides (UTP)TP)

: cDNA molecules with either a green or a red : cDNA molecules with either a green or a red fluoresence tagfluoresence tag

The principle of DNA microarraysThe principle of DNA microarrays

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These tagged cDNAs are then combined and hybridized to These tagged cDNAs are then combined and hybridized to the microarray plate the microarray plate

: containing tens of thousands of immobilized : containing tens of thousands of immobilized short DNA fragments of specified sequencesshort DNA fragments of specified sequences

After hybridization and washing to remove nonspecific binAfter hybridization and washing to remove nonspecific binding, the DNA microarray plate is subjected to excitation ding, the DNA microarray plate is subjected to excitation with lasers of different wavelengths with lasers of different wavelengths

: leading to distinct emissions from the Cy3 and Cy5 : leading to distinct emissions from the Cy3 and Cy5 labelled probeslabelled probes

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After detection by microscope scanning, the separate imaAfter detection by microscope scanning, the separate image files are exported to analysis softwarege files are exported to analysis software

: they are converted to pseudo-colored images and : they are converted to pseudo-colored images and a visual interpretation of expression changes is a visual interpretation of expression changes is provided by merging the 2 imagesprovided by merging the 2 images

The data are subjected to analysis with specific bioinformThe data are subjected to analysis with specific bioinformatic toolsatic tools

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These gene expression dataThese gene expression data

Referred to as signaturesReferred to as signatures: because the expression patterns are distinct and u: because the expression patterns are distinct and u

nique for each type of tumornique for each type of tumor

Can be used to histologically classify similar tumors inCan be used to histologically classify similar tumors into specific subtypesto specific subtypes

Providing clinically novel and relevant informationProviding clinically novel and relevant information

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Reflecting the origin and function of different cell typesReflecting the origin and function of different cell types cf. Histochemical approaches cf. Histochemical approaches : only discriminate between malignant and : only discriminate between malignant and nonmalignant cells on the basis of nonmalignant cells on the basis of morphologic appearancemorphologic appearance

Anticipated to provide in the immediate future a more Anticipated to provide in the immediate future a more accurate prognosis and prediction of response to indivaccurate prognosis and prediction of response to individual therapyidual therapy

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The molecular basis of cervical The molecular basis of cervical and endometrial carcinomasand endometrial carcinomas

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The major conventional parametersThe major conventional parameters

Clinical and/or surgical stageClinical and/or surgical stage Size and grade of tumorSize and grade of tumor Histologic typeHistologic type Lymphatic spreadLymphatic spread Vascular invasionVascular invasion

The prognosis of cervical & The prognosis of cervical & endometrial cancerendometrial cancer

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Additional parametersAdditional parameters

Cytogenetic abnormalitiesCytogenetic abnormalities Acquired mutations of several proto-oncogenes, tumor Acquired mutations of several proto-oncogenes, tumor

suppressor genes, other cell cycle regulatory moleculesuppressor genes, other cell cycle regulatory moleculess

-> Considered as candidate molecular events in the -> Considered as candidate molecular events in the pathogenesis and the eventual evolution of the pathogenesis and the eventual evolution of the tumor to metastasistumor to metastasis

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Human papilloma virus (HPV)Human papilloma virus (HPV)

The role of HPV infectionThe role of HPV infection : the development of preinvasive or invasive carcinoma o: the development of preinvasive or invasive carcinoma o

f the cervixf the cervix

Recent studies that use microarraysRecent studies that use microarrays

HPV 16 E6 oncoproteinHPV 16 E6 oncoprotein : Regulate differentiation-associated genes: Regulate differentiation-associated genes

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HPV 16 E7 proteinHPV 16 E7 protein : regulate several key modulators: regulate several key modulators

Signaling factorsSignaling factors Cell cycle regulatorsCell cycle regulators ChaperonesChaperones

: Escape immune surveillance: Escape immune surveillance

E2 viral proteinE2 viral protein : Delay mitotic progression in HPV-mediated: Delay mitotic progression in HPV-mediated tumorigenesis tumorigenesis

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The recently established microarray DNA technolThe recently established microarray DNA technologyogy

The detection and typing of HPV infection The detection and typing of HPV infection

Sensitive high-throughput screening test for the detectSensitive high-throughput screening test for the detection of latent HPVion of latent HPV

Essential insights on the mechanisms of multiple infeEssential insights on the mechanisms of multiple infections and the various genotypes of HPVctions and the various genotypes of HPV

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Single gene-based approaches for Single gene-based approaches for the investigation of endometrial the investigation of endometrial

carcinomascarcinomas

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No common molecular parameters have been forNo common molecular parameters have been formulated so far in endometrial cancermulated so far in endometrial cancer

K-ras proto-oncogene mutationK-ras proto-oncogene mutation No prognostic valueNo prognostic value : Because, no correlation to: Because, no correlation to

The stageThe stage The histologic typeThe histologic type The grade of the tumorThe grade of the tumor

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Early endometrial cancerEarly endometrial cancer : Specific for detecting submicroscopic : Specific for detecting submicroscopic myometrial strips infiltrated with tumor cellsmyometrial strips infiltrated with tumor cells

COX-2 expressionCOX-2 expression Lymph node metastasisLymph node metastasis Parametrial invasionParametrial invasion

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Functional genomics of cervical Functional genomics of cervical and endometrial cancerand endometrial cancer

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Early studies : a very limited researchEarly studies : a very limited research

The identification of genes related to radiosensitiveity The identification of genes related to radiosensitiveity of cervical squamous cell carcinomasof cervical squamous cell carcinomas

The identification of genes involved in the developmenThe identification of genes involved in the development of cervical carcinomast of cervical carcinomas

The identification of lovostatin-induced apoptosis-specThe identification of lovostatin-induced apoptosis-specific genesific genes

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The characterization of genes that are The characterization of genes that are transactivated by the PTEN tumor suppressor transactivated by the PTEN tumor suppressor gene in endometrial cellsgene in endometrial cells

# Limited number of samples# Limited number of samples # Small number of genes contained in the cDNA# Small number of genes contained in the cDNA microarraysmicroarrays → → not provided any conclusive pattern of not provided any conclusive pattern of expression or gene signature for the expression or gene signature for the neoplasianeoplasia → → not clarified the precise genes and the not clarified the precise genes and the various steps of carcinogenesisvarious steps of carcinogenesis

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New studies : used cDNA microarraysNew studies : used cDNA microarrays

: Systematic analysis of the pattern of expression : Systematic analysis of the pattern of expression during the various steps of cervical tumorigensisduring the various steps of cervical tumorigensis

Specific patterns of gene expression assigned to seveSpecific patterns of gene expression assigned to several cellular processesral cellular processes

① ① establish early enough during carcinogenesis establish early enough during carcinogenesis ② ② can discriminate normal cervical tissue andcan discriminate normal cervical tissue and LSIL from HSIL and cervical cancersLSIL from HSIL and cervical cancers

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Many of these genes are also expressed in stroma adjMany of these genes are also expressed in stroma adjacent to the cancer tissueacent to the cancer tissue

The extent of gene overexpression is increased during The extent of gene overexpression is increased during the progression from LSIL to HSIL and finally to cancer the progression from LSIL to HSIL and finally to cancer

→ → The identification of reliable biomarkers The identification of reliable biomarkers associated with every stage of tumor progressionassociated with every stage of tumor progression

→ → Eventually, leading to the improvement of early Eventually, leading to the improvement of early detectiondetection

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Gene array expression profiles of Gene array expression profiles of ovarian cancerovarian cancer

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4 major histologic types of ovarian carcinoma4 major histologic types of ovarian carcinoma

Clear cellClear cell EndometrioidEndometrioid MucinousMucinous SerousSerous

→ → Directly analyzed using oligonucleotide Directly analyzed using oligonucleotide microarraysmicroarrays

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Expression profiling patternsExpression profiling patterns

Mucinous and clear cell types can be readily distinguisMucinous and clear cell types can be readily distinguished from serous type, regardless of tumor stage and thed from serous type, regardless of tumor stage and typeype

Clear cell carcinomas seem to be more similar to a suClear cell carcinomas seem to be more similar to a subset of mucinous and endometrioid carcinomas than tbset of mucinous and endometrioid carcinomas than to serous carcinomaso serous carcinomas

Endometrioid carcinmas exhibit extensive overlap with Endometrioid carcinmas exhibit extensive overlap with the other histologic typesthe other histologic types

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Clear cell ovarian carcinomas exhibit an expression prClear cell ovarian carcinomas exhibit an expression profile distinct from other poor prognosis typesofile distinct from other poor prognosis types

: the 73 genes upregulated in clear cell: the 73 genes upregulated in clear cell → → consistent with the capacity of being consistent with the capacity of being chemotherapy-resistant chemotherapy-resistant → → hence of poor prognosishence of poor prognosis

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Feasible pattern distinction Feasible pattern distinction between normal and ovarian between normal and ovarian

cancercancer

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Alternative ovarian cancer modelAlternative ovarian cancer model

The clear distinction between normal human The clear distinction between normal human ovarian surface epithelial (HOSE) cell and ovarian surface epithelial (HOSE) cell and epithelial ovarian carcinoma (EOC)epithelial ovarian carcinoma (EOC)

Based on primary cells expanded in vitro either Based on primary cells expanded in vitro either from normal ovaries or from EOCfrom normal ovaries or from EOC

The elucidation of the molecular events The elucidation of the molecular events occurring at specific cell types and stages of occurring at specific cell types and stages of ovarian carcinogenesisovarian carcinogenesis

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The set of differentially expressed genes in the epithelThe set of differentially expressed genes in the epithelial ovarian carcinoma cell from this studyial ovarian carcinoma cell from this study

: previously associated with ovarian tumorigenesis: previously associated with ovarian tumorigenesis : novel genes -> in embryonic pattern formation: novel genes -> in embryonic pattern formation

Mxil (a single tumor suppressor gene)Mxil (a single tumor suppressor gene) : tissue-specific expression and upregulation in : tissue-specific expression and upregulation in normal ovarian cellnormal ovarian cell : downregulation in the EOC cell: downregulation in the EOC cell

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Which is the “right” normal Which is the “right” normal sample?sample?

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The continuous passage and the nonreproducible The continuous passage and the nonreproducible culture conditionsculture conditions

: tumor cell lines may not reflect the actual : tumor cell lines may not reflect the actual biologic events of the primary tumorbiologic events of the primary tumor

The loss of tumor markers during cultureThe loss of tumor markers during culture

The possibility of propagating selected The possibility of propagating selected subpopulation from the original primary tissuesubpopulation from the original primary tissue

The putative modification of gene expression by The putative modification of gene expression by the in vitro expansion conditionsthe in vitro expansion conditions

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The choice of normal ovarian controlThe choice of normal ovarian control

: A relevant critical issue in correctly identifying : A relevant critical issue in correctly identifying the differentially expressed genes in tumorsthe differentially expressed genes in tumors

The selection of the type of the normal control The selection of the type of the normal control cell type can influence the set of differentially cell type can influence the set of differentially expressed genes with a tumor sampleexpressed genes with a tumor sample

The lack of a universal control tissue sample The lack of a universal control tissue sample each time does not permit meaningful each time does not permit meaningful comparisons among similar studiescomparisons among similar studies

The type of normal tissue should be clearly The type of normal tissue should be clearly specifiedspecified

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Early events of ovarian Early events of ovarian carcinogenesiscarcinogenesis

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Lack of available early malignant ovarian tissuesLack of available early malignant ovarian tissues

: A specific problem for a systematic analysis of the biol: A specific problem for a systematic analysis of the biologic phenomena of the early ovarian carcinogenesisogic phenomena of the early ovarian carcinogenesis

Most of the ovarian tumors are discovered when they Most of the ovarian tumors are discovered when they have already proceeded beyond stage Ihave already proceeded beyond stage I

The required tissue size for the extraction of 10 to 50The required tissue size for the extraction of 10 to 50ug of total RNA for gene expression analysis exceeds tug of total RNA for gene expression analysis exceeds the available amount of premalignant ovarian tissuehe available amount of premalignant ovarian tissue

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To address these difficultiesTo address these difficulties

The short-term in vitro expansion of normal The short-term in vitro expansion of normal and and

malignant ovarian epithelial cells before RNA malignant ovarian epithelial cells before RNA

harvestingharvesting

The purification of ovarian epitheliumThe purification of ovarian epithelium

The amplification of the RNAThe amplification of the RNA

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Stage-specific expression patternsStage-specific expression patterns

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Genome-wide examination of chromosomeGenome-wide examination of chromosome

: The expression patterns of the different stages of tumo: The expression patterns of the different stages of tumor progression of ovarian cancerr progression of ovarian cancer

The patterns of 21 early stage : 17 late stage The patterns of 21 early stage : 17 late stage -> compared by using cDNA microarray analysis, -> compared by using cDNA microarray analysis, comparative genomic hybridizationcomparative genomic hybridization

Early stage : stage I/II, endometrial or serous Early stage : stage I/II, endometrial or serous carcinomascarcinomas Late stage : stage III/IV, serous carcinomasLate stage : stage III/IV, serous carcinomas

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A more recent studyA more recent study

: Identification of gene expression patterns of the 2 survi: Identification of gene expression patterns of the 2 survival groupval group

Advanced stage (III or IV), serous ovarian cancer Advanced stage (III or IV), serous ovarian cancer -> short (< 2yr) : long (> 7yr) survival-> short (< 2yr) : long (> 7yr) survival

Significant number of differentially expressed genes Significant number of differentially expressed genes : IL2 receptor, chemokine ligands (CCL4, CCL5), : IL2 receptor, chemokine ligands (CCL4, CCL5), several interferon pathway activitiesseveral interferon pathway activities : immune system functions : immune system functions : upregulation -> a favorable outcome: upregulation -> a favorable outcome

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Metastasis mechanisms revisitedMetastasis mechanisms revisited

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The pattern of aberrant expression in The pattern of aberrant expression in the early stages of ovarian the early stages of ovarian carcinogenesiscarcinogenesis

: The potential for metastasis is an inherent : The potential for metastasis is an inherent feature of early stage cancersfeature of early stage cancers

Important implication on future strategies for Important implication on future strategies for rational treatment and screeningrational treatment and screening

Aggressive treatment of poorly Aggressive treatment of poorly differentiated stage I ovarian cancersdifferentiated stage I ovarian cancers

The rationale for screening strategiesThe rationale for screening strategies

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These tumors seem These tumors seem to include subpopulations of cells exhibiting to include subpopulations of cells exhibiting

tissue-specific profiles predicting the site of tissue-specific profiles predicting the site of metastasismetastasis

to argue against the current model that to argue against the current model that metastasis is derived from rare cells residing metastasis is derived from rare cells residing within the tumorwithin the tumor

These new data alter radically our current model These new data alter radically our current model of tumor progression and metastasisof tumor progression and metastasis

: Sequential accumulation of numerous : Sequential accumulation of numerous mutations mutations

occurring on a rare subpopulation of tumor occurring on a rare subpopulation of tumor cells cells

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Therapy-related predictionsTherapy-related predictions

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The prediction of response to chemotherapyThe prediction of response to chemotherapy

: Recent studies have addressed the feasibility of gener: Recent studies have addressed the feasibility of generating novel reliable molecular markers using gene exprating novel reliable molecular markers using gene expression profilingession profiling

The paclitaxel-induced apoptosis pathways The paclitaxel-induced apoptosis pathways The p53-independent mitochondrial pathway The p53-independent mitochondrial pathway The stress reaction-induced pathwayThe stress reaction-induced pathway

-> Suppression of these pathways can contribute -> Suppression of these pathways can contribute to the acquisition of resistance to paclitaxelto the acquisition of resistance to paclitaxel

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Epothilone BEpothilone B

Nontaxane agent being active in paclitaxed-resistanNontaxane agent being active in paclitaxed-resistant cellst cells

The pattern of expression of ovarian cancer cell to The pattern of expression of ovarian cancer cell to Epothilone B Epothilone B

: triggering of stress-related signal : triggering of stress-related signal transduction pathways associated to transduction pathways associated to TNF-aTNF-a

This finding provides the impetus for further studies This finding provides the impetus for further studies to delineate the mechanisms of drug resistanceto delineate the mechanisms of drug resistance

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Future perspectivesFuture perspectives

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Microarray analysis Microarray analysis -> to characterize more efficiently human -> to characterize more efficiently human tumors at the gene expression leveltumors at the gene expression level

Revealing significantly and highly altered genes between tRevealing significantly and highly altered genes between tumor and normal tissueumor and normal tissue

-> to begin deciphering the pathways mostly -> to begin deciphering the pathways mostly affected in disease processaffected in disease process

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The diversification of the microarray platform expanding bThe diversification of the microarray platform expanding beyond DNA such as eyond DNA such as

proteinsproteins carbohydratescarbohydrates peptidespeptides nanotube precursors nanotube precursors

-> to add a significant dimension to our -> to add a significant dimension to our understanding of the complexity of understanding of the complexity of cellular machinerycellular machinery

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The evaluation of the biologically and clinically relevant geThe evaluation of the biologically and clinically relevant genes nes

-> should be mediated by the diverse bioinformatic -> should be mediated by the diverse bioinformatic protocols protocols

① ① novel classifiers with a capacity for accurate and novel classifiers with a capacity for accurate and robust cancer diagnosis robust cancer diagnosis ② ② novel statistical methods such as comparative novel statistical methods such as comparative metaprofiling and high- throughput tissue metaprofiling and high- throughput tissue microarraysmicroarrays

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Bioinformatic analysisBioinformatic analysis

: Incorporating the clinical and pathologic : Incorporating the clinical and pathologic information as well as the patient historyinformation as well as the patient history

-> We could begin to see trends important in -> We could begin to see trends important in better tumor classification, patient better tumor classification, patient diagnosis, and prognosisdiagnosis, and prognosis