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1、In Pursuit of Personalized Therapy for Breast Cancer: Gene Expression SignaturesJane S. Chawla, M.D.November 6, 2009Objectives Case: Chemotherapy or NOT Who should receive adjuvant chemotherapy? The birth of gene-expression signatures: Intrinsic breast cancer subtypes Gene Expression Signatures unde

2、r development 70-gene Signature 21-gene Signature 2-gene Signature 50-gene signature Case revisitedShould the patient receive chemotherapy?Clinical Case30 y/o WF with newly diagnosed IDC R breast MMG / Ultrasound 1.2 x 1.2 cm mass at 10 oclock + surrounding microcalcifications + several suspicious a

3、xillary LNs s/p bilateral mastectomies at OSH Surgical Path - 4.6 cm lesion consisting of IDC mixed with DCIS; intermediate grade; + lymphovascular invasion; 11 (-) LNs; ER+/PR+/HER-2-; T2N0M0 Oncotype DX - - low risk for recurrence Based on oncotype results her oncologist recommended adjuvant tamox

4、ifen x 5 years Pt presents for a second opinionHow else can we differentiate these tumors?Who Should Receive Adjuvant Chemotherapy? 65% of women with invasive breast cancer have LN(-) disease Adjuvant chemotherapy improves DFS and OS in pre- and postmenopausal women 70 with LN+/- breast cancer NSABP

5、 B-14 & B-20 showed the benefit of tamoxifen & chemo in ER+/LN- breast cancer Likelihood of distant mets at 10 years is about 15% 85% of pts are overtreated if chemo given to all patients Those with poor prognostic features benefit most from chemotherapy Main clinical prognostic factors: age

6、, tumor size, axillary LN status, tumor histology, grade, and hormone-receptor statusSotiriou, C. et al. NEJM, 2009.Luminal ALuminal BHER2+Basal-likeIntrinsic Breast CancerSubtypes described byPerou et al.Express amountsOf luminal cyto-Keratins & geneticMarkers of luminalEpithelial cells ofNorma

7、l tissueExpress levels of EGFR, c-kit, & growth factors like hepatocyte growth factor and IGFGene Expression Profiles or “Signatures” Composed of a selection of genes felt to provide prognostic or predictive information about tumors A number of gene expression signatures have been developed to h

8、elp identify those patients at highest risk for recurrent disease This may avoid chemotherapy administration to patients at low riskThe 70-gene Assay: MammaPrintMammaprint: Development of the 70-Gene Signature DNA microarray analysis of 78 breast primary tumors (untreated) Pts were 55 years of age w

9、ith T1-2/N0 disease Pts selected based on outcome: Distant metastases within 5 years Statistical analysis, “supervised classification,” identified 231 genes correlated with disease outcome Top 70 genes selected Genes that regulate cell cycle, invasion, metastasis, & angiogenesis Patients categor

10、ized as “good prognosis” or “poor prognosis.” Found to be a better predictor of distant metastases within 5 years than all clinical variables in this study Odds ratio (distant metastases): poor to good prognosis groups = 15Van t Veer, L. Nature, 2002.Retrospective Validation of the 70-Gene Signature

11、 295 women ages 52 with T1-2, LN-/+ breast cancer 226 ER+ / 69 ER- chemo 31%; hormonal 7%; both 7% 61 pts included in the analysis were used to develop 70-gene signatureVan de Vijver. NEJM, 2002.Prognosis Signature Predictive of End Points (10 years)995% 2.6%555% 4.4% 85 % 4.3%51 % 4.5%Probability o

12、f RemainingMetastasis-freeOverall SurvivalMultivariate AnalysisIndependent predictors of risk of distant metastasis as a 1st event 70-gene signature Tumor diameter Chemotherapy Lymph node status Vascular invasionAuthors report that gene signature is predictive of distant mets in LN+ pts Multivariate

13、 analysis argues that LN status isindependent of gene signature as a predictorVan de Vijver. NEJM, 2002.TRANSBIG Independent Retrospective Validation Study Retrospective evaluation of 302 pts from several sites in Europe (age60, T1-2, LN-) previously untreated Aim: to examine whether the 70-gene sig

14、nature had prognostic value independent of the best clinical risk classifications (St Gallens, Nottingham Prognostic Index, & adjuvant online)Buyse, M. J of NCI, 2006.Does 70-gene Signature have Independent Prognostic Value?Gene signature adds independent prognostic information to that provided

15、by various risk classificationsThe signature remained a statistically significant prognostic factor for time to distant metastases & OS even after adjustment for various risk classifications (HR 2.15 & 2.15, respectively) Buyse, M. J of NCI, 2006.Forrest Plot of HR for Time to Distant Metast

16、ases Although, average HR of 2.32 is significantthere was wide variation in the HRs among clinical sites. Therefore model is very sensitive to variation in patient populationClinicalSitesBuyse, M. J of NCI, 2006.Conclusions of Validation Studies Studies were retrospective Concern for overfitting wit

17、h supervised analysis of relatively small sample sizes and large numbers of genes The first validation study contained 61 patients from study on which the classifier was built Claims exceed evidence?Prospective Validation of Mammaprint: The MINDACT TrialAccrual started 2/07 and is expected to be fin

18、ished within 3 years6,000 LN- patientsHigh risk Chemo +EndocrineRisk assessed viaClinicopathologicalFactors (adjuvant)+ MammaprintLow risk EndocrineDiscordant cases:random assignmentto follow genomicvs clinicopathologicresultCardoso, F. JCO, 2008.The 21-gene Recurrence Score: Oncotype DXOncotype Dx:

19、 The 21-Gene Assay Designed to quantify the risk of distant recurrence in patients with LN(-), ER(+) tumors receiving tamoxifen RT-PCR was used to quantify gene expression from fixed, parafin-embedded tumor tissue 250 candidate genes selected based on published literature, genomic databases, & e

20、xperiments based on DNA arrays on fresh-frozen tissue Analyzed data from 3 independent studies (447 patients) including tamoxifen-only arm of NSABP trial B-20 to test relation b/w 250 genes and recurrence of breast cancer From these studies, 21 genes were selected that correlated with proliferation

21、and endocrine responseLevels of Gene Expression Determine Recurrence Score21-gene assay = 16 outcome-related genes + 5 reference genesHigher expression levels of“favorable” genes = RSHigher expression levels of“unfavorable” genes = RSA risk score is calculated from 0 -100Cutoff points chosen based o

22、nResults of NSABP trial B-20Sparano, J & Paik, S. JCO, 2008.Prospective Validation Study of 21-Gene Assay Analyzed tumor blocks of 668 patients on NSABP trial B-14 study that were randomized to tamoxifen RS was significantly correlated with relapse free interval & OS (p0.001) Rate of distant

23、 recurrence at 10 years: Low risk 6.8% Intermediate risk 14.3% High risk 30.5% (similar risk to LN+ patients)No distant recurrence (low risk) = 93.2%No distant recurrence (high risk) = 69.5%p0.001Paik, S. JCO, 2004.Other End PointsRS predicted distantrecurrence independent of age & tumor sizeThe

24、 likelihood of distant recurrence continuously as the RS Multivariate Cox AnalysisDistant Recurrence as a Function of RSPaik, S. JCO, 2004.Does RS Have Prognostic or Predictive Value?Oncotype was evaluated in both TAM (n=290) & placebo (n=355) arms of the NSABP B-14 trial to determine its progno

25、stic value in an untreated population Patients with low (p=0.02) and intermediate (p=0.04) RS derived benefit from tamoxifen, whereas those with high RS (p=0.82) did not Although sample size limited, seems to indicate that RS predicts greater benefit to TAMPopulation-based external validation study

26、of women with ER+/LN- breast cancer in The Northern California Kaiser Permanente tumor registry, age 75, untreated & TAM RS was associated with risk of breast cancer death in tamoxifen-treated (p=0.003) and untreated patients (p=0.03) Prognostic in TAM-treated and untreated ptsPaik, S. ASCO Abst

27、ract, 2005.Habel, L. Breast Cancer Research, 2006.Does the 21-Gene Assay Predict Response to Chemotherapy? NSABP B20 trial: 651 ER+/LN- pts randomized to chemo + TAM (n=424) v. TAM alone (n=227) (ALL PATIENTS) Chemo CMF or MF No distant recurrence - 92.2% (chemo + TAM) v. 87.7% (TAM) No local/distan

28、t relapse - 90.1% (chemo + TAM) v. 83.5% (TAM) Overall Survival 89.5% (chemo + TAM) v. 86.4% (TAM) The TAM alone arm was used in the training set in development of the 21-gene assayPaik, S. JCO, 2006.Kaplan-Meier Plots for Distant RecurrenceAll patientsLow Risk RS 18Intermediate Risk RS 18-30High Ri

29、sk RS 3188%60%92.2%87.7%Paik, S. JCO, 2006.Relative & Absolute Benefit of Chemotherapy as a Function of RS27.6%Low RS mean absolute decrease in distant recurrence rate of -1.1%High RS mean absolute decrease inDistant recurrence rate of 27.6%Intermediate RS no substantial benefit,But the uncertai

30、nty in the estimate cannotExclude a clinically important benefitPaik, S. JCO, 2006.Prospective Validation of Oncotype DX: The TAILORX TrialDowsett, M. & Dunbier, A. Clin Cancer Res, 2008.Low RS:HormonalTherapyHigh RS:Chemo +HormonalTherapyHormonal TherapyChemo + Hormonal11,248 ER+/LN- patientsTh

31、e Two-gene Ratio: HOXB13:IL17BRTwo-Gene Expression Ratio: HOXB13:IL17BR Developed to predict response to tamoxifen in ER+/PR+, early stage , LN+/- breast cancers Performed microarray gene expression analysis of tumors from 60 women treated with adjuvant tamoxifen Patients selected based on outcome:

32、early relapse or not RNA isolated from frozen tumor-tissue 9 genes were identified with p0.001 2 selected HOXB13 was overexpressed in tamoxifen recurrences & IL17BR was overexpressed in tamoxifen nonrecurrences HOX family of genes implicated in tumor invasion and metastases HOXB13 is expression

33、is often up-regulated in breast cancer cellsMa, X. Cancer Cell, 2004.HOXB13:IL17BR Predicts Response to TamoxifenHOXB13 & IL17BR outperformed other predictive markers (quant-itative ER and PR, ERBB2, & EGFR)HOXB13:IL17BR ratio was a better composite predictor of tamoxifen responseHOXB13:IL17

34、BR was an indepen-dent predictor of treatment outcome when controlled for tumor size & expression of PGR and ERBB2HOXB13:IL17BR ratio was highly correlated with clinical outcome in an independent 20 pt cohortMa, X. Cancer Cell, 2004.Lack of Consistency of 2-gene SignatureSTUDYPATIENTSHAZARD RATI

35、OSOUTCOMECUT POINTSGoetz et al.206 ER+, LN+/- adjuvant TAMRFS 1.98DFS 2.03OS 2.4(LN- pts)Prognostic in TAM-treated LN- pts onlyTAM -1.849Ma et al.852 pts (66% untreated & 34% TAM-treated )RFS 3.9 (p=.007) in TAM and untreated pts combinedPrognostic in ER+/LN- ptsUnable to compare TAM & untre

36、ated groupsTAM -0.06Untreated 1.0Jansen et al.1,252 LN+/-, 32% adj tx(44% hormonal, 53% chemo, & 3% both)DFS (LN-, no tx) 1.74 (p=.006)DFS (TAM) 2.97 (p.001)Prognostic in untreated, LN-Predicts response to TAMUntreated 1.0TAM 0.06Limitations of These Studies Extreme sensitivity to training set s

37、ince there is such variability in the cut point Does not seem to be effective in LN+ patients Needs prospective validationIntrinsic Subtypes: PAM50Intrinsic Subtypes: PAM50 Training set =189 breast tumor + 29 normal samples qRT-PCR An expanded gene set of 1,906 genes from other microarray studies an

38、alyzed by hierarchical clustering significant clusters correlated to known intrinisic subtypes narrowed to 50 significant genes (PAM50) Intrinsic subtypes are useful in predicting RFS of untreated patients & those stratified by ER status Cox models tested using intrinsic subtypes alone and toget

39、her with clinical variables (ER status, tumor size, & node status) ROR-S Subtypes alone ROR-C Subtype + clinical variables Models containing both subtype & clinical variables better than either clinical variables (p0.0001) or subtype alone (p0.0001)ROR-C Predicts RFS in Untreated LN- Patient

40、sOnly Luminal A group contained low risk patientsROR-C Scores Stratified by SubtypeKaplan-Meier Plots of RFS of RiskGroupsHigh RiskMedium RiskLow RiskRisk Groups Predict RFSPAM50 Predicts Response to Neoadjuvant T/FACROR-S v Probability of pCRLow-risk tumors (luminal A) chemo-insensitive tumors with

41、 probability of pCRPlateau chemo- resistance among high risk tumorsSensitivity 94% & NPV 97% for identifying nonresponders toNeoadjuvant chemoROCC of ROR-S v pCR Probability of pCR with ROR-SPAM50 Prognostic in TAM-treated Patients 786 ER+ TAM treated pts qRT-PCR, PAM50, and ROR calculation done

42、 Multivariate analysis Intrinsic subtype and ROR score were independently prognostic (Grade and HER2 were not) Low ROR score good outcomes (even if +LN) Found to be superior to immunohistochemical markers & adjuvant! Online as a prognosticatorEllis, M. ASCO, 2009.5 Gene Signatures ComparedIs The

43、re Concordance Between Gene Expression Profiles?295 Stage I/II ER +/-Analyzes done withAll pts & ER+ only165 untreated20 TAM20 TAM + chemo90 chemoFan, C. NEJM, 2006.Relapse-free SurvivalOverall SurvivalIntrinsicSubtypeRecurrenceScore70-GeneProfileWoundResponseTwo-GeneRatioAll models except the t

44、wo-gene ratio modelwere significant predictors of both RFS and OS.Summary of Gene Signatures70-gene Signature21-geneSignature2-GeneRatioIntrinsicSubtypesAnalysisApproachSupervisedSupervisedSupervisedUnsupervisedTissue TypeFresh or FrozenFormalin-Fixed, Parafin-embeddedFormalin-Fixed, Parafin-embedde

45、dFormalin-Fixed, Parafin-embeddedTechniqueDNA microarraysQ-RT-PCRQ-RT-PCRQ-RT-PCRPrognosticUntreated pts age1 cm LN-, ER+/HER2 tumorsResourcesDowsett, M. & Dunbier, A. Emerging Biomarkers and New Understanding of Traditional Markers in Personalized Therapy for Breast Cancer. Clinical Cancer Rese

46、arch 2008; 14: 8019-26.Sotiriou, C et al. Gene-Expression Signatures in Breast Cancer. New England Journal of Medicine 2009; 360: 790-800.Vant Veer, L et al. Gene Expression Profiling Predicts Clinical Outcome of Breast Cancer. Nature 2002; 415: 530-36.Van de Vijver, M. et al. A Gene-Expression Sign

47、ature as a Predictor of Survival in Breast Cancer. The New England Journal of Medicine 2002; 347: 1999-2009.Paik, S. et al. A Multigene Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative Breast Cancer. The New England Journal of Medicine 2004; 351: 2817-2826.Sparano, J and Paik, S. Deve

48、lopment of the 21-Gene Assay and Its Application in Clinical Practice and Clinical Trials. Journal of Clinical Oncology 2008; 26: 721-728.Paik, S. et al. Gene Expression and Benefit of Chemotherapy in Women With Node-Negative, Estrogen-Receptor-Positive Breast Cancer. Journal of Clinical Oncology 20

49、06; 24: 3726-3734.Paik, S. et al. Expression of the 21 genes in the Recurrence Score assay and tamoxifen clinical benefit in the NSABP study B-14 of node negative, estrogen receptor positive breast cancer. Journal of Clinical Oncology 2005; 23: suppl abstr 510.ResourcesMa, X. et al. A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell, 2004; 5: 607-16.Ma X. et al. The HOXB13:IL17BR Expression Index Is a Prognostic Factor in Early-Stage Breast Cancer. Journal of Clinical Oncol