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Update on Predic2ve Biomarkers in Breast Cancer
Hal Berman, MD PhD Department of Laboratory Medicine
and Pathobiology University of Toronto
University Health Network
Molecular Markers in Breast Cancer – 2007 ASCO guidelines
Evidence of clinical u.lity / recommended for use in rou.ne prac.ce:
Molecular Marker Indica2on ER, PR Hormonal therapy HER2 An2-‐HER2 therapy CA15-‐3 Monitoring treatment failure in the
metasta2c seSng CA27.29 Monitoring treatment failure in the
metasta2c seSng CEA Monitoring treatment failure in the
metasta2c seSng uPA Determina2on of prognosis in newly
diagnosed N0 breast cancer. PAI-‐1
Determina2on of prognosis in newly diagnosed N0 breast cancer.
Certain mul2parameter gene expression assays
TBD
Molecular Markers in Breast Cancer – 2007 ASCO guidelines
Insufficient evidence to support rou.ne use in clinical prac.ce
P53 Cathepsin D Cyclin E
DNA/ploidy by Flow Markers of prolifera2on
(Ki67, PCNA, cyclin D, p27, p21, TK, TOP2A) Proteomics
Certain mul2paramter assays Detec2on of BM micromets
Detec2on of circula2ng tumor cells
Impediments to clinical development of predic2ve biomarkers
• Predic2ve versus prognos2c
• Requirement for level I evidence
• The moving target of therapy
• Biological heterogeneity
• Pre-‐analy2cal
Tumor Marker U2lity Grading System (TMUGS)
* TMUGS-‐Plus – Es2ma2on of rela2ve strength of prognos2c factor (risk ra2o) or predic2ve factor (benefit ra2o).
Hayes et al JNCI 1996.
Obtaining level I evidence from archival 2ssue – The “Prospec2ve
Retrospec2ve” approach • Availability of adequate and representa2ve archival
2ssue from a previous prospec2ve clinical trial. • The test should be analy2cally / pre-‐analy2cally
validated for use in archival 2ssue. • Plan for biomarker evalua2on predetermined in
wri2ng. • Results must be validated from similar but separate
studies.
Simon, Paik, & Hayes. JNCI 2009, 101(21):1446-‐52.
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Predic2ve/Prognos2c Breast Cancer Mul2-‐marker Molecular Tests
• Oncotype DX (RT-‐PCR, 21 genes) • Mammaprint (Microarray, 70 genes) • MapQuant Dx (Microarray, 97 genes) • Mammostrat (IHC, 5 markers) • Endopredict (RT-‐PCR, 11 genes) • PAM50 (Microarray, 50 genes) • Roherdam Signature (Microarray, 76 genes) • Two gene ra2o (RT-‐PCR, HOXB13:IL17BR) • Molecular Grade Index (RT-‐PCR, 5 genes)
– TBCI (MGI + 2-‐gene ra2o) • Blueprint (Microarray, 80 genes)
Oncotype DX
• Prognos2c and predictve test based upon RT-‐PCR assay of 21-‐genes in FFPE 2ssue.
• Aliases: recurrence score (RS), 21-‐gene recurrence score (RS-‐21), Genomic Health recurrence score (GH-‐RS or GHI-‐RS).
• Model building: 250 candidate genes prospec2vely selected, evaluated in 3 randomized studies (B-‐20, Rush U, St. Joseph’s) to derive 21-‐gene panel.
Oncotype DX 21-‐Gene Recurrence Score (RS) Assay
RS = + 0.47 x HER2 Group Score - 0.34 x ER Group Score + 1.04 x Proliferation Group Score + 0.10 x Invasion Group Score + 0.05 x CD68 - 0.08 x GSTM1 - 0.07 x BAG1
16 Cancer and 5 Reference Genes From 3 Studies
Category RS (0 -100) Low risk RS <18 Int risk RS 18 - 30
High risk RS ≥ 31
Paik et al. N Engl J Med. 2004;351:2817-‐2826.
PROLIFERATION Ki-‐67 STK15 Survivin Cyclin B1 MYBL2
ESTROGEN ER PR Bcl2
SCUBE2
INVASION Stromelysin 3 Cathepsin L2
HER2 GRB7 HER2
“Unaligned” CD68 GSTM1 BAG1
REFERENCE Beta-‐ac2n GAPDH RPLPO GUS TFRC
Oncotype DX – Valida2on Studies • NSABP B14 (Hormonal benefit study – NEJM 2004;351:2817-‐26). – 668 ER+ N0 pa2ents treated with tamoxifen. – RS es2mates risk of distant recurrence (con2nuous) and overall survival.
• NSABP B-‐20 (Chemotherapy benefit study) – JCO 2006;24(23):3726-‐33. – 651 ER+ N0 pa2ents with tamoxifen +/-‐ CMF/MF chemotherapy.
– RS es2mates risk of distant recurrence. – RS predicts benefit of CMF/MF chemotherapy for reducing distant recurrence.
Oncotype DX – Valida2on Studies
• Kaiser Permanente (BCR 2006;8(3):1-‐15). – Case-‐control study – 790 of 4,964 studied, node nega2ve, no chemo.
– RS prognos2c of breast cancer death in ER-‐posi2ve tamoxifen treated and untreated pa2ents.
• MD Anderson (Clin Can Res. 2005, 11(9):3315-‐9) – 149 pa2ents, node nega2ve, ER pos (70%) and ER neg (30%), untreated.
– RS not prognos2c of distant recurrence in untreated pa2ents)
Oncotype DX – Valida2on Studies – ECOG 2197 (ER,PR central lab study – JCO 2008, 26(15):2473-‐81).
• Case-‐control 776 pa2ents (ER pos or neg, 0-‐3 nodes pos) all treated.
• ER and PR: local IHC, central TMA 1D5 ER and 636 PR, Oncotype ER and PR.
• High concordance. RT-‐PCR was more sensi2ve. RS was prognos2c for risk of recurrence.
– SWOG 8814 (Node posi2ve study – Lancet Oncol 2010, 11:55-‐65)
• 367 ER+ pa2ents treated with tamoxifen only versus tamoxifen plus CAF (cyclophosphamide, doxorubicin, fluorouracil).
• RS prognos2c in tamoxifen only group and predic2ve of CAF benefit.
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Oncotype DX – Prospec2ve Studies
– SWOG S1007 / RxPONDER (Node posi2ve study, RS <=25)
• Prospec2ve study screening > 9000 pa2ents to enroll ~4,000 women -‐ hormone receptor posi2ve, HER2 nega2ve, 1-‐3 posi2ve lymph nodes with RS <=25.
• Goal: Determine if there is benefit to adding chemotherapy to hormonal therapy in this cohort.
– TAILORx • To be discussed.
Oncotype DX – Approved Uses In newly diagnosed, ER+ N0 pa2ents: • Es2mate risk of recurrence in pa2ents treated with tamoxifen. • Iden2fy pa2ents who are likely to receive most therapeu2c benefit from tamoxifen. • Iden2fy pa2ents who receive lihle benefit from chemotherapy. • “The absolute benefit for those with a higher RS is likely to outweigh risk from treatment.”
Harris et al. JCO 2007, 25(33):5287-‐5312.
45 year old female 1.5 cm IDC, single focus pT1C N0
Nuclear Grade Quiz
• A) 1
• B) 2
• C) 3
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ER
PR HER2
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Independent QA of Oncotype DX HER2 Assessment (Dabbs et al. JCO Oct 11. 2011)
• 843 pa2ents from 3 central laboratories with available Oncotype DX results.
• 99% IHC/FISH neg. also neg. by Oncotype. • All IHC/FISH equivocal, neg. by Oncotype. • Of IHC/FISH posi2ve cases:
– 45% posi2ve – 33% equivocal – 39% nega2ve
Limita2on: Only 4% of cohort HER2 posi2ve (36 pa2ents) and 3% HER2 equivocal (23 pa2ents).
TAILORx Trial
• Prospec2ve randomized phase III study • Primary objec2ve – determine if hormonal therapy is not inferior to chemotherapy in women with RS 11-‐25.
• >10,000 ER+ N0 women to yield ~4800 with RS 11-‐25 (accrual completed – 11,233 / 6,907).
• Randomized to hormone Rx versus chemotherapy + hormone Rx.
• Es2mated final results ~ 2014-‐15.
TAILORx Oncotype DX – Technical Considera2ons
• Exclusions: – Tumors < 2 mm in maximal dimension – Tumor occupying < 5% of the sec2on
• Macrodissec2on to obtain enriched tumor 2ssue: – Sec2ons with non-‐tumor elements that were both sufficiently localized
to be amenable to macro-‐dissec2on and cons2tuted >50% of the overall 2ssue area of the sec2on
– Non-‐tumor elements defined as : smooth muscle, fibrosis, hemorrhage, normal breast stromal 2ssue, but not DCIS or LCIS or necrosis.
• Amount of 2ssue: – 3 x 10 micron sec2ons without dissec2on – 6 x 10 micron sec2ons with macro-‐dissec2on.
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Tumor heterogeneity -‐ Is it a serious concern?
Drury et al. J Clin Path 2010, 63:513-‐7
Tumor heterogeneity -‐ Is it a serious concern?
* N=19 Pa2ents
Kim and Paik Nat Rev Clin Oncol, 2010, 7:340-‐7.
Oncotype DX® 21-‐Gene Recurrence Score (RS) Assay
RS = + 0.47 x HER2 Group Score - 0.34 x ER Group Score + 1.04 x Proliferation Group Score + 0.10 x Invasion Group Score + 0.05 x CD68 - 0.08 x GSTM1 - 0.07 x BAG1
16 Cancer and 5 Reference Genes From 3 Studies
Category RS (0 -100) Low risk RS <18 Int risk RS 18 - 30
High risk RS ≥ 31
Paik et al. N Engl J Med. 2004;351:2817-‐2826.
PROLIFERATION Ki-‐67 STK15 Survivin Cyclin B1 MYBL2
ESTROGEN ER PR Bcl2
SCUBE2
INVASION Stromelysin 3 Cathepsin L2
HER2 GRB7 HER2
“Unaligned” CD68 GSTM1 BAG1
REFERENCE Beta-‐ac2n GAPDH RPLPO GUS TFRC
36 IDC, 2 ILC, 1 DCIS, 1 FA
Microarray (open bx)
+doxorubicin
16 w
Microarray (excision)
20 IDC
LABC Study
Excluded if different
(i.e. extrinsically variable)
The “intrinsic” gene set - missing the elephant (proliferation) in the room
Sorlie et al. PNAS 2003, 100(14):8418-‐23.
The intrinsic set revisited (Hu et al. BMC Genomics 7:96, 2006).
Luminal
HER2
IFN/STAT1
Basal
Prolifera2on
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Luminal A Luminal B Basal HER2
ER + + -‐ +/-‐
HER2 -‐ +/-‐ -‐ +
Prolifera2on -‐ + + +/-‐
Minimalist Molecular Subtypes IHC4 (ER, PR, HER2, Ki67)
Cuzick et al. JCO Oct. 11, 2011.
IHC4 Score vs GH-‐RS -‐ TransATAC
• 1,125 ER+ ATAC trial pa2ents (non-‐chemotherapy arms)
• GHI-‐RS and IHC4 computed • Distal recurrence primary endpoint • Prognos2c model -‐ IHC4 combined with classical variables assessed in separate 786 ER+ NoSngham cohort
Cuzick J et al. J Clin Oncol. 2011 Oct 11.
IHC4 Score vs GH-‐RS -‐ TransATAC
• ER, PR, HER2, and Ki-‐67 each provided independent prognos2c informa2on in the presence of classical variables.
• Informa2on in IHC4 was similar to GHI-‐RS and lihle addi2onal prognos2c value was seen in the combined use of both scores.
• IHC4 was validated in the NoSngham cohort.
Cuzick J et al. J Clin Oncol. 2011 Oct 11.
– Williams et al. Appl. Immuno. Mol. Morphol. 2011, 19:431-‐6.
• 133 pa2ents with Oncotype Dx tes2ng. • Ki-‐67 (MIB-‐1) and PPH3 tested, both significantly associated with RS and grade.
– Gwin et al. 2009 IJSP 17(4), 303-‐10. • 32 pa2ents, overall concordance between Ki-‐67 and RS. • However, some tumors with low RS but high Ki-‐67.
– Sahebjam et al. BJC 2011, 1-‐4. • 53 cases ER+, N0. • Clone 30-‐9 (Venta) • Strong Linear correla2on between Ki67 and RS.
Oncotype DX versus Ki-‐67
Sahebjam et. al. BJC 2011, 1-‐4
High
Intermediate
Low
10.0
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Wirapa2 et al. Breast cancer research 2008, 10(4):R65
* 2,833 breast invasive tumors with publically available gene expression and clinical data.
Prolifera2on and Predic2ve/Prognos2c Signatures
Wirapa2 et al. Breast cancer research 2008, 10(4):R65
* Hazard ra2o for distant recurrence free survival
Azambuja et al, BJC 2007, 96:1504-‐13. Azambuja et al, BJC 2007, 96:1504-‐13.
Ki-‐67
• Mouse monoclonal an2body iden2fied in 1983 by Gerdes et al. (Int J Cancer 31, 13-‐20) in human Hodgkins cell line.
• Detailed understanding of structure, kine2cs and subcellular localiza2on, but func2on remains poorly studied.
• Different an2bodies have been generated to the Ki-‐67 protein (i.e. MIB-‐1, MIB-‐3, MM-‐1, Ki-‐S5, SP-‐6, 30-‐9, 7B11, DVB-‐2).
Losa et al, Clinical Science 1998, 95, 129-‐35.
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Molino et al, AIMM 2002 Dec;10(4):304-9
Ki67 as a prognos2c marker – thresholds of posi2vity
* 38 studies
Prevent Overtreatment
Chemosensi.vity
Luminal A vs B threshold
From the Danish Breast Cancer Coopera2ve Tumour Biology Commihee Offersen et al. Histopathology 2003, 43:573-‐82.
Ki-‐67 Reproducibility
Ki67 Score Quiz
• A) < 20%
• B) 20-‐30%
• C) 30-‐40%
• D) 40-‐50%
• E) > 50% From the 10th European Congress on Telepathology and 4th Intena2onal Congression on Virtual Microscopy. Fasanella et al. Diag Path 2011, 6(S1):S7.
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JNCI, 103:1-‐9 Sep. 29 2011
KI67 Recommenda2ons
• MIB1 is the gold standard • Ki67 can be scored from core and excision bxs. • TMAs for clinical trials • Fixa2on standards equivalent to ER/PR/HER2. • Unstained < 14 days at room temp. • “Image analysis methods for Ki67 remain to be proven for use in clinical prac2ce.”
JNCI, 103:1-‐9 Sep. 29 2011
KI67 Recommenda2ons -‐ Scoring
• Nuclear staining of any intensity is posi2ve. • Count at least 500 malignant cells (preferably > 1000)
• At least three high-‐powered x40 objec2ve “representa2ve” of spectrum of staining.
• But “hot spots” should be included (A working party is working on the issue of hot spots).
• For prognos2c evalua2on, score invasive edge.
JNCI, 103:1-‐9 Sep. 29 2011
“Why can’t we use estrogen receptor, HER2, Ki-‐67, and tumor grade to guide treatment when breast cancer biology is essenFally dictated by estrogen receptor, HER2, and proliferaFon?” “This is an issue that has been argued over and over during the past decade. To me the answer is clear – yes, I think we can if we have reliable assays for estrogen receptor (ER), HER2, and tumor histological grade (or prolifera2on with Ki-‐67).” -‐ Soonmyung Paik, The Breast 20 (2011) S3, S87-‐91 .
