The Molecular Diversity of Luminal a Breast Tumors

The molecular diversity of Luminal A breast tumors

Giovanni Ciriello Computational Biology, MSKCC TCGA Annual Symposium Washington DC, 2012 Breast'Cancer'Heterogeneity

Breast Cancer Intrinsic Subtypes '

Breast Cancer '

Her2-negative Her2 - positive PAM50: Her2-enriched

ER - negative PAM50: Basal ER-positive

PAM50: Luminal B

PAM50: Luminal A ' Breast'Cancer'Heterogeneity

Breast Cancer Intrinsic Subtypes '

Breast Cancer '

Her2-negative Her2 - positive PAM50: Her2-enriched

ER - negative ' PAM50: Basal ' ER-positive

PAM50: Luminal B '

PAM50: Luminal A ' Breast'Cancer'Heterogeneity

Breast Cancer Intrinsic Subtypes '

Breast Cancer '

Her2-negative Her2 - positive PAM50: Her2-enriched

ER - negative PAM50: Basal ER-positive

PAM50: Luminal B PAM50 (Perou et al., 2000) PAM50: Luminal A Luminal(Heterogeneity

Integrated data from: TCGA 2012, Ellis et al. 2012, Banerji et al. 2012 Curtis et al. 2012 Russnes et al. 2010

~ 1500 Luminal tumors

Copy Number Alterations Somatic Mutations mRNA expression Luminal(Molecular(Heterogeneity

PAM50 vs CNA-clusters (Curtis et al., TCGA) Integrated data from: TCGA 2012,

Ellis et al. 2012, Basal Her2 LumA LumB Normal Banerji et al. 2012 IC10

Curtis et al. 2012 IC5

Russnes et al. 2010 IC3

IC1

IC4

IC8 ~ 1500 Luminal tumors

Curtis datset IC7

IC9

IC6

IC2

Copy Number Alterations CNA1

Somatic Mutations CNA3

mRNA expression CNA2

CNA5 TCGA datset CNA4

p < 10- 4 10 - 4 < p < 0.05 Luminal(Molecular(Heterogeneity

CNA-clusters Somatic Mutations (Curtis et al., TCGA) (TCGA, Ellis et al., Banerji et al) Integrated data from: TCGA 2012,

Ellis et al. 2012, Basal Her2 LumA LumB Normal Banerji et al. 2012 IC10 Her2- Curtis et al. 2012 IC5 enriched Russnes et al. 2010 IC3

IC1 Basal-like IC4

IC8 ~ 1500 Luminal tumors

Curtis datset IC7 Luminal B IC9

IC6

IC2 # of mutations Luminal A per sample Copy Number Alterations CNA1 # of SigGenes ( q<0.05) Somatic Mutations CNA3 0 20 40 60 80 mRNA expression CNA2

CNA5 TCGA datset CNA4

p < 10- 4 10 - 4 < p < 0.05 Luminal(A(Clinical(Heterogeneity

• Luminal A tumors have heterogenous outcome

METABRIC Dataset (Curtis et al.) 7 Haque et al., 2012 Days at death death at Days 0 2000 4000 6000 8000

Basal Her2 LumB LumA Luminal(Heterogeneity

• Luminal A tumors have heterogenous outcome

METABRIC Dataset (Curtis et al.) Haque et al., 2012 Days at death death at Days 0 2000 4000 6000 8000

Basal Her2 LumB LumA

Luminal A is molecularly and clinically heterogeneous Can we link outcome variability to molecular diversity? Luminal(A(Heterogeneity Figure 1

A) B) Luminal A Breast Tumors (TCGA Dataset) 50% T: TCGA data * C: Curtis data 40% a 30% * 20% %Samples 1q/16q b * * 10% * c 0 T C T C T C T C T C d * IGP p-value < 0.001 Quiet C) Copy Number e Copy NumberOverall survival Alterations (Curtis Dataset) f

Chr.8 identify five major subgroups

associated g

h High Copy Number % Surviving i

Mixed Copy Number Quiet 20 40 60 80 100 1q/16q Chr.8-associated Mixed Overlall Log-rank p = 0.015 Copy Number High CNH Log-rank p = 0.001 0

0 50 100 150 Chromosomes Months Survival Luminal(A(Heterogeneity Figure 1

A) B) Luminal A Breast Tumors (TCGA Dataset) 50% T: TCGA data * C: Curtis data 40% a 30% * 20% %Samples 1q/16q b * * 10% * c 0 T C T C T C T C T C d * IGP p-value < 0.001 Quiet C) Copy Number e Overall survival (Curtis Dataset) f Chr.8

associated g

h High Copy Number % Surviving i

Mixed Copy Number Quiet 20 40 60 80 100 1q/16q Chr.8-associated Mixed Overlall Log-rank p = 0.015 Copy Number High CNH Log-rank p = 0.001 0

0 50 100 150 Chromosomes Months Survival Genomic(Instability(correlates( with(poor(prognosis

A) B) CNH survival (Curtis et al.) CNH Survival (Russnes et al.)

CNH Others % Surviving % Surviving

CNH Others Log-rank p = 4.6E-05 Log-rank p = 0.003 0 20 40 60 80 100 0 20 40 60 80 100

0 2 4 6 8 10 12 14 , 0 2 4 6 8 10 12 14 , Years Survival , Years Survival , Genomic(Instability(correlates( with(poor(prognosis

A) B) CNH survival (Curtis et al.) CNH Survival (Russnes et al.)

CNH Others % Surviving % Surviving

CNH Others Log-rank p = 4.6E-05 Log-rank p = 0.003 0 20 40 60 80 100 0 20 40 60 80 100

0 2 4 6 8 10 12 14 , 0 2 4 6 8 10 12 14 , Years Survival , Years Survival ,

What are the molecular features of these tumors? CNH$Molecular$Features

A) 60% ** * Luminal A 50% ** Copy Number High 40% Other 30% ** ** p < 0.01 ** p < 0.05 20% * %Samples 10%

0% 5q 20q TP53 MYC (mut) (amp) (gain) (loss) PIK3CA(mut) B) C) Luminal A Samples AURKA

8q.24 PLK1 AURKA CDC25B CCNA2 AURKB CDK1 CCNA1 CDC25B/C BIRC5 PLK1

TP53BP1 POLH

POLI CDK1

MLH3 Cyclin-A/B POLK Differentially Expressed Genes Differentially Copy Number High Low High Mitosis CNH$Molecular$Features

A) 60% ** * Luminal A 50% ** Copy Number High 40% Other 30% ** ** p < 0.01 ** p < 0.05 20% * %Samples 10%

0% 5q 20q TP53 MYC (mut) (amp) (gain) (loss) PIK3CA(mut) B) C) Luminal A Samples AURKA

8q.24 PLK1 AURKA CDC25B CCNA2 AURKB CDK1 CCNA1 CDC25B/C BIRC5 PLK1

TP53BP1 POLH

POLI CDK1

MLH3 Cyclin-A/B POLK Differentially Expressed Genes Differentially Copy Number High Low High Mitosis

What characterizes the other Luminal A subtypes? Luminal(A(Somatic(Mutations

A) CNA-Quiet 1q/16q Chr.8-associated CNH Mixed

PIK3CA GATA3 AKT1 MAP3K1 TP53 Samples Somatic mutations

Chr. 8 - associated B) Co-occurrent (p<0.05) Copy Number Loss D) Chr. 8 Chr.16 Mutually Exclusive (p<0.05) Copy Number Gain Mutated genes PIK3CA MAP3K1 GATA3 Samples CNA 1p12 1q21 1q32 6p23 6q27 7p12 9p21 11p13 11q14 10p13 10q23 12q15 13q12 14q13 14q24 16q12 16q23 17p12 17q12 17q21 19q12 19q13 20p13 3p25.1 4p16.3 4q13.3 6q14.2 8p21.3 8p11.22 8p11.21 11p15.5 11q13.2 17p11.2 21q11.2 8q24.21 10q22.3 12p13.3 13q14.2 15q26.3 16q24.2 20q13.2 Chromosomes 8p11

Somatic mutations Amplification MAP3K1 C) PIK3CA Mutations GATA3 Mutations E) 25 20 MAP3K1 mutations

15 Mutation Type 10 10 Missense Splice-site

#mutations 5 5 In Frame Del. Nonsense 0 0 1q/16q Quiet Chr.8 CNH Mixed 1q/16q Quiet Chr.8 CNH Mixed #mutations Frame-shift PIK3CA Hotspots (E545K, H1047R/L) GATA3 Hotspots (e4-2 , P409fs) Other MAP3K1 mutated samples Luminal(A(Somatic(Mutations

A) CNA-Quiet 1q/16q Chr.8-associated CNH Mixed

PIK3CA GATA3 AKT1 MAP3K1 TP53 Samples Somatic mutations

Somatic mutations Amplification MAP3K1 C) PIK3CA Mutations GATA3 Mutations E) 25 20 MAP3K1 mutations

15 Mutation Type 10 10 Missense Splice-site

A) CNA-Quiet 1q/16q Chr.8-associated CNH Mixed

PIK3CA GATA3 AKT1 MAP3K1 TP53 Samples Somatic mutations

Somatic mutations Amplification MAP3K1 C) PIK3CA Mutations GATA3 Mutations E) 25 20 MAP3K1 mutations

15 Mutation Type 10 10 Missense Splice-site

Which pathways are most de-regulated? MEMo:%Mutual%Exclusivity%Modules

Mutations CNA User Defined Set

● Homdel Hetloss Diploid Genes connected by Gain

C24 Q1756 ● Amp ● ● Mutated ● ● ● Normal ● ● ● ● ● ● network proximity ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ●●●●● ● ● ● ● ● ● ● ●● ● ●● ● BRCA1

Cases Genes

Altered Not Altered .

G S G S G S

Gene A 54% Module t t + 1 Gene B Gene C 8% 8.5% Genes Samples

MCMC Switching Permutation Gene D - preserves #alterations per sample 30.5%t - preserves #alterations per gene

Ciriello et al., Genome Res. 2012 Mutual Exclusivity Module www.cbio.mskcc.org/memo Luminal(A(Pathways((MEMo)

A) 2 IGF1R ERBB2 4% 4%

PTEN NF1 6% 3%

PIK3CA KRAS 1% 49%

PIK3R1 2% MAP3K1 AKT1 14% 4% PAK1 8% MAP2K4 MAP2K3/6 MAP2K1/2 11%

JNK p38 ERK Survival Apoptosis Proliferation

Activating interaction Inhibiting interaction Inactivating Activating Alterations+to+NCor/SMRT+components

NCOR1 NCOR2 GPS2 ANKRD11 TBL1XR1 NCOR1 NCOR2 GPS2 ANKRD11 TBL1XR1

Fingerprint mRNA Heatmap CNA - Clusters 1q/16q Hom. Del Copy Number Quiet Chr.8-associated Somatic Low High mutation Copy Number High Mixed Alterations+to+NCor/SMRT+components

NCOR1 NCOR2 GPS2 ANKRD11 TBL1XR1 NCOR1 NCOR2 GPS2 ANKRD11 TBL1XR1

Fingerprint mRNA Heatmap CNA - Clusters 1q/16q Hom. Del Copy Number Quiet Chr.8-associated Somatic Low High mutation Copy Number High Mixed

Why are co-repressor complexes important in ER+ tumors? NCor/SMRT*are*required* for*Tamoxifen*effects

Co-repressors

Tamoxifen NCOR1 ER Proliferation NCOR2 GPS2 ANKRD11 TBL1XR1 NCOR1 NCOR2 GPS2 ANKRD11 TBL1XR1

Fingerprint mRNA Heatmap CNA - Clusters 1q/16q Hom. Del Copy Number Quiet Chr.8-associated Somatic Low High mutation Copy Number High Mixed NCor/SMRT*are*required* for*Tamoxifen*effects

Co-repressors Co-activators

Tamoxifen Tamoxifen NCOR1 ER ER Proliferation NCOR2 GPS2 ANKRD11 TBL1XR1 NCOR1 NCOR2 GPS2 ANKRD11 TBL1XR1

Fingerprint mRNA Heatmap CNA - Clusters 1q/16q Hom. Del Copy Number Quiet Chr.8-associated Somatic Low High mutation Copy Number High Mixed Loss$of$NCor/SMRT$predicts$resistance

Co-repressors Co-activators

Tamoxifen Tamoxifen NCOR1 ER ER Proliferation NCOR2 GPS2 ANKRD11 TBL1XR1 N-Cor ANKRD11 NCOR1 TBL1XR1 NCOR2 2% 3% GPS2 ANKRD11 NCOR1 GPS2 TBL1XR1 5% 2% p160/SRC

Fingerprint mRNA Heatmap CNA - Clusters SMRT 1q/16q Hom. Del Copy Number Quiet NCOR2 Chr.8-associated Somatic Low High mutation Copy Number High 1% Mixed ER Proliferation Conclusions

We dissected the genomics of Luminal A tumors in multiple datasets to explain their molecular and clinical heterogeneity Four major subtypes of Luminal A tumors Atypical Luminal subtype characterized by: • high genomic instability • p53 mutations • Aurora kinases up-regulation • poor prognosis Luminal A hallmark mutations are prevalent in tumors characterized by low copy number alterations We identified multiple rare, but mutually exclusive alterations associated with loss NCor/SMRT - These alterations may predict lack of response to endocrine therapy Thanks!

Chris Sander Chuck Perou Niki Schultz Katie Hoadley Rileen Sinha Anders Jacobsen Boris Reva Xiaohong Jing Wei-Qing Wang

...and the whole TCGA Breast AWG! Appendix(A

Luminal A Recurrently Mutated Genes (q < 0.05) data from TCGA, Ellis et al., Banerji et al

Her2- enriched

Basal-like

PIK3CA RUNX1 DGKG MAP3K1 CTCF SMCHD1 Luminal B GATA3 CBFB KRAS TP53 SF3B1 CCND3 # of mutations CDH1 MED23 NKAIN4 Luminal A per sample MLL3 WNT7A HIST2H2BE # of SigGenes ( q<0.05) MAP2K4 TBL1XR1 HIST1H3B NCOR1 TBX3 SHD 0 20 40 60 80 AKT1 GPS2 GPR32 PTEN FOXA1 Appendix(B

ER Positive Targets (BRCA_ER_POS) 0.8 A) 60% ** * Luminal A 50% ** Copy Number High 40% Other 30% ** ** p < 0.01 ** p < 0.05 20% * %Samples CNH tumors are ER+ 10% Enrichment score (ES) 0.0 0% 5q 20q TP53 MYC (mut) (amp) (gain) (loss) PIK3CA(mut) B) C) ClassA (positively correlated) ClassB (negatively correlated) Luminal A Samples -3 AURKA p < 10 , FDR = 0.03

8q.24 PLK1 AURKA CDC25B CCNA2 AURKB CDK1 CCNA1 CDC25B/C BIRC5 PLK1

TP53BP1 POLH

POLI CDK1

MLH3 Cyclin-A/B POLK Differentially Expressed Genes Differentially Copy Number High Low High Mitosis Appendix(B

8q.24 PLK1 AURKA CDC25B CCNA2 AURKB CDK1 CDC25B/C Luminal B have similar subgroup CCNA1 BIRC5 PLK1

TP53BP1 POLH Copy Number POLI CDK1

MLH3 Cyclin-A/B POLK High Differentially Expressed Genes Differentially 80% * Copy Number High Low High Mitosis 70% ** ... 60% E2F1 BCAT1 50% AURKA AURKB 40% ** * KIF2C * CENPA 30% CCNA2 CDCA5 %Samples 20% CDCA3 TAF2 10% CDC20 CDC25B 0% PLK1 5q ... 20q TP53 MYC (mut) (amp) (gain) (loss) PIK3CA(mut)

Luminal B Expressed Genes Differentialy High Copy Number High Other Low ** p < 0.01 Luminal B Samples * p < 0.05