Breast-tumour samples from TCGA (n = 792) were ordered according to intrinsic subtype and then by correlation to the subtype centroid.4 Gene-expression data for selected genes are shown. Yellow: higher than median gene expression; black: median; blue: lower than median.

Gene profile

Luminal A90% ER+, 89% PR+, 14% HER2+• Mostly diploid; few copy number changes; 1q gain and 16q loss• Low pathological grade; high morphological differentiation; low proliferation rates• Recurrently mutated genes (>5%): PIK3CA, CDH1, MAP3K1, GATA3, MAP2K4, FOXA1, TP53, RUNX1, CBFB, NBL1, CTCF, NCOR1, PTEN, CDKN1B, AKT1, TBX3, ARID1A, and NF1• Typically responsive to endocrine therapy• Usually less responsive to (neo)adjuvant chemotherapy• Highest number of recurrently mutated genes, but the lowest total number of mutations and copy number changes, suggesting that these alterations are likely to be driver mutations (see figure, black line indicates a non-silent mutation)

HER2-enriched38% ER+, 20% PR+, 72% HER2+• High levels of HER2/ERBB2 amplification • Mostly aneuploid with high chromosomal instability• Highest single nucleotide mutation rate,4 but small list of recurrently mutated genes (TP53 [71%] and PIK3CA [35%])• Linked to APOBEC-mediated mutational profile5

• Mostly high pathological grade, typically ER– (62%)• High rate of brain metastases• Typically responsive to (neo)adjuvant trastuzumab in combination with chemotherapy6

• Sensitive to adjuvant anthracyclines7 and taxanes8

• HER2-enriched tumours that are also clinically HER2+ show high levels of HER2 protein and phosphoprotein suggesting active HER2 signalling (see figure)

Luminal B98% ER+, 82% PR+, 24% HER2+• Mostly aneuploid, with many high-level focal amplifications (11q13 [Cyclin D1, 56%]; 8p11–12 [FGFR1, 23%]) •Recurrently mutated genes: PIK3CA, GATA3, PTEN, and TP53 • The quantitative level of PR is lower in luminal B relative to luminal A, while Ki-67 is higher in luminal B relative to luminal A3

• Typically responsive to endocrine therapy, possibly less so than luminal A cancers• Higher pCR rate to neoadjuvant chemotherapy9 and possibly more sensitive to adjuvant chemotherapy than luminal A cancers • A subset of luminal B tumours have a hypermethylated phenotype (see figure),4 based on genome-wide DNA methylation patterns

Clinical outcomesThe intrinsic subtypes of breast cancer can predict prognosis.1,2 Luminal A tumours tend to have the most favourable outcomes, while luminal B, HER2-enriched, and basal-like tumours have worse prognosis. At the clinical level, there is correlation between the three established clinical biomarkers—ER, PR, and HER2—and the intrinsic subtypes. Luminal A tumours tend to be ER+, PR+, HER2–. Luminal B tumours are likely to be ER+, PR+/–and sometimes HER2+.3 The HER2-enriched tumours are usually HER2+/ER–. The basal-like subtype tends to be triple negative (ER–, PR–, HER2–). Despite this correlation, the intrinsic subtypes cannot be accurately identified using these markers.3 Nonetheless, most luminal A and B tumours are ER+ and/or PR+, and thus candidates for endocrine therapy; most HER2-enriched cancers are HER2+ and thus candidates for anti-HER2 therapies, and most basal-like tumours are ER–, PR–, and HER2– and therefore candidates for chemotherapy regimens.

MTOR

INPP5D

INPP4B

PDK1

MAPK8MEK2

MEK1

HRAS

KRASNRAS

IGF2RKITMET

FGFR2ERBB3 ERBB4EGFR

NOTCH1

FGFR3 FGFR4NOTCH3 NOTCH4

3% mut15% amp

0.4% mut13% amp

2% mut13% amp

14% mut0.8% del

4% mut13% amp

4% mut3% del

9% mut2% del

MAP3K1

MAP2K4

NOTCH2 FGFR1ERBB2

GRB7CDH1

CDKN2B CDKN2CCDKN2A CDKN2D

CDKN1A CDKN1B

0.3% mut16% amp

0.4% mut14% amp

35% mut2% del

MDM4

2% amp

4% amp MDM2

CDK6CDK4CCND3CCND2CCND1

0.1% mut5% amp

0.3% mut16% amp

CDK2

CCNE2CCNE1

6% mut5% del

PIP2 PIP3

35% mut5% amp

2% mut2% del

PIK3CA

0.5% mut14% amp

AKT1

AKT2 AKT3

3% mut1% amp

2% amp

0.3% mut6% amp

SOS1

GRB2

BRAF

RAF1 ARAF

0.1% mut5% amp

ERK1

Germline mutations

2% mut

3% mut

2% mut

BRCA2

BRCA1

ATM

040% loss 40% gain

Samples altered

XBP1

MLL4 SETD2

PGR AR2% mut5% del

RB1

8% mut1% del

1% mut4% amp

4% mut0.5% del

ARID1AKMT2C KDM5A

FOXA1

4% mut2% amp

MYC

0.3% mut21% amp

GATA3

12% mut

PTEN

PIK3R1

ESR11% mut3% amp

0.4% mut7% amp

0.4% mut5% amp

RARA

TOP2A

E2F1E2F3

Top GWASloci

NF1

ERK2

TP53

1% mut6% amp

IGF1R

Common genetic alterations TCGA data on breast tumour DNA copy number and somatic mutations were used to identify the frequency of each genetic alteration across 792 patients (all cancer subtypes).4

Each gene is shaded according to the overall frequency of alteration. Orange indicates a high level of amplification and/or likely gain-of-function mutations; blue represents homozygous deletions and/or likely loss-of-function mutations.

Three germline mutation rates are shown—taken from a subset of 500 TCGA samples previously published.4

Basal-like8% ER+, 7% PR+, 7% HER2+• Includes 70–80% of TNBC10

• High metastasis rate11

• Associated with younger age; high frequency in those of African descent12

• Associated with BRCA1 germline mutations2

• TP53 is the only recurrently mutated gene (>10%)• Highest pCR rate to neoadjuvant chemotherapy9

• TILs are prognostic within this subtype13

• In a 12 tumour-type analysis, the basal-like subtype formed a unique group14 (distinct from other breast cancers), showing similar characteristics to ovarian cancer, squamous cancers of the lung, head and neck, with frequent TP53 mutations, amplification of 3q, loss of 4q and 5q (see figure)

◀

Basal-likeHER2-ELuminal A Luminal B

ESR1PGR

GATA3XBP1

FOXA1

HER2GRB7

EGFRFOXC1

ID4KIT

MYC

MKI67MYBL2AURKA

BUB1CENPF

KRT8KRT18

KRT5KRT6A

Lum

inal

gene

sH

ER2

ampl

icon

Bas

alge

nes

Pro

lifer

atio

nge

nes

Ker

atin

gene

s

Low High

PIK3CACDH1GATA3

MAP3K1MAP2K4

FOXA1AKT1PTENTP53

Basal-likeHER2-E

Luminal A Luminal B

DN

A m

ethy

latio

npr

obes

(n

= 5

74

)

High

Low

Hypermethylation phenotype in luminal B samples

DN

A m

ethylation

Reprinted with permission from © Elsevier from Cell 158, Hoadley et al. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin, 929–944 (2014).

Pro

babi

lity

0

0.8

0.6

0.4

0.2

1.0

Censored

Luminal A

Luminal B

Basal-like

HER2-E

0 48 96 120Time to distance metastasis (months)

144 16824 72

0 48 96 120Overall survival (months)

24 72

Pro

babi

lity

0

0.8

0.6

0.4

0.2

1.0

Chr

omos

ome Ampli�cation

Deletion

Neutral

Ovarian95% TP53 mut

Basal-like80% TP53 mut

Luminal and HER2-E24% TP53 mut

Squamous-like72% TP53 mut

Clinical ER

Clinical HER2

HER2 mRNA

HER2 protein

HER2 pY1248

Basal-likeHER2-E Luminal ALuminal B

IHC: ER

IHC: ER

IHC: HER2

IHC: KRT6A

Adapted with permission from © National Academy of Sciences, USA (2003).

Biomarker Potential treatment Evidence

Luminal A and B ER+ and/or PR+ by IHC Endocrine therapy Meta-analysisPIK3CA mutation PI3K inhibitors Data from phase I and II trialsFGFR1 ampli�cation FGFR inhibitors Data from phase I and II trialsGermline BRCA1/BRCA2 variants PARP inhibitors Data from phase I and II trialsGATA3 mutation Endocrine therapy Retrospective analysis of trialsMDM2 ampli�cation MDM2 inhibitors Preclinical evidenceTP53 mutation Sensitivity to chemotherapy Retrospective analysis of trialsHER2 mutation HER2 inhibitors Preclinical evidenceCCND1/ CDK4/CDK6 ampli�ed CDK4/6 inhibitors Preclinical evidenceAKT1 mutation AKT inhibitors Preclinical evidenceIGF1R ampli�cation IGFR inhibitors Preclinical evidence

HER2-enriched HER2 ampli�cation or HER2 IHC+ HER2 inhibitors Data from phase III trialsHER2 mutation HER2 inhibitors Preclinical evidencePIK3CA mutation PI3K inhibitors Data from phase I and II trialsPTEN mutation/loss PI3K/mTOR inhibitors Retrospective analysis of trialsPIK3R1 mutation PI3K inhibitors Preclinical evidenceFGFR4 ampli�cation FGFR inhibitors Preclinical evidence

Basal-like Germline BRCA1/BRCA2 mutation PARP inhibitors Data from phase I and II trialsNOTCH1/NOTCH3 ampli�cation/mutation γ-Secretase inhibitors Preclinical evidenceAKT3 ampli�cation AKT inhibitors Preclinical evidenceEGFR ampli�cation EGFR inhibitors Preclinical evidenceNF1 deletion, KRAS ampli�cation MEK inhibitors Phase I and II trials in other diseasesTP53 mutation Sensitivity to chemotherapy Retrospective analysis of trialsPIK3R1/PTEN/INPP4B mutation/loss PI3K inhibitors Preclinical evidenceMET ampli�cation/mutation MET inhibitors Preclinical evidence

Breast cancer intrinsic subtypesKatherine A. Hoadley, Fabrice Andre, Matthew J. Ellis and Charles M. Perou

Breast cancer consists of many diseases. This heterogeneity is visible at the histological, clinical, genetic and genomic level. Genomic studies have identified four intrinsic subtypes of breast cancer: basal-like, luminal A and B, and HER2-enriched. The basal-like tumours are identified by high expression of KRT5/6A, ID4, and FOXC1 (basal epithelial-like cluster). The luminal epithelial-like cluster is characterized by high expression of ER, GATA3, XBP1, and FOXA1. Luminal A tumours have the highest expression of luminal epithelial genes when compared

with luminal B tumours; luminal A and B tumours show, respectively, low and high proliferation rates. The HER2-enriched subtype, although expressing the luminal-epithelial cluster, is defined by amplification of genes on 17q12 including HER2/ERBB2. Recent studies have described the somatic mutations and DNA copy-number landscape of breast cancers, showing a good concordance between these genetic alterations and the genetic intrinsic subtypes. Here, we present an overview of the common genetic and genomic events seen in breast tumours.

Abbreviations amp amplificationAPOBEC apolipoprotein B mRNA editing enzymedel deletionER oestrogen receptorER+ oestrogen receptor positiveER– oestrogen receptor negativeHER2+ HER2 positiveHER2– HER2 negativeHER2-E HER2-enriched molecular subtypeIHC immunohistochemistryKRT cytokeratinmut mutation pCR pathological complete responsePR progesterone receptor PR+ progesterone receptor positive TCGA The Cancer Genome AtlasTIL tumour-infiltrating lymphocyteTNBC triple-negative breast cancer

References1. Perou C. M. et al. Molecular portraits of human breast tumours. Nature 406, 747–752 (2000).2. Sorlie, T. et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc. Natl Acad. Sci. USA 100, 8418–8423 (2003).3. Prat, A. et al. Prognostic significance of progesterone receptor-positive tumor cells within immunohistochemically defined luminal a breast cancer. J. Clin. Oncol. 31, 203–209 (2013).4. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012).5. Roberts, S. A. et al. An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat. Genet. 45, 970–976 (2013).6. Prat, A. et al. Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study. Clin. Cancer Res. 20, 511–521 (2014).7. Cheang, M. C. et al. Responsiveness of intrinsic subtypes to adjuvant anthracycline substitution in the NCIC.CTG MA.5 randomized trial. Clin. Cancer Res. 18, 2402–2412 (2012).

8. Hayes, D. F. et al. HER2 and response to paclitaxel in node-positive breast cancer. N. Engl. J. Med. 357, 1496–1506 (2007).9. Rouzier, R. et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin. Cancer Res. 11, 5678–5685 (2005).10. Prat, A. et al. Molecular characterization of basal-like and non-basal-like triple-negative breast cancer. Oncologist 18, 123–133 (2013).11. Harrell, J. C. et al. Genomic analysis identifies unique signatures predictive of brain, lung, and liver relapse. Breast Cancer Res. Treat. 132, 523–535 (2012).12. Huo, D. et al. Population differences in breast cancer: survey in indigenous African women reveals over-representation of triple-negative breast cancer. J. Clin. Oncol. 27, 4515–4521 (2009).13. Adams, S. et al. Prognostic value of tumor-Infiltrating lymphocytes in triple-negative breast cancers from two phase III randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199. J. Clin. Oncol. 32, 2959–2966 (2014).14. Hoadley, K. A. et al. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin. Cell 158, 929–944 (2014).

AffiliationsLineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, USA (K. A. Hoadley, C. M. Perou). Department of Medical Oncology, Gustave Roussy Cancer Centre, France (F. Andre). Lester and Sue Smith Breast Center, Baylor College of Medicine, Texas, USA (M. J. Ellis).

Edited by Alessia Errico. Designed by Laura Marshall.

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