HER2 Heterogeneity

Otto Metzger Filho, MDSusan F. Smith Center for Women’s Cancers

Dana-Farber Cancer InstituteHarvard Medical School

Boston, USA

HER2+ DISEASE: MAJOR CLINICAL ADVANCES and MAJOR DIAGNOSTIC CHALLENGES

1998

TrastuzumabApproved

2002

First PreoperativeTrials Reported Paving

The Way For Use inEarly Stage Disease

2005

Three LargeAdjuvant Trials

Reported

2005

LapatinibApproved

2007-2008

Initial Trialsof T-DM1,Neratinib

2010

PreoperativeTrials of

Dual Blockade

PertuzumabApproved

2012

2013

T-DM1Approved

1. HER2 assessment 2. Intratumor heterogeneity3. Intertumor heterogeneity

THREE

THEMES

Challenges in the diagnosis of HER2+ breast cancer

Poor reproducibility in measuring the target in the ALTTO trial (central vs local lab)

8000 women worlwide

Central lab Rest of the world :

discordant results

21 % 22 %

19 % 12 %

8 %PgR

13 %

26 %HER2 (IHC)

25 %HER2 (FISH)

Central labU.S. :

discordant results

ER ER PgR

HER2 IHC HER2 FISH

2007 Guidelines1

• Resection specimens preferred sample for

HER2 testing

• More representative sample of the patient’s

tumor, more tumor tissue for evaluation

2013 Guideline Update2

• Increasing use of core for testing

• Core biopsies can be used for initial test (likely better

pre-analytics)

• Repeat testing on the excision may be necessary if a HER2 result is negative on

the core in certain circumstances

Important Messages from 2013 HER2 Testing

Tumor Specimens to be Tested

1. Wolff AC, et al., Arch pathol lab med. 2007;131:18-43.

2. Wolff AC, et al. Arch pathol lab med. 2014;138:241-256.

Consider repeat core bx for equivocal cases when preoperative therapy is considered

2013 HER2 Testing in BC Guideline Update

Tumor Specimen Selection

Core samples may not be optimal in some situations

HeterogeneityHER2 IHC stain obtained by core

needle biopsy

Intratumoral Heterogeneity

(HER2 IHC)

Crush(HER2 IHC) Edge Artifact (HER2 IHC)

Chromosomal Abnormalities involving CEP17 (Aneusomy)Chromosome 17-polysomy is a rare event

• Polysomy 17 = increased copy number of HER2 & CEP17 signals

o Most frequently defined as average CEP17# >3 by ISH

o HER2/CEP17 <2 (not amplified)

• aCGH studies have shown true chromosome 17-polysomy is rare

• CEP17 copy number >3.0 in ISH is frequently related to gain or amplification of the centromeric regiono Typically high grade tumor and

HER2 IHC is (2+) or (3+)

Chromosome 17

HER2

CEP17

HER2 FISH

HER2 IHC

Hanna, et al., Mod Pathol. 2014 Jan;27(1):4-18, Tse, et al., J Clin Oncol. 2011 Nov 1;29(31):4168-74

• Co-amplification of CEP17 region is observed in many ISH assays with increased HER2 and CEP17 copy number

– May lead to a HER2/CEP17 ratio < 2.0 suggesting lack of HER2 amplification and discordant IHC/ISH results

– If the HER2 copy number is >6, the HER2 test result must be reported as Positive regardless of the HER2/CEP17 ratio

– HER2 amplification defined by ratio criterion (>2), HER2 copy number criterion( >6) or both

• Some labs may choose to repeat HER2 testing in the same specimen using an alternative chromosome 17 reference probe (another gene on chromosome 17 not expected to co-amplified with HER2) to help demonstrate an amplified ratio (>2)

2013 HER2 Testing in BC Guideline Update Changes to Testing Algorithm Help Address Chromosomal

Abnormalities involving CEP17 (Aneusomy)

Oncotype Dx data 36 HER2 positive cases by both IHC and FISH (amplified)

10 reported as positive

12 reported as equivocal

14 reported as negative

Therefore, there was a 39% (14/36) false negative rate. Unfortunately, some patients did not receive HER2 targeted therapy due to the negative Oncotype DX result.

4 had low RS and 7 had intermediate RS – likely incorrect due to wrong HER2 score

Dabbs, DJ, et al. J Clin Oncol 29:1-7, 2011.Bartlett, JMS et al. J Clin Oncol 29: 2011.

mRNA techniques should not be used to diagnose HER2+ breast cancer in clinical practice

Intratumor Heterogeneity

Definition of Heterogeneity

Heterogeneous amplification of HER2 includes the existence of 2 distinct clones of breast cancer cells with different patterns of gene amplification (usually 1 clone amplified and 1 clone non-amplified)

Viale et al. Modern Pathology 2013

The CAP guideline [1] suggests that all cases with between 5% and 50% of cells with HER2/CEP17 ratios greater than 2.2 be regarded as heterogeneous amplified

In the UK guideline [2] HER2 assessment should be followed by scanning the entire tumor section before selecting at least three separate tumor fields and counting the number of chromosome 17 (CEP17) and HER2 signals.

1. Vance, G.H., et al. Arch Pathol Lab Med, 2009. 133 (4): p. 611-2.2. Walker, R.A., et al. J Clin Pathol, 2008. 61 (7): p. 818-24

Guidelines Definition of HER2 heterogeneity

Example of HER2 heterogeneity analyzed by different methods

1. Viale et al. Modern Pathology 2013

IHC staining showing clusters of HER2+ cells (brown

staining)

Double FISH/IHC showing HER2 protein expression (green) and HER2 gene

amplification (red)

Dual-color in situ hybridization (HER2 black,

chromosome 17 red)

Figure 1: Heterogeneous tumor analyzed by different methods

• HER2 heterogeneity is most common in carcinomas that would be classified as equivocal by IHC:

IHC Heterogeneous cases

0 13%1+ 13%2+ 27% 3+ 11%

The overall frequency of heterogeneity was 15%.

Ohlschlegel, C, et al. J Clin Pathol 64:1112-1116, 2011.

The true prevalence of HER2 heterogeneity is unknown

~ 50%: high ratios (>4) IHC 3+ No heterogeneity (80 to 100%

positive cells).

~ 50%: low ratios (2 to 3)IHC often 2+Heterogeneous expression

The results depicted below describe what we see in clinical practice: Cases where HER2 positivity is clear and cases

where the interpretation of results is challenging

Ohlschlegel, C, et al. J Clin Pathol 64:1112-1116, 2011.

1. Interpretation of data from retrospective series is challenging

2. The impact of HER2 heterogeneity on response to anti-HER2 therapies is unknown

What is the clinical significance of HER2 heterogeneity?

?

Is HER2 heterogeneity providing us with a clear picture of

different biologic processes ?

Is HER2 heterogeneity a consequence of imperfect HER2

assessment techniques ?

HER2 heterogeneity

Main Messages - I

• Strict adherence to guidelines is necessary when evaluating HER2 status

• Close communication with pathologists is key

• HER2 assessment should be interpreted with caution and taking into consideration the clinical context. E.g. A dubious HER2 test in a tumor strongly ER+ and low histologic grade is different from a dubious HER2 test in a tumor with low ER+ PgR negativity and high histologic grade

• The diagnosis of HER2 heterogeneity is not easy and guidelines are in somehow confusing

• The prognostic and predictive implications of HER2 heterogeneity remains to be defined

• Additional studies are needed

N = 160 ptsCentrally-reviewed HER2+ early stage BC Stage II or III

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 weeks

MANDATORY Research biopsies performed in different geographic regions of the tumor and marked with different clips

MANDATORY specimen collection after the surgical procedure (i.e. mastectomy or breast conserving surgery)

6 cycles of T-DM1 + Pertuzumab

Legend Tumor area Bx site 1 and research clip Bx site 2 and research clip

Ongoing Effort to Elucidate the Impact of HER2 heterogeneity

Hypothesis: Anti-HER2 therapy may not be sufficient if HER2-negative subclones (i.e. HER2 heterogeneity) are present within a HER2+ tumor

PI Metzger and Krop

Core bx site 1

Core bx site 2

HER2 heterogeneity definition follows CAP guideline Central pathology assessment for heterogeneity will be performed by Dr. Giuseppe Viale

at the European Institute of Oncology, Milan Correlative science will be performed at Kornelia Polyak Lab, DFCI

Study Design

Residual tumor: similar evaluation for core bx site 1 and 2

50 cells/area

“Intertumor” Heterogeneity

Genomic Analysis of HER2+ BC reveals significant heterogeneityResults from BOLERO-1 and BOLERO-3 samples and TCGA

TCGA filtering: remove variants with 0 or 1 hits in COSMIC, remove amp/del of unknown significance (similar to FMI).Abbreviations: TCGA, The Cancer Genome Atlas.1. Cancer Genome Atlas Network. Nature. 2012;490:61-70.

• Common mutations are very similar between BOLERO-1 and BOLERO-3

• Genetic profiles of BOLERO-1 and BOLERO-3 samples are comparable with TCGA1

– Only 111 HER2+ samples from TCGA are available for comparison

TP53 and PIK3CA gene mutations are the most frequent alterations beyond HER2 amplification

22

FGFR1 amplification is highly and FGF3/4/19 amplification are moderately enriched in HER2+, HR+ BC

Locus/genes HR+ (%) HR– (%) FDR

TP53 54.6 86.7 0.0001

CCND1/FGF3,4,19 22.5 8.7 0.001

FGFR1/ZNF703 16.7 2.7 < 0.0001

GATA3 9.7 2.7 0.03

Abbreviations: FDR, False discovery rate

Hormone Receptor Positive and Negative HER2+ breast cancer have distinct pattern of somatic alterations

HER2+ Heterogeneity: Focus on PIK3CA

Expression of a mutant PIK3CA or loss of PTEN in breast cancer cell lines is associated with trastuzumab resistance, and this resistance can be reversed with a PI3K inhibitor

PI3K is the dominant signaling network downstream of amplified HER2 such that HER2 is unable to transform mouse embryonic fibroblasts that lack a functional PI3K p110α subunit

In vitro studies of p110α inhibitors as well as pan-PI3Ki’s in breast cancer cell lines reveal that HER2 overexpression predicts sensitivity to these agents regardless of PIK3CA mutation status or PTEN expression

Berns K. Cancer cell. 2007;12(4):395-402. Eichhorn PJ. Cancer Res. 2008;68(22):9221-30.

Migliaccio I. San Antonio Breast Cancer Symposium; 2008.Nagata Y. Cancer Cell. 2004;6(2):117-27.

Zhao JJ. Proc Natl Acad Sci U S A. 2006;103(44):16296-300. Serra V. Cancer Res. 2008;68(19):8022-30

• End points– PFS and OS– quality of life– biomarker analysis

1:1 HER2-positive

MBC(53% no prior chemo

10% prior trastuzumab)

Docetaxel + trastuzumab + placebo

Docetaxel + trastuzumab + pertuzumab N=800

What have we learned from CLEOPATRA? CLEOPATRA: Trial Design

HER2 blockade

CLEOPATRA: Shorter Median PFS Observed with Mutated PIK3CA

Placebo + T + D PERTUZUMAB + T + D

PIK3CA

statusn Events Median

(mo) n Events Median(mo)

Hazardratio

Mut 90 63 8.6 86 45 12.5 0.64

WT 191 101 13.8 190

83 21.8 0.67

Overall 406 242 12.4 402

191 18.5 0.62

The prognostic impact of PIK3CA mutations cannot be attributed to a specific mutation, nor to mutation(s) in a specific exon, based on the available data set.– 182 mutations detected overall (32%)– Number of mutations in exon 7 = 12; exon 9 = 39; exon 20 = 131

Baselga J et al. Proc SABCS 2012;Abstract S5-1

CLEOPATRA: Shorter Median PFS Observed with Mutated PIK3CA

While Treatment Effect is Maintained

Pla + T + D PERTUZUMAB + T + D

PIK3CA

statusn Events Median

(mo) n Events Median(mo)

Hazardratio

Mut 90 63 8.6 86 45 12.5 0.64

WT 191 101 13.8 190 83 21.8 0.67

Overall 406 242 12.4 402 191 18.5 0.62

The prognostic impact of PIK3CA mutations cannot be attributed to a specific mutation, nor to mutation(s) in a specific exon, based on the available data set.– 182 mutations detected overall (32%)– Number of mutations in exon 7 = 12; exon 9 = 39; exon 20 = 131

Baselga J et al. Proc SABCS 2012;Abstract S5-1

GeparQuinto GeparSixto

Neo-Altto CHERLOB

What is the impact of PIK3CA mutation status on response to neoadjuvant anti-HER2 therapy?A meta-analysis of 4 clinical trials

PIK3CA Mutation analysis (Exon 9 & 20) in the overall study cohort (n=967 pts)

78.3

7.2

14.5

PIK3CA wt PIK3CA mut exon 9PIK3CA mut exon 20

PIK3CA mutation rate 21.7%

The frequency of PIK3CA mutation is equally distributed among HR+ and HR- patients

Overall HER2+ HR+ HER2+ HR-

Overall 21.7% 21.7% 21.7%

GEPARstudies n=504

21.4% 21.3% 21.6%

Neo-ALTTO n=355

22.5% 22.7% 22.4%

CHERLOB n=108 20.4% 20.9% 19.5%

pCR rate according to PIK3CA mutation status overall and by HR status

Overall HR-ve HR+ve0%

10%

20%

30%

40%

50%

16.2%

27.2%

7.6%

29.6%

36.4%

24,2%

PIK3CA mut PIK3CA wt

N=967 N=424 N=543

P interaction = 0.036P<0.001 P=0.125 P<0.001

pCR

pCR in the mutants

pCR in the mutants

pCR in the mutants

pCR according to PIK3CA mutation and anti-HER2 treatment

PIK3CA mut PIK3CA wt0%

10%

20%

30%

40%

50%

11.3%16.9%

20.3%27.1%

16.7%

39.1%

L T L+T

pCR

N=210 N=757

P<0.001P=0.384

• The magnitude of benefit of dual anti-HER2 blockade is greater in the PIK3CA wt subset when compared to PIK3CA mut subset

• In the advanced setting, dual anti-HER2 blockade provided (trastuzumab + pertuzumab) added benefit regardless of PIK3CA mutation status

L T L+T

L = LapatinibT = Trastuzumab

32

Abbreviations: EVE, everolimus; PBO, placebo; PFS, progression free survival; TRAS, trastuzumab; VNB, vinorelbine.Figure reprinted from Lancet Oncology, Vol. 15, André F, O'Regan R, Ozguroglu M, et al. Everolimus for women with trastuzumab-resistant, HER2-positive, advanced breast cancer (BOLERO-3): a randomised, double-blind, placebo-controlled phase 3 trial, 580-591,

Copyright (2014), with permission from Elsevier.1. Andre F, et al. Lancet Oncol 2014; 15: 580-91.

• Locally advanced or metastatic HER2+ breast cancer

• No prior therapy for advanced or metastatic disease (except endocrine therapy

EVE + TRAS + PAC(n = 480)

PBO + TRAS + PAC(n = 239)

R2:1

BOLERO-3

• Locally advanced or metastatic HER2+ breast cancer

• Prior taxane required• TRAS resistant

EVE + TRAS + VNB(n = 284)

PBO + TRAS + VNB(n = 285)

R1:1

BOLERO-1

Preliminary results demonstrating that blockade of PI3K mTOR signaling pathway adds benefit in HER2+ BC

PBO WTEVE WT

P-value = 0.5

PBO MUTEVE MUT

P-value = 0.05

PIK3CA Wild type (69.7% in combined population)

Population Treatment N Events Median PFS mo HR (95% CI)

BOL-1PBO 48 31 17.08

1.13 (0.72 - 1.78)EVE 89 51 18.46

BOL-3PBO 82 59 6.57

1.08 (0.75 - 1.56)EVE 77 57 6.77

BOL-1 + BOL-3

PBO 130 90

1.1 (0.83 - 1.46)

EVE 166 108

PIK3CA Mutant (30.3% in combined population)

Population Treatment N Events Median PFS mo HR (95% CI)

BOL-1PBO 19 17 7.56

0.70 (0.38-1.29)EVE 39 30 11.96

BOL-3

PBO 42 37 5.680.65 (0.38 -

1.11)EVE 29 21 6.93

BOL-1 + BOL-3

PBO 61 54

0.67 (0.45 - 1)

EVE 68 51

Patients with PIK3CA mutation showed PFS benefit from everolimus whereas those with wildtype PIK3CA did not. Same trend in both studies

Abbreviations: EVE, everolimus; MUT, mutant; PBO, placebo; WT, wildtype.

Time, monthsTime, months

Pro

gres

sion

free

sur

viva

l pro

babi

lity

Pro

gres

sion

free

sur

viva

l pro

babi

lity

Pooled analysis: KM curve by treatment in the PIK3CA WT group Pooled analysis: KM curve by treatment in the PIK3CA MUT group

Predictive biomarkers of everolimus efficacy in HER2+ advanced breast cancer: Combined exploratory analysis from BOLERO-1 and BOLERO-3 Correlation of PFS and PIK3CA genotype

Correlation of PFS and PTEN expression

34

Abbreviations: EVE, everolimus; PBO, placebo.

Patients with low/no PTEN benefited significantly from everolimus, whereas those with normal PTEN did not. Consistent results in both studies

PTEN normal (85.6% in combined population)Populati

onTreatme

nt N Events

Median PFS mo HR (95% CI)

BOL-1PBO 90 53 13.80 1.02 (0.72 -

1.43)EVE 163 87 16.10

BOL-3PBO 110 87 6.74

1 (0.73 - 1.35)EVE

107 78 6.83

BOL-1 + BOL-3

PBO200 140

1 (0.8 - 1.26) EVE

270 165

PTEN low/loss (14.4% in combined population)Populati

onTreatmen

t N Events

Median PFS mo HR (95% CI)

BOL-1PBO 18 12 16.82 0.56 (0.26 -

1.21)EVE 31 17 23.46

BOL-3PBO

15 12 5.45

0.52 (0.22 - 1.22)

EVE15 11 9.53

BOL-1 + BOL-3

PBO33 24

0.54 (0.31 - 0.96)

EVE46 28

Time, months Time, months

Pro

gres

sion

free

sur

viva

l pro

babi

lity

Pro

gres

sion

free

sur

viva

l pro

babi

lity

PBO normalEVE normal

P-value = 0.97

PBO lowEVE low

P-value = 0.035

Pooled analysis: KM curve by treatment in the PTEN normal group Pooled analysis: KM curve by treatment in the PTEN low group

Main Messages - II

• HER2+ breast cancer can be sub-classified into several subtypes using genomic data

• Pre-clinical data have consistently demonstrated the importance of PI3K signaling in HER2+ disease

• Data from Bolero-1 and Bolero-3 studies showed a trend in favor of everolimus for HER2+ tumors with features of PI3K alteration

• Subsequent studies including “modern” PI3K inhibitors are needed

• A more refined evaluation of HER2 heterogeneity coupled with PIK3CA evaluation is needed

Patients with HER2 metastatic breast cancer (any line)

STUDY DESIGN: 3 + 3

Three independent cohorts

Prior pertuzumab and T-DM1 allowed

T-DM1 + placebo

Cohort A2 TRASTUZUMAB PERTUZUMAB TASELISIB PACLITAXEL

T-DM1 + placeboCohort A1 TRASTUZUMAB + PERTUZUMAB +

TASELISIB

Cohort B T-DM1 +TASELISIB

Phase Ib study of taselisib in combination with anti-HER2 agents

Expansion phase Two cohorts (A1/2 and B)

20 pts each Baseline bx required

PI Metzger and Krop

β

δ

α

Receptors

γ

Ki = 0.29 nM

Ki = 9.1 nM (31x)

Ki = 0.12 nM (0.4x)

Ki = 0.97 nM (3.3x)

Taselisib

PI3K (p110) class I isoforms

Published Online 24 August 2015

STAR-FISH (specific-to-allele PCR-FISH)

- Combined detection of single-nucleotide and copy number alterations in single cells in intact archival tissue

- Assessed the clinical impact of changes in the frequency and topology of PIK3CA mutations and HER2 amplification within HER2+ BC during neoadjuvant chemotherapy

Results: Intratumoral topology of HER2 amplification and/or PIK3CA mutation status

Thank you