her2 heterogeneity otto metzger filho, md susan f. smith center for women’s cancers dana-farber...
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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
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