1. HER2 Heterogeneity Otto Metzger Filho, MD
Susan F. Smith Center for Women’s Cancers
Dana-Farber Cancer Institute
Harvard Medical School
Boston, USA

2. HER2+ DISEASE: MAJOR CLINICAL ADVANCES and MAJOR DIAGNOSTIC CHALLENGES
1998
Trastuzumab
Approved
2002
First Preoperative
Trials Reported Paving
The Way For Use in
Early Stage Disease
2005
Three Large
Adjuvant Trials
Reported
Lapatinib
2007-
2008
Initial Trials
of T-DM1,
Neratinib
2010
Preoperative
Trials of
Dual Blockade
Pertuzumab
2012
2013
T-DM1
1. HER2 assessment
2. Intratumor heterogeneity
3. Intertumor heterogeneity
THREE
THEMES

3. Challenges in the diagnosis of HER2+ breast cancer

4. Poor reproducibility in measuring the target in the ALTTO trial (central vs local lab)
8000 women worlwide ER
PgR
13 % ER
PgR
8 %
21 %
22 %
Central lab
U.S. :
discordant
results
Central lab
Rest of the world :
discordant
results
26 %
HER2 (IHC)
25 %
HER2 (FISH)
HER2 IHC
HER2 FISH
19 %
12 %

5. Important Messages from 2013 HER2 Testing Tumor Specimens to be Tested
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
The 2007 guideline stated that the resection specimen was the preferred sample for HER2 testing. The thinking was that the re-sected tumor sample would provide more tumor tissue for analysis. Since that time, we have clearly seen an increasing use of the initial diagnostic needle core biopsy for breast predictive factor testing. One advantage of the needle core biopsy would be a negligible ischemic time for the tissue and rapid tissue fixation.
The panel felt that the core biopsy was an acceptable sample for the initial HER2 analysis, however repeat testing on the excision may be necessary if a HER2 result is negative on the core in certain circumstances:
Tumor is grade III
Amount of invasive tumor in the core biopsy is small
Resection specimen contains high grade carcinoma that is morphologically distinct from that in the core
There is doubt about specimen handling of the core biopsy:
Long ischemic time,
Short time in fixative,
Different/alternative fixative used, and/or
Test is suspected by the pathologist to be negative based on testing error
Some of the limitations of using core biopsies for HER2 testing are shown on the next slide.
Consider repeat core bx for equivocal cases when preoperative therapy is considered
1. Wolff AC, et al., Arch pathol lab med. 2007;131:18-43.
2. Wolff AC, et al. Arch pathol lab med. 2014;138:

6. 2013 HER2 Testing in BC Guideline Update Tumor Specimen Selection
Core samples may not be optimal in some situations
HER2 IHC stain obtained by core needle biopsy
Heterogeneity
Intratumoral Heterogeneity
(HER2 IHC)
Crush(HER2 IHC)
This slide explains the limitations of using needle core biopsies for HER2 testing in breast cancer.
Edge Artifact (HER2 IHC)

7. Chromosomal Abnormalities involving CEP17 (Aneusomy)
Chromosome 17-polysomy is a rare event
Chromosome 17
Polysomy 17 = increased copy number of HER2 & CEP17 signals
Most frequently defined as average CEP17# >3 by ISH
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 region
Typically high grade tumor and HER2 IHC is (2+) or (3+)
CEP17
HER2
HER2 FISH
Some breast cancers demonstrate an abnormal number of copies of chromosome 17 within the tumor cells. This is referred to as chromosome 17 polysomy in the literature. This is defined in ISH assays as an average of > 3 copies of chromosome 17 within tumor cells (CEP17 > 3); this exists in 5 – 10% of breast cancers (Tubbs et al. 2007, Downs-Kelly et al. 2005).
Using the average number of CEP17 to define polysomy assumes that the number of CEP17 signals directly reflects the number of copies of chromosome 17 in the tumor cells and those tumors with increased CEP17 signals display chromosome 17 polysomy. Each extra copy of chromosome 17 in such cases would be expected to have a copy of the HER2 gene, which results in an overall increase in the average number of copies of HER2 genes. The HER2/CEP17 ratio corrects the number of HER2 copies for the increased number of chromosome 17 and the resulting ratio may be greater than or less than 2. For such cases, a ratio of < 2 has been interpreted as non-amplified for HER2. It is interesting that patients with a HER2/CEP17 ratio > 2 appear to benefit from HER2 targeted therapy regardless of the chromosome 17 copy number (Perez et al. 2010).
Recent pangenomic studies have demonstrated that true polysomy 17 is rare (Tse et al. 2011, Yeh et al Mod Pathol, Moelans et al Breast Cancer Res Treat) and in some cases, CEP17 may undergo gene amplification events in the same way as HER2 or other gene loci. Breast tumors with a HER2/CEP17 ratio <2, where the average HER2 copy number is > 6 and the average CEP17 copy number is > 3 may, in fact, represent HER2 positive cancers with amplification of both HER2 and CEP17 (Tse et al. 2011). In support of this, cases with these findings frequently are high grade and show over-expression of HER2 protein (2+ or 3+) by IHC.
HER2 IHC
Hanna, et al., Mod Pathol Jan;27(1):4-18, Tse, et al., J Clin Oncol Nov 1;29(31):

8. 2013 HER2 Testing in BC Guideline Update Changes to Testing Algorithm Help Address Chromosomal Abnormalities involving CEP17 (Aneusomy)
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)
Although increases in CEP17 copy number have been interpreted as representing polysomy 17, recent pangenomic studies have demonstrated that true polysomy 17 is rare (Tse et al. 2011, Yeh et al Mod Pathol, Moelans et al Breast Cancer Res Treat) and in some cases, CEP17 may undergo gene amplification events in the same way as HER2 or other gene loci. Breast tumors with a HER2/CEP17 ratio <2, where the average HER2 copy number is > 6 and the average CEP17 copy number is > 3 may, in fact, represent HER2 positive cancers with amplification of both HER2 and CEP17 (Tse et al. 2011).
Based on this emerging literature, the ASCO/CAP panel concluded in the 2013 update that if the average HER2 copy number was > 6 then the HER2 test result must be reported as positive, regardless of the HER2/CEP17 ratio. Furthermore, in such cases, repeat ISH analysis for HER2 with an alternative chromosome 17 reference probe in such cases (such as RARA, or TP53) will frequently demonstrate a HER2-to-chromosome 17 ratio that is in the amplified range, supporting the idea that the HER2 gene and the centromere is co-amplified in these cases. It is important to carefully review all the pathologic features (grade, proliferative index, ER/PR results and HER2 IHC) for such cases before making a final determination of the HER2 status. Clearly, more investigation of the clinical outcomes for such patients is needed, particularly for patients whose HER2 status may be reclassified as amplified rather than non-amplified due to polysomy 17.

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

10. Intratumor Heterogeneity

11. 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

12. Guidelines Definition of HER2 heterogeneity
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.
Vance, G.H., et al. Arch Pathol Lab Med, (4): p
Walker, R.A., et al. J Clin Pathol, (7): p

13. Example of HER2 heterogeneity analyzed by different methods
Figure 1: Heterogeneous tumor analyzed by different methods
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)
Viale et al. Modern Pathology 2013

14. The true prevalence of HER2 heterogeneity is unknown
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: , 2011.

15. 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
~ 50%: high ratios (>4)
IHC 3+
No heterogeneity (80 to 100% positive cells).
~ 50%: low ratios (2 to 3)
IHC often 2+
Heterogeneous expression
Ohlschlegel, C, et al. J Clin Pathol 64: , 2011.

16. What is the clinical significance of HER2 heterogeneity?
Interpretation of data from retrospective series is challenging
The impact of HER2 heterogeneity on response to anti-HER2 therapies is unknown
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

17. 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

18. Ongoing Effort to Elucidate the Impact of HER2 heterogeneity
N = 160 pts
Centrally-reviewed HER2+ early stage BC
Stage II or III
6 cycles of T-DM1 + Pertuzumab 
weeks
MANDATORY Research biopsies performed in different geographic regions of the tumor and marked with different clips
Legend
Tumor area
Bx site 1 and research clip
Bx site 2 and research clip
MANDATORY specimen collection after the surgical procedure (i.e. mastectomy or breast conserving surgery)
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

19. Study Design Core bx site 1
Residual tumor: similar evaluation for core bx site 1 and 2
50 cells/area
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 19 19

20. “Intertumor” Heterogeneity

21. Genomic Analysis of HER2+ BC reveals significant heterogeneity Results from BOLERO-1 and BOLERO-3 samples and TCGA
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
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.

22. Hormone Receptor Positive and Negative HER2+ breast cancer have distinct pattern of somatic alterations
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
<
GATA3
9.7
0.03
Abbreviations: FDR, False discovery rate

23. 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):
Eichhorn PJ. Cancer Res. 2008;68(22):
Migliaccio I. San Antonio Breast Cancer Symposium; 2008.
Nagata Y. Cancer Cell. 2004;6(2):
Zhao JJ. Proc Natl Acad Sci U S A. 2006;103(44):
Serra V. Cancer Res. 2008;68(19):

24. HER2 blockade
What have we learned from CLEOPATRA? CLEOPATRA: Trial Design
Docetaxel + trastuzumab
+ placebo
HER2-positive MBC
(53% no prior chemo
10% prior trastuzumab)
1:1
Docetaxel + trastuzumab
+ pertuzumab
End points
PFS and OS
quality of life
biomarker analysis
N=800

25. CLEOPATRA: Shorter Median PFS Observed with Mutated PIK3CA
Placebo + T + D
PERTUZUMAB + T + D
PIK3CA
status n
Events
Median
(mo)
Hazard
ratio
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
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

26. CLEOPATRA: Shorter Median PFS Observed with Mutated PIK3CA While Treatment Effect is Maintained
Pla + T + D
PERTUZUMAB + T + D
PIK3CA
status n
Events
Median
(mo)
Hazard
ratio
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
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

27. A meta-analysis of 4 clinical trials
What is the impact of PIK3CA mutation status on response to neoadjuvant anti-HER2 therapy?
A meta-analysis of 4 clinical trials
GeparQuinto
GeparSixto
Neo-Altto
CHERLOB

28. PIK3CA Mutation analysis (Exon 9 & 20) in the overall study cohort (n=967 pts)
PIK3CA mutation rate 21.7%

29. The frequency of PIK3CA mutation is equally distributed among HR+ and HR- patients
Overall
HER2+ HR+
HER2+ HR-
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%

30. pCR rate according to PIK3CA mutation status overall and by HR status
Studienkonzept G5
pCR in the mutants
pCR in the mutants
 pCR in the mutants

31. pCR according to PIK3CA mutation and anti-HER2 treatment
L T L+T
wt: Lap n=10 (18.6%), Trast n=19 (33.3%), All n=89 (37.1%)
Mt: Lap n=19 (17.6%), Trast n=10 (18.2%), All n= 40 (17.0%)
Ucrmdrehen zuerst mutiert, dann wt
p Rate überprüfen
L = Lapatinib
T = Trastuzumab
N=210
N=757
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

32. Preliminary results demonstrating that blockade of PI3K mTOR signaling pathway adds benefit in HER2+ BC
BOLERO-1
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) R
2:1
BOLERO-3
Locally advanced or metastatic HER2+ breast cancer
Prior taxane required
TRAS resistant
EVE + TRAS + VNB
(n = 284)
PBO + TRAS + VNB
(n = 285) R
1:1
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, , Copyright (2014), with permission from Elsevier.
1. Andre F, et al. Lancet Oncol 2014; 15:

33. 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
Patients with PIK3CA mutation showed PFS benefit from everolimus whereas those with wildtype PIK3CA did not. Same trend in both studies
Pooled analysis: KM curve by treatment in the PIK3CA WT group
Pooled analysis: KM curve by treatment in the PIK3CA MUT group
PBO WT
EVE WT
P-value = 0.5
PBO MUT
EVE MUT
P-value = 0.05
Progression free survival probability
Progression free survival probability
Time, months
Time, months
PIK3CA Mutant (30.3% in combined population)
Population
Treatment N
Events
Median PFS mo
HR (95% CI)
BOL-1
PBO 19 17
7.56
0.70 ( )
EVE 39 30
11.96
BOL-3 42 37
5.68
0.65 ( ) 29 21
6.93
BOL-1 + BOL-3 61 54
0.67 ( ) 68 51
PIK3CA Wild type (69.7% in combined population)
Population
Treatment N
Events
Median PFS mo
HR (95% CI)
BOL-1
PBO 48 31
17.08
1.13 ( )
EVE 89 51
18.46
BOL-3 82 59
6.57
1.08 ( ) 77 57
6.77
BOL-1 + BOL-3
130 90
1.1 ( )
166
108
Abbreviations: EVE, everolimus; MUT, mutant; PBO, placebo; WT, wildtype.

34. Correlation of PFS and PTEN expression
Patients with low/no PTEN benefited significantly from everolimus, whereas those with normal PTEN did not. Consistent results in both studies
Pooled analysis: KM curve by treatment in the PTEN normal group
Pooled analysis: KM curve by treatment in the PTEN low group
PBO normal
EVE normal
P-value = 0.97
PBO low
EVE low
P-value = 0.035
Progression free survival probability
Progression free survival probability
Time, months
Time, months
PTEN low/loss (14.4% in combined population)
Population
Treatment N
Events
Median PFS mo
HR (95% CI)
BOL-1
PBO 18 12
16.82
0.56 ( )
EVE 31 17
23.46
BOL-3
1 5
5.45
0.52 ( ) 11
9.53
BOL-1 + BOL-3
3 3 24
0.54 ( )
4 6 28
PTEN normal (85.6% in combined population)
Population
Treatment N
Events
Median PFS mo
HR (95% CI)
BOL-1
PBO 90 53
13.80
1.02 ( )
EVE
163 87
16.10
BOL-3
110
6.74
1 ( )
10 7 78
6.83
BOL-1 + BOL-3
20 0
140
1 ( ) 
27 0
165
Abbreviations: EVE, everolimus; PBO, placebo.

35. 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

36. Phase Ib study of taselisib in combination with anti-HER2 agents
T-DM1 + placebo
Cohort A1 TRASTUZUMAB + PERTUZUMAB + TASELISIB
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
Cohort B T-DM1 +TASELISIB 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
Expansion phase
Two cohorts (A1/2 and B)
20 pts each
Baseline bx required
Results in 2014?
PI Metzger and Krop

37. 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
Published Online 24 August 2015

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

39. Thank you