Predictors of HER2 FISH amplification
in immunohistochemistry score 2+
infiltrating breast cancer:
a single institution analysis
Maria Vittoria Dieci1, Elena Barbieri1, Stefania Bettelli², Federico
Piacentini1, Guido Ficarra², Sara Balduzzi1, Massimo Dominici1,
PierFranco Conte1, Valentina Guarneri1
1Department
of Oncology, Hematology and Respiratory Diseases,
and ²Department of Pathology, University Hospital, Modena, Italy
Background
Herbst. Int J Radiat Oncol Biol Phys. 2004;59(suppl):21; Roskoski. Biochem Biophys Res Commun. 2004;319:1; Rowinsky. Annu Rev Med. 2004;55:433
Background (cont’d)
 HER2 amplification is the primary mechanism for overexpression
 HER2 is amplified in 18-20% of infiltrating breast cancers
 HER2 overexpression is associated with higher risk of recurrence and
death, relative resistance to endocrine therapy, apparent lesser benefit
from certain chemotherapeutic regimens
 The MoAb antiHER2 Trastuzumab was FDA-approved in 1998 for
treatment of metastatic disease
 Adjuvant trastuzumab reduces the risk of recurrence and death by 1/2
and 1/3, respectively, in patients with early stage, high-risk HER2 positive
breast cancers
 One year trastuzumab costs: $70,000-$110,000
The Need For Accurate Testing
 Various strategies to determine or confirm HER2
expression have been used in clinical trials
 Correct HER2 assessment is critical for patients
and clinicians since anti-HER2 therapies are
effective in HER2+ disease only
HER2 expression
Normal 0
Normal 1+
Normal
Normal
Abnormal 2+
Abnormal 3+
Abnormal low
amplification
Abnormal high
amplification
IHC Images by Kornstein, MD, Medical College of Virginia
From FDA to ASCO/CAP
 For IHC: the cut-off for 3+ score was raised from 10% to
30% of invasive tumor cells with uniform and intense
membrane staining
 For FISH amplification: a HER2/CEP17 ratio greater
than 2.2, rather than equal or greater than 2.0
 Recent studies have shown that the concordance
between FISH and IHC is higher when the cut-off level to
define HER2 3+ score is > 30%
As a consequence:
 a higher proportion of breast cancer specimens are scored
IHC 2+
 but only 24% of the IHC 2+ tumors have gene amplification
when tested by FISH
 the request for FISH assay is increasing
Mass. Proc Am Soc Clin Oncol. 2000; Shah, Hum Pathol 2010
Endpoint
To evaluate if routinely assessed pathologic
parameters such as tumor grade, hormone
receptor status and Ki67 are able to
predict FISH results in patients with an
equivocal IHC HER2 score 2+
Tissue specimens
• 480 infiltrating breast cancer with IHC
2+ and evaluable HER2/CEP17 ratio
ANTIBODY: Novocastra Laboratories, clone CB11 until June 2008 and Ventana, clone 4B5 from
July 2008
PROBE: PathVysion HER-2 DNA Probe Kit (Vysis Inc., Downers Grove, IL)
• 96% were primary surgical samples or core
biopsy for patients undergoing neoadjuvant
therapy
• 4% were samples from bilateral breast cancer
or metastases biopsy
Tumor characteristics
Samples
480
Hormone Receptor expression
Negative (ER and PgR)
Positive (ER and/or PgR)
Not evaluable
44
433
3
9.2%
90,2%
0.6%
56
420
4
11.7%
87.5%
0.8%
133
345
2
27.7%
71.9%
0.4%
156
311
13
23.5% (0-98)
32.5%
64.8%
2.7%
153
268
59
31.9%
55.8%
12.3%
Estrogen Receptor expression
Negative (<10%)
Positive (>10%)
Not evaluable
Progesteron Receptor expression
Negative (<10%)
Positive (>10%)
Not evaluable
Ki67 expression
Low proliferation (<15%)
High proliferation (>15%)
Not evaluable
Median Ki67 expression (range)
Histologic Grade
G1-2
G3
Not evaluable
Distribution of FISH results
ASCO/CAP
FDA
Negative
(<1.8)
332
(69.2%)
Negative
(<2.0)
348
(72.5%)
Borderline
(1.8-2.2)
48
(10.0%)
Positive
(>2.2)
100
(20.8%)
Positive
(>2.0)
132
(27.5%)
Distribution of FISH results by G, HR and Ki67
(ASCO/CAP criteria)
G1-2
G3
HR -ve
HR +ve
Low Ki67
High Ki67
N
Negative
(<1.8)
Borderline
(1.8-2.2)
Positive
(>2.2)
153
126
(82%)
13
(9%)
14
(9%)
268
166
(62%)
30
(11%)
72
(27%)
44
33
(75%)
3
(6.8%)
8
(18%)
433
297
(68.6%)
45
(10.4%)
91
(21%)
156
117
(75%)
20
(13%)
19
(12%)
206
(66%)
26
(8%)
79
(25%)
311
p
value
0.000
0.638
0.003
Distribution of FISH results by G, HR and Ki67
(FDA criteria)
G1-2
N
Negative
(<2.0)
Positive
(>2.0)
153
126
(82%)
27
(18%)
G3
268
178
(66%)
90
(34%)
HR –ve
44
35
(79.5%)
9
(20.5%)
HR +ve
433
311
(72%)
122
(28%)
Low Ki67
156
123
(79%)
33
(21%)
214
(69%)
97
(31%)
High Ki67
311
p
value
0.000
0.274
0.022
OR for FISH positive test
ASCO/CAP and FDA criteria
ASCO/CAP
OR for
FISH
positivity
G1-2
Ref.
95% CI
1.97-6.72
G3
Low Ki67
G1-2
Ref.
G3
Ref.
2.45
FDA
OR for
FISH
positivity
0.000
3.64
Low Ki67
1.42-4.22
High Ki67
p
value
p
value
1.45-3.83
0.001
1.07-2.65
0.023
2.35
Ref.
0.001
High Ki67
95% CI
1.68
Discussion & Conclusions
 While the association between HER2 status and pathological and
molecular characteristics has been largely documented in literature,
the possibility to predict HER2 amplification in HER2 equivocal (IHC
2+) cases has been less extensively studied
 This is the largest single-institution series of HER2 IHC equivocal
breast cancer specimens tested by FISH and analyzed to identify
potential predictors for FISH amplification
 This is the first report on the role of Ki67 index in such contest
 On a series of 480 HER2 equivocal samples, both tumor grade and
proliferation were found to be significantly associated with HER2
FISH amplification, according to the FDA and ASCO/CAP guidelines