Quantitative Measurement of Human Epidermal Growth Factor Receptor-2 (HER2) Protein
Expression in ‘Classical’ and ‘Non-classical’ FISH Categories: a Comparative Study
Jian Shen, MD PhD1, Brandon Buscaglia,BS1, Hideki Goda, PhD, Loralee McMahon, MS, HTASCP1, Takako Natori, PhD2, Bradley Turner, MD1, Hisatake Okada, MR2, Yasushi Nakano, PhD2 and David G. Hicks , MD1
1Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA
2 Konica Minolta INC., Bio Health Care Business Development Division, Corporate R&D Headquarters, No. 1 Sakura-machi, Hino-shi Tokyo 191-8511, Japann (Average)
159% (Range)

Age(63.4)

Diagnosis (DX)
IDC
145
ILC
12Mixed2Nuclear Grade
17252
386
ER Allred Score
0-2 (-)
37
3-8 (+)
130

Parameter
ER/PR Statusn (Average)(27-89+)ER+/PR+

105

66.9

91.2
7.5
1.3

ER+/PRER-/PR+
ER-/PRHER2 IHC Score

173
32

10.8
1.9
20.4

0
1+
2+
3+
Tumor Size (cm)>52-5

1519102
23

9.4
11.9
64.2
14.5

1664

12.8
51.2

4.8
35.9
59.3
22.2
77.8

PR Allred Score
0-2 (-)
513-8 (+)
1161-2<130.569.5% (Range)

35
10200

450400180

Concordant Pos. PID

Concordant Pos. PID

160350300
250200150100140120100806040502000028.0
8.0Results

Discordant Neg. PID

Discordant Neg. PID

PID/ROI100µm2

HER2 FISH testing results in breast cancer can be
categorized based on the HER2/CEP17 ratio andHER2 gene copy number according to the ASCO/CAPguideline. Cases with both a HER2/CEP17 ratio ≥ 2and HER2 gene copy number ≥ 6 are considered‘classical amplified’ (group 1 and 2). The HER2 FISH
amplified cases with only either HER2/CEP17 ratio ≥2 or HER2 gene copy number ≥ 6 are considered‘non-classical’ amplified such as ‘low-level amplified’(group 3), “monosomy” (group 4 ), ‘polysomy ratio
negative’ (group 5 ) (Fig 3). Press et al., has reported
that ‘non-classical’ FISH amplified patients did notappear to have the same benefit from targeted therapy
in terms of DFS or OS as ‘classical’ amplifiedpatients. In the current study, we attempt to encompass
all possible differentiable FISH categories that could
be considered a unique subset. We expanded theHER2 FISH categories by adding ‘polysomy ratio
positive’ (group 2) (subgroup the “classical amplified”
cases with CEP17 > 2.7) (Table 2, Fig 3). We thenused streptavidin coated Phosphor-integrated dot
fluorescent nanoparticles (PID), a novel quantitative
methodology, to measure HER2 protein receptor
expression in each HER2 FISH category. We lookedfor the PID protein expression levels in different FISH
categories. We compared the ‘non-classical’ FISH
amplified categories to ‘classical’ amplified and‘classical’ non-amplified categories to determine
which categories were comparable (Table 2, Fig 1, 3).

Parameter
N TotalPID/CellBackground

51015
HER2 Copy #20051015
HER2 Copy #20

Figure 2: Discordant and concordant cases in “polysomy ratio positive’ category (group 2)

Table 1: Patient demographics and pathologic feature (N = 159)
Nuclear Grade (%)

Polysomy Ratio Negative(Group 5)

IHC Score (%)

Polysomy Ratio Positive (Group 2)
Low HER2 PID
High HER2 PID(Discordant cases)(Concordant cases)
1415
16.6 (SD 6.3)
202.6 (SD 50.3)(Group #)Study FISH Categories

NMeanAge123ER+ (%)

PR+ (%)

0-1+2+3+N
PID score @cell20
16.0 (SD 7.5)

Classic Amplified

22

61.2

0 (0.0)6 (30.0)

14 (70.0)

14 (63.6)

9 (40.9)

0 (0.0)9 (40.9)

13(59.1)

HER2 copy number (SD)

7.1 (1.8)

10.5 (4.5)

18.5 (SD 2.3)

(2)

Polysomy Pos. Ratio

29

65.7

1 (3.7)

3 (11.1)

23 (85.2)

23 (79.3)

20 (69.0)

3 (10.3)

18 (62.1)

8 (27.6)

CEP17 copy number

4.7

3.33.8(3)Low-Level Amplified

27

65.2

0 (0.0)7 (30.4)

16 (69.6)

19 (73.1)

19 (73.1)

3 (11.1)

24 (88.9)

0 (0.0)

Monosomy9

53.8

2 (25.0) 4 (50.0)

2 (25.0)

9 (100.0)

7 (77.8)

1 (11.1)

8 (88.9)

0 (0.0)(5)

Polysomy Neg. Ratio

20

63.8

0 (0.0)3 (17.6)

14 (82.4)

17 (85.0)

16 (80.0)

0 (0.0)19 (95.0)

1 (5.0)(6)

Equivocal

23

65.7

1 (4.8)12 (57.1)

8 (38.1)

18 (81.9)

18 (81.9)

1 (4.3)22 (95.7)

0 (0.0)

Ratio (SD)
Tumor size
Tumor grade
IHC 0-1 (%)
IHC 2 (%)
IHC 3 (%)

1.5 (0.2)
2.7
3.0
0 (0.0)19 (95.0)
1 (5.0)3.3 (1.5)
2.8
2.8
3 (21.4)
11 (78.6)
0 (0.0)5.3 (1.7)
3.7
2.8
0 (0.0)7 ( 46.7)
8 (53.3)(7)

Classic Non-Amplified

2962.13 (10.3) 17 (58.6)

9 (31.0)

22 (75.9)

19 (65.5)

26 (89.7)

2 (6.9)1 (3.4)(1)(4)

Table 2: Patient pathologic characteristics broken down in FISH categories.

Table 3: The Patient pathologic characteristics in FISH HER2 categories with polysomy (Group2 and 5). The discordant and concordant cases in group 2 can be sub-grouped.

HER2/CEP17 Ratio ≥ 2Group 1
ClassicalAmplified

Group 2Polysomy Pos.

Group 3
Low-Level
Amplified

HER2/CEP17 Ratio < 2Group 4
MonosomyGroup 5PolysomyGroup 6Equivocal(HER2 copy
number < 4)

(HER2 copy
number ≥ 6,
CEP17 copy
number ≥ 2.7)

(HER2 copy
number ≥ 4 and <6)Group 7
Classic NonAmplified(HER2 copy
number ≥ 6,
CEP17 copynumber ≥ 2.7)

(HER2 copy
number ≥ 4 and <6.0)

FISH +FISH +FISH +FISH +FISH+FISH Eq

FISH -PID +Mean: 167.7
SD: 148.5

PID +Mean: 112.6
SD: 126.1

PID –Mean: 15.4
SD: 10.2

PID –Mean: 12.5
SD: 3.8

PID –Mean: 16.3
SD: 7.5

PID –Mean: 8.5
SD: 6.5

PID –Mean: 6.3
SD: 7.3(HER2 copy
number ≥ 6,
CEP17 copy
number < 2.7)

(HER2 copy
number < 4)

Materials and Methods
159 invasive breast cancer cases were retrospectively
selected from the University of Rochester Medical
Center’s (URMC) FISH database based upon thediffering HER2 FISH results (Table 1). The cases had
previously undergone IHC and FISH analysis. PID
staining and quantitation was completed on all 159
cases for both PID/Cell and PID/ROI100µm2.

PID +Mean: 202.6
SD: 50.3

Figure 1 : HER2 protein expression in different FISH HER2 categories measured by PIDs

PID Mean: 16.6
SD: 6.3

Figure 3: Flow diagram of different FISH categories and corresponding PID results

The ‘non-classical’ amplified (groups 3, 4, 5) andequivocal categories (groups 6) were all found to have
low-levels of protein expression similar to group 7(‘classical’ non-amplified) categories. Their HER2
protein expression PID scores are below theexperimental threshold values, which were predictive ofa lower likelihood of a good pathologic response (RCB0 or 1) to Trastuzumab based neoadjuvant therapy
proposed in the previous study. (Figure 1).
The “polysomy ratio positive’ category (group 2)
showed that HER2 protein expression PID scores of thisgroup are comparable to the “classical” amplified
categories with significant variability (Fig 1 and 2). The
discordant (low PID) ‘polysomy ratio positive’ cases
showed distinct pathologic features such as HER2 IHC,average HER2/CEP17 ratio and average HER2 genecopy number that were comparable to ‘polysomy ratio
negative’ cases (Table 3, Fig 3).

Conclusions‘Non-classical’ FISH amplified categories (group 3, 4 ,5) and equivocal category (group 6) were all comparable
to the ‘classical’ non-amplified FISH category whenmeasured by PID. This suggests that these ‘nonclassical’ FISH amplified and equivocal categories may
be less likely to respond to targeted HER2 therapy. The“polysomy ratio positive’ category (group 2) examinedin this study is considered ”classical” amplified in thecurrent ASCO/CAP guideline report and most clinicalstudies. Group 2 PID results showed a subgroup of casesthat had low HER2 protein expression levels, whichindicate that there are a small percentage of polysomy“classical” amplified breast cancer cases may be lesslikely to respond to HER2 targeted therapy. These
controversial cases warrant follow up studies with alarger patient cohort and may be better categorized using
both FISH and quantitative HER2 protein analysis suchas PID.

1 - The authors of this abstract have indicated that they have no conflicts of interest that relate to the content of this abstract.
2 - The authors of this abstract have indicated the following conflicts of interest that relate to the content of this abstract: Dr.
Hideki Goda, Dr. Takako Natori, Hisatake Okada and Dr. Yasushi Nakano are employees of Konica Minolta.