Supplementary information

HER-2 and membrane EGFR protein expression and gene amplification

One core was lost in HER-2 protein and gene analyses and we obtained data on 112 samples for membrane EGFR and 110 samples for FISH EGFR. Cores were either lost, fragmented, did not have invasive tumor or signals in FISH were suboptimal due to under or over digestion. Overexpression of HER-2 and membrane EGFR (as detected by both EGFR antibodies used) was observed in 27 (23%) and 2 (2%) cases, respectively (Supplementary Table 1). In the same group of samples 21 (18%) had HER-2 gene amplification and 2 (2%) showed EGFR amplification (Supplementary Table 1). HER-2 protein expression showed significant correlation with ER, PR (p=0.0192 and p=0.0496 respectively) and border line significance with Ki-67 (p=0.0551). On the other hand HER-2 amplification showed significant correlation with histological grade (p=0.0214), ER (p=0.0013), PR (p=0.0263) and Ki-67 (p=0.0360) (Supplementary Table 1). Expression of membrane EGFR correlated with histological grade (p=0.00001), ER (p=0.00001), PR (p=0.00008) and Ki-67 (p=0.0016), while EGFR amplification correlated with histological grade (p=0.0460) and Ki-67 (p=0.0350) (Supplementary Table 1). A statistically significant correlation was observed between protein expression and gene amplification for HER-2 and membrane EGFR (Supplementary Table 2; p<0.00001 and p<0.0035, respectively). There was no statistically significant correlation between HER-2 and membrane EGFR status (data not shown).

Supplementary Table 1. Correlations between HER-2 and membrane EGFR status and clinicopathological parameters in invasive ductal breast cancer

HER-2 / Membrane EGFR*
IHC / FISH / IHC / FISH
Parameters / 0 / 1+ / 2+ / 3+ / P / - / + / P / 0 / 1+ / 2+ / 3+ / P / - / + / P
Overall / 71 / 18 / 11 / 16 / 95 / 21 / 100 / 10 / 1 / 1 / 108 / 2
Tumor size / NS / NS / NS / NS
T1 (≤ 2 cm) / 38 / 10 / 6 / 8 / 53 / 10 / 57 / 3 / 0 / 1 / 58 / 1
T2 (2 - 5 cm) / 29 / 6 / 4 / 7 / 36 / 10 / 37 / 7 / 1 / 0 / 44 / 1
T3 (> 5 cm) / 4 / 2 / 1 / 1 / 6 / 1 / 6 / 0 / 0 / 0 / 6 / 0
Grade / NS / 0.0214 / 0.00001 / 0.04604
I / 20 / 6 / 3 / 1 / 29 / 2 / 29 / 1 / 0 / 0 / 30 / 0
II / 38 / 9 / 6 / 8 / 50 / 10 / 57 / 1 / 0 / 0 / 56 / 0
III / 13 / 3 / 2 / 7 / 16 / 9 / 14 / 8 / 1 / 1 / 22 / 2
LN mets / NS / NS / NS / NS
0 / 39 / 10 / 5 / 9 / 53 / 10 / 53 / 5 / 1 / 1 / 57 / 1
1-3 / 11 / 3 / 5 / 2 / 16 / 5 / 21 / 0 / 0 / 0 / 21 / 0
≥4 / 21 / 5 / 1 / 5 / 26 / 6 / 26 / 5 / 0 / 0 / 30 / 1
ER / 0.0192 / 0.0013 / 0.00001 / NS
- / 24 / 8 / 3 / 12 / 32 / 15 / 34 / 10 / 1 / 1 / 44 / 2
+ / 47 / 10 / 8 / 4 / 63 / 6 / 66 / 0 / 0 / 0 / 64 / 0
PR / 0.0496 / 0.0263 / 0.00008 / NS
- / 22 / 6 / 4 / 11 / 31 / 12 / 31 / 9 / 1 / 1 / 40 / 2
+ / 49 / 12 / 7 / 5 / 64 / 9 / 69 / 1 / 0 / 0 / 68 / 0
NPI / NS / NS / NS / NS
Good (< 3.4) / 37 / 10 / 5 / 6 / 51 / 8 / 53 / 2 / 0 / 1 / 53 / 1
Moderate (3.4-5.4) / 19 / 4 / 3 / 5 / 25 / 6 / 26 / 4 / 1 / 0 / 31 / 0
Poor (>5.4) / 15 / 4 / 2 / 4 / 19 / 6 / 20 / 4 / 0 / 0 / 23 / 1
LVI / NS / NS / NS / NS
- / 12 / 3 / 4 / 3 / 16 / 6 / 20 / 0 / 0 / 1 / 20 / 1
+ / 34 / 13 / 4 / 10 / 48 / 12 / 51 / 6 / 1 / 0 / 56 / 1
Ki-67 / 0.0551 / 0.0360 / 0.00168 / 0.03503
<10% / 32 / 6 / 7 / 3 / 45 / 4 / 47 / 1 / 0 / 0 / 47 / 0
10-30% / 26 / 8 / 0 / 8 / 34 / 8 / 38 / 3 / 1 / 0 / 42 / 0
>30% / 12 / 3 / 3 / 4 / 14 / 8 / 15 / 6 / 0 / 1 / 19 / 2

*Same level of membrane EGFR expression was detected in examined tumors by both EGFR antibodies used.

IHC:immunohistochemistry; FISH: fluorescent in situ hybridization;Grade: histological grade; LN mets: lymph node metastasis; ER:estrogen receptor; PR: progesterone receptor; NPI:Nottingham prognostic index; LVI: lymphovascular invasion; NS: not significant.

Correlations were calculated using Fisher’s exact test.

Supplementary Table 2. Correlation between IHC and FISH for HER-2, membrane EGFR

Parameters / IHC / FISH / Fisher's
exact test (p)
Not amplified / Amplified
HER-2 / 0 / 71 / 0 / < 0.00001
1+ / 17 / 1
2+ / 6 / 4
3+ / 0 / 16
Membrane EGFR / 0 / 98 / 0 / 0.0035
1+ / 10 / 0
2+ / 0 / 1
3+ / 0 / 1

IHC: immunohistochemistry; FISH: fluorescent in situ hybridization.

SupplementaryTable 3. Survival analysis –strong cyclin D1 expression with matching parameters

Status / Paired / Not paired / N / Sign* / Matching parameters / Cyclin D1 strong / Fisher's
exact test (p)
Died / 41 / 40 / 117 / -1 / ER + / + / 0.0422
Survived / 26 / 10
Died / 40 / 41 / 117 / -1 / PR + / + / 0.0145
Survived / 27 / 9
Died / 61 / 20 / 117 / -1 / Grade I / + / 0.0192
Survived / 34 / 2

*Sign: if its “+” it is higher probability that paired will die than to survive

if its “-” it is higher probability that paired will survive than to die

Paired: number of samples that had been positive for cyclin D1 strong expression and had matching specific parameter

Not paired: number of samples that did not have both, positive cyclin D1 strong expression and matching specific parameter

ER: estrogen receptor; PR: progesterone receptor; Grade I: histological grade I.