Relationship between HER2 gene status and selected potential biological features related to trastuzumab resistance and its influence on survival of breast cancer patients undergoing trastuzumab adjuvant treatment

Figures & data

Table 1 Clinicopathological characteristics of invasive ductal breast cancer patients (n=117)

Parameter	Category	N (%)
pTa	1	50 (45.0)
	2 + 3	61 (55.0)
pNb	0	50 (43.1)
	1	38 (32.7)
	2	14 (12.1)
	3	14 (12.1)
Gradeb	2	37 (31.9)
	3	79 (68.1)
Surgery	BCS	40 (34.2)
	Madden	70 (59.8)
	Mastectomy + sentinel node	2 (1.7)
	BCS + lymphadenectomy	5 (4.3)
Chemotherapy	Not administered	1 (0.9)
	TAC	1 (0.9)
	ACT	40 (34.1)
	AC	70 (59.8)
	FAC	5 (4.3)
Hormonal therapy	Not administered	50 (42.7)
	Administered	67 (57.3)
Radiotherapy	Not administered	15 (12.8)
	Administered	102 (87.2)

Note:

a Data unavailable in six cases,

b data unavailable in one case.

Abbreviations: AC, doxorubicin and cyclophosphamide; ACT, doxorubicin, cyclophosphamide, and paclitaxel; BCS, breast-conserving surgery; FAC, 5-fluorouracil, doxorubicin, and cyclophosphamide; Madden, Madden mastectomy; TAC, docetaxel, doxorubicin, and cyclophosphamide.

Figure 1 Visualization of HER2 amplification (FISH) and HER2 protein expression (IHC). (A) HER2 expression score 2+, (B) HER2 expression score 3+, (C) example of low level of HER2 gene amplification, square outlined by dotted line is magnified in left bottom corner, (D) example of high level of HER2 gene amplification, (E) example of HER2 amplification with polysomy, and (F) HER3 expression. (A, B, and F) 20× magnification, (C, D, and E) 100× magnification.

Abbreviations: FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor type 2; IHC, immunohistochemistry.

Table 2 Detailed information on immunohistochemical procedures

Antigen	Clone	Manufacturer	Antigen retrieval	Dilution	Number of positive/all stained cases
PTEN	138G6	Cell Signaling Technologya	TRS, pH=6.1 DAKO, 50 min, 96°C	1:75e	105/109
IGF-1Rβ	D406W	Cell Signaling Technologya		1:100e	95/106
MUC4	1G8	BioGenexb		1:60f	90/107
HER3	SP71	Thermo Fisher Scientificc		1:100f	93/112
HER4	Polyclonal	Thermo Fisher Scientificc	TRS, pH=6.1 DAKO, 20 min, 96°C	1:100f	113/113
EGFR	H11	DAKOd	Proteinase K, 10 min, RT	1:200f	17/111

Notes:

a Cell Signaling Technology, Danvers, MA, USA;

b BioGenex, Fremont, CA, USA;

c Thermo Fisher Scientific, Fremont, CA, USA;

d DakoCytomation Denmark A/S, Glostrup, Denmark. Incubation with primary antibody:

e 1 h at 37°C;

f overnight, 4°C.

Abbreviations: EGFR, epidermal growth factor receptor; RT, room temperature.

Table 3 Relations between studied parameters

Parameter	Category	N	HER2 copy number per cell mean ± SD	HER2/CEP17 ratio mean ± SD	Polysomy
					Absent N	Present N
pT	1	50	16.66 ± 8.95	6.96 ± 4.31	38	12
	2 + 3	61	16.64 ± 8.16	6.41 ± 3.74	40	21
pN	0	50	16.75 ± 8.24	6.59 ± 3.99	36	14
	1	38	17.90 ± 8.28	7.12 ± 4.17	23	15
	2	14	15.32 ± 8.38	6.47 ± 3.21	12	2
	3	14	14.53 ± 8.31	6.34 ± 4.01	11	3
G	2	37	14.94 ± 7.56	6.19 ± 3.89	26	11
	3	79	17.32 ± 8.57	6.86 ± 3.94	56	23
ER/PR	− and −#	48	20.71 ± 7.76	8.38 ± 3.92	32	16
	+ or +	69	13.73 ± 7.41***	5.51 ± 3.50***	51	18
ER	−	49	20.54 ± 7.77	8.37 ± 3.88	33	16
	+	68	13.75 ± 7.50***	5.47 ± 3.51***	50	18
PR	−	59	19.86 ± 7.78	8.06 ± 3.78	40	19
	+	58	13.27 ± 7.44***	5.27 ± 3.59***	43	15
HER2 IHC	2+	29	7.32 ± 2.47	3.17 ± 1.52	24	5
	3+	86	19.53 ± 7.19***	7.87 ± 3.82***	59	27
EGFR	−	94	16.00 ± 8.23	6.54 ± 3.92	67	27
	+	17	18.52 ± 8.00	7.01 ± 2.80	11	6
PIK3CA mutations##	Absent	81	16.94 ± 8.24	6.92 ± 4.25	58	23
	Present	15	13.32 ± 7.27	5.76 ± 2.75	11	4
Polysomy	Absent	83	15.53 ± 8.29	7.53 ± 4.16
	Present	34	19.20 ± 7.75*	4.62 ± 2.20***

Notes:

*** P<0.001,

* P=0.026 (from Kruskal–Wallis ANOVA test with Bonferroni correction). The statistically significant differences are indicated using bold font.

# “+” immunopositive cases, “−” immunonegative cases;

## “Absent” tumors without H1047R or E545K PIK3CA mutations “Present” tumors with H1047R or/and E545K PIK3CA mutations.

Abbreviations: EGFR, epidermal growth factor receptor; ER/PR, estrogen/progesterone receptor; HER2, human epidermal growth factor receptor type 2; IHC, immunohistochemistry; N, number of cases; PIK3CA, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit.

Table 4 MFS according to studied parameters

Parameter	N Number of patients with distant metastasis/without distant metastasis	p-value	MFS (%)
pN
0 + 1	5/83	0.007	93.3
2 + 3	6/22		72.2
HER3 (H-score)
≤25	0/32	0.018	100.0
>25	11/69		83.2
EGFR
Immunonegativity	8/85	0.347	89.7
Immunopositivity	3/14		82.3
Polysomy
Absent	8/75	0.865	90.3
Present	3/31		88.2
PIK3CA mutations#
Absent	9/72	0.604	86.5
Present	1/14		93.3
HER2/CEP17 ratio
≤4	0/36	0.062	100
>4	11/70		85.5
HER2/CEP17 ratio/HER3
≤4 or ≤25	0/57	0.001	100
>4 and >25	11/45		78.7

Notes:

# “Absent” tumors without H1047R or E545K PIK3CA mutations; “Present” tumors with H1047R or/and E545K PIK3CA mutation. The statistically significant differences are indicated using bold font.

Abbreviations: EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor type 2; MFS, metastasis-free survival.

Figure 2 Metastasis-free survival curves of patients with invasive ductal breast cancer stratified by (A) HER2/CEP17 ratio, (B) expression of HER3, and (C) combined HER2/CEP17 ratio with HER3 expression.

Abbreviation: HER2, human epidermal growth factor receptor type 2.