Formalin fixation on HER-2 and PD-L1 expression in gastric cancer: A pilot analysis using the same surgical specimens with different fixation times

Table 1 Clinicopathological characteristics and immunohistochemistry results according to used formalin solution n (%)

	All cases (n = 32)	10% formalin (n = 22)	1% NBF (n = 10)	P
Age (mean ± SD)	74.5 ± 7.8	73.3 ± 8.0	77.1 ± 7.0	0.2020
Gender				0.7731
Male	18 (56.3)	12 (54.6)	6 (60.0)
Female	14 (43.7)	10 (45.4)	4 (40.0)
T-Stage				0.6793
T1b2	6 (18.8)	3 (13.6)	3 (30.0)
T2	9 (28.1)	6 (27.3)	3 (30.0)
T3	5 (15.6)	4 (18.2)	1 (10.0)
T4a	12 (37.5)	9 (40.9)	3 (30.0)
Lauren's classification
Intestinal	18 (56.2)	12 (54.6)	6 (60.0)	0.7731
Diffuse / mixed	14 (43.8)	10 (45.4)	4 (40.0)
HER-2 IHC
Complete negative1	24 (75)	18 (81.8)	6 (60.0)	0.1966
Final positive by DISH	5 (15.6)	2 (9.1)	3 (30.0)	0.1311
PD-L1 positivity
Cut off 1%	20 (62.5)	14 (63.4)	6 (60.0)	0.8439
Cut off 10%	13 (40.6)	8 (36.4)	5 (50.0)	0.4666
Cut off 50%	7 (21.9)	4 (18.2)	3 (30.0)	0.4535

¹The cases that all four specimens of different fixation time were assessed as score 0.

IHC: Immunohistochemistry; NBF: Neutral buffered formalin; DISH: Dual color in situ hybridization; HER-2: Human epidermal growth factor receptor 2; PD-L1: Programmed death-ligand 1.

Table 2 Whole data of immunohistochemistry according to fixation time

Case No.	Formalin	HER-2 IHC score; P = 0.7713 (6 h-48 h vs 1 wk)1				PD-L1 expression (%); P = 0.46 (6 h-48 h vs 1 wk)1
		6 h	24 h	48 h	1 wk	6 h	24 h	48 h	1 wk
1	10% F	0	0	0	0	0	0	0	0
2	10% F	0	0	0	0	10	10	10	1-9
3	10% F	0	0	0	0	0	0	0	0
4	10% F	0	0	0	0	80	80	90	90
5	10% F	0	0	0	0	0	0	0	1-9
6	10% F	0	0	0	0	0	0	0	0
7	10% F	0	0	0	0	0	0	0	0
8	10% F	0	0	0	0	0	0	1-9	1-9
9	10% F	0	0	0	0	0	1-9	0	1-9
10	10% F	0	0	0	0	40	30	30	40
11	10% F	0	0	0	0	1-9	0	10	10
12	10% F	3+	3+	3+	3+	1-9	1-9	1-9	0
13	10% F	0	0	0	0	0	0	0	0
14	10% F	1+	2+	0	0	100	1-9	30	60
15	10% F	0	0	0	0	0	0	0	0
16	10% F	0	0	0	0	1-9	10	1-9	1-9
17	10% F	0	0	0	0	0	0	0	0
18	10% F	0	0	0	0	20	60	20	40
19	10% F	0	0	0	0	1-9	1-9	1-9	0
20	10% F	2+	0	0	0	80	80	20	1-9
21	10% F	0	0	0	0	1-9	0	1-9	1-9
22	10% F	1+	1+	2+	2+	0	0	0	0
23	10% NBF	0	0	0	0	0	0	0	0
24	10% NBF	0	0	0	0	0	0	0	0
25	10% NBF	0	1+	2+	1+	10	0	10	0
26	10% NBF	2+	2+	2+	2+	0	0	0	0
27	10% NBF	2+	1+	1+	3+	20	70	30	1-9
28	10% NBF	0	0	0	0	0	0	0	0
29	10% NBF	0	0	0	0	1-9	1-9	0	1-9
30	10% NBF	0	0	0	0	100	100	100	100
31	10% NBF	3+	3+	3+	3+	0	1-9	10	1-9
32	10% NBF	0	0	0	0	70	80	90	90

¹Simultaneous tests for linear hypotheses based on a linear mixed effects model.

F: Formalin; NBF: Neutral buffered formalin; HER-2: Human epidermal growth factor receptor 2; PD-L1: Programmed death-ligand 1.

Table 3 Human epidermal growth factor receptor 2 immunohistochemistry and dual color in situ hybridization results in Human epidermal growth factor receptor 2-immunoreactive cases (n = 8)

Case No.	Formalin	HER-2 IHC score; P = 1.000 (6 h-48 h vs 1 wk)1				DISH; HER2/CEP17ratio (average HER2 copy number); P = 0.6989 (6 h-48 h vs 1 wk)1				Final assessment
		6 h	24 h	48 h	1 wk	6 h	24 h	48 h	1 wk
12	10% F	3+	3+	3+	3+	11.4 (33.7)	10.9 (28.5)	7.1 (20.4)	4.8 (17.4)	+
14	10% F	1+	2+	0	0	1.2 (3.0)	1.3 (3.3)	1.3 (2.2)	1.3 (2.2)	-
20	10% F	2+	0	0	0	0.8 (2.1)	1.2 (2.8)	1.3 (2.1)	1.3 (2.0)	-
22	10% F	1+	1+	2+	2+	1.6 (3.4)	1.9 (4.4)	2.1 (2.5)	2.1(2.8)	+
25	10% NBF	0	1+	2+	1+	1.0 (1.9)	1.1 (2.2)	1.8 (3.1)	1.4 (2.5)	-
26	10% NBF	2+	2+	2+	2+	2.6 (5.2)	3.7 (6.5)	3.5 (6.6)	3.3 (5.7)	+
27	10% NBF	2+	1+	1+	3+	1.1 (2.9)	1.4 (2.8)	1.3 (2.1)	2.2 (4.9)	+
31	10% NBF	3+	3+	3+	3+	4.7 (19.8)	6.7 (21.2)	4.7 (15.7)	7.2 (21.2)	+

¹Simultaneous tests for linear hypotheses based on a linear mixed effects model.

F: Formalin; NBF: Neutral buffered formalin; DISH: Dual color in situ hybridization; HER-2: Human epidermal growth factor receptor 2; PD-L1: Programmed death-ligand 1.

Table 4 Programmed death-ligand 1 expression results of the programmed death-ligand 1 immunoreactive cases (n = 20)

Case No.	Used formalin	PD-L1 expression; P = 0.4605 (6 h-48 h vs 1 wk) 1				Assessment (Cut-off 1%)	Assessment (Cut-off 10%)	Assessment (Cut-off 50%)
		6 h	24 h	48 h	1 wk
2	10% F	10	10	10	1-9	+	+	-
4	10% F	80	80	90	90	+	+	+
5	10% F	0	0	0	1-9	+	-	-
8	10% F	0	0	1-9	1-9	+	-	-
9	10% F	0	1-9	0	1-9	+	-	-
10	10% F	40	30	30	40	+	+	-
11	10% F	1-9	0	10	10	+	+	-
12	10% F	1-9	1-9	1-9	0	+	-	-
14	10% F	100	1-9	30	60	+	+	+
16	10% F	1-9	10	1-9	1-9	+	+	-
18	10% F	20	60	40	40	+	+	+
19	10% F	1-9	1-9	1-9	0	+	-	-
20	10% F	80	80	20	1-9	+	+	+
21	10% F	1-9	0	1-9	1-9	+	-	-
25	10% NBF	10	0	10	0	+	+	-
27	10% NBF	20	70	30	1-9	+	+	+
29	10% NBF	1-9	1-9	0	1-9	+	-	-
30	10% NBF	100	100	100	100	+	+	+
31	10% NBF	0	1-9	10	1-9	+	+	-
32	10% NBF	70	80	90	90	+	+	+

¹Simultaneous tests for linear hypotheses based on a linear mixed effects model.

F: Formalin; NBF: Neutral buffered formalin.

Table 5 Details of long-fixation cases

	Case No.	Formalin	Min. expression (6 h - 1 wk fixation)	Max. expression (6 h - 1 wk fixation)	Fixation period (mo)	Results after long fixation
HER-2:
	12	10% F	Score 3+	Score 3+	19	Score 2+
	22	10% F	Score 1+	Score 2+	3	Score 0
	26	10% NBF	Score 2+	Score 2+	20	Score 0
	27	10% NBF	Score 1+	Score 3+	18	Score 0
	31	10% NBF	Score 3+	Score 3+	16	Score 3+
PD-L1:
	20	10% F	1%-9%	80%	28	0%
	30	10% NBF	100%	100%	16	90%
	32	10% NBF	70%	90%	14	70%

F: Formalin; NBF: Neutral buffered formalin; HER-2: Human epidermal growth factor receptor 2; PD-L1: Programmed death-ligand 1.