The Programmed Cell Death Ligand 1 and Lipocalin 2 Expressions in Primary Breast Cancer and Their Associations with Molecular Subtypes and Prognostic Factors

Figures & data

Table 1 Distribution of Cases According to Molecular Subtypes and Prognostic Factors

Molecular Subtypes	Number of Cases (%)	Prognostic Factors
		HG I N (%)	HG II N (%)	HG III N (%)	Nec. (+) N (%)	LNM (+) N (%)	LVI (+) N (%)	TILs≥%5 (%)
Luminal A	36 (40.5)	1 (2.8)	32 (88.9)	3 (8.3)	2 (5.5)	10 (27.8)	5 (13.9)	0
Luminal B	36 (40.5)	0	14 (38.9)	22 (61.1)	3 (8.3)	11 (30.5)	9 (1.9)	5 (13.9)
HER2+	4 (4.4)	0	0	4 (100)	1 (25)	1 (25)	1 (25)	0
TNBC	13 (14.6)	0	1 (7.7)	12 (92.3)	5 (38.5)	5 (38.5)	2 (15.4)	7 (53.8)

Notes: The headings are given in bold.

Abbreviations: Luminal A, ER+, PR+, HER2-, Ki-67 < %20; Luminal B, ER+, PR ±, HER2- Ki-67 ≥%20 and ER+, PR ±, HER2+, any Ki-67; HER2+, human epidermal growth factor receptor 2 positive, ER-, PR-, HER2+, Ki-67 ≥%20; TNBC, triple negative breast cancer, ER-, PR-, Ki-67 ≥% 20; HG, histological grade; Nec, necrosis; LNM, lymph node metastasis; TILs, tumor-infiltrating lymphocytes; LVI, lymphovascular invasion; N, number.

Figure 1 Lipocalin 2 (LCN2) staining scored with the Allred scoring in different breast cancerous tissues. (a) Cytoplasmic staining with Allred score 1 in ductus epithelium and stroma of normal breast tissue (blue arrow), (b) Cytoplasmic and membranous staining with Allred score 3 in tumor cells in a case with Luminal A, (c) Allred score 4 in tumor cells in a case with Luminal A, (d) Allred score 5 in tumor cells in a case with Luminal B, (e) Allred score 6 in tumor cells in a case with Luminal B, (f) Allred score 7 in a case with TNBC, (g) Allred score 8 LNC2 expression in a case with tumor cells of TNBC, (h) LCN2 positive control (colon adenocarcinoma). All figures are at 100 µm magnification.

Table 2 Distribution of Cases into Molecular Subtypes and Prognostic Factors According to LCN2 Expression by Allred Score

Molecular Subtypes and Prognostic Factors	LCN2 (Allred Score)
	3 N (%)	4 N (%)	5 N (%)	6 N (%)	7 N (%)	8 N (%)
Luminal A	25 (69.4)	8 (22.2)	3 (8.3)	0	0	0
Luminal B	6 (16.7)	11 (30.5)	7 (19.4)	3 (8.3)	5 (13.9)	4 (11.1)
HER2 (+)	1 (25)	1 (25)	0	0	1 (25)	1 (25)
TNBC	1 (7.7)	1 (7.7)	0	4 (30.7)	2 (15.4)	5 (38.4)
Ki-67 < 20%	26 (72.2)	7 (19.4)	3 (8.3)	0	0	0
Ki-67 ≥ 20%	8 (15.1)	13 (24.5)	7 (13.2)	8 (15.1)	8 (15.1)	9 (17)
HG I	0	1 (100)	0	0	0	0
HG II	26 (55.3)	12 (25.5)	6 (12.7)	3 (6.4)	0	0
HG III	7 (17.9)	7 (17.9)	4 (8.1)	8 (16.3)	6 (12.2)	9 (18.3)
Necrosis (+)	3 (27.3)	0	1 (9.1)	1 (9.1)	1 (9.1)	5 (45.4)
LNM (+)	8 (29.6)	6 (22.2)	5 (18.5)	1 (3.7)	5 (18.5)	2 (7.4)
LVI (+)	7 (41.2)	2 (11.7)	3 (17.6)	1 (5.9)	2 (11.7)	2 (11.7)
TILs ≥5%	1 (8.3)	1 (8.3)	1 (8.3)	5 (41.7)	2 (16.7)	2 (16.7)

Notes: The headings are given in bold.

Abbreviations: LCN2, lipocalin 2; TNBC, triple negative breast cancer; HG, histological grade; LNM, lymph node metastasis; LVI, lymphovascular invasion; TILs, tumor-infiltrating lymphocytes; N, number.

Table 3 Comparison of LCN2 Allred Score Expressions in Molecular Subtypes and Prognostic Factors Based on Positive versus Negative Status Among All Cases

Molecular Subtypes and Prognostic Factors	LCN2 (Allred Score)
	Positive^a Median (Min-Max)	Negative^b Median (Min-Max)	P value^c	LCN2 ≥ 6^d (%)
Luminal A	3 (3–5)	5 (3–8)	<0.0001^k	0
Luminal B	5 (3–8)	3 (3–8)	0.19^k	33.3%
HER2 (+)	5.5 (3–8)	4 (3–8)	N.D.	50%
TNBC	6 (3–8)	4 (3–8)	<0.01^k	84.6%
HER2(+) + TNBC	6 (3–8)	4 (3–8)	<0.01^k	76.5%
Ki-67 Index (+)	5 (3–8)	3 (3–5)	<0.0001^m	47.2%
HG I	N.D.	N.D.	N.D.	N.D.
HG II	3 (3–7)	6 (3–8)	<0.0001^k	6.1%
HG III	6 (3–8)	3 (3–7)	<0.0001^k	56.1%
Necrosis (+)	7 (3–8)	4 (3–8)	<0.05^m	63.6%
LNM (+)	4 (3–8)	4 (3–8)	0.34^m	29.6%
LVI (+)	4 (3–8)	4 (3–8)	0.90^m	29.4%
TILs ≥5%	6 (3–8)	4 (3–8)	<0.001^m	75%

Notes: The headings are given in bold. ^aRespective positive cases for either molecular subtype or prognostics factors among all cases. ^bRespective negative cases for either molecular subtype or prognostics factors among all cases. ^cP values between negative and positive cases among all cases were determined by non-parametric Mann–Whitney U-test (m) after the analysis of Gaussian distribution. When groups were more than two Kruskal–Wallis test followed by Dunn’s multiple comparisons (k) was used. P values less than 0.05 are considered statistically significant. m, Mann–Whitney U-test; k, Kruskal–Wallis test. ^dPercentage of LCN2 scores ≥6 cases among respective subtypes and prognostic factors.

Abbreviations: LCN2, lipocalin 2; HER, TNBC, triple negative breast cancer; HG, histological grade; LNM, lymph node metastasis; LVI, lymphovascular invasion; TILs, tumor-infiltrating lymphocytes; N.D., could not be determined due to low sample number.

Table 4 Distribution of Cases into Molecular Subtypes and Prognostic Factors According to PD-L1 Expression Based on CPS Cut-Offs

Molecular Subtypes and Prognostic Factors	PD-L1 (CPS Cut-Offs)
	CPS <1 (Negative) N (%)	CPS ≥1 N (%)	CPS ≥10 N (%)
Luminal A	27 (75)	9 (25)	0
Luminal B	13 (36.1)	13 (36.1)	10 (27.8)
HER2 (+)	1 (25)	2 (50)	1 (25)
TNBC	2 (16.7)	4 (33.3)	7 (58.3)
Ki-67 ≥ 20%	15 (28.3)	19 (35.8)	19 (35.8)
HG I	1 (100)	0	0
HG II	31 (66)	15 (31.9)	1 (2.1)
HG III	11 (26.8)	13 (31.7)	17 (41.5)
Necrosis (+)	3 (27.3)	2 (18.2)	6 (54.5)
LNM (+)	13 (48.4)	11 (40.7)	3 (1.1)
LVI (+)	10 (58.8)	5 (29.4)	2 (11.8)
TILs ≥5%	1 (8.3)	0	11 (91.7)

Notes: The headings are given in bold.

Abbreviations: PD-L1, programmed cell death ligand 1; CPS, combined positive score; TNBC, triple negative breast cancer; HG, histological grade; LNM, lymph node metastasis; LVI, lymphovascular invasion; TILs, tumor-infiltrating lymphocytes; N, number.

Table 5 Distribution of Cases into Molecular Subtypes and Prognostic Factors According to PD-L1 Expression

Molecular Subtypes and Prognostic Factors	PD-L1
	Positive^a (CPS ≥1) Median	Negative^b (CPS <1) Median	P value^c	% PD-L1 Positivity (CPS ≥1) (%)^d
Luminal A	0 (0–3)	2 (0–100)	<0.001^k	25%
Luminal B	1 (0–80)	0 (0–100)	1^k	63.9%
HER2 (+)	1.5 (0–100)	1 (0–80)	N.D.	75%
TNBC	12 (0–70)	0 (0–100)	<0.05^k	84.6%
HER2(+) + TNBC	5 (0–100)	0 (0–80)	<0.05^k	82.3%
Ki-67%	2 (0–100)	0 (0–3)	<0.0001^m	69.8%
HG I	0	0	0	0%
HG II	0 (0–15)	2.5 (0–100)	<0.0001^m	34%
HG III	3 (0–100)	0 (0–15)	<0.0001^m	73.2%
Necrosis (+)	10 (0–100)	0 (0–70)	<0.05^m	72.7%
LNM (+)	1 (0–30)	1 (0–100)	0.28^m	51.8%
LVI (+)	0 (0–100)	1 (0–80)	0.36^m	41.2%
TILs ≥5%	45 (5–75)	0 (0–100)	<0.0001^m	91.7%

Notes: The headings are given in bold. ^aRespective positive cases for either molecular subtype or prognostics factors among all cases. ^bRespective negative cases for either molecular subtype or prognostics factors among all cases. ^cP values between negative and positive cases among all cases were determined by non-parametric Mann–Whitney U-test (m) after the analysis of Gaussian distribution. When groups were more than two Kruskal–Wallis test followed by Dunn’s multiple comparisons (k) was used. P values less than 0.05 are considered statistically significant. m, Mann–Whitney U-test; k, Kruskal–Wallis test. ^dPercentage of PD-L1 CPS scores among respective subtypes and prognostic factors.

Abbreviations: PD-L1, programmed cell death ligand 1; CPS, combined positive score; TNBC, triple negative breast cancer; HG, histological grade; LNM, lymph node metastasis; LVI, lymphovascular invasion; TILs, tumor-infiltrating lymphocytes; N.D., could not be determined due to low sample number.

Figure 2 PD-L1 staining scored with CPS. Representative CPS score (CPS 80) of the same area in a TNBC tumor, (a) H&E staining 500 µm and 100 µm in inlet, (b) PD-L1 staining 500 µm and 100 µm in inlet, (c) Tonsil tissue PD-L1 positive control, (d) Normal breast tissue PD-L1 negative control.

Table 6 Association of LCN2 and PD-L1 Staining Scores with Prognostic Factors and Molecular Subtypes

Molecular Subtypes and Prognostic Factors	Pearson’s Correlation Test LCN2 vs PD-L1
	P value^a	r^b
Luminal A	0.64	0.08
Luminal B	0.006	0.45**
TNBC	N.D.	N.D.
HER2 (+) and TNBC	0.1	0.40
Ki-67 ≥ 20%	0.000	0.46**
HG I and HG II	0.05	0.27
HG III	0.001	0.49**
Necrosis (+)	N.D.	N.D.
LNM (+)	0.18	0.26
LVI (+)	0.09	0.42
TILs ≥5%	0.51	0.20

Notes: The headings are given in bold. ^aPearson’s correlation test was performed between LCN2 and PD-L1 scores of respected positive cases. P values less than 0.05 are considered statistically significant. ^br values between 0 to 1 is considered are considered correlation significant. ** Significant correlation with p <0.01.

Abbreviations: LCN2, lipocalin 2; PD-L1, programmed cell death ligand 1; TNBC, triple negative breast cancer; HG, histological grade; LNM, lymph node metastasis; LVI, lymphovascular invasion; TILs, tumor-infiltrating lymphocytes; N.D., could not be determined due to low sample number.

Figure 3 Expression of LCN2 and PD-L1 in Triple-Negative Breast Cancer (TNBC). (a) Representative H&E staining of TNBC, (b) LCN2 staining of tumor cells (Allred score 7 with cytoplasmic staining of the same area in a, (c) PD-L1 staining in tumor cells and lymphocytes (CPS 50, same area in (a and b). All figures are at 100 µm magnification.