Tumor infiltrating lymphocytes in triple negative breast cancer receiving neoadjuvant chemotherapy

Table 1 Clinical and pathological general features

Features	n = 98 (%)
Median age	49 ± 9
Clinical stage
II	13 (13.3)
III	85 (86.73)
Histologic grade
II	23 (23.5)
III	74 (75.5)
Subtype histologic
Ductal	95 (96.9)
Lobular	2 (2.1)
Neoadjuvant chemotherapy
AC	9 (9.2)
AC-Taxane	86 (87.7)
Taxane alone	3 (3.1)
Surgery
Tumorectomy	29 (29.6)
Mastectomy	65 (66.3)
Unknown	4 (4.1)
Inflammatory
No	69 (70.4)
Yes	29 (29.6)
pCR
No	69 (70.4)
Yes	29 (29.6)
Recurrence
No	56 (57.1)
Yes	42 (42.9)
Death
No	53 (54.1)
Yes	45 (45.9)

AC: Adriamycin/cyclophosphamide; AC-Taxane: Adriamycin/cyclophosphamide, followed by treatment with taxane; pCR: Pathologic complete response.

Table 2 Tumor infiltrating lymphocytes in pre-neoadjuvant-chemotherapy and post-neoadjuvant-chemotherapy samples (median ± interquartile range)

TIL population	(Me ± IQR)
Pre-NAC TIL
Median percentage TIL H and E full-face (n = 91)	40 ± 20
Median percentage TIL H and E TMA (n = 30)	20 ± 15
Median counting TIL CD3 (n = 28)	244.5 ± 253.8
Median counting TIL CD4 (n = 19)	48 ± 107.5
Median counting TIL CD8 (n = 28)	102 ± 98.8
Median counting TIL FOXP3 (n = 29)	22 ± 35
Post-NAC TIL
Median percentage TIL H and E (n = 80)	20 ± 15
Median percentage TIL H and E TMA (n = 58)	10 ± 5
Median counting TIL CD3 (n = 68)	156.5 ± 200.8
Median counting TIL CD4 (n = 54)	12 ± 27.5
Median counting TIL CD8 (n = 70)	75.5 ± 93.5
Median counting TIL FOXP3 (n = 70)	7.5 ± 14.5

NAC: Neoadjuvant-chemotherapy; TIL: Tumor-infiltrating-lymphocyte; TMA: Tissue microarrays; Me ± IQR: Median ± interquartile range.

Table 3 Comparison between tumor-infiltrating-lymphocytes evaluated in full-face and tissue microarrays sections

Features	H and E in the full-face section			H and E in TMA section
	n	Me ± IQD	P-value2	n	Me ± IQD	P-value3
Pre-NAC	91			30
Age			0.1427			0.6313
≤ 49	50	40 ± 20		19	20 ± 15
> 49	41	20 ± 15		11	20 ± 25
pCR			0.02511			0.2227
No	63	30 ± 20		15	30 ± 25
Yes	28	40 ± 17.5		15	20 ± 10
Inflammatory			0.6401			0.03871
Yes	27	40 ± 15		8	10 ± 6.5
No	64	35 ± 20		22	30 ± 15
DFS			0.00761			0.1601
< 32 mo	46	20 ± 20		20	15 ± 13.8
≥ 32 mo	45	40 ± 20		10	25 ± 25
OS			0.03341			0.7214
< 41 mo	51	30 ± 20		22	20 ± 15
≥ 41 mo	40	40 ± 20		8	20 ± 15
Post-NAC	80			58
Age			0.8547			0.5684
≤ 49	43	20 ± 15		31	10 ± 15
> 49	37	20 ± 15		27	10 ± 5
Inflammatory			0.4582			0.1299
Yes	26	15 ± 10		16	10 ± 0
No	54	20 ± 15		42	10 ± 15
pCR			0.6331			0.1299
No	63	20 ± 15		44	10 ± 7.5
Yes	17	20 ± 10		14	10 ± 7.5
DFS3			0.2450			0.2573
< 32 mo	35	20 ± 15		22	10 ± 0
≥ 32 mo	28	20 ± 15		22	10 ± 15
OS3			0.5973			0.6948
< 41 mo	40	20 ± 15		27	10 ± 10
≥ 41 mo	23	20 ± 15		17	10 ± 5

¹P < 0.05;

²U Mann Whitney test;

³Only cases with incomplete pathological response in post NAC. DFS: Disease free survival; Me ± IQD: Median ± interquartile deviation; OS: Overall survival; pCR: Pathologic complete response; NAC: Neoadjuvant-chemotherapy.

Table 4 Comparison of tumor-infiltrating-lymphocyte evaluated in pre- vs post-neoadjuvant-chemotherapy samples

Population of lymphocytes	n	Pre-NAC Me ± IQR	Post-NAC Me ± IQR	P-value2	P corrected value3
Percentage
H and E whole slide	73	40 ± 15	20 ± 15	< 0.001	< 0.0021
H and E TMA	16	30 ± 17.5	15 ± 15	0.4321
Absolute counting
CD3	22	244.5 ± 315.5	255.5 ± 267	0.8583
CD4	7	14 ± 94	32 ± 43	0.6721
CD8	21	127 ± 193.5	156 ± 90.5	0.7544
FOXP3	21	18 ± 31	12 ± 19.5	0.0917

¹P < 0.05;

²Wilcoxon signed rank test;

³Bonferroni correction. NAC: Neoadjuvant-chemotherapy; TMA: Tissue microarrays; Me ± IQR: Median ± interquartile range.

Table 5 Relationship between percentage and absolute counting methodologies of tumor-infiltrating-lymphocyte subpopulations

Population of lymphocytes	n	rho2	P-value	P corrected value3
Pre-NAC
CD3	27	0.7182	< 0.001	< 0.0041
CD4	17	0.5071	0.0378	0.1512
CD8	27	0.6064	0.0008	0.00321
FOXP3	26	0.7192	< 0.001	< 0.0041
Post-NAC
CD3	55	0.7733	< 0.001	< 0.0041
CD4	30	0.6129	0.0003	0.00121
CD8	55	0.7338	< 0.001	< 0.0041
FOXP3	47	0.5387	0.0001	0.00041

¹P < 0.05;

²Spearman correlation coefficient test;

³Bonferroni correction. NAC: Neoadjuvant-chemotherapy.