Tumor-infiltrating Treg, MDSC, and IDO expression associated with outcomes of neoadjuvant chemotherapy of breast cancer

Figures & data

Figure 1. The association between tumor-infiltrating CD33⁺MDSC, Foxp3⁺Treg, IDO expression, and IDO expression in CD33⁺MDSCs. (a1) CD33 in cancer tissues was expressing in normal cells between cancer cell nests dispersed on membrane and few in cytoplasm in cancer tissues (× 400, red arrows); (a2) negative expression of CD33 in breast cancer tissues (× 400); (a3) negative expression of CD33 in normal breast tissues, which were set as the negative control (NC); (b1) IDO in cancer tissues mainly expressed in tumor cells and some karyocytes dispersed in stroma with brownish yellow staining in cytoplasm (× 400, red arrows); (b2) negative expression of IDO in breast cancer tissues (× 400); (b3) negative control; (c1) in breast cancer tissues, Foxp3 expression in lymphocytes dispersed in stroma with brownish dark yellow staining in nucleus (× 400, red arrows); (c2) negative expression of Foxp3 in breast cancer tissues (× 400); (c3) negative control; (d) co-expression of IDO and CD33 in tumor tissues was determined using immunohistochemical double-staining method; (d1) IDO and CD33 negative staining of breast cancer tissues; (d2) CD33 positive and IDO negative cells (IDO⁻CD33⁺) were stained red on membrane (red arrows); (d3) IDO positive and CD33 negative cells (IDO⁺CD33⁻) were stained purple in cytoplasm (red arrows); (d4) IDO⁺CD33⁺MDSCs were stained red on membrane and purple in cytoplasm (× 400, red arrows); (e1) from the total of 54 patients, 33 patients were positive for CD33⁺MDSCs, 27 patients were positive for IDO expression (IDO positive, orange; negative, blue), and 22 patients were both positive for CD33⁺MDSCs and IDO expression. By Spearman's rank correlation test, it was found that CD33⁺MDSCs were positively correlated with IDO expression (r² = 0.418, P = 0.002); (e2) 31 patients were positive for Foxp3⁺Tregs (Foxp3⁺Treg positive, orange; negative, blue), 33 patients were positive for CD33⁺MDSCs, 24 patients were positive for both CD33⁺MDSCs and Foxp3⁺Tregs. By Spearman's rank correlation test, it was found that CD33⁺MDSCs were positively correlated with Foxp3⁺Tregs (r² = 0.388, P = 0.004); (e3) 31 patients were positive for Foxp3⁺Tregs, 27 patients were positive for IDO expression, 22 patients were positive for both Foxp3⁺Treg and IDO expression (IDO positive, orange; negative, blue). By Spearman's rank correlation test, it was found that IDO expression was positively correlated with Foxp3⁺Tregs (r² = 0.487, P = 0.000); (f) 31 patients were positive for Foxp3⁺Tregs, 22 patients were positive for IDO expression in CD33⁺MDSCs, 20 patients were positive for both Foxp3⁺Tregs and IDO expression in CD33⁺MDSCs. By Spearman's rank correlation test, IDO⁺CD33⁺MDSCs were positively correlated with Foxp3⁺Tregs (r² = 0.528, P = 0.000).

Table 1. The association between tumor-infiltrating CD33⁺MDSC, Foxp3⁺Treg, IDO expression and clinicopathological features prior NCT.

		CD33^+MDSC				Foxp3^+Treg				IDO
Clinicopathological features		−	+	x ^2	P	−	+	x ^2	P	−	+	x ^2	P
n		21	33			23	31			27	37
Age	<50	8	18	1.391	0.275	12	14	0.260	0.784	15	11	1.187	0.414
	≥50	13	15			11	17			12	16
Menopausal status	Pre-	13	15	1.391	0.275	11	17	0.260	0.784	13	15	0.297	0.786
	Post-	8	18			12	14			14	12
History of gravidity	No	4	3	1.128	0.411	5	2	2.735	0.122	6	1	4.103	1.000
	Yes	17	30			18	29			21	26
Family history of malignance	No	14	26	0.982	0.355	19	21	1.520	0.347	21	19	0.386	0.757
	Yes	7	7			4	10			6	8
Clinical T stage	T1, T2	16	7	15.864	0.000	16	7	11.921	0.001	17	6	9.164	0.005
	T3, T4	5	26			7	24			10	21
Clinical N stage	N0	7	6	1.612	0.328	9	4	4.969	0.051	10	3	4.964	0.054
	N1-N3	14	27			14	27			17	24
Clinical stage	I, II	6	8	0.125	0.758	8	6	1.636	0.226	9	5	1.543	0.352
	III, IV	15	25			15	25			18	22
Histological type	IDBC	17	27	0.003	1.000	22	22	5.332	0.032	23	21	0.491	0.728
	Other	4	6			1	9			4	6
ER	−	9	15	0.035	1.000	11	13	0.186	0.784	16	8	4.800	0.054
	+	12	18			12	18			11	19
PR	−	13	14	1.948	0.264	11	16	0.076	1.000	14	13	0.074	1.000
	+	8	19			12	15			13	14
Her-2	−	15	18	1.539	0.261	16	17	1.205	0.398	19	14	1.948	0.246
	+	6	15			7	14			8	13
Ki67	−	10	12	0.673	0.571	13	9	4.133	0.054	14	8	2.761	0.166
	+	11	21			10	22			13	19
P53	−	7	16	1.205	0.398	8	15	0.999	0.407	13	10	0.682	0.583
	+	14	17			15	16			14	17
Luminal type	Luminal	16	26	0.751	0.687	18	24	0.176	0.916	19	23	4.381	0.112
	Her-2	1	3			2	2			4	0
	Basal-like	4	4			3	5			4	4

IDBC: Invasive ductal breast cancer.

Table 2. The association between co-expression of IDO/CD33 and clinicopathological features prior NCT.

Clinicopathological features		n	IDO^−CD33^−	IDO^−CD33^+	IDO^+CD33^−	IDO^+CD33^+	x ^2	P
Total		54	16	11	5	22
Age	<50	26	7	8	1	10	4.437	0.218
	≥50	28	9	3	4	12
Menopausal status	Pre-	28	9	4	4	11	2.798	0.424
	Post-	26	7	7	2	10
History of gravidity	No	7	3	3	1	0	5.967	0.133
	Yes	47	13	8	4	22
Family history of maligant	No	40	11	10	3	16	2.396	0.494
	Yes	14	5	1	2	6
Clinical T stage	T1, T2	23	14	3	2	4	19.627	0.000
	T3, T4	31	2	8	3	18
Clinical N stage	N0	13	6	4	1	2	5.234	0.155
	N1-N3	41	10	7	4	20
Clinical stage	I, II	14	4	5	2	3	4.430	0.218
	III, IV	40	12	6	3	19
Histological type	IDBC	44	13	10	4	17	0.914	0.822
	Other	10	3	1	1	5
ER	−	24	7	9	2	6	8.893	0.031
	+	30	9	2	3	16
PR	−	27	10	4	3	10	2.200	0.532
	+	27	6	7	2	12
Her-2	−	33	11	8	4	10	4.037	0.122
	+	21	5	3	1	12
Ki67	−	22	8	6	2	6	3.090	0.378
	+	32	8	5	3	16
P53	−	23	6	7	1	9	3.231	0.357
	+	21	10	4	4	13
Luminal type	Luminal	42	12	7	4	19	8.921	0.178
	Her-2	4	1	3	0	0
	Basal-like	8	3	1	1	3

IDBC: Invasive ductal breast cancer.

Table 3. Univariate analysis of clinicopathological features with respect to breast cancer patients’ response to NCT.

			Clinical response				Pathological response
Clinicopathological features		Total	CR+PR(n)	PD+SD(n)	x ^2	P	pCR (n)	No-pCR (n)	x ^2	P
Age	<50	26	13	13	2.605	0.183	5	21	0.017	1.000
	≥50	28	20	8			5	23
Menopausal status	Pre-	28	20	8	2.605	0.183	5	23	0.017	1.000
	Post-	26	13	13			5	21
History of gravidity	No	7	5	2	0.360	0.693	2	5	0.539	0.601
	Yes	47	28	19			8	39
Family history of malignance	No	40	24	16	0.080	1.000	6	34	1.266	0.424
	Yes	14	9	5			4	10
Clinical T stage	T1, T2	23	18	5	4.958	0.047	8	15	7.024	0.012
	T3, T4	31	15	16			2	29
Clinical N stage	N0	13	8	5	0.001	1.000	5	8	4.513	0.048
	N1-N3	41	25	16			5	36
Clinical stage	I+II	14	8	6	0.125	0.758	3	11	0.106	0.708
	III+IV	40	25	15			7	33
Pathological T stage	pT0, T1, T2	43	30	13	6.656	0.015	8	34	0.814	0.667
	pT3, T4	11	3	8			2	10
Pathological N stage	pN0	9	5	4	0.140	0.708	0	9	2.455	0.183
	pN1-N3	45	28	17			10	35
Pathological TNM stage	I+II	18	10	8	0.351	0.569	3	15	0.061	1.000
	III+IV	36	23	13			7	29
Histological type	IDBC	44	27	17	0.006	0.936	7	37	1.072	0.370
	Other	10	6	4			3	7
ER	−	24	19	5	5.926	0.015	5	19	0.153	0.736
	+	30	14	16			5	25
PR	−	27	20	7	3.818	0.093	6	21	0.491	0.728
	+	27	13	14			4	23
Her-2	−	33	18	15	1.539	0.261	9	24	4.310	0.069
	+	21	15	6			1	20
Ki67	−	22	15	7	0.781	0.410	5	17	0.436	0.723
	+	32	18	14			5	27
P53	−	23	13	10	0.355	0.584	3	20	0.796	0.489
	+	21	20	11			7	24
Luminal type	Luminal	42	22	20	6.237	0.044	6	36	2.520	0.284
	Her-2	4	4	0			1	3
	Basal-like	8	7	1			3	5
Chemotherapy regime	TAC/TEC	39	26	13	1.834	0.100	6	33	1.747	0.418
	TA/TE	13	6	7			3	10
	Other	2	1	1			1	1
Chemotherapy cycles	2 cycles	39	27	12	3.895	0.065	5	34	3.021	0.119
	≥3 cycles	15	6	9			5	10

IDBC: Invasive ductal breast cancer; For categorical variables, the x² value and P value were calculated by Chi-square tests and Fisher's exact tests.

Table 4. Univariate analysis of immunological features with respect to breast cancer patients’ response to NCT.

			Clinical response				Pathological response
Clinicopathological features		Total	CR+PR(n)	PD+SD(n)	x ^2	P	pCR (n)	No-pCR (n)	x ^2	P
Foxp3^+Treg	−	23	18	5	4.958	0.047	7	16	3.770	0.078
	+	31	15	16			3	28
CD33^+MDSC	−	21	17	4	5.692	0.023	7	14	4.998	0.035
	+	33	16	17			3	30
IDO	−	27	21	6	6.312	0.024	7	20	1.964	0.293
	+	37	12	15			3	24
IDO^+CD33^+MDSC	IDO^−/CD33^−	16	15	1	10.578	0.014	5	11	6.095	0.107
	IDO^−/CD33^+	11	6	5			2	9
	IDO^+/CD33^−	5	2	3			2	3
	IDO^+/CD33^+	22	10	12			1	21

Table 5. Multivariate analysis of immunological and clinicopathological features with respect to breast cancer patients’ clinical response to NCT.

Features		β	SE	Wald	df	P	OR(95% CI)
CD33^+MDSC	−						1
	+	4.040	1.501	7.246	1	0.007	56.843(3.000∼1077.076)
IDO	−						1
	+	3.286	1.518	4.687	1	0.030	26.743(1.365∼523.973)
IDO^+CD33^+MDSC	Other						1
	IDO^+CD33^+	4.981	2.010	6.139	1	0.013	145.553(2.831∼7483.474)
Pathological T stage	pT0, T1, T2						1
	pT3, T4	2.659	1.020	6.791	1	0.009	14.287(1.933∼105.574)
ER	−						1
	+	1.705	0.968	3.100	1	0.078	5.502(0.825∼36.709)

β:regression coefficient; SE: standard error; Wald: wald chi-square; df: degree of freedom; OR:odds ratio.

Table 6. Multivariate analysis of immunological and clinicopathological features with respect to breast cancer patients’ pathological response to NCT.

Features		β	SE	Walds	df	P	OR(95% CI)
CD33^+MDSC	−						1
	+	0.260	0.960	0.073	1	0.787	1.297(0.198∼8.505)
Clinical T stage	pT0, T1, T2						1
	T3, T4	2.334	1.138	4.206	1	0.040	10.322(1.109∼96.078)
Clinical N stage	N0						1
	N1-N3	1.997	0.965	4.279	1	0.039	7.366(1.111∼48.852)

β:regression coefficient; SE: standard error; Wald: wald chi-square; df: degree of freedom; OR:odds ratio.

Figure 2. The survival curves for breast cancer patients treated by NCT. (a) PFS curves arranged by clinical stage (P = 0.021); (b) OS curves arranged by clinical T stage (P = 0.001); (c) PFS curves arranged by tumor-infiltrating CD33⁺MDSCs (P = 0.225); (d) OS curves arranged by tumor-infiltrating CD33⁺MDSCs (P = 0.121); (e) PFS curves arranged by IDO expression (P = 0.378); (f) OS curves arranged by IDO expression (P = 0.508); (g) PFS curves arranged by tumor-infiltrating Foxp3⁺Tregs (P = 0.979); (h) OS curves arranged by tumor-infiltrating Foxp3⁺Tregs (P = 0.022); (i) PFS curves arranged by IDO expression in CD33⁺MDSCs (P = 0.354); (j) OS curves arranged by IDO expression in CD33⁺MDSCs (P = 0.523).

Table 7. COX regression for prognostic significance of immunological features for breast cancer patients.

Features			β	SE	Wald	df	P	HR(95% CI)
PFS	Foxp3^+Treg	−						1
		+	−0.156	0.887	0.031	1	0.861	0.856(0.151∼4.866)
	CD33^+MDSC	−						1
		+	2.826	1.506	3.523	1	0.061	16.873(0.882∼322.662)
	IDO	−						1
		+	0.626	1.254	0.250	1	0.617	1.871(0.160∼21.841)
	IDO^+CD33^+MDSC	Other						1
		IDO^+CD33^+	−1.963	1.846	1.131	1	0.288	0.140(0.004∼5.236)
OS	Foxp3^+Treg							1
		+	10.437	3.874	7.259	1	0.007	34107.438(17.192∼664331.905)
	CD33^+MDSC	−						1
		+	−0.448	1.349	.110	1	0.740	0.639(0.045∼8.988)
	IDO	−						1
		+	6.788	4.342	2.444	1	0.118	887.195(0.179∼67700.393)
	IDO^+CD33^+MDSC	Other						1
		IDO^+CD33^+	1.689	3.104	.296	1	0.586	5.412(0.012∼2374.547)

β:regression coefficient; SE: standard error; Wald: wald chi-square; df: degree of freedom; HR:hazard ratio.

To get adjusted hazard ratios, COX regression was applied for adjusting covariates, which included Clinical T stage, Clinical N stage, Clinical stage, Pathological T stage, Pathological N stage, Pathological stage, Histological type, ER status, PR status, Her-2, Luminal type, Clinical response, Pathological response, Surgery method, Post-operative chemotherapy, Post-operative radiation therapy, Post-operative hormone therapy.

Kaufmann M, von Minckwitz G, Smith R, Valero V, Gianni L, Eiermann W, Howell A, Costa SD, Beuzeboc P, Untch M, et al. International expert panel on the use of primary (preoperative) systemic treatment of operable breast cancer: review and recommendations. J Clin Oncol. 2003;21(13):2600–8. doi:10.1200/JCO.2003.01.136. PMID:12829681.

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