The association of clinical outcome and peripheral T-cell subsets in metastatic colorectal cancer patients receiving first-line FOLFIRI plus bevacizumab therapy

Figures & data

Figure 1. Therapy-induced changes in the ratio of CD4⁺ effectors to regulatory T cells (Tregs), and in the immunosuppressive activity of Tregs in patients with colorectal cancer. (A) Waterfall plot of the change in the ratio between CD4⁺ effector T cells and regulatory T cells (CD4⁺:Treg ratio) in the course of FOLFIRI therapy in patients (n = 23) with first-line metastatic colorectal cancer. (B) Enough peripheral blood mononuclear cells (PBMCs) from 13 patients were available to measure suppressive activity of Tregs. Waterfall plot of the change in suppressive activity of Tregs after FOLFIRI therapy, compared to baseline, in patients with first-line metastatic colorectal cancer. Peripheral blood samples were collected at baseline and after 30 d of therapy.

Table 1. Patient demographics and clinical characteristics.

Parameter		Median (% or IQR)
Age (years)		71 (55–83)
Gender	Male	11 (48%)
	Female	12 (52%)
Primary tumor	Colon	19 (83%)
	Rectal	4 (17%)
Grade	1	0 (0%)
	2	16 (70%)
	3	7 (30%)
ECOG	0	16 (70%)
	1	7 (30%)
	3	0 (0%)
Mucinous/Non-mucinous	Mucinous	7 (30%)
	Non-mucinous	16 (70%)
Metastases	Liver only	8 (35%)
	Other tissues only	8 (35%)
	Liver and other tissues	7 (30%)
CEA		6.7 (3.1–22.5)
CA19.9		23 (5.3–108.5)
ALP		210 (130–268)
LDH		172 (133–293)

The median and frequency (%) or interquartile range (IQR) for all parameters are shown.

ECOG, Eastern Cooperative Oncology Group, NCI; CEA, carcinoembryonic antigen; ALP, alkaline phosphatase; LDH, lactate dehydrogenase.

Table 2. Evaluation of immune cell subsets in colorectal cancer patients prior to and after 30 d of standard-of-care anticancer therapy.

	Baseline	Post 30 d	N
Number of PBMCs (x10^3/µL)	2.1 (1.9–2.6)	2.1 (1.5–2.7)	23
CD4^+ T cells (%)	47 (31–53)	47 (39–52)	23
Number of CD4^+ T cells (x10^3/µL)	0.9 (0.6–1.1)	1.0 (0.7–1.3)	23
CD8^+ T cells (%)	24 (15–30)	22 (19–26)	23
Number of CD8^+ T cells (x10^3/µL)	0.5 (0.4–0.8)	0.5 (0.3–0.6)	23
Treg of CD4^+ T cells (%)	5.8 (4.7–8.0)	5.7 (4.6–6.7)	23
Treg of PBMCs (%)	2.6 (2.1–3.3)	2.6 (1.5–3.2)	23
Number of Tregs (x10^3/µL)	0.06 (0.04–0.07)	0.06 (0.03–0.07)	23
CD4^+ Teff:Treg Ratio	17.0 (12.4–21.3)	17.6 (14.9–21.7)	23
CD8^+ Teff:Treg Ratio	9.9 (6.1–15.0)	8.7 (5.7–16.0)	23
CD4^+:CD8^+ Ratio	1.9 (1.1–3.1)	2.0 (1.4–3.2)	23
Treg suppression (%)	44 (34.5–51)	39 (28.0–58.0)	13

Twenty-three first-line metastatic colorectal cancer patients were enrolled and treated with FOLFIRI therapy. PBMCs were collected at baseline and post 30 d of therapy, and subjected to flow cytometry analysis as described in Methods section. Values are the median (interquartile range). Wilcoxon test between baseline and post 30 d showed no statistical differences for any of the immune cell subsets studied, or for the Treg suppression assay.

PBMCs, peripheral blood mononuclear cells; Treg, regulatory T cells.

Figure 2. Therapy-induced changes in the ratios of CD8⁺ effectors to regulatory T cells in patients with colorectal cancer. Waterfall plot of the change in the ratio between CD8⁺ effector T cells and regulatory T cells (CD8⁺:Treg ratio) in the course of FOLFIRI therapy in patients with first-line metastatic colorectal cancer (n = 23). Peripheral blood samples were collected at baseline and after 30 d of therapy.

Figure 3. Association between the frequency of regulatory T cells (Tregs) in peripheral blood mononuclear cells (PBMCs) at baseline and progression-free survival (PFS) after FOLFIRI therapy in patients with colorectal cancer. Patients were categorized based on their frequency of Tregs (< or > 25% of PBMCs) before therapy. A Kaplan–Meier curve is shown for these two groups and the association with PFS. The median PFS for patients with <2.5% Tregs pre-treatment was 23.3 months, and for patients with >2.5% Tregs pre-treatment 10.7 months (p = 0.037, n = 23, 3 patients were censored).

Figure 4. Association between the change in regulatory T cell (Treg) frequency in peripheral blood mononuclear cells (PBMCs) after FOLFIRI therapy and overall survival (OS) in patients with colorectal cancer. Patients were categorized based on any decrease or increase in Treg frequency after 30 d of FOLFIRI therapy. (A) A Kaplan–Meier curve is shown for these two groups and the association with OS. The median OS for patients with decreased Tregs after 30 d treatment was 43.3 mo, and for patients with increased Tregs after treatment, OS was 23.3 mo (p = 0.036, n = 23). The change in Treg frequency was found to be an independent predictive variable for OS by stepwise multiple regression analysis. (B) The median decrease in Treg frequency was −26%. (C) The median increase in Treg frequency was +23%.

Table 3. Clinical characteristics of patients enrolled on the study

Pt	Age	Gender	Primary	Mucinous/ Non-mucinous	ECOG	Grade	Metastases 1.liver 2.liver+other 3.other	CEA Pre	CA19.9 Pre	Best Response by RECIST	PFS (mo)	OS (mo)	MSI status	CD3^+	CD8^+	CD4^+
1	73	F	colon	Non Muc	1	2	3	94.6	6803	PR	55.3	55.3	MSS	moderate	mild	moderate
2	80	F	colon	Non Muc	0	2	1	6.6	20.8	SD	11.4	38.3	MSS	moderate	mild	moderate
3	70	F	colon	Muc	0	2	2	14.7	40.1	PR	8.6	11.4	MSS	moderate	mild	mild
4	81	M	colon	Non Muc	1	2	3	3.1	8.1	PD	1.8	14.1
5	74	M	colon	Muc	0	3	1	15.5	57.9	SD	9.7	25.7	MSI	mild	mild	mild
6	63	F	colon	Non Muc	0	3	3	6.7	23.2	PR	42.7	42.7	MSS	severe	moderate	moderate
7	79	F	colon	Non Muc	0	2	3	1	3.8	SD	21.3	43.9	MSS	mild	mild	mild
8	56	M	colon	Non Muc	0	3	1	17	108.5	PR	7.8	13.5	MSS	severe	mild	moderate
9	68	F	colon	Muc	1	2	1	22.5	410.1	PD	4.7	27.2
10	55	F	colon	Non Muc	0	2	1	1389	10000	SD	5.9	8.1
11	83	F	colon	Muc	1	3	1	0.8	0.6	SD	13.2	40.9	MSS	moderate	mild	mild
12	57	F	rectal	Non Muc	0	2	2	26.5	1473	PR	10.7	10.7
13	41	M	rectal	Non Muc	0	2	3	3.3	8.8	PD	3	9	MSI	mild	absent	mild
14	71	M	rectal	Non Muc	0	2	1	2.7	5.3	PR	15.5	43.3	MSS	moderate	mild	mild
15	66	M	rectal	Non Muc	0	3	3	6.6	22.9	PR	37.9	28.9	MSS	severe	mild	moderate
16	77	M	colon	Non Muc	0	2	2	5.2	9.8	PR	34.1	37.2
17	62	F	colon	Muc	0	3	3	26.8	2412	PR	22	55.9	MSS	moderate	mild	mild
18	66	M	colon	Non Muc	0	2	1	1.8	4.7	PR	30.1	51.6	MSS	moderate	mild	mild
19	72	M	colon	Muc	1	2	2	1.7	4.5	PR	23.3	23.3	MSS	moderate	mild	mild
20	75	M	colon	Non Muc	0	3	2	12.6	34.5	SD	9.9	31
21	69	F	colon	Muc	0	2	3	6.9	25.5	PR	12.4	47.2
22	76	F	colon	Non Muc	1	2	2	6.2	13.4	SD	12.7	18.7
23	76	M	colon	Non Muc	1	2	2	125	0.1	PD	3.9	6.9	MSS	moderate	mild	mild

ECOG, Eastern Cooperative Oncology Group, NCI; CEA, carcinoembryonic antigen; RECIST, Response Evaluation Criteria in Solid Tumors; PR, partial response; SD, stable disease; PD, progressive disease; PFS, progression-free survival; OS, overall survival; MSI, microsatellite instability; MSS, microsatellite stability.

Table 4. Multiple regression analysis of variables associated with overall survival.

Variable	Standardized regression coefficient	Standard error	p value
Tregs at baseline	0.506	0.175	0.015
LDH	0.628	0.220	0.016
Change in Tregs at 30 d	0.443	0.194	0.043
Mucinous vs. non-mucinous	−0.252	0.176	0.181
Metastases	−0.223	0.167	0.209
ALP	−0.197	0.188	0.315
Age	0.164	0.184	0.392
Grade	−0.118	0.170	0.500
CEA	−0.066	0.344	0.851
Gender	0.033	0.192	0.866
CA19.9	0.005	0.341	0.988
Forward Stepwise Method
LDH	0.500	0.139	0.002
Tregs at baseline	0.467	0.142	0.004
Change in Tregs at 30 d	0.454	0.143	0.006
Mucinous vs. non-mucinous	−0.249	0.139	0.091
Metastases	−0.200	0.139	0.168

Metastases were categorized as 1. Liver vs. 2. liver + other sites vs. 3. Other sites.

Tregs, regulatory T cells; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; CEA, carcinoembryonic antigen.