Tumor lymphocyte immune response to preoperative radiotherapy in locally advanced rectal cancer: The LYMPHOREC study

Figures & data

Figure 1. Example of slides of FoxP3 and CD8 labeling A and C: CD8+ labeling, B and D: FoxP3+ labeling, A and B patient N°1; C and D: patient N°2, red arrow: sTILs (stromal Tumor-Infiltrating Lymphocytes), black arrow: iTILS (intraepithelial Tumor-Infiltrating Lymphocytes).

Table 1. Characteristics of patients included in LYMPHOREC study.

	N = 237
Age, years
Median [min-max]	66.9 [28.7–85.5]
Gender
Male	159 (67.1%)
Female	78 (32.9%)
Histopathology type
Poorly differentiated adenocarcinoma	14 (5.9%)
Moderately differentiated adenocarcinoma	114 (48.1%)
Highly differentiated adenocarcinoma	104 (43.9%)
Others	5 (2.1%)
Tumor stage
1	2 (0.8%)
2	10 (4.2%)
3	190 (80.2%)
4	21 (8.9%)
Unknown	14 (5.9%)
Node stage
0	88 (37.1%)
1	130 (54.9%)
Unknown	19 (8.0%)
Metastatic stage
0	211 (89.0%)
1	18 (7.6%)
Unknown	8 (3.4%)
Radiotherapy
Long course (≤ 2 Gy / fraction)	174 (73.4%)
Short course (> 2 Gy / fraction)	60 (25.3%)
Unknown	3 (1.3%)
Concomitant Chemotherapy
Yes	111 (46.9%)
Capecitabine alone	41 (36.94%)
Folic Acid + 5FU	32 (28.83%)
Oxaliplatin + Capecitabine	25 (22.52%)
Platin + 5FU	5 (4.5%)
5FU alone	4 (3.6%)
Folfox	3 (2.7%)
Capecitabine + Bevacizumab	1 (0.9%)
No	122 (51.5%)
Unknown	4 (1.7%)
Adjuvant Chemotherapy
Yes	73 (30.8%)
No	158 (66.7%)
Unknown	6 (2.5%)
Median follow up (years)	8.1 (95% CI^c = [7.4–8.8])
Median PFS^a (years)	6.3 (95% CI = [4.3–8.3]
Median OS^b (years)	7.9 (95% CI = [6.4–0.9])

^a PFS: Progression Free Survival;

^b OS: Overall Survival;

^c 95% confidence interval.

Table 2. Effect of neo-adjuvant treatments on TILs quantity.

	Before treatment (biopsy)	After treatment (surgical sample)	Delta (after – before ttt)
Median [min-max]	N = 135	N = 237	N = 132	P value
TILs^a CD8+	44.5 [0.0–290.7]	47.0 [1.8–421.3]	2.4 [−214.7–186.3]	0.496
iTILs^b CD8+	4.05 [0.0–87.2]	1.8 [0.0–97.0]	−1.0 [−79.6–64.2]	0.120
sTILs^c CD8+	35.7 [0.0–203.5]	1.8 [0.0–97.0]	3.0 [−135.1–182.0]	0.217
TILs FoxP3+	32 [0.0–253.3]	15.5 [0.0–141.3]	−8.6 [−209.0–101.0]	<0.001
iTILs FoxP3+	0 [0.0–20.7]	0.0 [0.0–6.6]	0.0 [−18.1–6.6]	<0.001
sTILs FoxP3+	32 [0.0–245.7]	14.9 [0.0–141.3]	−8.0 [−206.8–101.0]	<0.001
TILs CD8+ / FoxP3+	1.2 [0.0–317.3]	3.1 [0.1–121.3]	0.7 [−310.7–56.8]	0.008
iTILs CD8+ / FoxP3+	2.6 [0.0–50.9]	1.6 [0.0– 97.0]	−0.3 [−31.4–53.4]	0.753
sTILs CD8 +/ FoxP3+	1.1 [0.0– 200.7]	2.7 [0.1–103.1]	0.7 [−200.5–64.4]	0.012

^a TIL: Tumor- Infiltrating Lymphocytes;

^b iTIL: intraepithelial Tumor-Infiltrating Lymphocytes;

^c sTIL: stromal Tumor-Infiltrating Lymphocytes.

Table 3. Effect of radiotherapy scheme on TILs.

	lc-preopRT^d	sc-preopRT^e
	(N = 170)	(N = 60)	P value
TILs^a CD8+	64.4 (54.7)	57.4 (61.1)	0.325
iTILs^b CD8+	7.8 (14.3)	8.2 (14.2)	0.732
sTILs^c CD8+	56.6 (47.8)	49.2 (52.8)	0.253
TILs FoxP3+	24.7 (27.9)	23.4 (19.3)	0.370
iTILs FoxP3+	0.4 (1.1)	0.1 (0.5)	0.437
sTILs FoxP3+	24.3 (27.5)	23.3 (19.2)	0.327
TILs CD8+ / FoxP3+	8.2 (15.0)	4.3 (5.9)	0.027
iTILs CD8+ / FoxP3+	6.7 (13.0)	7.6 (13.3)	0.589
sTILs CD8 +/ FoxP3+	7.4 (13.2)	3.9 (5.8)	0.019

^a TIL: Tumor-Infiltrating Lymphocytes;

^b iTIL: intraepithelial Tumor-Infiltrating Lymphocytes;

^c sTIL: stromal Tumor-Infiltrating Lymphocytes;

^d lc-preopRT: long course preoperative radiotherapy;

^e sc-preopRT: short course preoperative radiotherapy.

Table 4. Relation between TILs at the baseline and after preopRT and progression free survival.

		PFS^b
		Univariate Analysis			Multivariate Analysis^g
	TILs^a parameters	HR	95% CI^c	P value	HR	95% CI	P value
At baseline (N = 135)	CD8+ TILs			0.432
	<21.3	1 (ref.)	[0.687–2.446]
	]21.3; 44.5]	1.296	[0.695–2.541]
	]44.5; 74.3]	1.329	[0.398–1.642]
	> = 74.3	0.809
	FoxP3+ TILs			0.116
	<1.0	1 (ref.)	[0.308– 1.150]
	]1.0; 32.0]	0.595	[0.615– 2.028]
	]32.0; 82.7]	1.116	[0.300– 1.163]
	> = 82.7	0.591
	FoxP3+ iTILs^d			0.810
	<1.4	1 (ref.)	[0.542–1.615]
	> = 1.4	0.935
	FoxP3+ sTILs^e			0.115
	<0.7	1 (ref.)	[0.456–1.669]
	]0.7; 32.0]	0.873	[0.766–2.672]
	]32.0; 79.1]	1.431	[0.309–1.309]
	> = 79.1	0.636
	CD8+/FoxP3+ TlLs			0.199
	<0.7	1 (ref.)	[0.353–1.366]
	]0.7; 2.6]	0.694	[0.349–1.354]
	]2.6; 8.0]	0.687	[0.674–2.282]
	> = 8.0	1.240
After preoRT^f (N = 232)	CD8+ TILs			0.011			0.081
	<24.6	1 (ref.)	[0.485–1.221]		1	[0.500–1.343]
	]24.6; 47.0]	0.770	[0.330–0.885]		0.820	[0.370–1.055]
	]47.0; 83.0]	0.540	[0.276–0.768]		0.625	[0.303–0.890]
	> = 83.0	0.461			0.519
	FoxP3+ TILs			0.003			0.059
	<6.5	1 (ref.)	[0.416–1.076]		1	[0.521–1.502]
	]6.5; 15.5]	0.669	[0.369–0.971]		0.884	[0.395–1.140]
	]15.5; 36.5]	0.599	[0.226–0.635]		0.671	[0.273–0.849]
	> = 36.5	0.379			0.481
	CD8+/FoxP3+ TlLs			0.176
	<1.4	1 (ref.)	[0.771–2.114]
	]1.4; 3.1]	1.276	[0.644–1.878]
	]3.1; 6.5]	1.100	[1.024–2.771]
	> = 6.5	1.684
Delta after preopRT minus at baseline (n = 132)	CD8+ TILs			0.734
	<−24.8	1 (ref.)	[0.643–2.400]
	]−24.8; 2.4]	1.242	[0.425–1.721]
	]2.4; 34.1]	0.855	[0.547–2.113]
	> = 34.1	1.075
	FoxP3+ TILs			0.304
	<−62.1	1 (ref.)	[0.811–3.229]
	]−62.1; −8.6]	1.618	[0.773–3.055]
	]−8.6; 14.2]	1.537	[0.477–2.038]
	> = 14.2	0.986
	CD8+/FoxP3+ TlLs			0.531
	<−3.4	1 (ref.)	[0.352–1.331]
	]−3.4; 0.7]	0.685	[0.344–1.243]
	]0.7; 3.4]	0.654	[0.359–1.361]
	> = 3.4	0.699
	CD8+/FoxP3+ iTlLs			0.015			0.049
	<−3.8	1 (ref.)	[1.527–7.740]		1	[1.244–7.392]
	]−3.8; −0.3]	3.438	[1.544–7.822]		3.033	[1.399–7.758]
	]−0.3; 2.5]	3.475	[1.127–6.062]		3.295	[1.049–6.437]
	> = 2.5	2.614			2.598

^a TIL: Tumor-Infiltrating Lymphocytes;

^b PFS: Progression Free Survival;

^c 95% confidence interval;

^d iTIL: intraepithelial Tumor-Infiltrating Lymphocytes;

^e sTIL: stromal Tumor-Infiltrating Lymphocytes;

^f preopRT: preoperative radiotherapy;

^g Cox multivariate models have been adjusted for chemotherapy, T, N, M and the delay between radiotherapy and surgery.

Table 5. Relation between TILs at the baseline and after preopRT and overall survival.

		OS^b
		Univariate Analysis			Multivariate Analysis^g
	TILs^a parameters	HR	95% CI^c	P value	HR	95% CI	P value
At baseline (N = 135)	CD8+ TILs			0.119
	<21.3	1 (ref.)	[0.544–2.744]
	]21.3; 44.5]	1.222	[0.401–2.254]
	]44.5; 74.3]	0.950	[0.046–1.079]
	> = 74.3	0.224
	FoxP3+ TILs			0.016			0.040
	<1.0	1 (ref.)	[0.218–0.898]		1	[0.235–1.047]
	]1.0; 32.0]	0.443	[0.605 −2.004]		0.497	[0.612–2.330]
	]32.0; 82.7]	1.101	[0.231–1.002]		1.194	[0.187–1.060]
	> = 82.7	0.481			0.446
	FoxP3+ iTILs^d			0.938
	<1.4	1 (ref.)
	> = 1.4	1.023	[0.574–1.825]
	FoxP3+ sTILs^e			0.027			0.035
	<0.7	2.017	[0.921– 4.419]		1	[0.340–1.452]
	]0.7; 32.0]	1.310	[0.592– 2.896]		0.702	[0.728–2.983]
	]32.0; 79.1]	2.724	[1.294–5.733]		1.474	[0.170–1.104]
	> = 79.1	1 (ref.)			0.433
	CD8+/FoxP3+ TlLs			0.175
	<0.7	1 (ref.)	[0.353–1.366]
	]0.7; 2.6]	0.694	[0.349–1.354]
	]2.6; 8.0]	0.687	[0.674–2.282]
	> = 8.0	1.240
After preoRT^f (N = 232)	CD8+ TILs			0.106
	<24.6	1 (ref.)	[0.521–1.415]
	]24.6; 47.0]	0.858	[0.367–1.043]
	]47.0; 83.0]	0.619	[0.321–0.949]
	> = 83.0	0.552
	FoxP3+ TILs			0.054
	<6.5	1 (ref.)	[0.426–1.199]
	]6.5; 15.5]	0.715	[0.394–1.124]
	]15.5; 36.5]	0.665	[0.267–0.802]
	> = 36.5	0.463
	CD8+/FoxP3+ TlLs			0.549
	<1.4	1 (ref.)	[0.830–2.378]
	]1.4; 3.1]	1.405	[0.741–2.232]
	]3.1; 6.5]	1.287	[0.826–2.447]
	> = 6.5	1.422
Delta after preopRT minus at baseline (n = 132)	CD8+ TILs			0.689
	<−24.8	1 (ref.)	[0.717–3.051]
	]−24.8; 2.4]	1.480	[0.513–2.331]
	]2.4; 34.1]	1.093	[0.631–2.780]
	> = 34.1	1.325
	FoxP3+ TILs			0.200
	<−62.1	1 (ref.)	[0.961–4.250]
	]−62.1; −8.6]	2.021	[0.725–3.347]
	]−8.6; 14.2]	1.558	[0.520–2.463]
	> = 14.2	1.132
	CD8+/FoxP3+ TlLs			0.308
	<−3.4	1 (ref.)	[0.275–1.141]
	]−3.4; 0.7]	0.560	[0.300–1.165]
	]0.7; 3.4]	0.591	[0.412–1.570]
	> = 3.4	0.804
	CD8+/FoxP3+ iTlLs			0.014			0.024
	<−3.8	1 (ref.)	[1.405–8.753]		1	[1.102–8.113]
	]−3.8; −0.3]	3.507	[1.808–11.18]		2.990	[1.715–11.609]
	]−0.3; 2.5]	4.495	[1.195–7.818]		4.463	[1.114–8.424]
	> = 2.5	3.057			3.063

^a TIL: Tumor-Infiltrating Lymphocytes;

^b OS: Overall Survival;

^c 95% confidence interval;

^d iTIL: intraepithelial Tumor-Infiltrating Lymphocytes;

^e sTIL: stromal Tumor-Infiltrating Lymphocytes;

^f preopRT: preoperative radiotherapy;

^g Cox multivariate models have been adjusted for chemotherapy, T, N, M and the delay between radiotherapy and surgery.

Figure 2. Effect of delta CD8+ / FoxP3+ iTILs on survival Kaplan-Meier curves for PFS (a) and for OS (b) stratified according to quartiles of delta CD8+ / FoxP3+ iTILs (intraepithelial Tumor-Infiltrating Lymphocytes).