Neoadjuvant radiochemotherapy decreases the total amount of tumor infiltrating lymphocytes, but increases the number of CD8+/Granzyme B+ (GrzB) cytotoxic T-cells in rectal cancer

Figures & data

Table 1. Patients' characteristics.

	nRCTx (n = 130)		control group (n = 30)
	n/median (range)	%	n/median (range)	%	p
Sex
male	96	73.8	20	66.7	n.s.
female	34	26.2	10	33.3
Age (y)	61.0 (36–82)		67.6 (22–76)
≤ 65	88	67.7	12	40.0	0.005
> 65	42	32.3	18	60.0
NCTx
5-FU	117	90.0	—	—	—
others	13	10.0	—	—	—
Accumulated radiation dose [in Gy]
46.8	1	0.8	—	—	—
50.4	125	96.2	—	—	—
55.8	4	3	—	—	—
Death
Yes	45	34.6	13	43.3	n.s.
No	85	65.4	17	56.7
Metastases in follow up
Yes	39	30.0	7	23.3	n.s.
liver	5	12.8	1	14.3
lung	7	17.9	0	0.0
others	6	15.4	0	0.0
No	91	70.0	23	76.7
Local recurrence
Yes	10	7.7	2	6.7	n.s.
No	120	92.3	28	93.3
Follow up [months]	59.8 (0.3–155.5)		106.9 (12.9–163.4)

Abbreviations: n.s.: not significant (P>0.05); 5-FU: 5-fluorouracil; nCTx: neoadjuvant chemotherapy; Gy: Gray.

Table 2. Pathological characteristics.

	nRCTx (n = 130)		control group (n = 30)
	n/median (range)	%	n/median (range)	%	p
ypT
1	7	5.4	3	10.0	n.s.
2	36	27.7	9	30.0
3 a	47	36.2	12	40.0
3b	35	26.9	6	20.0
4	5	3.8	0	0.0
ypN
0	83	63.8	22	73.3	n.s.
1	31	23.8	5	16.7
2	16	12.4	3	10.0
lymph nodes investigated	14.0 (3–35)		14.0 (4–56)		n.s.
CM
0	120	92.3	24	80.0	0.043
1	10	7.7	6	20.0
Microsatellites^#
stable	123	94.6	28	93.3	n.s.
instable	5	3.8	2	6.7
L
0	108	83.1	19	63.3	0.016
1	22	16.9	11	36.7
V
0	122	92.3	22	73.3	0.003
1	10	7.7	8	26.7
Pn
0	98	75.4	21	70.0	n.s.
1	32	24.6	9	30.0
Tumor regression grade (TRG)
0	1	0.8	—	—	—
1	33	25.4	—	—	—
2	77	59.2	—	—	—
3	19	14.6	—	—	—

Abbreviations: n.s.: not significant (P>0.05); ypT: size and extent of primary tumor (y: posttherapy stage; p: pathological stage); ypN: involvement of regional lymph node (y: posttherapy stage; p: pathological stage); cM: presence or absence of distant metastasis (clinical stage); L: lymphatic invasion; V: vessel invasion; Pn: perineural invasion;

# two cases were not evaluable because of insufficient tumor cell content.

Figure 1. Pathway of the immune cell quantification. IHC: immunohistochemistry.

Table 3. Immune cell infiltrate.

	nRCTx (n = 130)	control group (n = 30)
	n/median (range)	n/median (range)	p
CD3+ stroma	260.8 (51.3–719.3)	526.3 (173.7–964.3)	<0.001
CD3+ epithel	3.0 (0–35)	8.5 (1–55)	<0.001
CD3+/MIB1+ stroma	1.7 (0–37.7)	0.7 (0–19.7)	n.s.
CD3+/MIB1+ epithel	0.0 (0–4)	0.0 (0–3)	n.s.
CD8+ stroma	86.5 (0–451.3)	154.0 (53.3–267.3)	<0.001
CD8+ epithel	2.0 (0–31)	5.5 (0–48)	0.002
GrzB+/CD8+ stroma	43.8 (0–240)	48.8 (17.0–195.7)	n.s.
GrzB+/CD8+ epithel	2.0 (0–29)	5.0 (0–22)	0.028
CD8/GrzB//CD8 stroma	0.52 (0–1)	0.32 (0.21–0.78)	<0.001
CD8/GrzB//CD8 epithel	0.82 (0–1)	0.57 (0–1)	n.s.

Abbreviations: n.s.: not significant (P>0.05); CD: cluster of differentiation; MIB: mindbomb E3 ubiquitin protein ligase 1; GrzB: granzyme B.

Figure 2. Boxplots of CD3⁺/CD8⁺ in tumor stroma (A) and in tumor epithel (B); Data distribution of CD8⁺/GrzB⁺ and CD8⁺ cells in tumor stroma (C) and tumor epithel (D). CD: cluster of differentiation; GrzB: granzyme B.

Table 4. Differences of immune expression in preoperative biopsy and tumor.

	Biopsy (median (range))	Tumor (median (range))	p
CD3+ stroma	387.3 (85.7–850.3)	252.5 (94.7–719.3)	n.s.
CD3+ epithel	9.0 (0.0–34.0)	3.0 (0–23)	n.s.
CD3+/MIB1+ stroma	4.8 (0.3–29.0)	2.3 (0–19)	n.s.
CD3+/MIB1+ epithel	0.5 (0.0–3.0)	0.0 (0–4)	n.s.
CD8+ stroma	65.0 (25.0–156.7)	103.5 (21.7–253.7)	0.036
CD8+ epithel	3.0 (0–17)	2.0 (0–21)	n.s.
GrzB+/CD8+ stroma	26.7 (12.7–79.7)	38.5 (0–167.3)	0.011
GrzB+/CD8+ epithel	2.0 (0–12)	2.0 (0–19)	n.s.
GrzB/CD8//CD8 stroma	0.60 (0–0.71)	0.46 (0.27–0.59)	0.007
GrzB/CD8//CD8 epithel	0.85 (0–1)	0.77 (0–1)	n.s.

Abbreviations: n.s.: not significant (P>0.05); CD: cluster of differentiation; MIB: mindbomb E3 ubiquitin protein ligase 1; GrzB: granzyme B.

Table 5. Survival analysis.

	OS			RFS
	univariate	multivariate		univariate	multivariate
	p#	HR (95% CI)	p##	p#	HR (95% CI)	p##
gender (m)	n.s.		n.s.	n.s.		n.s.
age (>65y)	0.014	2.18 (1.14–4.20)	0.019	n.s.		n.s.
ASA (3)	0.005	3.37 (1.64–6.93)	0.001	n.s.	1.84 (1.00–3.39)	0.050
ypT (3/4)	0.039		n.s.	n.s.		n.s.
ypN (+)	0.024	2.15 (1.05–4.40)	0.036	0.008		n.s.
cM (1)	0.002	3.45 (1.40–8.48)	0.007	<0.001	4.54 (1.90–10.85)	0.001
MSI-H	0.015		n.s.	n.s.		n.s.
L (1)	n.s		n.s.	0.023		n.s.
V (1)	n.s.		–	n.s.		n.s.
Pn (1)	0.005		n.s.	0.019		n.s.
CD3+ stroma	n.s.		n.s.	n.s.		n.s.
CD3+ epithel	0.020		n.s.	n.s.		n.s.
CD3+/MIB1+ stroma	n.s.		n.s.	n.s.		n.s.
CD3+/MIB1+ epithel	n.s.		n.s.	n.s.		n.s.
CD8+ stroma	n.s.		n.s.	n.s.		n.s.
CD8+ epithel	n.s.		n.s.	n.s.		n.s.
GrzB+/CD8+ stroma	n.s.		n.s.	n.s.		n.s.
GrzB+/CD8+ epithel	n.s.		n.s.	n.s.		n.s.
GrzB+/CD8+//CD8+ stroma	n.s.		n.s.	n.s.		n.s.
GrzB+/CD8+//CD8+ epithel	n.s.	0.51 (0.27–0.96)	0.037	n.s.		n.s.

Abbreviations: n.s.: not significant (P>0.05); OS: overall survival; RFS: recurrence free survival; HR: hazard ratio; CI: confidence interval; ASA: American Society of Anesthesiologists; ypT: size and extent of primary tumor (y: posttherapy stage; p: pathological stage); ypN: involvement of regional lymph node (y: posttherapy stage; p: pathological stage); cM (1): presence of distant metastasis (clinical stage); MSI-H: microsatellite instability – high; L: lymphatic invasion; V: vessel invasion; Pn: perineural invasion; CD: cluster of differentiation; MIB: mindbomb E3 ubiquitin protein ligase 1; GrzB: granzyme.

Table 6. Tumor regression grade (TRG) system according to (6).

Tumor Regression Grade	Histopathological findings
Grade 0:	No regression.
Grade 1:	Minor regression, residual tumor outgrowing fibrosis.
Grade 2:	Moderate regression, low tumor cell fibrosis in 26% to 50% of the tumor mass.
Grade 3:	Major regression, very few tumor cells in fibrotic tissue with or without mucous substance.
Grade 4:	Total regression, no viable tumor cells, only fibrotic mass.

Tumor regression grade (TRG) system according to Dworak et al.