Human papilloma virus load and PD-1/PD-L1, CD8⁺ and FOXP3 in anal cancer patients treated with chemoradiotherapy: Rationale for immunotherapy

Figures & data

Table 1. Correlation of CD8⁺, PD-1 and PD-L1 with clinicopathologic parameters in the entire cohort.

Parameter	No.	CD8 low n (%)	CD8 high n (%)	p value	PD-1 low n (%)	PD-1 high n (%)	p value	PD-L1 low n (%)	PD-L1 high n (%)	p value
Age
≤ 59 y	75	48 (48)	27 (53)	0.730	38 (49)	37 (51)	1	45 (45)	30 (60)	0.119
> 59 y	75	51 (52)	24 (47)		39 (51)	36 (49)		55 (55)	20 (40)
Gender
male	66	46 (46)	20 (39)	0.488	36 (47)	30 (41)	0.514	45 (45)	21 (42)	0.862
female	84	53 ( 54)	31 (61)		41 (53)	43 (59)		55 (55)	29 (58)
T-stage
T1/2	105	66 (66)	40 (78)	0.133	53 (69)	51 (71)	0.859	71 (71)	34 (68)	0.709
T3/T4	45	34 (34)	11 (22)		24 (31)	21 (29)		29 (29)	16 (32)
N-stage
N0	97	57 (58)	40 (78)	0.012	50 (65)	47 (64)	1.00	64 (64)	33 (66)	0.858
N1–3	53	42 (42)	11 (22)		27 (35)	26 (36)		36 (36)	17 (34)
Grading
G1/2	114	72 (76)	42 (82)	0.429	60 (79)	54 (77)	0.881	80 (82)	34 (71)	.232
G3	25	19 (20)	6 (12)		13 (17)	12 (17)		15 (15)	10 (21)
Gx	7	4(4)	3 (6)		3 (4)	4 (6)		3 (3)	4 (8)
HPV-16 load
HPV-16 low	67	51 (56)	16 (36)	0.043	47 (68)	20 (30)	< 0.001	50 (55)	17 (39)	.098
HPV-16 high	68	40 (44)	28 (64)		22 (32)	46 (70)		41 (45)	27 (61)
p16I^NK4a
p16 low	73	54 (55)	19 (37)	0.058	45 (58)	28 (38)	0.015	48 (48)	25 (50)	0.863
p16 high	77	45 (45)	32 (63)		32 (42)	45 (62)		52 (52)	25 (50)
CD8^+
CD8 low					58 (75)	41 (56)	0.016	74 (74)	25 (50)	.006
CD8 high					19 (25)	32 (44)		26 (26)	25 (50)
PD-1
PD-1 low	77	58 (59)	19 (37)	0.016				63 (63)	14 (28)	< 0.001
PD-1 high	73	41 (41)	32 (63)					37 (37)	36 (72)
PD-L1
PD-L1 low	100	74 (75)	26 (51)	0.006	63 (82)	37 (51)	< 0.001
PD-L1 high	50	25 (25)	25 (49)		14 (18)	36 (49)
FOXP3
FOXP3 low	77	62 (63)	15 (29)	< 0.001	50 (65)	27 (37)	0.001	62 (62)	15 (30)	< 0.001
FOXP3 high	73	37 (37)	36 (71)		27 (35)	46 (63)		38 (38)	35 (70)
pCasp-8
pCasp-8 low	80	59 (60)	21 (41)	0.039	46 (60)	34 (47)	0.14	53 (53)	27 (54)	1.00
pCasp-8 high	70	40 (40)	30 (59)		31 (40)	39 (53)		47 (47)	23 (46)

Abbreviations: HPV, human papilloma virus; pCasp-8: phosphorylated Caspase-8 (pT273).

Significant p values have been marked with bold.

Figure 1. Prognostic impact of (A) CD8⁺ and (B) PD-1 and (C) PD-L1 expression on cumulative incidence of local recurrence and disease-free survival, as indicated. Analysis was based on the dichotomized total score in patient tumor samples (cut-off according to median value of total score).

Table 2. Univariate and multivariate analyses of prognostic factors in patients with ASCC.

Parameter	Univariate		Multivariate
	p value	HR	95% CI	p value
Cumulative incidence of local recurrence
Age (>/≤ 59 y)	0.710
Sex (female/male)	0.004	0.39	0.16 to 0.96	0.040
T-category (T3/4 / T1/2)	0.010	1.48	0.59 to 3.72	0.404
N-category (N1–3/N0)	0.004	2.29	0.99 to 5.30	0.054
HPV16 load (>/≤ median)^*	0.002	0.28	0.10 to 0.77	0.014
p16 (>/≤ median)^*	0.002	0.32	0.14 to 0.77	0.011
CD8 (>/≤ median)^*	0.007	0.25	0.07 to 0.83	0.023
PD-1 (>/≤ median)^*	0.003	0.29	0.12 to 0.72	0.007
PD-L1 (>/≤ median)^*	0.017	0.27	0.08 to 0.90	0.033
FOXP3 (>/≤median)^*	0.025	0.44	0.18 to 1.05	0.065
pCasp-8 (>/≤ median)^*	0.013	0.48	0.20 to 1.17	0.105
Disease-free survival
Age (>/≤ 59 y)	0.269
Sex (female/male)	0.004	0.44	0.23 to 0.82	0.010
T-category (T3/4 / T1/2)	0.060
N-category (N1–3/N0)	0.010	1.56	0.84 to 2.87	0.156
HPV16 load (>/≤ median)^*	0.001	0.40	0.20 to 0.79	0.009
p16 (>/≤ median)^*	0.088
CD8 (>/≤ median)^*	0.008	0.42	0.20 to 0.87	0.020
PD-1 (>/≤ median)^*	0.007	0.46	0.25 to 0.86	0.014
PD-L1 (>/≤ median)^*	0.063
FOXP3 (>/≤median)^*	0.013	0.54	0.29 to 1.00	0.050
pCasp-8 (>/≤ median)^*	0.001	0.49	0.26 to 0.94	0.031
Overall survival
Age (>/≤ 59 y)	0.050	2.32	1.13 to 4.73	0.021
Sex (female/male)	0.002	0.32	0.15 to 0.68	0.003
T-category (T3/4 / T1/2)	0.012	1.62	0.81 to 3.24	0.171
N-category (N1–3/N0)	0.022	1.11	0.51 to 2.43	0.789
HPV16 load (>/≤ median)^*	0.005	0.44	0.20 to 0.95	0.036
p16 (>/≤ median)^*	0.674
CD8 (>/≤ median)^*	0.040	0.52	0.23 to 1.19	0.120
PD-1 (>/≤ median)^*	0.039	0.54	0.26 to 1.10	0.090
PD-L1 (>/≤ median)^*	0.250
FOXP3 (>/≤median)^*	0.114
pCasp-8 (>/≤ median)^*	0.011	0.51	0.24 to 1.07	0.073

* Due to multicollinearity for HPV16, p16, CD8, PD-1, PD-L1, FOXP3 and caspase-8, and a limited number of events, multivariate analyses were performed separately including only one molecular parameter in conjunction with sex, T- and N-category each time. Only parameters found to be significant in the univariate analysis were included in the multivariate one. HRs and Cis originate from the respective, separate multivariate analysis.

Significant p values have been marked with bold.

Figure 2. Prognostic impact of (A) combined total CD8⁺/PD-1 and (B) combined CD8⁺/PD-L1 expression on cumulative incidence of local recurrence and disease-free survival, as indicated. Analysis was based on the dichotomized total score in patient tumor samples (cut-off according to median value of total score).

Figure 3. Prognostic impact of (A) HPV viral load and (B) p16 expression on cumulative incidence of local recurrence and disease-free survival, as indicated. Analysis was based on the dichotomized total score in patient tumor samples (cut-off according to median value).

Figure 4. Prognostic impact of (A) combined HPV viral load/CD8⁺ expression and (B) combined HPV viral load/PD-1 expression and (C) combined HPV viral load/PD-L1 expression and (D) combined HPV viral load/FOXP3 expression on cumulative incidence of local recurrence and disease-free survival, as indicated. Analysis was based on the dichotomized total score in patient tumor samples (cut-off according to median value of total score).