High expression of PD-1 ligands is associated with kataegis mutational signature and APOBEC3 alterations

Figures & data

Figure 1. Distribution of common molecular alterations in PD-L1/2 overexpressed tumors (part A). Molecular alterations were ordered by descending frequency in the subset studied (n = 469 tumors), except for MSI-High as too many tumors were not evaluated for this marker. Each column represents a tumor. Dark green: tumor exempt of alteration. Light green: tumor not presenting the mutation. Gray: alteration not evaluated in the tumor. Purple: total number of mutations and kataegis-related mutations observed in each tumor, represented in percentile (minimum: light purple, maximum: dark purple). Frequency of alterations in comparison to non PD-1 ligand overexpressed tumors (part B). The table presents frequencies of alterations in both PD-1 ligand overexpressed and non-overexpressed tumors, p values of the univariate analysis and p values obtained in the final model of prediction for PD-1 ligand overexpression. Median alterations counts were 66.5 total mutations and 0 kataegis-related mutations per tumor. PD-L1 3′-UTR data were extracted from Ref. ¹¹. This factor is displayed for descriptive purpose only and was not included in the analysis. Abbreviations. APOBEC = apolipoprotein B editing complex; MMR = mismatch repair; MSI = microsatellite instability; MSI-high = microsatellite instability high; ns = non-significant; 3′-UTR = 3′ untranslated region.

Table 1. Multivariate analysis of association^a between all factors and PD-1 ligand mRNA overexpression, using single factors (model A) or relevant combined factors (model B).

	A. Model using only single factorsR^2 = 25.9%			B. Model using single and combined factors^b R^2 = 24.2%
Subgroups and factors	p value	OR	CI 95%	p value	OR	CI 95%
POLYMERASES δ and ϵ
POLD1 mutated	0.0172	2.2	[1.2–4.3]	0.0374	2.1	[1.0–4.1]
MUTATION BURDEN
Kataegis estimate	0.0210	1.3	[1.0–1.7]	0.0237	1.3	[1.0–1.7]
PD-1 LIGAND
PD-L1 amplified^c	<0.0001	3.8	[2.9–4.9]
PD-L2 amplified^c	<0.0001	3.8	[2.9–4.9]
Any PD-1 ligand amplified				<0.0001	3.6	[2.8–4.6]
AID/APOBEC FAMILY
APOBEC3B mutated	0.0267	3.4	[1.2–10.2]
APOBEC3D mutated	0.0224	3.8	[1.2–11.7]
APOBEC3H mutated	0.0257	4.0	[1.2–13.4]
Any APOBEC3 mutated				0.0027	2.4	[1.3–4.1]
APOBEC3A overexpressed	<0.0001	4.2	[2.9–6.0]
APOBEC3C overexpressed	<0.0001	2.3	[1.5–3.4]
APOBEC3G overexpressed	0.0002	2.1	[1.4–3.2]
Any APOBEC3 overexpressed				<0.0001	2.7	[2.1–3.4]
LYMPHOCYTE FACTORS
CD3G overexpressed	<0.0001	2.7	[1.8–4.0]	<0.0001	3.2	[2.2–4.7]
CD4^+ overexpressed	0.0005	2.0	[1.4–2.9]	<0.0001	2.2	[1.5–3.2]
CD8A overexpressed	0.0004	2.1	[1.4–3.2]	0.0210	1.6	[1.1–2.3]
NCAM1 overexpressed	0.0149	1.9	[1.1–3.3]	0.0135	2.0	[1.1–3.3]
CD14 overexpressed	<0.0001	3.0	[2.0–4.4]	<0.0001	2.6	[1.8–3.8]
CD33 overexpressed	<0.0001	2.7	[1.8–4.0]	<0.0001	2.4	[1.6–3.6]
IFNγ overexpressed	<0.0001	3.1	[2.1–4.5]	<0.0001	2.6	[1.8–3.8]

^a Alterations with a p value ≤0.25 in univariate analysis were selected for multivariate analysis, respecting each model (i.e., using only single factors or single and relevant combined factors). A Fifth-corrected logistic regression with backward elimination was performed until all factors influencing the model present a p value ≤0.05. Only factors with p value ≤0.05 for a positive association in multivariate analysis are shown.

^b Combined factors as outlined in Table S2. For instance, any MMR member altered, any APOBEC3 member overexpressed, any PD-1 ligand mutated and total number of CNV represent combined factors.

^c PD-L1 amplification and PD-L2 amplification are co-linear variables.

Abbreviations: AID = activation-induced deaminase; APOBEC = apolipoprotein B editing complex; CI 95% = 95% confidence interval; CNV = copy number variation; MMR = mismatch repair; OR = odds ratio.

Figure 2. Graphical model of conditional dependences between PD-1 ligand overexpression, APOBEC3 mutation, APOBEC3 overexpression, kataegis and mutational burden predictors. Arrows represent dependent relationships between factors. Each dependent variable (in red) can be modeled by the association of independent factors pointing in its direction (multivariate models using combined factors, p ≤ 0.05). Confirmed bilateral relationships between major factors are represented in red. Common microsatellite and genomic instability factors are represented in blue. Additional genomic alterations are represented in dark gray and marker of lymphocyte activation (IFNγ overexpression) is represented in green. Associations with others APOBEC members and lymphocyte markers are not shown in this diagram. Abbreviations. APOBEC = apolipoprotein B editing complex; CNV = copy number variations; MMR = mismatch repair; MSI = microsatellite instability; MSI-high = microsatellite instability high.

Table 2. Multivariate analysis of association^a between all factors and tumor mutational burden (model A) or kataegis-signature (model B).

	A. Mutation burden model^b R^2 = 30.3%			B. Kataegis model^b R^2 = 30.0%
Subgroups and factors	p value	OR	CI 95%	p value	OR	CI 95%
MICROSATELLITE STATUS
MSI-high^c	<0.0001	54.7	[10.9–274.8]	ns	—	—
MISMATCH REPAIR
Any MMR altered	<0.0001	1.6	[1.4–1.8]	0.0133	1.2	[1–1.4]
POLYMERASES δ and ϵ
POLD1 mutated	0.0002	4.0	[1.9–8.1]	ns	—	—
POLE mutated	<0.0001	5.7	[3.3–9.8]	ns	—	—
MUTATION BURDEN
Number of CNV	<0.0001	2.0	[1.8–2.3]	0.0005	1.4	[1.1–1.6]
Number of mutations	—	—	—	<0.0001	14.1	[11.8–16.7]
Kataegis estimate	<0.0001	14.1	[11.8–16.8]	—	—	—
PD-1 LIGAND
Any PD-1 ligand mutated	0.0222	5.9	[1.3–26.6]	0.0124	2.8	[1.3–6.4]
Any PD-1 ligand amplified	ns	—	—	0.0070	1.3	[1.1–1.5]
Any PD-1 ligand overexpressed	ns	—	—	0.0099	1.4	[1.1–1.9]
AID/APOBEC FAMILY
AICDA mutated	0.0076	5.1	[1.5–16.9]	ns	—	—
APOBEC1 mutated	0.0129	4.5	[1.4–14.6]	ns	—	—
Any APOBEC3 mutated	<0.0001	4.8	[2.6–8.8]	ns	—	—
APOBEC4 mutated	0.0004	13.3	[3.2–56.5]	ns	—	—
APOBEC1 amplified	<0.0001	1.4	[1.3–1.7]	ns	—	—
APOBEC2 amplified	<0.0001	1.5	[1.3–1.8]	ns	—	—
Any APOBEC3 amplified	ns	—	—	<0.0001	1.6	[1.3–1.9]
APOBEC4 amplified	0.0043	1.2	[1.1–1.4]	<0.0001	1.4	[1.2–1.6]
Any APOBEC3 overexpressed	ns	—	—	0.0016	1.3	[1.1–1.5]
LYMPHOCYTE MARKERS
Lymphocyte infiltrate^d	0.0005	1.4	[1.2–1.6]	<0.0001	1.2	[1.1–1.5]
CD8A overexpressed	0.0214	1.4	[1–1.8]	ns	—	—

^a Alterations with a p value ≤0.25 in univariate analysis were selected for multivariate analysis, respecting each model (i.e., using only single factors or single and relevant combined factors). A Fifth-corrected logistic regression with backward elimination was performed until all factors influencing the model present a p value ≤0.05. Only factors with p value ≤0.05 for a positive association in multivariate analysis using combined models are shown.

^b Combined factors as outlined in Table S2. For instance, any MMR member altered, any APOBEC3 member overexpressed, any PD-1 ligand mutated and total number of CNV represent combined factors.

^c MSI-status was dichotomized as MSI-high versus MSI-not high (MSS or MSI-low) and unknown versus MSI-not high (MSS or MSI-low).

^d Lymphocyte infiltration factor was dichotomized as positive versus negative and unknown versus negative.

Abbreviations: AID = Activation-induced deaminase; APOBEC= apolipoprotein B editing complex; CI 95% = 95% confidence interval; CNV = copy number variation; MMR = mismatch repair; MSI = microsatellite instability; MSI-High = microsatellite instability high; MSI-Low = microsatellite instability low; MSS = microsatellite stable; ns = non-significant; OR = Odds Ratio.

Table 3. Multivariate analysis of association^a between all factors and APOBEC3 members (APOBEC3A/B/C/D/F/G/H) mRNA overexpression (model A) or mutation (model B).

	A. APOBEC3 Overexpression model^b R^2 = 14.0%			B. APOBEC3 Mutation model^b R^2 = 20.1%
Subgroups and factors	p value	OR	CI 95%	p value	OR	CI 95%
MICROSATELLITE STATUS
MSI-high^c	ns	—	—	<0.0001	5.4	[3–9.4]
POLYMERASES δ and ϵ
POLE mutated	ns	—	—	<0.0001	6.4	[4.2–9.8]
MUTATION BURDEN
Number of mutations	ns	—	—	<0.0001	6.7	[3.9–11.4]
Kataegis estimate	0.0048	1.2	[1.1–1.5]	ns	—	—
PD-1 LIGAND
Any PD-1 ligand mutated	ns	—	—	0.0246	3.0	[1.2–7.7]
Any PD-1 ligand overexpressed	<0.0001	2.6	[2–3.2]	0.0067	2.0	[1.2–3.4]
AID/APOBEC FAMILY
APOBEC1 mutated	ns	—	—	0.0009	4.0	[1.8–9.1]
APOBEC2 mutated	ns	—	—	0.0057	4.7	[1.6–14.1]
APOBEC1 amplified	0.0461	1.2	[1–1.3]	ns	—	—
Any APOBEC3 amplified	<0.0001	2.3	[1.9–2.7]	ns	—	—
AICDA overexpressed	<0.0001	2.2	[1.5–3.3]	ns	—	—
LYMPHOCYTE MARKERS
CD3G overexpressed	<0.0001	2.0	[1.5–2.8]	ns	—	—
CD4^+ overexpressed	0.0131	1.5	[1.1–2]	ns	—	—
CD8A overexpressed	<0.0001	3.5	[2.6–4.8]	ns	—	—
MS4A1 overexpressed	0.0040	1.8	[1.2–2.8]	ns	—	—
IL3RA overexpressed	0.0025	1.6	[1.2–2.1]	ns	—	—
CD33 overexpressed	<0.0001	2.1	[1.5–2.9]	ns	—	—
IFNγ overexpressed	<0.0001	3.7	[2.7–5]	ns	—	—

^a Alterations with a p value ≤0.25 in univariate analysis were selected for multivariate analysis, respecting each model (i.e., using only single factors or single and relevant combined factors). A Fifth-corrected logistic regression with backward elimination was performed until all factors influencing the model present a p value ≤ 0.05. Only factors with p value ≤0.05 for a positive association in multivariate analysis using combined models are shown.

^b Combined factors as outlined in Table S2. For instance, any MMR member altered, any APOBEC3 member overexpressed, any PD-1 ligand mutated and total number of CNV represent combined factors.

^c MSI-status was dichotomized as MSI-high versus MSI-not high (MSS or MSI-low) and unknown versus MSI-not high (MSS or MSI-low).

Abbreviations: AID = Activation-induced deaminase; APOBEC= apolipoprotein B editing complex; CI 95% = 95% confidence interval; CNV = copy number variation; MMR = mismatch repair; MSI = microsatellite instability; MSI-High = microsatellite instability high; MSI-Low = microsatellite instability low; MSS = microsatellite stable; ns = non-significant; OR = Odds Ratio.

Figure 3. Causal hypothesis of mutation burden and immune tolerance co-occurrence in human tumors. Pathogen concept (Part A). Both PD-1 ligand and APOBEC enzymes are induced after viral infection, via interferon gamma (IFNγ) secretion. The defensive anti-viral function of APOBEC3 results in off-target alterations within the host genome, which can be repaired by DNA repair processes, unless alteration of these functions. Carcinogen concept (Part B). Both PD-1 ligand and APOBEC enzymes are regulated by the same factors, like exogenous activators. PKC has been linked to APOBEC3 and PD-L1 overexpression in distinct studies.³⁵ PKC can be activated by exogenous substances such as phorbol, which acts as a co-carcinogen and T-lymphocyte activator.³⁴ Clonal selection concept (Part C). Co-occurrence of kataegis-mutation process and expression of immune checkpoints such as PD-L1/2 leads to the accumulation of mutations and the tumor progression. In the presence of high level of mutation burden, only tumors that overexpress PD-L1 (and hence evade the immune system) survive. Other mechanisms of immune escape, not studied herein, could also be surgical. Abbreviations. APOBEC = apolipoprotein B editing complex; MMR = mismatch repair.

Kataoka K, Shiraishi Y, Takeda Y, Sakata S, Matsumoto M, Nagano S, Maeda T, Nagata Y, Kitanaka A, Mizuno S et al. Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers. Nature 2016; 534:402-6; PMID:27281199; http://dx.doi.org/10.1038/nature18294

 PubMed Web of Science ®Google Scholar

Jang B-C, Park Y-K, Choi I-H, Kim S-P, Hwang J-B, Baek W-K, Suh M-H, Mun K-C, Suh S-I. 12-O-tetradecanoyl phorbol 13-acetate induces the expression of B7-DC, -H1, -H2, and -H3 in K562 cells. Int J Oncol 2007; 31:1439-47; PMID:17982670; http://dx.doi.org/10.3892/ijo.31.6.1439

 Google Scholar

Madsen P, Anant S, Rasmussen HH, Gromov P, Vorum H, Dumanski JP, Tommerup N, Collins JE, Wright CL, Dunham I et al. Psoriasis upregulated phorbolin-1 shares structural but not functional similarity to the mRNA-editing protein apobec-1. J Invest Dermatol 1999; 113:162-9; PMID:10469298; http://dx.doi.org/10.1046/j.1523-1747.1999.00682.x

 PubMed Web of Science ®Google Scholar