Hindawi

Journal of Oncology
Volume 2019, Article ID 3865279, 10 pages
https://doi.org/10.1155/2019/3865279

Research Article
Bioinformatic Analysis Reveals an Immune/
Inflammatory-Related Risk Signature for Oral Cavity Squamous
Cell Carcinoma

Shuang Bai ,1,2 Ying-Bin Yan,1 Wei Chen,1 Ping Zhang,1 Tong-Mei Zhang,1
Yuan-Yuan Tian,1 and Hao Liu 1
1
Department of Oral and Maxillofacial Surgery, Stomatological Hospital of Nankai University, Tianjin Stomatological Hospital,
No. 75, Dagu Road, Heping District, Tianjin 300041, China
2
Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, No. 22,
Zhongguancun South Avenue, Haidian District, Beijing 100081, China

Correspondence should be addressed to Shuang Bai; cmubaishuang@hotmail.com and Hao Liu; tjshhaoliu@hotmail.com

Received 1 August 2019; Revised 6 October 2019; Accepted 5 November 2019; Published 13 December 2019

Guest Editor: Qigen Fang

Copyright © 2019 Shuang Bai et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
High-throughput gene expression profiling has recently emerged as a promising technique that provides insight into cancer
subtype classification and improved prediction of prognoses. Immune/inflammatory-related mRNAs may potentially enrich
genes to allow researchers to better illustrate cancer microenvironments. Oral cavity squamous cell carcinoma (OC-SCC) exhibits
high morbidity and poor prognosis compared to that of other types of head and neck squamous cell carcinoma (HNSCC), and
these differences may be partially due to differences within the tumor microenvironments. Based on this, we designed an immune-
related signature to improve the prognostic prediction of OC-SCC. A cohort of 314 OC-SCC samples possessing whole genome
expression data that were sourced from The Cancer Genome Atlas (TCGA) database was included for discovery. The GSE41613
database was used for validation. A risk score was established using immune/inflammatory signatures acquired from the training
dataset. Principal components analysis, GO analysis, and gene set enrichment analysis were used to explore the bioinformatic
implications. When grouped by the dichotomized risk score based on the signature, this classifier could successfully discriminate
patients with distinct prognoses within the training and validation cohorts (P < 0.05 in both cohorts) and within different
clinicopathological subgroups. Similar somatic mutation patterns were observed between high and low risk score groups, and
different copy number variation patterns were also identified. Further bioinformatic analyses suggested that the lower risk score
group was significantly correlated with immune/inflammatory-related biological processes, while the higher risk score group was
highly associated with cell cycle-related processes. The analysis indicated that the risk score was a robust predictor of patient
survival, and its functional annotation was well established. Therefore, this bioinformatic-based immune-related signature
suggested that the microenvironment of OC-SCC could distinguish among patients with different underlying biological processes
and clinical outcomes, and the use of this signature may shed light on future OC-SCC classification and therapeutic design.

1. Introduction but do not overlap. There are differences in the associated

Oral cavity squamous cell carcinoma (OC-SCC) is the most
common malignancy of the head and neck region (excluding
nonmelanoma skin cancer). There has also been a recent
dramatic rise in the incidence of oropharyngeal squamous
cell carcinoma (OP-SCC) [1]. Anatomically, the mouth and
oropharynx are separate areas that are adjacent to each other
mortality, etiology, risk factors, and even the biomarkers of
squamous cell carcinomas located within these two major
sites.
Many risk factors can contribute to OC-SCC. Tobacco is
classified as a group 1 carcinogen for OC-SCC. Currently,
tobacco smokers exhibit a 3.43 fold relative risk for OC-SCC
compared to that of nonsmokers [2], and they possess a fully
2 Journal of Oncology
adjusted HR of 1.7 [3]. Alcohol has been established as an
independent risk factor, and studies of nonsmokers have
demonstrated a strong correlation and dose-response re-
lationship between alcohol consumption and OC-SCC [4].
Additionally, the combined effect of smoking and alcohol
consumption was greater if the relationship was multipli-
cative [5]. HPV, as a major etiological factor, contributes
disproportionally to the formation and prognosis of squa-
mous cell carcinoma formation at different sites within the
head and neck region [6]. Specifically, the high-risk genotype
HPV-16 accounts for the vast majority (about 90% to 95%)
of HPV-positive OP-SCC, while the HPV type is more
variable in OC-SCC [7, 8]. Additionally, a far more favorable
outcome exists for HPV positive compared to that for HPV-
negative OP-SCC [9].
The above three major risk factors may contribute to
carcinogenesis through different biological processes or
pathways [10–12]; however, the dysregulation of immune or
inflammatory responses is mutually shared among the
carcinogenic mechanisms. Moderate alcohol consumption
or alcohol abuse can suppress multiple arms of the immune
response [13, 14]. Cigarette smoke exposure significantly
affects the immune system, impairing the ability of the host
to produce appropriate immune and inflammatory re-
sponses, ultimately leading to smoking-related pathologies
[15]. These concepts are consistent with the notion that head
and neck cancer is an intrinsically immune-suppressing
disease [16]. For HPV-positive SCC, more intense PD-L1+,
CD4+, and CD8+ T-cell infiltration correlates with a better
outcome [17]. Increasing evidence supports the idea that
evoked immune or inflammatory responses may elicit an-
titumor effects concerning OC-SCC development [18, 19].
Our current study aimed to develop a key gene signature
that is representative of immune and inflammatory re-
sponses that could be correlated with patient prognosis, and
we incorporated a bioinformatics-based approach associated
with clinical covariates to achieve our aim. Following this
principle, we performed a combined analysis to identify a
robust gene signature, and we established a risk score sys-
tem. Further bioinformatic analyses revealed that the risk
score exhibited excellent prognostic value for stratifying
patients irrespective of mutation or copy number variation
(CNV) patterns, and this risk score was highly associated
with cell-cycle processes.
2. Materials and Methods
2.1. Datasets. Whole genome mRNA expression RNA-seq
(20101 genes) data, somatic mutation data, copy number
variation data, and all corresponding clinical information
from the TCGA-HNSC dataset [20] (http://cancergenome.
nih.gov/) were downloaded for use as the training cohort.
The following dataset was obtained for validation: GSE41613
(23520 genes) [21] (http://www.ncbi.nlm.nih.gov/geo/
query/acc.cgi?acc�GSE41613). The training dataset com-
prised 314 OC-SCC patients while the validation dataset
comprised 97 OC-SCC patients. The patient characteristics
are summarised in Table S1. Two gene sets (immune re-
sponse, M14329 and inflammatory response, M13657) [22]
were extracted from the Molecular Signatures Database v6.1
(http://http://software.broadinstitute.org/gsea/msigdb/
index.jsp) and were combined to integrate the immune/
inflammatory-related gene set.
2.2. Statistical Analysis. Overall survival time (OS) was
defined as the interval from the date of diagnosis until
death or until the last follow-up. The prognostic value for
patients possessing high or low expression of a certain gene
or score (higher or lower than the median value) was
calculated using the Kaplan–Meier method with the two-
sided log-rank test by package “survival” of R. Univariate
and multivariate COX regression analysis was also per-
formed using package “survival” of R. Chi-squared test and
Fisher’s exact test were used to compare the frequencies
between groups. A two-tailed Student’s t-test was per-
formed to compare two groups of numerical values.
Analysis of variance (ANOVA) was used to analyze the
differences among group means. The median absolute
deviation (MAD) calculated in R. Pearson correlation
analysis was used to evaluate the association between two
variables and was calculated by R function “cor.test.” The
statistical analysis was performed using the software of R
version 3.4 for Windows. The statistical significance was
established at the level of P < 0.05.
2.3. Bioinformatic Analysis. The R “Limma” package, a
package that can perform the differential expression analyses
of RNA sequencing (RNA-seq) data [23], was used to
identify differentially expressed genes (DEGs) based on a
threshold of false discovery rate (FDR) of less than 0.05. The
packages “gaia,” “maftool,” and “circlize” were used to
generate mutation and CNV plots [24]. The R “TCGAbio-
links” package was employed to investigate relevant bi-
ological implications [25]. The biological phenotype was
further verified by gene set enrichment analysis (GSEA) [22].
Normalized enrichment score (NES) and false discovery rate
were used to determine the statistical significance. The R
“ESTIMATE” package that was fitted for our data was used
to calculate ImmuneScore, StromalScore, and tumor purity
[26].
3. Results
3.1. Different Immune/Inflammatory Phenotypes of OC-SCC
Tumor. The gene expression and clinical data for 314 pa-
tients were obtained from the TCGA database (Table S1).
Previous research defined four RNA subtypes of the TCGA
cohort, including atypical, basal, classical, and mesenchymal
[20]. The combined gene set that was representative of
immune/inflammatory response (1224 genes) was used to
illustrate the immune microenvironment of the OC-SCC
tumor. Principal components analysis based on the 1224
genes revealed a different distribution pattern regarding OC-
SCC tumor subtypes. A mutually exclusive pattern was
observed within the mesenchymal, basal, and classical
subtypes, while the atypical subtype lacked a clear distri-
bution pattern (Figure 1(a)). As clinical differences between
Journal of Oncology 3

–0.08
–0.1
0 –0.075
0.1
–0.07
0.2

–0.1

PC3
0

0.1

0.2

PC
2
PC

Basal 1 Atypical
Mesenchymal Classical
(a)

Go_inflammatory_response (454 genes) The cancer genome atlas dataset (20101 genes) Go_immune_response (1100 genes)

1224 common genes

Heterogeneity analysis (MAD > 1.0)

668 genes

Univariate Cox regression (P < 0.01)

β-value of
18 genes
each gene

Risk score = expression value of each gene × β-value of each gene

(b)

Figure 1: Different immune/inflammatory patterns of OC-SCC subtypes and the workflow of the signature establishment. (a) Principal
components analysis of immune/inflammatory genes among four subtypes of OC-SCC. (b) Workflow of the signature establishment.

the four tumor subtypes were previously established, we
sought to construct a gene signature to further explore the
immune phenotype of OC-SCC tumors.
3.2. Identification of an Immune/Inflammatory Signature for
Prognosis Prediction in OC-SCC. Taking into account the
differential distribution of immune/inflammatory genes
among OC-SCC subtypes, we sought to identify the gene or
group of genes that were of significant prognostic value. As
the PCA exhibited a heterogeneous expression pattern
among OC-SCC patients, genes that were homogeneously
expressed (MAD ≤ 1) were excluded from further analysis.
Univariate Cox regression analysis was used to explore the
prognostic value of the resulting genes. Eighteen genes
(CD27, CD79B, CMA1, CCR4, CCR7, CNR2, CTLA4,
CTSG, GZMM, IL16, MASP1, SAA1, CCL11, TNFAIP3,
BATF, IL19, PGLYRP4, and TREML1) were identified to be
associated with prognosis in OC-SCC patients (Figures 1(b)
and 2(a)).
Next, the risk score method was employed based on
gene expression levels (coefficient of each gene was the
beta value in the univariate Cox regression model of each
gene; Table S2), and the associations between TCGA
4 Journal of Oncology

Color key TCGA OSCC

Training cohort 100

Overall survival (%)

–2 0 2
Row Z-score
Risk score 50 P = 1.13e – 05
Tumor stage
RNA subtype
Methylation
Gender 0
SAA1 0 2000 4000 6000
TNFAIP3
CCR7 Low risk (n = 157)
CD79B High risk (n = 157)
PGLYRP4
CD27 (b)
BATF
CCL11
CTLA4
IL19 GSE41613 HPV negative
CCR4
TREML1 100

Overall survival (%)

MASP1
CMA1
GZMM P = 0.0162
IL16
CNR2 50
CTSG
Tumor stage RNA subtype Methylation
III/IV AT Hypermethylated
0
I/II BA Hypomethylated 0 1000 2000 3000
Gender CL CpG hypermet
Male ME Normal-like Low risk (n = 48)
Female High risk (n = 49)

(a) (c)

Figure 2: Overview of the eighteen-gene-based risk score and its prognostic value across different cohorts. (a) Heatmap depicting the gene
expression values of the eighteen genes comprising the signature of the training cohort. Columns representing each sample that were sorted
by increasing value of the risk score. Rows representing the expression value of each gene. (b) Kaplan–Meier survival analyses based on the
median cutoff of the risk score within the training dataset. (c) Kaplan–Meier survival analyses based on the median cutoff of the risk scores
within the validation dataset.

results and two functional gene sets were explored. A
strong correlation was identified between the risk score
and the ssGSEA score in the immune/inflammatory re-
sponse gene set (R � − 0.693, P < 0.0001; Figure S1A). The
resulting 18 genes were also mutually correlated, and this
supported the integrity of the signature genes (Figure S1B
and S1C). Patients were divided into high-risk and low-
risk groups based on the median cutoff value of the risk
score. This classifier could stratify patients according to
distinct prognosis within the training cohort (median
OS � 804 vs 2166 days; P � 1.13e − 05; Figure 2(b)) and
within the validation cohort (P � 0.0162; Figure 2(c)).
3.3. Distribution and Prognostic Value of the Risk Score among
Subgroups of OC-SCC. The patients within the training
cohort were further stratified based on several clinico-
pathological factors, including age, gender, stage, RNA
subtype, and methylation subtype. We found that in patients
classified at a higher stage, the classical and hypomethylated
subtypes exhibited a higher risk score (Figure S2A). Based on
the median cutoff value of risk score in the training cohort,
the patients were divided into either a high- or low-risk
group within each subgroup to query the prognostic value. A
nearly universal result was achieved among most of the
subgroups (Figure S2(B–J)), demonstrating that an elevated
risk score was strongly correlated with poor prognosis and
vice versa. Similar results were observed for the validation
GSE41613 cohort (Figure S3). Univariate and multivariate
Cox regression analyses also indicated that the risk score
provided an independent prognostic factor after adjusting
for other clinical covariates (Table 1). Of note, tobacco use,
alcohol consumption, and HPV infection, when assessed as
initiating risk factors for OC-SCC, did not provide signif-
icant HR value in either univariate or multivariate Cox
analyses. We further explored the relationship between
tobacco use, alcohol consumption, or HPV infection and the
risk score, and we found that there were no significant
differences.
3.4. Mutation and CNV Patterns of Different Subgroups of the
Risk Score. To further investigate the impact of the risk score
at the DNA level, TCGA cases possessing available somatic
mutation and copy number variation (CNV) information
were analyzed. Based on the increasing risk score, cases were,
Journal of Oncology
Table 1: COX regression analysis of the risk score and other
characteristics in the TCGA OC-SCC cohort.
Univariate Multivariate
Cox Cox
Variables regression regression
HR P HR P
Risk score (high vs low) 2.09 1.7e − 05 1.99 0.0002
Age (>60 vs ≤60) 1.15 0.3963 1.31 0.1580
Gender (female vs male) 1.08 0.6698 1.10 0.6357
Tobacco history (yes vs no) 1.29 0.2099 1.27 0.2740
Alcohol history (yes vs no) 1.07 0.6958 1.01 0.9507
HPV status (positive vs negative) 0.88 0.7502 0.86 0.7138
Stage (III/IV vs I/II) 2.23 0.0006 2.10 0.0023
HR: hazard ratio.
respectively, divided into four subgroups, and the most
representative subgroups (lower quantile risk score group,
n � 78 and higher quantile risk score group, n � 78) were
selected.
An analysis of the 20 most frequently mutated genes
within either subgroup was performed (Figure 3). Well-
known mutated genes such as TP53, a somatic mutation
detected in 60–80% of OSCC [27], were a genome guardian
and played a pivotal role in regulating the cell cycle, cellular
differentiation, DNA repair, and apoptosis and showed no
significant mutation difference. There was also no significant
difference regarding FAT1 mutation, which played a role in
regulating the migration and invasion of OSCC cells through
the localization of β-catenin [28]. Only frequent mutations
in NSD1 (P � 0.034) were significantly enriched in cases
with higher risk score. Subsequently, CNV data were in-
vestigated, and our results revealed similar overall variant
counts between OC-SCC at both lower and higher risk
scores (mean 108.2 variants vs 111.6 variants; P � 0.8713).
There were some differences in the gene-level CNV land-
scape; however, the frequently deleted genomic regions were
located at the 9p21.3 region encompassing the CDKN2A/
CDKN2B (mean deletion − 0.002 vs − 0.138, P � 0.015). The
7p11.2 region encompassing EGFR (mean amplification
0.083 vs 0.212, P � 0.016) was frequently amplified for cases
with higher risk scores (Figure 3).
3.5. High Risk Score OC-SCC Exhibited Cell-Cycle-Related
Gene Function While Low Risk Score Exhibited Immune/In-
flammatory Responses. To further explore the prognostic
value, similar patterns of mutation, and the CNV of the risk
score, GO analysis was performed to assess the functional
aspects. Pearson correlation score (R) was calculated for each
gene within the training cohort. GO results based on 1355
negatively correlated (R < − 0.4) genes suggested that these
genes were highly enriched in immune/inflammatory re-
sponses (Figure 4(a)). Additionally, GO results based on
1632 positively correlated (R > 0.2) genes suggested that
these genes were highly enriched in cell-cycle-related pro-
cesses (Figure 4(b)). We next performed gene set enrichment
analysis for further validation. GSEA revealed that a lower
risk score was associated with processes or pathways closely
related to immune/inflammatory responses (Figure 4(c)),
5
and a higher risk score was highly correlated with cell-cycle-
related processes (Figure 4(d)).
3.6. Association between the Risk Score and Tumor Purity.
It is established that OC-SCC tissues contain abundant
nontumor cells within their microenvironment. Thus, tumor
purity could reflect the tumor and nontumor compartments
of the OC-SCC tissue. As analyzed above, the risk score was
closely associated with immune/inflammatory responses.
Based on this, we further explored the correlation between
tumor purity and the risk score. The risk score exhibited a
high negative correlation with ImmuneScore and Stromal-
Score and a positive correlation with tumor purity (Fig-
ure 5). The results obtained using the validation cohort
GSE41613 were in accordance with those obtained using the
training cohort (Figure S4). These results further validated
the idea that the risk score provided a robust predictor of
OC-SCC immune/inflammatory responses; however, the
dichotomized median value of either of the purity-related
scores was unable to achieve a significant prognostic value.
4. Discussion
As the most common malignancy of the head and neck, OS-
SCC is diagnosed using histopathological criteria and is
staged using the TNM system [1]. Several studies have
provided high-resolution images of the OC-SCC molecular
landscape, and these images revealed significant changes that
may contribute to the pathogenesis and biology of this
disease [20]. Risk factors such as tobacco use, alcohol
consumption, and HPV infection have been proposed as
initiating risk factors for OC-SCC [29–31]; however, there is
little consensus on how immune/inflammatory responses
could affect OS-SCC subtypes. The development of mean-
ingful signatures to determine the immune status of patients
provides an attractive therapeutic approach to this disease,
as these signatures not only promise to be powerful prog-
nostic biomarkers, but if properly applied, they also stratify
patients to increase the likelihood of positive outcomes in
response to immunotherapy. In our study, we demonstrated
that the four subtypes of OC-SCC represent distinct im-
mune/inflammatory phenotypes, and based on this, we
established a gene signature that could stratify patients to
provide different prognoses.
For our signature, we investigated the DNA level sce-
nario and discovered similar mutational and CNV patterns
that were present in either high or low risk score groups. This
result of the mutated genes suggested that it was the in-
flammatory responses and microenvironment that mainly
contribute to the different prognosis rather than well-known
tumorigenesis progresses. Only frequent mutations in NSD1
were significantly enriched in cases with higher risk scores. It
was reported that NSD1 was more mutated in laryngeal and
pharyngeal squamous cell carcinoma (L/P-SCC) than in
OC-SCC [32], and given the role of NSD1 as a chromatin
modifier, these mutations could contribute to cancer for-
mation through a combination of rare germline variants and
somatic loss-of-heterozygosity (LOH) [33].
6 Journal of Oncology

12 10
10 8
8
6 6
4 0 10 20 30 40 50 4
2 2 0 10 20 30 40 50
0 0
69 TP53 77 TP53
35 TTN 35 TTN
29 CDKN2A 30 FAT1
23 FAT1 22 CDKN2A
18 NOTCH1 17 SYNE1
18 PIK3CA 16 LRP1B
15 PCLO 14 NOTCH1
13 MUC16 14 CASP8
13 CSMD3 14 CASMD3
12 USH2A 14 DNAH5
12 SYNE1 14 PCLO
(%)

(%)
12 CASP8 13 PIK3CA
12 FLG 12 MUC16
10 LRP1B 12 KMT2D
10 KAT4 10 FLG
10 KMT2D 10 PLEC
9 DNAH5 9 NSD1
9 HUWE1 8 FAT4
5 AHNAK 8 HRAS
5 HRAS 8 HUWE1
3 PLEC 6 AHNAK
1 NSD1 3 USH2A

Nonsense_Mutation Splice_Site Frame_Shift_Ins Nonsense_Mutation Splice_Site Frame_Shift_Ins

Missense_Mutation Multi_Hit In_Frame_Ins Missense_Mutation Multi_Hit In_Frame_Ins
Frame_Shift_Del In_Frame_Del Frame_Shift_Del In_Frame_Del

(a) (b)
14 15 16 15 16
90 M B 0 MB 90 M B 0 M 14 90MB 0
MB 9 0 MB 0 M
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4 4

Mutations CNVs Mutations CNVs

Missense_Mutation Amp Missense_Mutation Amp
Frame_Shift_Ins Del
Frame_Shift_Ins Del
Nonsense_Mutation

(c) (d)

Figure 3: Different mutation and copy number variation patterns of the risk score. (a, b) An analysis of the 20 most mutated genes of either
subgroup ((a), lower risk score; (b), higher risk score) was performed. Columns are sorted by samples with increasing risk score. (a) The sum
of mutations in each of the sample categories is indicated by the legend; (b) the sum of the mutations in each gene is indicated by the legend.
(c, d) The overall recurrent copy number variation (CNV) profile in order of increasing risk score ((c), lower risk score; (d), higher risk
score).

Aneuploidy, also known as somatic copy number al- results of these analyses indicated that a lower risk score
terations, is widespread in human cancers and has been was correlated with immune/inflammatory responses,
proposed to drive tumorigenesis. Aneuploidy was pre- and a higher risk score was associated with cell-cycle-
viously demonstrated to correlate with tumor cell pro- related processes. These results could partially explain
liferation and reduced immune processes [33]. In our that patients possessing a higher risk score demonstrated
study, overall copy number variant counts in high or low a worse prognosis, and patients with more active im-
risk score cases were not significantly different. This mune/inflammatory responses exhibited a better out-
suggested that our signature could infer immune/in- come. The high correlation between the risk score and the
flammatory responses irrespective of aneuploidy differ- tumor purity also suggested the presence of more immune
ence; however, some key genes did exhibit different cell infiltration in lower risk score cases. We also per-
variation patterns. The deletion of CDKN2A, a tumor formed ssGSEA analysis to evaluate the relationship
suppressor gene that functions in G1 cell cycle control, was between our risk score and the tumor purity as defined by
associated with poor prognosis and low survival rate in expression data. ImmuneScore, StromalScore, ESTI-
OC-SCC [34], and EGFR amplification was shown to be MATE score, and tumor purity were all significantly
associated with advanced clinical stage in OC-SCC pa- correlated with the risk score, and of these scores, the
tients [35]. The result of the aneuploidy difference re- ImmuneScore exhibited the highest correlation. Of note,
garding CDKN2A and EGFR was in accordance with the different from other cancer types such as glioma [36] and
worse prognosis higher risk score represented. colon cancer [37], the purity-related scores or purity
We performed GO and GSEA analyses to further alone were unable to distinguish among different prog-
validate the functional annotation of our signature. The nostic patient groups [38].
Journal of Oncology 7

Percent of enriched gene set

0 20 40 60 80 100 120

Immune response
Inflammatory response
Defense response
Regulation of lymphocyte activation
Positive regulation of immune system process
Leukocyte activation
Response to wounding
Regulation of cell activation
Positive regulation of cell activation
Cell activation
Regulation of leukocyte activation
Positive regulation of leukocyte activation
Chemotaxis
Taxis
Lymphocyte activation
Regulation of T-cell activation
Locomotory behavior
Behavior
Positive regulation of lymphocyte activation
T-cell activation
Antigen processing and presentation of peptide
or polysaccharide antigen via MHC class II
Positive regulation of response to stumulus
Cellular defense response
Cellular metal ion homeostasis
Cellular calcium ion homeostasis

0 20 40 60
–log10 (FDR)

(a)
Percent of enriched gene set
0 10 20 30 40

RNA processing translation

G−protein coupled receptor protein signaling pathway
Cell cycle
Cell cycle process
M phase of mitotic cell cycle
Mitosis
Nuclear division
DNA metabolic process
Mitotic cell cycle
Cell surface receptor linked signal transduction
RNA splicing
Nuclear mRNA splicing via spliceosome
RNA splicing via transesterfication
Reactions with bulged adenosine as nucleophile
RNA splicing via transesterification reactions
mRNA metabolic process
M phase
Ribonucleoprotein complex biogenesis
mRNA processing
Cell cycle phase
Organelle fision
DNA repair
Neurological system process
Cell division
ncRNA processing

0 5 10 15 20
–log10 (FDR)

(b)
Enrichment plot: Enrichment plot: Enrichment plot:
HALLMARK_INFLAMMATORY_RESPONSE KEGG_T_CELL_RECEPROR_SIGNALING_PATHWAY GO_LYMPHOCYTE_COSTIMULATION
0.0 0.0 0.0
Ranked list metric (Signal2Noise() Enrichment score (ES)

Ranked list metric (Signal2Noise() Enrichment score (ES)

Ranked list metric (Signal2Noise() Enrichment score (ES)

NES = –1.743 NES = –1.879 NES = –2.024

–0.1 –0.1 –0.1
FD 0.001 FDR < 0.001 FDR < 0.001
–0.2 –0.2 –0.2
–0.3 –0.3 –0.3
–0.4 –0.4 –0.4
–0.5 –0.5 –0.5
–0.6 –0.6 –0.6
–0.7 –0.7 –0.7

0.25 “1” (positively correlated) 0.25 “1” (positively correlated) 0.25 “1” (positively correlated)
0.00 0.00 0.00
–0.25 Zero cross at 9620 –0.25 Zero cross at 9620 –0.25 Zero cross at 9620
–0.50 –0.50 –0.50
–0.75 –0.75 –0.75
“0” (negatively correlated) “0” (negatively correlated) “0” (negatively correlated)
0 2.5000 5.0000 7.5000 10.000 12.500 15.000 17.500 20.000 0 2.5000 5.0000 7.5000 10.000 12.500 15.000 17.500 20.000 0 2.5000 5.0000 7.5000 10.000 12.500 15.000 17.500 20.000
Rank in ordered dataset Rank in ordered dataset Rank in ordered dataset

Enrichment profile Enrichment profile Enrichment profile

Hits Hits Hits
Ranking metric scores Ranking metric scores Ranking metric scores

(c)
Figure 4: Continued.
8 Journal of Oncology

Enrichment plot: Enrichment plot: Enrichment plot:

HALLMARK_MYC_TARGETS_V2 KEGG_DNA_REPLICATION GO_GENTROMERE_COMPLEX_ASSEMBLY
0.8
Enrichment score (ES)

Enrichment score (ES)

Enrichment score (ES)

0.5 NES = 2.338 0.6 NES = 2.445 0.7 NES = 3.110
FDR < 0.001 0.5 FDR < 0.001 0.6 FDR < 0.001
0.4
0.4 0.5
0.3 0.3 0.4
0.2 0.3
0.2
0.2
0.1
0.1 0.1
0.0 0.0
0.0
Ranked list metric (Signal2Noise()

Ranked list metric (Signal2Noise()

Ranked list metric (Signal2Noise()

0.25 “1” (positively correlated) 0.25 “1” (positively correlated) 0.25 “1” (positively correlated)
0.00 0.00 0.00
–0.25 Zero cross at 9620 –0.25 Zero cross at 9620 –0.25 Zero cross at 9620
–0.50 –0.50 –0.50
–0.75 “0” (negatively correlated) –0.75 “0” (negatively correlated) –0.75 “0” (negatively correlated)
0 2.5000 5.0000 7.5000 10.000 12.500 15.000 17.500 20.000 0 2.5000 5.0000 7.5000 10.000 12.500 15.000 17.500 20.000 0 2.5000 5.0000 7.5000 10.000 12.500 15.000 17.500 20.000
Rank in ordered dataset Rank in ordered dataset Rank in ordered dataset

Enrichment profile Enrichment profile Enrichment profile

Hits Hits Hits
Ranking metric scores Ranking metric scores Ranking metric scores

(d)

Figure 4: Biological annotation of the risk score. (a) GO results based on 1355 negatively correlated (R < − 0.4) genes. (b) GO results based
on 1632 positively correlated (R > 0.2) genes. (c) GSEA results of the lower risk score. (d) GSEA results of the higher risk score.

6000 <2.2e – 16 0.078 0.014 0.016 ANNOVA 2.4e – 11 ANOVA 1.6e – 05

6000
ImmuneScore

4000
4000

2000 2000

R = –0.707 0
0 P < 0.0001

–50 –40 –30 –20 –10

Low risk score

High risk score

Low risk score

Age < 60

Age ≥ 60

Female

Basal

Classical
Male

CpG island
Nesenchymal

Atypical
Stage I/II

Normal-like
Stage III/IV

Hypermethylated
Hypomethylated
Risk score

(a)
<2.2e – 16 0.63 0.59 0.46 ANNOVA 2.2e – 16 ANOVA 0.0017
4000 5000
StromalScore

2000 2500

0 0
R = –0.662
P < 0.0001
–2000
–50 –40 –30 –20 –10
Low risk score

High risk score

Age < 60

Age ≥ 60

Basal

Classical

Low risk score

Female

Male

CpG island
Nesenchymal

Atypical
Stage I/II

Normal-like
Stage III/IV

Hypermethylated
Hypomethylated

Risk score

(b)
Figure 5: Continued.
Journal of Oncology 9

1.00 <2.2e – 16 0.22 0.11 0.093 ANNOVA 2.2e – 16 ANOVA 2.6e – 04

0.75
0.75
Purity

0.50
0.50

0.25 0.25
R = 0.750
P < 0.0001
0.00 0.00
–50 –40 –30 –20 –10

Low risk score

High risk score

Age < 60

Age ≥ 60

Female

Classical

Low risk score

Male

CpG island
Nesenchymal

Atypical

Basal
Stage I/II

Normal-like
Stage III/IV

Hypermethylated
Hypomethylated
Risk score

(c)

Figure 5: Association between the risk score and tumor purity. (a) Correlation between the ImmnueScore and the risk score and the
distribution of the ImmnueScore among subgroups of OC-SCC within the training cohort. (b) Correlation between the StromalScore and
the risk score and the distribution of the StromalScore among subgroups of OC-SCC within the training cohort. (c) Correlation between
tumor purity and the risk score and the distribution of tumor purity among subgroups of OC-SCC in the training cohort.

5. Conclusion Supplementary Materials

Taken together, we identified and validated an eighteen- Figure S1: association between the risk score and the im-
gene-based immune/inflammatory signature that mune genes and association among genes in the signature.
exhibited independent prognostic significance for pa- Figure S2: distribution and prognostic value of the risk score
tients with OC-SCC and reflected the overall intensity of across different subgroups of the training cohort. Figure S3:
immune/inflammatory responses within the tumor mi- distribution and prognostic value of the risk score across
croenvironment. Our study offers new insights regarding different subgroups of the validation cohort. Figure S4:
the OC-SCC immune microenvironment and immune- association between the risk score and tumor purity in the
related therapy for this disease. Evaluating this signature validation cohort. Table S1: demographic characteristics in
may help to elucidate the complex role of tumor im- the training cohort and validation cohort. Table S2: risk
mune/inflammatory responses in this disease and will score formula. (Supplementary Materials)
provide new insight into clinical management and drug
design.
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