Genomics 113 (2021) 1988–1998

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Genomics
journal homepage: www.elsevier.com/locate/ygeno

Original Article

MIN score predicts primary response to infliximab/adalimumab and

vedolizumab therapy in patients with inflammatory bowel diseases☆
Yuan Shi a, 1, Wei He b, 1, Ming Zhong a, *, Minhao Yu a, *
a
Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
b
Department of Pathology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

A R T I C L E I N F O A B S T R A C T

Keywords: Infliximab/adalimumab (IFX/ADA) and vedolizumab (VDZ) are the most widely used biologics in inflammatory
Inflammatory bowel disease bowel diseases. Current models used to predict their efficacies are restricted to either Crohn’s disease or ul­
Infliximab cerative colitis or to only one type of biologic, which makes them limited in external validation. We therefore
Response predictors
designed a comprehensive comparison among these models to identify the most meaningful predictors for patient
responses. Several biomarkers and models were compared for their abilities to predict both IFX/ADA and VDZ
responses by receiver operating characteristic curves. Least absolute shrinkage and selection operator regression
was adopted to determine a simplified gene signature. Verification was performed in biopsy samples by
immunohistochemical staining. The GIMATS module (based on counts of IgG plasma cells, inflammatory
monocytes, activated T cells, and stromal cells) had the best overall performance for response prediction in both
biologics (IFX/ADA, AUC = 0.720–0.853; VDZ, AUC = 0.661–0.728). Based on this module, patients were
equally divided into 3 groups: M type (GIMATS-low, metabolism), with a preference for IFX/ADA; I type
(GIMATS-high, immune), with a preference for VDZ; and N type (GIMATS-medium, normal), with no preference
for either treatment. Furthermore, to improve clinical utility, a simplified 6-gene model, MIN score, was
established to determine the baseline expression of G0S2, S100A9, SELE, CHI3L1, MMP1 and CXCL13 and
function as a substitute for GIMATS module. Our study suggested that the classification of metabolic or immune
type by MIN score was valuable for IBD diagnosis to assist with selection of IFX/ADA and VDZ.

1. Introduction
Inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ul­
cerative colitis (UC), are progressive inflammatory disorders in the
gastrointestinal tract. The aetiology of IBD remains unclear, but agree­
ment has been reached on the crucial role of immune dysfunction as well
as other factors, such as environmental exposure, genetic susceptibility
and intestinal flora disturbance.
Currently, the mainstream view is that the imbalance between pro-
inflammatory Th (T helper) 17 and anti-inflammatory Treg (regulato­
ry) cell differentiation plays a significant part in the pathogenesis of
abnormal intestinal immunity in IBD [1] and promotes the action of
molecules such as tumour necrosis factor α (TNF-α), interleukin-1 beta
(IL-1β), IL-6, and IL-23 to accelerate the development of mucosal
injuries [2]. As a result, therapies for IBD have been developed to block
these processes.
The anti-TNF-α antibodies, including infliximab or adalimumab
(IFX/ADA), and the gut-selective therapy, such as vedolizumab (VDZ),
are two of the most widely used biologics for treatment in both CD and
UC, but approximately one-third of patients receiving IFX/ADA treat­
ment will show primary non-response, while another one-third will
undergo secondary loss of response [3]. The efficacies of these therapies
are hard to predict. Moreover, given that both IFX/ADA and VDZ have
significant therapeutic effects compared with placebo [4,5], physicians’
prescriptions can influence the treatment outcome. For patients with
IFX/ADA treatment failure, an equal dosage of VDZ shows reduced ef­
ficacy compared to that in those naïve to IFX/ADA [6,7], indicating the
need for prospective guidelines for first-line decision-making.

☆
This work was supported by the National Natural Science Foundation of China (81702300, 81873555). The authors declared no financial disclosures or conflicts
of interest.
* Corresponding authors at: 160 Pujian Rd, Shanghai, China.
E-mail addresses: drzhongming@sjtu.edu.cn (M. Zhong), fishmeangood@163.com (M. Yu).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ygeno.2021.04.011
Received 8 October 2020; Received in revised form 8 February 2021; Accepted 5 April 2021
Available online 16 April 2021
0888-7543/© 2021 Elsevier Inc. All rights reserved.
Y. Shi et al.
Though controversial, many studies have reported predictors of
biological efficacy from the perspective of immunologic features, patient
characteristics (e.g., age, gender, body mass index, etc.) and disease
progression. Both the innate and adaptive immune systems have been
studied. Genes related to bacterial component recognition and cytokine
signalling are important for the IFX/ADA response [8]. Several genes,
including TNF receptor superfamily 11B (TNFRSF11B, osteoprotegerin),
stanniocalcin-1 (STC1), prostaglandin-endoperoxide synthase 2
(PTGS2), interleukin 13 receptor alpha 2 (IL13RA2), interleukin 11 (IL-
11) [9] and oncostatin M (OSM) [10], were identified as potential bio­
markers of IFX/ADA resistance. In addition, a few studies have shown
that the expression of integrin α4β7 [11], the level of IL-6 in circulating
blood [12], and a 4-gene signature of regulator of G protein signalling 13
(RGS13), Dachsous cadherin-related 2 (DCHS2), cilia and flagella asso­
ciated protein 91 (MAATS1) and Piwi like RNA-mediated gene silencing
1 (PIWIL1) at baseline [13] could predict the response to VDZ.
However, most of these models have been restricted to either a single
disease (CD or UC) or only one type of biologic, presenting challenges for
application to clinical practices. Therefore, we explored public cohort
data with a number of methodologies, including ESTIMATE (Estimation
of stromal and immune cells in malignant tumour tissues using expres­
sion data) and the GIMATS (IgG plasma cells, inflammatory mono­
nuclear phagocytes, activated T cells, and stromal cells) module, to
discover the decisive predictors for biologic selection and to provide
clues regarding the underlying mechanisms of the treatment response.
2. Methods
2.1. Study population
The flowchart of this study was shown in Fig. S1. All data for bio­
informatic analyses were obtained from the GEO (GSE12251, GSE16879
and GSE73661, www.ncbi.nlm.nih.gov/geo) and SRA databases
(ERP115036, SRP077046, SRP100787, ERP114636, and ERP113396)
according to the following inclusion criteria: organism: Homo sapiens;
expression profiling by array; samples with IBD; samples obtained from
mucosal biopsy (Table S1). Sample exclusion schemes were shown in
Fig. S2.
Besides, mucosal biopsies of patients with (n = 4) or with no (n = 4)
response to IFX therapy were obtained from Renji Hospital, School of
medicine, Shanghai Jiao Tong University, for immunohistochemical
staining (IHC). Baseline clinical characteristics of these patients were
summarized in Table S2. Ethics approvals of collection and preservation
of these samples were obtained from Institute Research Ethics Com­
mittee of Renji Hospital, and informed consents were signed following
the Declaration of Helsinki [14].
2.2. The expression profiles of RNA sequencing (RNA-seq) and
microarray
Quality assessments of ERP115036, SRP077046, SRP100787,
ERP114636 and ERP113396 were performed using FastQC software
(version 0.11.7) (http://www.bioinformatics.babraham.ac.uk/project
s/fastqc/). To quantify gene expression, reads of all RNA-seq samples
were mapped to filtered assembled transcriptomes with the Kallisto
(version 0.43.0) R package [15]. Gene expression values were presented
either as transcript per million (TPM) for model/marker calculation or
as raw counts for differentially expressed genes (DEGs) analysis. Batch
effects were moved by the sva (version 3.34.0) R package [16].
Raw CEL data were imported into R through the Affy (version 1.54.0)
R package, and robust multi-array (RMA)-based correction was per­
formed to determine the comparability of expression values among all
samples [17]. For merged microarray dataset (GSE12251, GSE16879,
GSE73661), batch effects were moved by the sva (version 3.34.0) R
package.
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Genomics 113 (2021) 1988–1998
2.3. Calculation of immune signatures and models
Previously, several models have been established to facilitate quan­
tified evaluations of immune infiltration according to distinct genetic
features of immune cells.
The ESTIMATE algorithm was introduced to determine fractions of
stromal and immune cells using gene expression signatures by R package
(version 1.0.13) [18]. Immune signatures were constituted with genes
involved in both infiltration of immune cells and the process of
haematopoiesis.
Specific tumour-infiltrating lymphocytes (TILs) were evaluated
through two approaches in this study, gene set enrichment analysis
(GSEA) and deconvolution. For the first mentioned of two, the 28
immune-related signatures, as described by Charoentong et al. [19],
expressed in neither cancer cell lines nor normal tissues. The single
sample GSEA (ssGSEA) was used to decompose cellular profiles and
generate 28 subpopulations of TILs via GSVA (version 1.20.0) R pack­
ages [20]. The expression level of each gene was normalized across all
patients using z-score. In addition, one of the most popular deconvolu­
tion methods, CIBERSORT (https://cibersort.stanford.edu/), were
employed to calculate the score of 22 cell types using characteristics of
547 marker genes [21]. Since these two methods produced similar re­
sults, we only compared the abilities of the 28 TILs subpopulations with
other biomarkers to predict patients’ response towards IFX/ADA and
VDZ. Scores of 22 cell types were used for further explanations of
mechanisms underlying GIMATS -high and -low subgroups.
The GIMATS module score was calculated based on IgG plasma cells,
inflammatory mononuclear phagocytes, activated T cells, and stromal
cells. It was derived by single-cell analyses of inflamed tissues from anti-
TNF-α-treated ileum CD patients. Genes listed as Subtype Set-high and
-low group were displayed in Table S3. Datasets were transformed to z-
scores. Transformed genes that belonged to Subtype Set-high and -low
were averaged and projected onto the GIMATS module and “No module”
as described by Martin et al. [22].
2.4. DEGs analysis
DEGs analysis was performed with the DESeq2 (version 1.38.0) R
package through the tximport (version 1.0.3) R package [23,24], using
the raw count produced by Kallisto. P-values were adjusted (adj.p) with
the Benjamini and Hochberg method. DEGs with a fold change >1 and
an adj.p < 0.05 between two groups were considered to have statistical
significance.
The clusterProfiler (version 3.14.3) R package was used to match
biological pathways for genes that found to be up- or down-regulated in
Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrich­
ment analyses with a cut-off value of an adj.p < 0.05 [25].
The weighted gene co-expression network analysis (WGCNA) was
performed to construct a co-expression network of specific genes in IBD
samples via the WGCNA (version 1.69) R package [26]. The minimum
module size was 30, and the module detection sensitivity of deepSplit
was 2, while the cut-off height for the merging of the modules was 0.2,
meaning that modules whose eigengenes had correlation coefficients
above 0.8 were merged.
2.5. Definition of outcomes
Treatment efficacies were evaluated under endoscopy by either
Simple Endoscopic Score for CD (SES-CD) [27] or Mayo Endoscopic
Subscore (MES) [28] with a histological activity system [29] at an in­
terval of 4–6 weeks. SES-CD represented the sum of scores for ulcer size,
ulcerated surface, affected surface, and luminal narrowing in each in­
testinal segment. In CD patients, the response was defined as the
achievement of a SES-CD ≤ 2. Meanwhile, MES was one of the most
widely used indexes for UC, in which patients were given 0 (normal or
inactive disease) to 3 (spontaneous bleeding or ulceration) according to
Y. Shi et al. Genomics 113 (2021) 1988–1998

Fig. 1. The top 5 biomarkers for IFX/ADA treatment response prediction in microarray (A) and RNA-seq datasets (B). The AUCs of different biomarkers for IFX/ADA
treatment response prediction (C). The top 5 biomarkers for VDZ treatment response prediction in microarray (D) and RNA-seq datasets (E). The AUCs of different
biomarkers for VDZ treatment response prediction (F). The logistic regression of the low, median and high GIMATS module score for IFX/ADA or VDZ treatment
response (G). In (C) (F), reference lines represented boundaries between top 5 predictors and the rest in corresponding datasets.
Abbreviations: ADA, adalimumab; AUC, area under the curve; CI, confident interval; GIMATS, IgG plasma cells, inflammatory mononuclear phagocytes, activated T
cells, and stromal cells; IFX, infliximab; OR, odds ratio; RNA-seq, RNA sequencing; ROC, receiver operating characteristic curve; VDZ, vedolizumab

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Fig. 2. Estimated abundances of immune cells (A) and accumulations of monocyte/macrophage subgroups (B) among different GIMATS module groups. Abbrevi­
ations: NK, natural killer; GIMATS, IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells.
their clinical presentation and mucosal appearance. For UC patients, the
response was considered to be complete mucosal healing when the MES
was ≤1 and the histological score was Grade 0–1, indicating only
structural change or chronic inflammation presented in haematoxylin-
eosin sections.
2.6. Protein-protein interaction (PPI) network hub genes
To explore gene interactions, the search tool STRING 10.5
(http://string-db.org) [30] was employed to establish a DEGs PPI
network, which was drawn by Cytoscape (version 3.6.1) [31]. A com­
bined score > 0.7 was set as the cut-off point. The maximal clique
centrality (MCC) algorithm of CytoHubba (version 0.1) was used to
explore PPI network hub genes [32]. ClueGO (version 2.5.4) facilitated
the visualization of the results [33].
2.7. Immunohistochemistry
Biopsy samples from IBD patients were fixed in 4% para­
formaldehyde followed by paraffin embedding. After deparaffinization
of the tissue sections, antigen unmasking was performed with heat-
induced epitope retrieval in 10 mM citrate buffer for 10 min. Next, the
sections were incubated with 3% hydrogen peroxide for 30 min and
blocked with 5% BSA for 1 h at room temperature. Serial tissue sections
were stained with primary antibodies against G0S2 (12091–1-AP, Pro­
teintech), CXCL13 (10927–1-AP, Proteintech), MMP1 (10371–2-AP,
Proteintech), CHI3L1 (12036–1-AP, Proteintech), E-selectin (20894–1-
AP, Proteintech) or S100A9 (26992–1-AP, Proteintech) and a goat anti-
rabbit IgG H&L (HRP) secondary antibody (ab6721, Abcam) and
counterstained with haematoxylin. Sections were visualized by a Nikon
microscope equipped with a Nikon camera and Vision 4.1 software in
three different fields at 20× and 40× magnification for each sample.
Patients’ response status to IFX was blind to both experimenters and
researchers who interpreted staining results.
2.8. Statistics and analysis
To extract the core subset of data to determine the GIMATS module, a
linear regression technique was used based on the least absolute
shrinkage and selection operator (LASSO) algorithm as implemented in
the glmnet (version 2.0–16) R package [34]. The 500-bootstrap resam­
pling procedure was used in the validation set to build confidence in­
tervals for receiver operating characteristic curves (ROC). Logistic
regression was performed using the stats (version 3.4.1) R packages. All
statistical tests were two-sided, and p-values less than 0.05 were
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Genomics 113 (2021) 1988–1998
considered statistically significant. Other R packages adopted in this
study included ggplot2 (version 3.3.2) [35], pheatmap (version 1.0.12)
[36], corrplot (version 0.84) [37], survminer (version 0.4.6) [38], pROC
(version 1.16.1) [39], forestplot (version 1.9) [40] and rms (version
6.0–1) [41].
3. Results
3.1. The comparison between markers to predict the response of IFX/ADA
or VDZ
The enrichment levels of 28 immune signatures, 2 related immune
models (the ESTIMATE score and GIMATS module), and the expression
of several genes, TNFRSF11B, STC1, PTGS2, IL13RA2, IL11, and OSM,
were previously identified as predictive biomarkers in IFX/ADA
response. They were assessed for prediction abilities by ROC in merged
microarray cohort (GSE12251, GSE16879 and GSE73661) and the RNA-
seq cohort (ERP113396) (Table S4).
The top 5 immune signatures with the highest area under the curve
(AUC) values in microarray data were shown in Fig. 1A: IL13RA2 (AUC
= 0.927), immature dendritic cells (AUC = 0.895), TNFRSF11B (AUC =
0.873), GIMATS module (AUC = 0.853) and STC1 (AUC = 0.850).
Furthermore, in the RNA-seq dataset (Fig. 1B), OSM exhibited the
highest AUC of 0.752, followed by IL11 (AUC = 0.726), GIMATS (AUC
= 0.720), PTGS2 (AUC = 0.720) and neutrophil cells (AUC = 0.718). In
general, only the GIMATS module showed satisfactory robustness and
validity in both datasets (Fig. 1C).
To evaluate the capabilities to predict VDZ response, immune models
and some other predictive markers, RGS13, DCHS2, MAATS1, and
PIWIL1, were utilized in the microarray (GSE73661) and RNA-seq
cohort (ERP114636). Exact AUC values of all the features were pre­
sented in Table S5. For the microarray data (Fig. 1D), the GIMATS
module and natural killer cells showed the best performance, followed
by activated dendritic cells (AUC = 0.634), mast cells (AUC = 0.634)
and gamma delta T cells (AUC = 0.629). Besides, in the RNA-seq cohort
(Fig. 1E), the top 5 signatures were type 2 T helper cells (AUC = 0.754),
the GIMATS module (AUC = 0.728), activated CD4+ T cells (AUC =
0.726), the Stromal score (AUC = 0.716), and natural killer T cells (AUC
= 0.716).
The data above demonstrated that the GIMATS module had the best
overall performance in both IFX/ADA and VDZ treatment response
prediction (Fig. 1F).
Y. Shi et al. Genomics 113 (2021) 1988–1998

Fig. 3. Biologics response correlated with distinct functional pathways. The KEGG analysis of differential gene expressions between GIMATS-high and -low groups
(A). The dendrogram of nine distinct modules defined by the WGCNA (B). Module-trait relationship revealed both positive and negative correlation with clinical
traits and modules. Each row in the table corresponded to a module and each column to a specific clinical trait. Values listed in the heatmap showed the correlation
coefficient and P-value within parentheses. The colour of the cell indicated the correlation coefficient between the traits: red indicates a high positive correlation and
blue a high negative correlation (C).
Abbreviations: KEGG, Kyoto Encyclopaedia of Genes and Genomes; WGCNA, weighted gene co-expression network analysis; GIMATS, IgG plasma cells, inflammatory
mononuclear phagocytes, activated T cells, and stromal cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

3.2. The GIMATS module provides assistance for treatment selection
Patients from each dataset were divided into three equal groups
according to their GIMATS module values and assessed by the logistic
regression model (Fig. 1G, Tables S6–7). Of note, compared to patients
with low GIMATS module scores, those with high GIMATS module
scores showed a significantly worse response to IFX/ADA therapy
[microarray: odds ratio (OR) = 0.01, 95% confidence interval (CI) =
0.00–0.06, p < 0.001; RNA-seq: OR = 0.17, 95%CI = 0.03–0.79, p =
0.032) but tended to have better clinical outcomes when receiving VDZ
(microarray: OR = 6.00, 95%CI = 0.72–129.52, p = 0.138; RNA-seq: OR
= 6.60, 95%CI = 1.50–34.76, p = 0.017).
On the other hand, the response rate to IFX/ADA or VDZ seemed to
show no significant difference in patients with a median GIMATS
module score, who may therefore benefit from either biologic.
3.3. Estimated abundance of immune cells among the different GIMATS
groups
The gene expression profile obtained from the merged RNA-seq co­
horts (ERP115036, SRP077046, SRP100787, ERP114636, and
ERP113396, n = 216) was used to calculate the estimated abundance of
22 types of tumour-infiltrating immune cells in the 3 GIMATS groups by
the CIBERSORT algorithm (Fig. 2A). Apart from the cell populations in
the GIMATS module (inflammatory macrophages, activated dendritic
cells and highly activated T cells), monocytes and M0 macrophages were
also observed to be highly accumulated in the GIMATS-high group
compared with those in the GIMATS-low group (p = 0.010, <0.001,
respectively, Fig. 2B; Table S8), while the abundance of M2
macrophages decreased (p < 0.001). This analysis confirmed the
different activation patterns of the innate immune system among
GIMATS groups.
3.4. Differentially expressed genes between the high and low GIMATS
groups
To further identify the underlying mechanisms of these features,
DEGs between the GIMATS -high and -low patients were determined
based on the thresholds of |log2(fold-change)| > 1 and p < 0.05,
revealing 2006 upregulated and 1332 downregulated genes (Table S9).
Moreover, KEGG analysis was conducted, and Fig. 3A summarized
the results for identified signatures. The GIMATS-high group was char­
acterized by the enrichment of immune-associated pathways, including
cytokine-cytokine receptor interaction, IL-17, chemokine and TNF sig­
nalling, whereas in the GIMATS-low group, metabolic pathways were
predominantly enriched, such as retinol, drug and xenobiotic meta­
bolism. In addition, 36.9%, 43.4%, and 19.7% of CD patients were
divided into the GIMATS-low, − median, and -high groups, respectively,
while 28.1%, 25.0%, and 46.9% of the UC patients belonged to the
GIMATS-low, − median, and -high groups, respectively. UC patients
were more likely to be assigned to the GIMATS-high group (Fig. S3,
Fisher’s test p < 0.001).
3.5. Weighted gene coexpression network analysis (WGCNA) identified
the hub genes of each GIMATS group
Biological components and processes are expected to be regulated in
a coordinated manner, resulting in the coexpression of genes that are

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Y. Shi et al. Genomics 113 (2021) 1988–1998

Fig. 4. Protein-protein interaction (PPI) networks established with 50 hub genes in each GIMATS module. The venn diagram of the intersection of GIMATS-related
DEGs and genes from the GIMATS-related WGCNA module (A, D and G). The top 50 hub genes from the PPI network ranked by maximal clique centrality (MCC)
algorithm (B, E and H). Edges represented protein-protein associations. The red nodes represented genes with high MCC sores, while the yellow node represented
genes with low MCC sores. The relationship between DEGs and GO terms with a p-value <0.05 in ClueGO network diagrams (C, F and I). Nodes represented pathways
that were significantly enriched. Size of coloured nodes indicated the enrichment significance. The edges were kappa scores that measured the interrelatedness of
nodes. A thick edge that represented annotated genes had previously been experimentally verified to be part of that term/pathway. The thin one represented that
most genes were inferred by electronic annotation (IEA). Some edges were bended for a better visualization.
Abbreviations: DEGs, differentially expressed genes; GO, gene ontology; GIMATS, IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and
stromal cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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(caption on next page)

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Fig. 5. A 6-gene response prediction model, MIN score, based on LASSO regression (A). Satisfactory linear dependences of MIN score in both training (B, microarray,
n = 95) and validation (C, RNA-seq, n = 216) cohorts. The logistic regression of the M, N, I type for IFX/ADA or VDZ treatment response (D). Abbreviations: CD,
Crohn’s disease; UC, ulcerative colitis; GIMATS, IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells; Inf.Mac, inflammatory
macrophage; AC.DC, activated dendritic cell; pDC, plasmacytoid dendritic cell; ACKR1.Ecs, ACKR1 + endothelial cell; AC.Fib, activated fibroblast; res.Macs, resident
macrophage; ILCs, innate lymphoid cells; T1cyTRM, type 1 cytokines tissue-resident memory T cell; enteric_neu, enteric neurons; CD36EC, CD36+ endothelial cell;
ADA, adalimumab; IFX, infliximab; VDZ, vedolizumab; AUC, area under the curve; LASSO, least absolute shrinkage and selection operator; RNA-seq, RNA sequencing
associated with the same protein complex or biological pathway.
Therefore, WGCNA was applied to classify similarly expressed genes into
modules and identify the protein-based coexpression networks in our
study. Nine distinct modules were generated through a hierarchical
clustering tree, as shown in the dendrogram (Fig. 3B). A total number of
718 genes in the turquoise module were found to be related to the
GIMATS-low signature (r = 0.60, p < 0.001, Fig. 3C, Fig. 4A-C), among
which 451 genes were significantly downregulated.
To further explore the interactions among these genes, we selected
the top 50 genes (hub genes) (Table S10), ranked by the connectivity
within the corresponding module, to draw the PPI network. It could be
seen that metabolic processes associated with the PPAR and adipocy­
tokine signalling pathways as well as fatty acid beta-oxidation were the
core of the relationships within the network (Fig. 4A).
In addition, the green and brown modules were found to be associ­
ated with the GIMATS-high module (r = 0.69 and 0.61, p < 0.001 and <
0.001, respectively, Fig. 3C). Within them, 122 and 166 genes were
significantly upregulated. The top 50 hub genes were identified in each
group. Genes associated with the IL-17 signalling pathway, cytokine-
cytokine receptor interaction and leukocyte migration showed remark­
able enrichment in the green module (Fig. 4D-F), while genes involved
in extracellular matrix organization, ECM-receptor interaction, blood
vessel development and endodermal cell differentiation were enriched
in the brown module (Fig. 4G-I).
The data above demonstrated that the GIMATS-low group was
typically associated with active metabolic processes, and therefore, we
named it the metabolic type (M type); the GIMATS-high group was
identified as the immune type (I type) due to its close association with
immune activation. The remaining GIMATS-median group was consid­
ered to be the normal type (N type).
3.6. Establishment of a simplified 6-gene model by LASSO regression
The GIMATS module exhibited an ability to classify IBD patients into
M, I and N type, who can probably benefit from IFX/ADA or VDZ or both
therapies, respectively. To make it more convenient for practical utili­
zation, we performed a sparse regression analysis according to a unified
score based on the expression of the associated genes.
Gene expressions in the training cohort of 95 patients (GSE12251,
GSE16879 and part of GSE73661) were analysed with a statistical
regression algorithm based on LASSO. To prevent over-fitting and data
leakage during the process of model establishment, we used 5-fold cross
validation. The training data (microarray, n = 95) was shuffled
randomly and split into 5 groups. For each unique group, we defined
itself as a test set and the remaining groups as one training set. The
training set was used to establish LASSO model, which was then un­
derwent evaluations in the test set. The average accuracy was 0.863.
Finally, an optimized 6-gene signature (MIN score) was yielded as a
weighted sum of expression levels of 6 genes for each patient (Fig. 5A,
Tables S6–7). The MIN score was calculated according to the following
formula: MIN score = G0S2 × 0.157 + S100A9 × 0.610 + SELE×0.268
+ CHI3L1 × 0.896 + MMP1 × 0.500 + CXCL13 × 0.564.
The Spearman correlation between the MIN score and GIMATS
module score was 0.974 (Fig. 5B). The merged RNA-seq cohort
mentioned above was adopted as a validation cohort. As shown in
Fig. 5C, the validation cohort also demonstrated good linear dependence
(Spearman correlation = 0.869, p < 0.001). The ROC of the MIN score to
match the GIMATS module in validation cohort was illustrated in Fig. S4
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Genomics 113 (2021) 1988–1998
(AUC = 0.958, 95%CI = 0.927–0.982).
To evaluate the predictive performance of MIN score, ROC and
precision-recall curves were shown in Fig. S5 and Table S11. Moreover,
a logistic regression model was used to examine the predictive value of
MIN score (Fig. 5D). IBD patients were divided into three equal cohorts.
Notably, compared with patients with low MIN score (GIMATS M type),
those with a high MIN score, which corresponded to the GIMATS I type,
had poorer rates of response to IFX/ADA therapy (microarray: OR =
0.01, 95%CI = 0.00–0.06, p < 0.001; RNA-seq: OR = 0.06, 95%CI =
0.01–0.31, p = 0.003), but were more likely to respond to VDZ
(microarray: OR = 6.00, 95%CI = 0.72–129.52, p = 0.138; RNA-seq: OR
= 9.53, 95%CI = 2.04–57.78, p = 0.007). Patients with a median MIN
score (N type) displayed no remarkable preference for either of the two
medications.
This 6-gene panel was further confirmed in our IBD patient cohort
using immunohistochemistry (IHC). As expected, the results showed
that G0S2, CXCL13, MMP1, CHI3L1, SELE and S100A9 staining were
much stronger in biopsies from patients with poor response to IFX
(Fig. 6). We found that CHI3L1 and CXCL13 were widely detected in
biopsies from patients with poor response, but were barely seen in those
with good response. Of note, SELE was expressed solely in endothelial
cells of specimens from poorly responded patients and was relatively
weak in well responded patients’ samples. Likewise, MMP1 and G0S2
expression were found in endothelial cells. In contrast, S100A9 was
predominantly expressed in infiltrating immune cells, which was much
more obvious in specimens from patients with a good response.
Collectively, these results suggested that the MIN score could be used to
predict the response of IBD patients to IFX therapy.
4. Discussion
In this study, we applied various immune-related models to predict
the IBD biological response. Among the validated signatures, GIMATS
module showed satisfactory prediction precision for both IFX/ADA and
VDZ datasets. Therefore, further analyses were focused on GIMATS
module and its related features of gene expression. Since GIMATS
module was originally generated in CD patients, we proved that it can be
extended to general scenarios that also include UC patients. In addition,
the LASSO algorithm was used to reduce the data dimensionality and
select the best weighting coefficients, resulting in a simple but practical
6-gene signature, MIN score, which optimizes the clinical feasibility for
pre-treatment testing.
As a scoring system based on the inflammatory status, a low MIN
score reveals the presence of relatively weak inflammation in the bio­
psied mucosa. Intriguingly, functional analyses found that the corre­
sponding group of patients (M type) usually showed more active
metabolic activities, highlighting the significant role of metabolism,
especially fatty acid catabolic processes, in the pathogenesis of IBD and
the function of TNF-α inhibitors.
Intense intestinal inflammation is associated with elevated serum
levels of free fatty acids, which provide additional rich energy supplies
to immune cells at the sites of inflammation despite the presence of
glucose [42], while the increases in the pro-inflammatory cytokine TNF-
α are also able to induce a shift towards abnormal lipid profiles in in­
flammatory conditions, the process of which can be partially reversible
through TNF-α blockade [43]. These alterations in energy homeostasis
were found more in CD than in UC [44], which is consistent with the
differential expression of peroxisome proliferator-activated receptors
Y. Shi et al. Genomics 113 (2021) 1988–1998

Fig. 6. Represented IHC staining results of biopsy derived from IBD patients well (A) or poor (B) response to IFX. Scale bar, 100 um. Abbreviations: IBD, inflam­
matory bowel disease; IFX, infliximab; IHC, immunohistochemistry.

(PPARs) [45], members of the steroid receptor superfamily that play
vital roles in both inflammation and fibrosis formation [46]. Although
the 3 subtypes, PPARα, PPARγ, and PPARβ/δ, are different in terms of
their distribution, all of them participate in the regulation of fatty acid
metabolism [47]. Therefore, the activation of the PPAR pathway can
probably cause the innate immune system to assume an IFX/ADA-
sensitive status and provide clues to physicians regarding the selection
of biologics.
In addition, researchers have long identified the function of intesti­
nal microbiota as a bridge between immunity and lipid metabolism, and

1996
Y. Shi et al.
the abundance of intestinal flora that produce short-chain fatty acids
(SCFAs) was also closely related to treatment outcome. SCFAs are the
main source of energy for intestinal epithelial cells and participate in the
regulation of intestinal haemostasis. SCFA-producing microflora act as
an epithelial barrier of metabolic and immune function, the imbalance
of which can lead to the onset of IBD and the existence of persistent
inflammation [48]. Studies have shown that, in both CD and UC, a
sustained response to IFX/ADA or likelihood of disease recurrence was
significantly correlated with the high abundance of intestinal SCFA-
producing bacteria (e.g., Clostridiales and F. prausnitzii) at baseline
[49–51].
On the other hand, patients who had higher MIN scores (I type)
tended to respond better to VDZ than IFX/ADA, and the functional
analysis was mainly focused on immunologic changes. Although the
pharmacologic target of VDZ, integrin α4β7, is located on both innate
and adaptive immune cells, a limited impact on intestinal T cells has
been observed during VDZ treatment [52], highlighting the significance
of the innate immune system in the action of medication.
The baseline accumulation of polarized macrophages was highly
correlated with the I-type response in our findings, as validated in other
studies that observed a switch from M1-type to M2-type macrophages in
patients who achieved VDZ-induced clinical remission [53]. This re­
flected a shift in homeostasis towards an anti-inflammatory state rather
than overactivation of the immune system. Additionally, cytokine sig­
nalling pathways, including the IL-17 pathway that was ranked among
the top 3 most remarkable pathways in our results, are known for their
function in the promotion of the development of IBD. IL-17 is produced
by Th17 cells, which, under normal circumstances, are located in the
mucosa to protect the host from invasive microorganisms. When
induced by cytokines such as IL-1β and IL-6, the imbalance in Th17
signalling as well as Th or Treg differentiation leads to disease pro­
gression [2]. Although there are already some models of cytokines used
for IFX/ADA prediction [54], little evidence has been found to link the
IL-17 pathway to the VDZ response. However, in these complicated
processes, IL-6 also serves as a pro-inflammatory and anti-apoptosis
factor through activation of signal transducer and activator of tran­
scription (STAT) 3 in T cells [55]. An increase in the baseline IL-6 level
and its tendency to decrease over the first 6 weeks had significant power
to predict VDZ-induced mucosal healing [12].
In our study, certain approaches were used to prevent over-fitting
and data leakage issues. Primarily, we performed LASSO regression to
optimize gene selections, and adopted one internal and two external
validation sets. During the process of model establishment, 5-fold cross
validation was employed to enhance robustness, after which, MIN score
was also tested in external RNA-seq validation as well as an independent
IHC cohort. The outcomes supported our previous conclusion.
However, there were still limitations. First, this study was based on
public datasets and bioinformatic tools, and the correlations would
require further experiments to verify if the exact causal relationships
exist. LASSO only works based on several assumptions. Considering its
limitations in usage, variable sparsity and irrepresentable condition
should be checked before processing the data [56]. In this study, the
conduction of LASSO regression was followed by multiple validations to
ensure its proper usage. Although we used the IHC experiment in an
attempt to utilize our model in the real clinical practice, it is undeniable
that the sample size was too small to draw a convincing conclusion. We
called for a new independent validation set in the future to confirm our
findings. Second, prescriptions of ADA and VDZ were unavailable in our
centre, therefore no biopsy sample was obtained for validation experi­
ments in response prediction of ADA/VDZ. Third, most clinical trials
included in the data sources in our research had an active follow-up
interval of 6 weeks, which was much shorter than the typical internal
(3–6 months if no emergencies happened) [57]. Future prospective
studies should have a more reasonable timetable for clinical observation
or used other sensitive but non-invasive tests for response assessment.
Fourth, it is a challenge to apply prognosis biomarkers (e.g. gene
1997
Genomics 113 (2021) 1988–1998
expression) in daily practices for community hospitals with limited
medical resources. Besides, given that different genetic assays would
produce different information on gene mutations and fusions, expres­
sion levels of genes seem less stable to guide clinical procedures. Hence,
in this study, we established the MIN score and validated it using
platform-varied data to ascertain the robustness. We believe that, with
the further reduction of sequencing cost and the emerging of next-
generation technologies, genetics biomarkers would be found highly
valuable in predicting treatment outcomes. Compared to GIMATS
module, this 6-gene scoring system developed for therapy decision is
simple, efficient and affordable with alternative laboratory techniques
of RNA-seq, microarray, real-time polymerase chain reaction and IHC.
In conclusion, our findings regarding the predictive value of the MIN
score provided new insights to assist with treatment choices in the
future. IBD patients were divided into 3 groups based on their baseline
MIN score, the M type, I type and N type groups, reflecting the disease
characteristics. The M and I types may favour treatment with IFX/ADA
and VDZ, respectively, while the N type can probably benefit from either
treatment. This grouping emphasized the significant role of metabolism
and immune response analysis at the time of IBD diagnosis instead of the
differentiation between CD and UC.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ygeno.2021.04.011.
Funding
This work was supported by the National Natural Science Foundation
of China (81702300, 81873555).
Ethics and data availability statements
Ethical approval was obtained from Institute Research Ethics Com­
mittee of Renji Hospital. Informed consent was signed for experimen­
tation with human subjects. The data underlying this article are
available in the article and in its online supplementary material.
Supplementary tables have been uploaded to the following link:
https://www.dropbox.com/s/tgnz52c0uq71u23/Revised%
20Supplementary%20tables%20for%20MIN%20score.xlsx?dl=0.
Brief summary
The 6-gene MIN score is able to classify the metabolic or immune
type of inflammatory bowel disease patients at diagnosis and assists with
the first-line selection of infliximab/adalimumab or vedolizumab
therapy.
Declaration of Competing Interest
The authors declared no conflict interest.
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