Neuropathology 2023

doi:10.1111/neup.12944

Original Article

Increased expression of leucine-rich α-2 glycoprotein
1 as a predictive biomarker of favorable
progression-free survival in meningioma
Mayuko Moritsubo,1 Takuya Furuta,1 Junko Miyoshi,1,2 Satoru Komaki,1,2 Kiyohiko Sakata,2Hiroaki Miyoshi,1 Motohiro Morioka,2 Koichi Ohshima1 and Yasuo Sugita1,3
Departments of 1Pathology, 2Neurosurgery, Kurume University School of Medicine and 3Department of Neuropathology,
St. Mary’s Hospital, Kurume, Japan
Most meningiomas, which are frequent central nervous
system tumors, are classiﬁed as World Health Organization(WHO) grade 1 because of their slow-growing nature.
However, the recurrence rate varies and is difﬁcult to predict using conventional histopathological diagnoses.
Leucine-rich α-2 glycoprotein 1 (LRG1) is involved in cellsignal transduction, cell adhesion, and DNA repair and is apredictive biomarker in different malignant tumors; however, such a relationship has not been reported in meningiomas. We examined tissue microarrays of histologicalsamples from 117 patients with grade 1 and 2 meningiomas
and assessed their clinical and pathological features, including expression of LRG1 protein. LRG1-high meningiomas
showed an increased number of vessels with CD3-positive
cell inﬁltration (P = 0.0328) as well as higher CD105-positive
vessels (P = 0.0084), as compared to LRG1-low cases. They
also demonstrated better progression-free survival (hazard
ratio [HR] 0.11, 95% conﬁdence interval [CI] 0.016–0.841)
compared to LRG1-low patients (P = 0.033). Moreover,multivariate analysis indicated that high LRG1 expression
was an independent prognostic factor (HR, 0.13; 95% CI,
0.018–0.991; P = 0.049). LRG1 immunohistochemistry may
be a convenient tool for estimating the prognosis of meningiomas in routine practice. Further studies are required to elucidate the key role of LRG1 in meningioma progression.
Key words: LRG1, menignioma, pathology, prognosis,
progression.

Correspondence: Takuya Furuta, MD, PhD, Department of Pathology, Kurume University School of Medicine, 67 Asahimachi,
Fukuoka 830-0011, Kurume, Japan. Email: tfuruta@med.kurume-u.ac.jp
Received 21 June 2023; revised 07 September 2023; accepted 11
September 2023.

© 2023 Japanese Society of Neuropathology.

INTRODUCTION
Meningioma is the most common primary intracranialtumor, accounting for 36% of all central nervous system
tumors that originate from arachnoid cells. According to theﬁfth edition of the World Health Organization Classiﬁcationof the Tumors of the Central Nervous System (WHO
CNS5), these tumors are categorized into 15 subtypes andthree grades of malignancy.1 Meningiomas are mostly
slow-growing, and their recurrence rate varies from 7 to 25%,thus making their prognosis using conventional pathologicaldiagnosis difﬁcult. A subset of meningiomas contain different genes that regulate tumor growth in vitro andin vivo2,3; however, their role as prognostic factors has
not been fully revealed.
Leucine-rich α-2 glycoprotein 1 (LRG1) is a member ofthe leucine-rich repeat protein family, which is involved incell signal transduction, cell adhesion, and DNA repair.
LRG1 is expressed in the hematopoietic stem cells, andafter their differentiation into neutrophilic granulocytes,4
LRG1 is involved in inﬂammation through activation of
the transforming growth factor beta and Smad1/5/8
pathways.5–7 In fact, serum LRG1 concentration is a biomarker of disease activity in ulcerative colitis and rheumatoid
arthritis.4,5 It also promotes pathogenic neovascularization
through endothelial transforming growth factor beta signaling in the presence of CD105 (endoglin).6
Studies have shown that LRG1 can be used as a prognosis marker in carcinomas such as hepatocellular carcinoma,8ovarian cancer,9 and non-small-cell lung cancer.10 Furthermore, our group has reported that LRG1 overexpression isan independent prognostic factor in glioblastomas.11
However, the association between clinicopathologicalfeatures and the expression of LRG1 has never been
investigated in meningiomas. In this study, we evaluatedthe expression of LRG1 in meningioma using immunohistochemistry and validated its role as a prognostic marker.

M Moritsubo et al.

MATERIALS AND METHODSClinical sample and histology
In this study, tissues from 121 patients with WHO grade
1 and 2 meningiomas were obtained through surgery andstored in the database of the Department of Pathology,
Kurume University. Histological analysis was based on the
WHO criteria.1 We used a tissue microarray (TMA)
containing each of the 121 specimens in a formalin-ﬁxed
parafﬁn block, where each sample was placed in a
2.5-mm-diameter space. The research ethics committee of
Kurume University approved the use of patient materials
and clinical information (No. 345), and the study was inaccordance with the Declaration of Helsinki.

Evaluation of immunohistochemical staining of
LRG1, CD3, CD20, and CD105
Slide-mounted formalin-ﬁxed, parafﬁn-embedded tissue
sections (2.5 μm in thickness) from the TMA were used aspreviously described.11 For immunohistochemistry analysis, sections were stained with antibodies against LRG1
(rabbit polyclonal, Sigma-Aldrich, St. Louis, MO, UnitedStates; 1:300 dilution), CD3 (Dako, Japan Co, Kyoto,
Japan; 1:50 dilution), CD20 (Dako; 1:5 dilution), andCD105 (Dako; 1:50 dilution). To evaluate tumor angiogenesis, CD105-positive vessels were counted.

The genes with P-values less than 0.05 and log2 foldchanges of < 1 or >1 were deﬁned as characteristic genes.

Molecular genetic analyses
Genomic DNA was isolated from the same sample as
extracted RNA from the relevant FFPE tissue followed byextraction using a GeneRead DNA FFPE Kit (QIAGEN,
Hilden, Germany). Telomerase reverse transcriptase (TERT)
promoter 5p15.33 was ampliﬁed by polymerase chain reaction (PCR) as described previously.11 No mutation was
found, conﬁrming the diagnosis of meningioma grade 1 or 2.

Statistical analysis
The clinicopathological characteristics of the patients werecompared using the χ2-test or Fisher’s exact test.
Progression-free survival was deﬁned as the time from thedate of diagnosis to the date of recurrence or last followup. The Kaplan–Meier method was used to estimate
progression-free survival distribution, and the log-rank test
was performed to evaluate signiﬁcant differences. Univariate and multivariate Cox proportional regression models
were used to evaluate the prognostic factors. Statisticalsigniﬁcance was set at P < 0.05. JMP version 16.0 was usedin all statistical analyses.

RESULTS
Deﬁnition of immunohistochemical LRG1-score
and cutoff value

Clinicopathological characteristics of patients withmeningioma

Two blinded pathologists (MM and MJ) independently
evaluated the expression of LRG1. Using the same
method as stated previously,11 the intensity of LRG1
staining of individual tumor cells was graded from 0 to 3(0, negative; 1, weak or focal positive; 2, moderate;
3, strong and diffuse positive). Based on the results, thestudy population was divided into two groups: low expression (score 0,1) and high expression (score 2,3) (Fig. 1).
For evaluation of tumor angiogenesis, CD105-positive vessels were counted in all the ﬁelds of each TMA at highmagniﬁcation (400). To evaluate inﬂammatory cell inﬁltration, CD3 and CD20-positive lymphocytes were counted
in all TMA ﬁelds. Cutoff values for high CD105-positive
vessel and CD3- and CD20-positive cell inﬁltration were set
above the median value per specimen.

The clinicopathological characteristics of the 117 patientsare summarized in Table 1. The median age was 61 (range
22–85) years. A total of 31 men and 86 women were
included. The follow-up period ranged from 0.23 to
114.8 months with a median value of 24.1 months.
According to the WHO classiﬁcation,1 distribution ofhistology was ﬁbrous (28.2%), transitional (22.2%),
meningothelial (24.8%), microcystic (5.1%), psammomatous(5.1%), angiomatous (4.3%), metaplastic (2.6%), secretory(0.9%), atypical (6.0%), and chordoid (0.9%). Eighty-nineof 117 patients (66.7%) had Simpson grade 1 or 2 in termsof surgical resection completeness,13 while 50.4% (59/117) ofpatients had tumors located at the skull base. Twenty-six
cases (22.2%) were categorized as LRG1-high.

Gene expression proﬁling

Clinicopathological comparison between
LRG1-high and -low patients

We extracted RNA from formalin-ﬁxed, parafﬁn-embedded
tissue sections of four LRG1-high and four low samples
using the RNeasy Micro Kit (Qiagen, Hilden, Germany).
This was followed by microarray as described previously.12

LRG1-high meningiomas showed CD3-positive cell inﬁltration (P = 0.033) and more CD105-positive vessels(P = 0.008) in comparison to LRG1 low cases. There were no
signiﬁcant differences in age, sex, histology, Simpson grade,© 2023 Japanese Society of Neuropathology.

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3

Fig 1 Representative images of meningioma specimen on HE staining (A–D) and leucine-rich α-2 glycoprotein 1 (LRG1) staining(E–H). A transitional meningioma case with LRG1 score 0 (A, E), a ﬁbrous meningioma case with LRG1 score 1 (B, F), a meningothelial
meningioma case with LRG1 score 2 (C, G), and a transitional meningioma case with LRG1 score 3 (D, H). Scale bars: 50 μm (A–H).

location, MIB-1 index, and CD20-positive cell inﬁltration
between these two groups (Table 1).

Progression-free survival curves in patients withmeningioma according to LRG1 expression
Patients with high LRG1 expression had better
progression-free survival compared to LRG1-low patients(log-rank, P = 0.010, Fig. 2A). Moreover, grade 1
LRG1-high meningioma showed better progression-freesurvival in comparison to LRG1-low patients (log-rank,
P = 0.015, Fig. 2B). Univariate analysis was performed
using degree of Simpson grade ≤2 (HR, 0.20; 95% CI,
0.092–0.435; P < 0.001), non-skull base (HR, 0.74; 95% CI,
0.348–1.556; P = 0.422), and Ki-67 labeling index <4%(HR, 0.40; 95% CI, 0.054–2.923; P = 0.366) (Table 2).
Multivariate analysis using these variables indicated that© 2023 Japanese Society of Neuropathology.

high LRG1 expression was an independent prognostic factor (HR, 0.13; 95% CI, 0.018–0.991; P = 0.049, Table 2).

RNA microarray expression analysis
Following appropriate normalization and standardization
procedures, the data on each chip were compared witheach other by using a hierarchical clustering method. In
the LRG1-high tumors, 763 probe sets were identiﬁed
as upregulated or downregulated on a heat map(Fig. 3A). The expression-altered genes were located on
the whole genome (Fig. 3B). The ﬁrst 10 genes showing
the highest upregulation or lowest downregulation arelisted in Table 3, respectively. Several genes associatedwith angiogenesis or inﬂammation were upregulated(Table S1). Pathway enrichment analysis using Metascape14 showed that LRG1-high specimens contained

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LRG1 as a predictive biomarker in meningiomaM Moritsubo et al.

Table 1 Clinicopathological characteristics of patients (n = 117)All

Median age, y (range)
FemaleHistology
FibrousMeningothelial
Transitional
Microcystic
Psammomatous
Angiomatous
Metaplastic
Secretory
AtypicalChordoid
Simpson grade
12
3
4
Location
Skull-baseHistological ﬁndings
MIB-1 index, % (range)
increased CD105 vessels†
increased CD3-positive T-cell‡increased CD20-positive B-cell§

LRG1
Low

High

P*

61 (22–85)
73.5% (86/117)

61 (22–85)
76.9% (70/91)

59 (34–85)
61.5% (16/26)

0.88
0.135

28.2% (33/117)
22.2% (26/117)
24.8% (29/117)
5.1% (6/117)
5.4% (6/117)
4.3% (5/117)
2.6% (3/117)
0.9% (1/117)
6.0% (7/117)
0.9% (1/117)

27.5% (25/91)
23.1% (21/91)
26.4% (24/91)
3.3% (3/91)
5.8% (5/91)
3.3% (3/91)
2.2% (2/91)
1.1% (1/91)
6.6% (6/91)
1.1% (1/91)

30.1% (8/26)
19.2% (5/26)
19.2% (5/26)
11.5% (3/26)
3.9% (1/26)
7.7% (2/26)
3.9% (1/26)
0% (0/26)
3.9% (1/26)
0% (0/26)

0.761

27.4% (32/117)
39.3% (46/117)
16.2% (19/117)
17.1% (20/117)

23.1% (21/91)
39.6% (36/91)
16.5% (15/91)
20.9% (19/91)

42.3% (11/26)
38.5% (10/26)
15.4% (4/26)
3.9% (1/26)

0.095

50.4% (59/117)

51.7% (47/91)

42.3% (11/26)

0.506

1.0 (0–20)
31.6% (37/117)
22.2% (26/117)
39.3% (46/117)

1.0 (0–8.0)
25.3% (23/91)
17.6% (16/91)
37.4% (34/91)

1.0 (0–20)
53.9% (14/26)
38.5% (10/26)
46.2% (12/26)

0.516
0.008
0.033
0.496

Abbreviation: LRG1, leucine-rich α-2 glycoprotein 1. *Fisher’s exact test; †≥11.6 (median); ‡≥79.2 (median); §≥6.8 (median).

genes associated with ribosomal proteins, genes regulating apoptosis, and DNA damage response genes mediated by TP53 (Fig. S1). Of note, no-CDKN2A/B

homozygous deletion was found. No mutation in TERT
promoter was identiﬁed, conﬁrming the diagnosis ofmeningioma grade 1 or 2.

Fig 2 Kaplan–Meier curves of progression-free survival in patients with meningioma. Patients with high leucine-rich α-2 glycoprotein 1(LRG1) expression had better progression-free survival compared to LRG1-low patients: (A) grade 1 and 2 meningiomas, P = 0.010;(B) grade 1 meningiomas, P = 0.015.

© 2023 Japanese Society of Neuropathology.

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45

Table 2 Prognostic factors affecting the PFS of patients with meningioma
Univariate

Multivariate

VariableHR

95% CIPHR

95% CIP

Not skull baseMIB-1 index <4%CD3 lymphocyte
CD105 vessel
Simpson grade ≤2
High LRG1

0.74
0.40
0.43
0.76
0.20
0.11

0.348–1.556
0.054–2.923
0.130–1.432
0.352–1.651
0.092–0.435
0.016–0.841

0.422
0.366
0.170
0.491
<0.001
0.033

0.22
0.13

0.099–0.474
0.018–0.991

0.00010.049

Note: The signiﬁcant variables has been in bold. LRG1, leucine-rich α-2 glycoprotein 1.

DISCUSSION
In this study, we demonstrated for the ﬁrst time that high
LRG1 expression in patients with low-grade meningioma
is an independent prognostic factor for favorable
progression-free survival. High LRG1 expression correlated with increased inﬁltration of CD3-positive T-cells
and proliferation of CD105-positive vessels.

The level of expression of LRG1 is associated with
progression in various tumors, including colorectal carcinoma, gastric cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, head and neck carcinoma,
and glioblastoma.8–11 Of these, in the case of head andneck squamous cell carcinoma and glioblastomas, high
LRG1 expression was a favorable prognostic factor. The

Fig 3 Gene expression proﬁles and clustering. The gene expression of leucine-rich α-2 glycoprotein 1 (LRG1)-high meningiomas wascompared with that of LRG1-low meningiomas by using the hierarchical clustering method (A). In LRG1-high meningiomas, 763 probe
sets were identiﬁed as upregulated or downregulated (B).

© 2023 Japanese Society of Neuropathology.

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LRG1 as a predictive biomarker in meningiomaM Moritsubo et al.

Table 3 Genes differentially regulated in LRG1 high or low meningioma
Gene symbol

Gene nameUp-regulated genes in LRG1 high meningioma compared with LRG1 low meningioma
1
ETV5
Ets variant 5
2
LDHA
Lactate dehydrogenase A
3
RPL30
Ribosomal protein L30
4
RPL35A
Ribosomal protein L35a5
ERI1
Exoribonuclease 1
6
NPIPB4
Nuclear pore complex interacting protein family,
member B4
7
GABARAPGABA(A) receptor-associated protein
8
RPS29
Ribosomal protein S29
9
NDUFB1
NADH dehydrogenase 1 beta subcomplex
10
TAF1D
TATA box binding protein associated factor 1D
Down-regulated genes in LRG1 high meningioma compared with LRG1 low meningioma
1
CFAP54
Cilia and ﬂagella associated 54
2
ARHGAP21
Rho GTPase activating protein 21
3
MGST3
Microsomal glutathione S-transferase 34
DHX15
DEAH (Asp-Glu-Ala-His) box helicase 15
5
NPR2
Natriuretic peptide receptor 2
6
NBPF15
Neuroblastoma breakpoint family, member 15
7
ZNF418
Zinc ﬁnger protein 418
8
SLIT2
Slit guidance ligand 2
9
NCS1
Neuronal calcium sensor 1
10
PPEF2
Protein phosphatase, EF-hand calcium binding domain
2

Location

Exp. Value

3q27.2
11p15.1
8q22.2
3q29
8p23.1
16p12.2

7.46226365
6.58567127
5.85758845
5.61779313
5.07252592
4.44884654

17p13.1
14q21.3
14q31.3
11q21

4.25306479
4.13068964
4.11329393
4.05961149

12q23.1
10p12.1
1q24.1
4p15.2
9p13.3
1q21.1
19q13.43
4p15.31
9q34.114q21.1

0.48650797
0.49153549
0.49475796
0.49707456
0.58995875
0.61717278
0.61776091
0.62028042
0.63012589
0.63686081

Abbreviation: LRG1, leucine-rich α-2 glycoprotein 1.

biological roles of LRG1 in tumor aggressiveness are
not fully understood, and the mechanism might differ
between different types of tumor.
The positive correlation between LRG1 expression andCD3-positive cell inﬁltration indicated that LRG1 may be
associated with the immune milieu of meningiomas.
Immune cell inﬁltration is commonly seen in other tumors,such as glioblastoma, primary central nervous system lymphoma, and metastatic brain tumors.15–18 Furthermore,
several studies have reported a relationship between meningioma and the immune microenvironment, such as CD20 inﬁltration and PD-L1 expression.17,18 CD3-positive cellinﬁltration is controversial, negative correlation between
CD3-positive cells and the tumor grade in meningiomas,18
which may reﬂect tumor aggressiveness. Similarly, in ourstudy, CD3-positive cell inﬁltration also showed better
progression-free survival.
Moreover, high expression of LRG1 was associatedwith a greater frequency of CD105-positive vessels. We
also performed CD34 immunostaining to detect all bloodvessels, and the CD105/CD34 ratio was 5.1%. No association
between CD34-positive vessels and LRG1 or histologicalvariants, including angiomatous variants, was identiﬁed,suggesting that an increasing number of CD105-positive
vessels reﬂected neoplastic angiogenesis (data not shown).
Lower expression of CD105, a marker of angiogenesis, demonstrates better overall survival in several types ofneoplasia,19,20 but no signiﬁcant relation is observed inmeningiomas.21 High CD105-positive vessels have also beenreported to be correlated with a higher Ki-67 index.21,22 Our
study also indicated similar results regarding the CD105 andKi-67 index (P = 0.063, data not shown), but neither was asigniﬁcant progressive factor.
Molecular genetic proﬁling of meningiomas has beenreported, while similar studies regarding LRG1 are lacking. Next-generation sequencing and methylation analysis
have revealed the risk of recurrence of meningiomas.
Mutations in genes such as TRAF7, KLF4, SMO, AKT1,
and chromosome 5 gain were observed in the three benigngroups; however, LRG1 and associated genes were notincluded in that study.23 We could not detect these genomic abnormalities in gene expression analysis (Table 3,
Fig. 3). The decrease of the genes in copy number onchromosome 19, where LRG1 is encoded, is a high-risk
loss,24 indirectly suggesting the relationship between low
expression of LRG1 and poor prognosis. Pathway enrichment analysis showed that LRG1-high specimens contained genes associated with ribosomal proteins and DNA
damage response genes mediated by TP53 (Table 3). As
discussed above, inﬂammation and angiogenesis may be
associated with LRG1, resulting in ribosomal stress25 andthereby regulating LRG1 expression. We performed
immunohistochemical analysis using antibodies against
p53 and caspase 3 but did not ﬁnd a relationship between
the expression of LRG1 and these proteins because of thelimited number of samples (data not shown).
© 2023 Japanese Society of Neuropathology.

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6

Malignant (WHO grade 3) meningiomas were excluded
from our study because of the limited availability of samples (only 11 patients). Due to the use of TMA, each single core might not reﬂect the heterogeneity of LRG1
expression in tumors. LRG1, CD105, and TGF-β were notincluded in the microarray design. To conﬁrm RNA
expression, customized probes and real-time PCR are
required in future research. An increased number ofsamples, such as frozen material and malignant meningioma, are needed to validate the biological function of
LRG1 in all meningioma grades. Therefore, it can be concluded that the overexpression of LRG1 is a predictor ofgood progression-free survival in low-grade meningiomas.
LRG1 has been suggested to prevent tumor progression
through tumor immunity. Immunohistochemistry for
LRG1 might be a powerful prognostic marker that can beeasily and rapidly performed in routine practice for lowgrade meningiomas with diverse recurrence rates. Further
studies may highlight a key role of LRG1 in the oncogenesis and progression of meningiomas, including malignant
meningiomas.

AUTHOR CONTRIBUTIONS
Takuya Furuta and Yasuo Sugita were involved in the
study conceptualization. Hiroaki Miyoshi and Koichi
Ohshima were involved in methodology. Mayuko
Moritsubo, Satoru Komaki, and Junko Miyoshi analyzed
and investigated the study. Mayuko Moritsubo wrote theoriginal draft. Takuya Furuta, Satoru Komaki, and Kiyohiko
Sakata wrote, reviewed, and edited the article. Motohiro
Morioka, Koichi Ohshima, and Yasuo Sugita supervised thestudy. All authors have read and approved the ﬁnal manuscript. The authors thank Prof. Kenta Murotani for assistancewith statistical analysis. This manuscript was formatted andedited by Editage (https://www.editage.com).

ACKNOWLEDGMENTS
This study was supported in part by an Uchimura grantfrom Kurume University for the Promotion of female
researcher, Fukuoka Public Health Promotion Organization Cancer Research Fund (2022-A12), and JSPS
KAKENHI Grant Number JP23K14490.

DISCLOSURE
The authors have nothing to disclose.

ETHICS STATEMENT
Approval of the research protocol: Yes.
Informed Consent: Yes.
Registry and the Registration No. of the study/trial: 345 (TheResearch Ethics Committee, Kurume University).
© 2023 Japanese Society of Neuropathology.

7
Animal Studies: N/A.
Research involving recombinant DNA: N/A.

DATA AVAILABILITY STATEMENTThe underlying data can be accessed at reasonable request
to the corresponding author.

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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher’s website: http://
onlinelibrary.wiley.com/doi/10.1111/neup.12944/suppinfo.
Fig. S1. Of the upregulated genes, Metascape14 automatically reported analyses. Leucine-rich α-2 glycoprotein
1 (LRG1)-high specimens contained genes associated withribosomal proteins, genes regulating apoptosis, and DNA
damage response genes mediated by TP53.
Table S1. Upregulated genes in leucine-rich α-2 glycoprotein 1 (LRG1) high meningioma compared with LRG1
low meningioma.

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