Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

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Taiwanese Journal of Obstetrics & Gynecology

journal homepage: www.tjog-online.com

Original Article

Molecular profiling of TAM tyrosine kinase receptors and ligands

in endometrial carcinoma: An in silico-study
€
Dilara Fatma Akin a, *, Didem Ozkan b

a €
Nigde Omer Halisdemir University, Faculty of Medicine, Medical Biology, Nigde, Turkey
b
Istanbul Okan University, Vocational School of Health Service, Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: TAM Receptors (TYRO3, AXL, and MerTK) and their ligands on tumor-associated macrophages
Accepted 29 September 2022 are promising therapeutic targets for most solid cancers. However, in endometrial cancer, the most
common invasive gynecologic malignancy, the TAM receptor-mediated activation pathway, its molecular
Keywords: mechanisms, and its pathophysiology are unknown. The goal of this research; to uncover the compre-
Endometrial carcinoma hensive genetic profile of TAM receptors and ligands in endometrial cancer.
€
TAM reseptOr
Material and methods: Mutation and expression profiles of the Uterine Corpus Endometrial Carcinoma
GAS6
(UCEC) cohort (n ¼ 509) were obtained using bioinformatics tools providing data from The Cancer
Macrophage
Mutation
Genome Atlas (TCGA). PolyPhen-2 and SNAP tools were used to predict the oncogenic/pathogenic
Gene expression properties of the identified mutations for UCEC. STRING network analysis was performed to better un-
derstand the functional relationships of the mutant proteins in cellular processes. Furthermore to the
mutation profile, gene expression and survival profiles were also determined. Finally, the correlation
between target genes and macrophage infiltration was investigated using the tool TIMER.
Results: A total of 229 mutations were detected in 6 genes, and 81 missense mutations are pathogenic. In
the UCEC cohort, the expression level of MerTK, AXL, GAS6, and PROS1 was statistically significantly
lower in the patient group, while the expression level of CD47 was higher in the patient group than in the
healthy group (p < 0.01). Proteineprotein interaction analysis identified target genes, SRC protein
responsible for important cellular mechanisms such as cell proliferation, adhesion and migration, ITGB3,
ITGAV and THSB1 proteins involved in endothelial mesenchymal transition and tumor metabolism
reprogramming, and FOLR1 involved in DNA replication and damage repair.
Conclusion: We believe that TAM receptors and their ligands may be attractive molecular targets for the
treatment of endometrial carcinoma because they act as pleiotropic inhibitors of immune cells, effec-
tively regulate phagocytic clearance of apoptotic cells, and make the tumor microenvironment a more
suitable niche for the tumour.
© 2023 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction tumor-promoting functions such as tumor initiation, growth,

Cancer progression is strongly influenced by the tumor micro-
environment (TME), which is composed of host and cancer cell
components [1,2]. The TME is composed of many different host cell
types, including blood vessels, extracellular matrix, fibroblasts, B
cells, and T cells. Tumor-associated macrophages are the critical
component of the TME [2e4]. Overall, these macrophages have
* Corresponding author. Dilara Fatma Akın-Balı, Nigde Omer Halisdemir Uni-
versity, Faculty of Medicine, Medical Biology, Nigde, Turkey.
E-mail addresses: dilarabali@ohu.edu.tr, dilarafatmaakin@gmail.com (Dilara
Fatma Akin).
angiogenesis, metastasis, immunosuppression, and resistance to
therapy [2e5]. Tumor-associated macrophages are promising for
cancer therapy because they play a therapeutic role in promoting
cancer progression and concomitant immunosuppression through
their TAM receptors (TYRO3, AXL, and MerTK) and are also an
abundant cell type in the tumor microenvironment [4,6]. Further-
more to these functions, TAM receptors also play a role in macro-
phage polarization and clearance of apoptotic cells, a tumor-
promoting process called efferocytosis [7,8]. In particular, tumor-
associated macrophages with a phenotype between M1 and M2
polarization are the most abundant immune cells and play a key
role in regulating the tumor microenvironment [4,9]. The M2-

https://doi.org/10.1016/j.tjog.2022.09.010
1028-4559/© 2023 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
 €
Dilara Fatma Akin and D. Ozkan
polarized population (pro-tumor phenotype) is larger at cancer
margins than the M1-polarized population (anti-tumor phenotype)
and plays an important role in tumor progression. Tumor-
associated macrophage infiltration (mainly of M2 phenotype)
generally indicates poor prognosis in some malignancies such as
breast, ovarian, and endometrial cancers [9e11]. M2 macrophages
support the tumor immunosuppressive microenvironment by
releasing anti-inflammatory cytokines such as IL-20, TGF-b, and
HGF or by preventing the release of intracellular antigens from
apoptotic cancer cells through efferocytosis [11e13]. The dysfunc-
tion of macrophages plays an important role in the development of
endometriosis. In EC tissues, M1 macrophages, with anti-
tumorogenic characteristic, provide pro-inflammatory response
and secrete TH1 cytokines. On the other hand, M2 macrophages,
with pro-tumorigenic characteristic, provide anti-inflammatory
response and secrete TH2 cytokines and support angiogenesis and
invasion. Specifically, CD163 þ M2-macrophages accumulated to a
greater extent in patients with EC. Density of CD163þ, CD68þ TAM
and M2 TAM was also associated with higher risk of myometrial
invasion, microvessel density, angiogenesis, invasion of lymphatic
space, lymph node metastasis, and, higher histologic grade, and
high expression of Ki-67 and p53 [9e15]. TAM Receptors on
tumour-associated macrophages bind the “eat me” signal phos-
phatidylserine on apoptotic cell membranes using Gas6 and protein
S as bridging ligands [4e8,16]. Apoptosis plays a critical role in
maintaining tissue homeostasis and represents a normal function to
eliminate excess or dysfunctional cells [17]. This process is essential
for the maintenance of tissue homeostasis. Apoptosis resistance in
developing EC is thought to play a role in tumor progression. This
possible resistance in TME promotes tumor growth, metastasis, and
evasion of anti-tumor immunity [18]. Therefore, targeting tumor-
associated macrophages and their functions are potential thera-
peutic strategies because of their great importance in disease pro-
gression. Endometrial carcinoma (EC) is one of the most common
gynecologic malignancies in women, which seriously threatens
women's health [19]. Although many people are affected by this
disease, the molecular mechanisms involved in the pathophysi-
ology of this gynecologic cancer are largely unknown and under-
studied. Although TAM receptors and their ligands (PROS1, GAS6)
are potential therapeutic targets on tumour-associated macro-
phages, the role of these receptors in EC has not yet been defined.
The aim of this study was to determine the pathogenic mutations in
the genes encoding TAM receptor and their specific ligands, which
are therapeutic candidates for the treatment of endometrial cancer,
and to predict their possible functional effects by determining their
expression profiles and to fathom the impact of these elements on
the pathogenesis and progression of EC .
Materials and methods
Downloading patient data
The UCEC (n:509) data set was downloaded from the cBioPortal
online database and the study's raw data accessible by means of
cBioPortal. Demographic and clinical data of the TCGA UCEC cohort
were summarizedin Table 1.
Genotyping analysis
The cBio Cancer Genomics Portal is an open-access tool that uses
data from TCGA to provide mutation data [20]. The UCEC was
chosen as the type of cancers of interest on the web interface to
examine mutations in MerTK, AXL, TYRO3, CD47, PROS1, and GASP6
genes in UCEC patients presented in the portal. The selected TCGA
data set comprised the genome sequencing data of 509 UCEC
312
Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324
patients. In this regard, comprehensive mutation profile analyzes of
TAM receptors and ligands genes were performed using OncoPrint,
Cancer Types Summary, and Mutation tabs provided by cbioportal.
These tools provide an overview of genomic alterations in partic-
ular genes affecting particular individual samples.
Functional impact analysis of detected mutations
To determine the pathogenicity of mutations identified in MerTK,
AXL, TYRO3, CD47, PROS1, and GASP6 genes, we used the scores
provided by the PolyPhen-2, SIFT and the COSMIC databases. The
PolyPhen-2 predicts the probability of the missense mutation being
damaging based on a combination of all these features and ensures
both a qualitative prediction (probably damaging, possibly damaging,
benign or unknown) as a score for the user [21]. The SIFT algorithm
predicts whether an amino acid substitution affects protein function
based on sequence homology and the physical properties of amino
acids [22]. Screening of detected mutations in different cancer types
was performed with the COSMIC database [23].
Gene expression and survival analysis
GEPIA is an advanced interactive network supporting normal
and tumor tissue samples gene expression profiling and interactive
analyses [24]. The effect of MerTK, AXL, TYRO3, CD47, PROS1, and
GASP6 on survival according to high/low m-RNA expression levels
in UCEC (n:171) patients was performed using the GEPIA database.
Moreover, the TIMER-2 gene expression module was used to
compare the expression levels of mutant and wild-type individuals
for each gene to determine the effect of the detected mutations on
m-RNA expression levels, with the findings presented as log2 fold
changes for each gene.
Proteineprotein interaction analysis
The STRING database is used to evaluate proteineprotein
interaction information [25]. The predicted interactions of MerTK,
AXL, TYRO3, CD47, PROS1, and GASP6 proteins were performed by
the STRING database, which defines the direct (physical) and indi-
rect (functional) relationships between the proteins.
Tumor immune estimation resource (TIMER) analysis
TIMER is a comprehensive open access on-line database that can
analyze immune invasion levels and differences in gene expression
levels in different tumor tissues [26]. The correlation between
immune cells associated with MerTK, AXL, TYRO3, CD47, PROS1, and
GASP6 was analyzed with Spearman correlation analysis using the
TCGA UCEC cohort (n ¼ 545) in TIMER database.
Statistical analysis
All statistical analyses used in the evaluation of study data were
performed on bioinformatics tools. KaplaneMeier curves for over-
all survival. Low- and high-expression groups were compared using
the log-rank test. The gene expression levels of wild-type and
mutant individuals were determined using the Wilcoxon test on
the TIMER-2 program. In all tests, p < 0.05 was considered to
indicate a statistically significant difference .
Results
Results of genotyping analysis
According to the results of genotyping analysis of 509 UCEC
cohort in MerTK, AXL, TYRO3, CD47, PROS1, and GASP6, the gene with
 €
Dilara Fatma Akin and D. Ozkan Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

the most mutations was determined as AXL (10%) and the one with MerTK analysis
the least was determined as CD47 (4%). Missense, nonsense,
frameshift, splice region mutations, deep deletions, and gene am- A total of 36 different mutations (31 missense, 3 nonsense, 1
plifications were detected in target genes. A total of 229 mutations frame shift, and 1 splice region mutation) have been identified in
were identified in 7 genes (192 missense mutations, 17 nonsense, 10 MerTK. The p. R737*, p. E770*, and p. R732* nonsense mutations
splice region, 7 frameshift, and 3 fusion gene mutations). These localized to the kinase domain can lead to premature termination
mutations are listed in detail in Table 2. Mutations were detected in of the polypeptide and cause the formation of a truncated protein.
104 (20.4%) of 509 individuals. Furthermore, a deep deletion There is also the p. G58Sfs*4 mutation that disrupts the reading
resulting in homozygous loss was detected in the AXL and TYRO3, frame at the early level of the protein. Because the p. H483 ¼
whereas gene amplification abnormalities were detected in all mutation is located in the splice region, which is 100% conserved
target genes except TYRO3. When we look at the frequency of car- between species in the evolutionary process, this mutation likely
rying mutations in target genes in UCEC cohort, MerTK; CD47, causes an abnormality in MerTK expression. The frequency of so-
MerTK; AXL, AXL; TYRO3, MerTK; TYRO3, TYR03; CD47 and AXL; matic mutations detected in this gene in UCEC cohort was 6%.
CD47 was statistically significant (p < 0.01). TYRO3, AXL, and MerTK
are receptor tyrosine kinases that play a role in the clearance of AXL analysis
apoptotic remnants formed as a result of physiological processes and
negative regulation of innate immune responses. They have 3 major Fifty-three different mutations (48 missense, 2 nonsense, 2
conserved homologous domains: fibronectin type III (FNIII), immu- frame shift, 1 splice region mutation) have been detected in AXL.
noglobulin (Ig)-like, transmembrane (TM), and kinase domains. TAM The frame shift mutations p. H292Pfs*47 and p. V213Sfs*37, which
receptors and ligands were schematized in Fig. 1. The localization of disrupt the reading frame, are located on the FNIII and immuno-
mutations detected in the domains of proteins belonging to the globulin (Ig)-like domains. The nonsense mutations p. G49* and p.
study genes in UCEC cohort is shown in Fig. 2A and B. S442* are capable of causing premature termination of the poly-
peptide and formation of a truncated protein. The frequency of
somatic mutations detected in this gene in UCEC cohort was 10%.
Table-1 The intracellular tyrosine kinase domain of AXL contains the
Clinico-pathological and Demographic data on the 509 Patients with UCEC cohort.
sequence KW (I/L)A (I/L)ES (a.a 714e720), which is conserved
Characteristic Patient data n: 509 (%) among all RTKs of the TAM family. No mutations were detected in
Gender this domain [25].
Female 509
Diagnosis age, years 63.8 (range, 31e90) TYRO3 analysis
Sample Type
Primary 509
Overall Survival Status
In UCEC study group, 25 mutations (18 missense, 5 nonsense, 2
Living 447 (83.6) splice regions) were identified in the TYRO3 gene. The mutations in
Deceased 87 (16.4) the splice regions p. G527 ¼ and p. V761 ¼ at the kinase domain and
Median Follow-up 54.94 the nonsense mutations p. E540*, p. E564*, and p. E528* have the
Tumor Type
property to cause insufficient function and truncated protein pro-
Endometrioid Endometrial Adenocarcinoma 399 (75.4)
Serious Endometrial Adenocarcinoma 109 (20.6) duction. The frequency of somatic mutations detected in this gene
Mixed Serious and Endometriod 21 (4) was 5% in UCEC cohort.
Endometrial Adenocarcinoma
ICD-10 Classification CD47 analysis
C54.1 520 (98.3)
C54.9 4 (0.8)
C54.3 3 (0.6) CD47, also known as integrin-associated protein, is a trans-
C54.0 2 (0.4) membrane protein encoded by the CD47 gene [26]. The frequency
Molecular Type of fusion genes (RNF183-CD47, RASGRP1-CD47, and SPON1-CD47)
CN high 163 (30.8) in CD47 gene was 0.5% and 15 missense mutations were deter-
CN low 147 (27.8)
mined. The frequency of somatic mutations detected in CD47 gene
MSI 148 (28.0)
POLE 49 (9.3) was 4% in UCEC cohort.
NA 22 (4.2)
Histologic Grade GAS6 analysis
G1 97 (18.3)
G2 119 (22.5)
G3 302 (57.1) The GAS6 encodes a protein consisting of 678 amino acids that
High Grade 11 (2.1) has just been included in the vitamin K-dependent protein family
Status of clinic genetic abnormalities [29]. A total of 34 different mutations in the GAS6 gene were
PTEN mutations 337 (65.2)
identified in the UCEC cohort. There were 3 frame-shift, 2 splice, 1
PIK3CA mutations 259 (50.1)
ARID1A mutations 227 (43.9)
nonsense, and 28 missense mutations discovered. When the
TTN mutations 206 (39.8) structure of GAS6 is examined, it is found to consist of 3 distinct
TP53 mutations 198 (37.3) regions: region A (amino-terminal end), region B (neck), and re-
Status of target genetic abnormalities gion C (carboxy-terminal end). Region B contains 4 epidermal
MerTK mutations 6
growth factor like domains. The p. X197_splice mutation has been
AXL mutations 10
TYRO3 mutations 5 identified in this region and has the property of causing inade-
PROS1 mutations 9 quate function and truncated transcript production. The nonsense
CD47 mutations 4 mutation p. R308* was discovered in the G-like 1 domain of
GAS6 mutations 8
laminin. The mutation p. Q551 ¼ in the splicing region is located
G: Grade; CN, copy number; MSI, microsatellite instability. in the LG2 domain. The GAS6; MerTK and GAS6; AXL and GAS6;
POLE: polymerase ε CD47 co-mutation was found to be statistically significant
313
 Table-2

Dilara Fatma Akin and D. Ozkan

Characteristics of mutations detected in MerTK, AXL, TYRO3, CD47, GAS6 and PROS1 in TCGA UCEC cohort.

No Gene Nucleotide Accession Number Alteration type Localization AA position Clinical significance
alteration
Poly-Phen2 (score) SIFFT (score) COSMIC/FATHMM
prediction

M-1 MERTK c.718G > A rs868703580/COSV54927603 Missense Exon-4 E240K Benign (0.10) Tolerated (1.00) Neutral (0.05)
M-2 MERTK c.1390G > A rs148833587/COSV54925754 Missense Exon-9 V464I Benign (0.10) Tolerated (0.72) Neutral (score 0.01)
M-3 MERTK c.232G > A rs764637670/COSV54926788 Missense Exon-2 V78I Benign (0.01) Tolerated (0.75) Neutral (score 0.02)

€
M-4 MERTK c.1449C > T rs540628681 Splice Exon-9 H483 ¼ e e e
M-5 MERTK c.79G > A COSV54925176 Missense Exon-2 E27K Benign (0.00) Tolerated (0.08) Neutral (score 0.05)
M-6 MERTK c.2060G > A rs775443181/COSV54929415 Missense Exon-15 R687Q Probably Deleterious (0.02) Pathogenic (score 0.99)
Damaging (0.99)
M-7 MERTK c.207G > T rs200120223/COSV54935093 Missense Exon-2 Q69H Benign (0.00) Tolerated (0.21) Neutral (score 0.02)
M-8 MERTK c.386C > A COSV54926114 Missense Exon-2 S129Y Probably Tolerated (0.66) Pathogenic (score 0.82)
Damaging (0.96)
M-9 MERTK c.2779G > A COSV54932626 Missense Exon-19 G927R Benign (0.03) Deleterious (0.05) Pathogenic (score 0.84)
M-10 MERTK c.1067G > T COSV54924890 Missense Exon-7 S356I Probably Deleterious (0.02) Neutral (score 0.27)
Damaging (0.95)
M-11 MERTK c.1187A > C COSV54925647 Missense Exon-8 E396A Benign (0.00) Tolerated (0.33) Neutral (score 0.33)
M-12 MERTK c.1656G > T COSV54925653 Missense Exon-11 K552N Probably Deleterious (0.00) Pathogenic (score 0.88)
Damaging (0.91)
M13 MERTK c.344G > A e Missense Exon-2 C115Y Probably Deleterious (0.00) NA
Damaging (1.00)
M-14 MERTK c.2394G > A e Missense Exon-18 M798I Probably Deleterious (0.00) NA
Damaging (1.00)
M-15 MERTK c.2194C > T rs890258715 Nonsense Exon-17 R732* e e e
M-16 MERTK c.2179C > T e Nonsense Exon-16 R727* e e e
M-17 MERTK c.2429A > G rs746238212 Missense Exon-18 Y810C Probably Deleterious (0.00) NA
Damaging (1.00)
314

M-18 MERTK c.937C > T e Missense Exon-6 P313S Possibly Deleterious (0.05) NA
Damaging (0.62)
M-19 MERTK c.1481C > T rs760743544/COSV54935252 Missense Exon-10 P494L Probably Deleterious (0.02) Pathogenic (score 0.75)
Damaging (0.93)
M-20 MERTK c.1866G > T e Missense Exon-13 K622N Probably Deleterious (0.00) NA
Damaging (1.00)
M-21 MERTK c.326A > C COSV54933423 Missense Exon-2 K109T Benign (0.05) Tolerated (0.15) Neutral (score 0.29)
M-22 MERTK c.2440C > A COSV54931474 Missense Exon-18 L814I Benign (0.08) Tolerated (0.18) Neutral (score 0.26)
M-23 MERTK c.2602G > T e Missense Exon-19 A868S Benign (0.04) Tolerated (0.18) NA

Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

M-24 MERTK c.1863G > T e Missense Exon-13 M621I Possibly Deleterious (0.00) NA
Damaging (0.89)
M-25 MERTK c.2308G > T e Nonsense Exon-17 E770* e e e
M-26 MERTK c.877C > T rs369655379 Missense Exon-6 R293C Possibly Tolerated (0.18) NA
Damaging (0.59)
M-27 MERTK c.472G > A rs780214104 Missense Exon-2 A158T Possibly Deleterious (0.14) NA
Damaging (0.64)
M-28 MERTK c.171_177del e Frame shift Exon-2 G58Sfs*4 e e e
M-29 MERTK c.2384C > T rs1326116803 Missense Exon-18 T795M Probably Deleterious (0.00) NA
Damaging (1.00)
M-30 MERTK c.1896G > T e Missense Exon-14 E632D Benign (0.40) Deleterious (0.01) NA
M-31 MERTK c.1775T > A COSV54926800 Missense Exon-12 I592N Possibly Deleterious (0.00) Pathogenic (score 0.97)
Damaging (0.86)
M-32 MERTK c.845-1G > A e Splice Exon-14 X282_splice e e e
M-33 MERTK c.2104A > G e Missense Exon-16 K702E Possibly Deleterious (0.00) NA
Damaging (0.74)
M-34 MERTK c.1436T > G rs145142422 Missense Exon-9 F479C Benign (0.32) Deleterious (0.01) NA
M-35 MERTK c.1465G > T rs140650643 Missense Exon-10 A489S Benign (0.03) Tolerated (0.24) NA
M-36 MERTK c.7C > T rs952265047 Missense Exon-1 P3S Benign (0.04) Tolerated (0.04) e
M-37 AXL c.874dup COSV56563342 Frame shift Exon-7 H292Pfs*47 e e e
M-38 AXL c.449T > C rs200505600 Missense Exon-4 V150A Deleterious (0.00) NA
 Dilara Fatma Akin and D. Ozkan
Probably
Damaging (1.00)
M-39 AXL c.1030C > T rs148886744 Missense Exon-8 R344W Probably Tolerated (0.11) NA
Damaging (0.96)
M-40 AXL c.211C > T rs778883675/COSV56573369 Missense Exon-2 R71W Probably Deleterious (0.03) Pathogenic (score 0.79)
Damaging (0.96)
M-41 AXL c.502C > T rs778883675/COSV56571617 Missense Exon-4 P168S Probably Deleterious (0.00) Pathogenic (score 0.91)
Damaging (1.00)
M-42 AXL c.854C > T rs201003955/COSV56565179 Missense Exon-7 S285L Benign (0.00) Tolerated (0.72) Neutral (score 0.04)

€
M-43 AXL c.2287C > T rs746364612 Missense Exon-19 R763C Benign (0.08) Deleterious (0.02) NA
M-44 AXL c.1375G > A rs138698106 Missense Exon-11 V459M Possibly Tolerated (0.06) NA
Damaging (0.85)
M-45 AXL c.1317G > T e Missense Exon-11 K439N Benign (0.00) Tolerated (0.72) NA
M-46 AXL c.1519C > T rs1366012932 Missense Exon-12 R507W Probably Deleterious (0.03) NA
Damaging (0.99)
M-47 AXL c.1956C > A e Missense Exon-17 F652L Probably Deleterious (0.00) NA
Damaging (1.00)
M-48 AXL c.266G > A COSV56574078 Missense Exon-2 G89D Benign (0.23) Deleterious (0.30) NA
M-49 AXL c.1501C > T rs771047905 Missense Exon-12 R501C Probably Deleterious (0.00) NA
Damaging (0.98)
M-50 AXL c.1135- e Splice e X379_splice e e e
M-51 AXL c.2176T > C e Missense Exon-18 Y726H Probably Deleterious (0.00) NA
Damaging (0.99)
M-52 AXL c.1145A > G COSV56563604 Missense Exon-9 D382G Probably Deleterious (0.01) Pathogenic (score 0.96)
Damaging (0.94)
M-53 AXL c.2274T > G e Missense Exon-19 I758M Probably Tolerated (0.26) NA
Damaging (0.99)
M-54 AXL c.142C > T rs774132867/COSV56563807 Missense Exon-2 R48W Probably Deleterious (0.02) NA
Damaging (0.98)
M-55 AXL c.2398C > T rs1036430672 Missense Exon-20 R800W Probably Deleterious (0.00) NA
315

Damaging (0.99)
M-56 AXL c.2300G > A rs200271277 Missense Exon-19 R767H Probably Deleterious (0.00) NA
Damaging (0.91)
M-57 AXL c.637del e Frame shift Exon-5 V213Sfs*37 e e e
M-58 AXL c.1428G > T e Missense Exon-11 K476N Possibly Tolerated (0.44) NA
Damaging (0.88)
M-59 AXL c.602C > T e Missense Exon-5 S201F Probably Deleterious (0.00) NA
Damaging (0.96)
M-60 AXL c.925G > A rs769316836 Missense Exon-7 V309M Probably Deleterious (0.00) NA

Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

Damaging (0.96)
M-61 AXL c.68C > T rs754135882 Missense Exon-1 A23V Benign (0.00) Tolerated (0.27) NA
M-62 AXL c.145G > T Nonsense Exon-2 G49* e e e
M-63 AXL c.140C > T rs1260076571 Missense Exon-2 A47V Benign (0.30) Deleterious (0.00) NA
M-64 AXL c.1338C > A e Missense Exon-11 F446L Benign (0.06) Tolerated (0.51) NA
M-65 AXL c.1526C > T e Missense Exon-12 T509I Possibly Tolerated (1.00) NA
Damaging (0.51)
M-66 AXL c.1789G > A rs537492526 Missense Exon-15 V597I Benign (0.09) Deleterious (0.03) NA
M-67 AXL c.2323C > A e Missense Exon-19 L775M Probably Deleterious (0.00) NA
Damaging (0.99)
M-68 AXL c.1381G > T rs781747516 Missense Exon-11 A461S Possibly Tolerated (0.41) NA
Damaging (0.78)
M-69 AXL c.80C > A e Missense Exon-1 P27H Benign (0.00) Tolerated (0.26) NA
M-70 AXL c.388C > T e Missense Exon-3 P130S Possibly Deleterious (0.01) NA
Damaging (0.62)
M-71 AXL c.352T > G COSV56568910 Missense Exon-3 L118V Benign (0.00) Tolerated (0.01) Neutral (score 0.02)
M-72 AXL c.2639C> e Missense Exon-20 A880V Benign (0.10) Tolerated (0.07) NA
M-73 AXL c.1176G > C e Missense Exon-9 E392D Benign (0.14) Tolerated (0.12) NA
M-74 AXL c.424C > A e Missense Exon-4 L142M Possibly Tolerated (0.12) NA
Damaging (0.51)
(continued on next page)
 Table-2 (continued )

Dilara Fatma Akin and D. Ozkan

No Gene Nucleotide Accession Number Alteration type Localization AA position Clinical significance
alteration
Poly-Phen2 (score) SIFFT (score) COSMIC/FATHMM
prediction

M-75 AXL c.1778A > G e Missense Exon-15 D593G Benign (0.00) Deleterious (0.04) NA
M-76 AXL c.685C > T rs140448864 Missense Exon-6 R229C Benign (0.19) Tolerated (0.04) NA
M-77 AXL c.1108G > A e Missense Exon-8 A370T Possibly Tolerated (0.44) NA
Damaging (0.51)

€
M-78 AXL c.1325C > A e Nonsense S442* e e e
M-79 AXL c.509A > G e Missense Exon-4 D170G Benign (0.07) Tolerated (0.24) NA
M-80 AXL c.1862C > A e Missense Exon-6 P621H Probably Deleterious (0.00) NA
Damaging (0.99)
M-81 AXL c.2111G > A rs748730668 Missense Exon-18 R704H Probably Deleterious (0.00) NA
Damaging (1.00)
M-82 AXL c.2280C > A e Missense Exon-19 D760E Possibly Tolerated (0.60) NA
Damaging (0.80)
M-83 AXL c.94G > A e Missense Exon-2 A32T Benign (0.10) Tolerated (0.33) NA
M-84 AXL c.952T > A e Missense Exon-7 S318T Probably Deleterious (0.00) NA
Damaging (1.00)
M-85 AXL c.766A > G rs749047403 Missense Exon-6 T256A Benign (0.01) Tolerated (0.35) NA
M-86 AXL c.1798C > T e Missense Exon-15 L600F Probably Deleterious (0.00) NA
Damaging (0.94)
M-87 AXL c.208C > A e Missense Exon-2 L70I Probably Tolerated (0.17) NA
Damaging (1.00)
M-88 AXL :c.2039T > C e Missense Exon-18 L680P Probably Deleterious (0.00) NA
Damaging (1.00)
M-89 AXL c.760C > T e Missense Exon-6 P254S Possibly Tolerated (0.09) NA
Damaging (0.88)
M-90 TYRO3 c.1355G > A rs760003502 Missense Exon-10 R452Q Benign (0.02) Deleterious (0.05) NA
316

M-91 TYRO3 c.386A > G e Missense Exon-3 Q129R Benign (0.18) Deleterious (0.04) NA
M-92 TYRO3 c.2277G > T rs1323346870 Missense Exon-18 E759D Benign (0.10) Tolerated (1.00) NA
M-93 TYRO3 c.1783C > T rs148583498 Missense Exon-15 R595C Benign (0.21) Tolerated (0.10) NA
M-94 TYRO3 c.1363C > T rs768075649 Missense Exon-10 R455W Possibly Deleterious (0.00) NA
Damaging (0.80)
M-95 TYRO3 c.2155G > A rs748781112 Missense Exon-18 G719R Probably Deleterious (0.00) NA
Damaging (1.00)
M-96 TYRO3 c.1354C > T rs774745326 Nonsense Exon-10 R452* e e e
M-97 TYRO3 c.1905C > A e Missense Exon-16 F635L Possibly Deleterious (0.00) NA

Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

Damaging (0.47)
M-98 TYRO3 c.644G > A rs1340865770 Missense Exon-5 R215H Probably Deleterious (0.00) NA
Damaging (0.99)
M-99 TYRO3 c.1219C > T Nonsense Exon-9 Q407* e e e
M-100 TYRO3 c.1784G > A rs1278003650 Missense Exon-15 R595H Probably Tolerated (0.05) NA
Damaging (0.98)
M-101 TYRO3 c.1618G > T Nonsense Exon-13 E540* e e e
M-102 TYRO3 c.1690G > T rs1158812678 Nonsense Exon-14 E564* e e e
M-103 TYRO3 c.2249G > A e Missense Exon-18 R750H Probably Deleterious (0.00) NA
Damaging (1.00)
M-104 TYRO3 c.2050G > T e Missense Exon-17 D684Y Probably Deleterious (0.00) NA
Damaging (1.00)
M-105 TYRO3 c.2283G > T e Splice Exon-19 V761 ¼ e e e
M-106 TYRO3 c.1582G > T rs1436858116 Nonsense Exon-13 E528* e e e
M-107 TYRO3 c.749A > G e Missense Exon-6 D250G Possibly Tolerated (0.40) NA
Damaging (0.47)
M-108 TYRO3 c.1849G > A rs769504685 Missense Exon-15 A617T Benign (0.11) Tolerated (0.22) NA
M-109 TYRO3 c.2325G > T e Missense Exon-19 Q775H Possibly Tolerated (0.34) NA
Damaging (0.76)
M-110 TYRO3 c.2182C > T rs148896634 Missense Exon-18 R728C Probably Deleterious (0.00) NA
Damaging (1.00)
 Dilara Fatma Akin and D. Ozkan
M-111 TYRO3 c.1781G > A e Missense Exon-15 G594D Probably Deleterious (0.01) NA
Damaging (0.97)
M-112 TYRO3 c.1597C > T rs909036764 Missense Exon-13 R533W Probably Deleterious (0.00) NA
Damaging (1.00)
M-113 TYRO3 c.1581A > G Splice Exon-13 G527 ¼ e e e
M-114 TYRO3 c.884G > A rs1446651175 Missense Exon-7 S295N Possibly Tolerated (0.21) NA
Damaging (0.89)
M-115 CD47 RNF183-CD47 e Fusion e e e e e
M-116 CD47 RASGRP1-CD47 e Fusion e e e e e

€
M-117 CD47 SPON1-CD47 e Fusion e e e e e
M-118 CD47 c.459A > G e Missense Exon-3 I153M Possibly Deleterious (0.00) NA
Damaging (0.66)
M-119 CD47 c.692C > T rs1176172930 Missense Exon-6 A231V Benign (0.12) Tolerated (0.78) NA
M-120 CD47 c.785C > T rs754405285/COSV62528392 Missense Exon-7 A262V Benign (0.01) Tolerated (0.90) Neutral (score 0.46)
M-121 CD47 c.647C > A COSV62527071 Missense Exon-5 S216Y Possibly Deleterious (0.00) Pathogenic (score 0.92)
Damaging (0.66)
M-122 CD47 c.632G > A COSV62526727 Missense Exon-5 G211D Probably Deleterious (0.00) Pathogenic (score 0.83)
Damaging (0.92)
M-123 CD47 c.379A > G rs867685532/COSV62528783 Missense Exon-2 I127V Benign (0.00) Tolerated (1.00) Neutral (score 0.01)
M-124 CD47 c.900A > C COSV62526904 Missense Exon-8 Q300H Probably Tolerated (0.21) NA
Damaging (0.94)
M-125 CD47 c.160G> COSV62526906 Missense Exon-2 V54I Benign (0.01) Tolerated (0.08) Neutral (score 0.01)
M-126 CD47 c.735G > C COSV62526662 Missense Exon-6 Q245H Probably Tolerated (0.06) NA
Damaging (1.00)
M-127 CD47 c.969A > C e Missense Exon-11 E323D Benign (0.23) Deleterious (0.01) NA
M-128 CD47 c.141G > T e Missense Exon-2 E47D Benign (0.34) Tolerated (0.07) NA
M-129 CD47 c.607T > G e Missense Exon-5 S203A Benign (0.07) Tolerated (0.10) NA
M-13 CD47 c.272T > C e Missense Exon-2 L91S Probably Deleterious (0.00) NA
Damaging (1.00)
M-131 CD47 c.533C > A e Missense Exon-4 A178D Benign (0.01) Tolerated (0.20) NA
317

M-132 CD47 c.799C > T e Missense Exon-7 H267Y Benign (0.02) Tolerated (0.11) NA
M-133 GAS6 c.2016del e Frame shift Exon-15 V673Wfs*85 e e NA
M-134 GAS6 c.1421C > T e Missense Exon-12 T474M Benign (0.01) Deleterious (0.04) NA
M-135 GAS6 c.449del e Frame shift Exon-5 G150Afs*49 e e NA
M-136 GAS6 c.1261C > A e Missense Exon-11 L421M Probably Deleterious (0.01) NA
Damaging (0.99)
M-137 GAS6 c.1925G > A e Missense Exon-15 G642D Probably Deleterious (0.00) NA
Damaging (1.00)
M-138 GAS6 c.2016dup e Missense Exon-15 V673Rfs*37 e e NA

Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

M-139 GAS6 c.486G > T e Missense Exon-6 Q162H Benign (0.02) Tolerated (0.14) NA
M-140 GAS6 c.212G > T e Missense Exon-2 S71I Benign (0.44) Deleterious (0.00) NA
M-141 GAS6 c.1990G > A e Missense Exon-15 D664N Probably Deleterious (0.02) NA
Damaging (1.00)
M-142 GAS6 c.422T > G e Missense Exon-2 F141C Possibly Tolerated (0.17) NA
Damaging (0.63)
M-143 GAS6 c.1531G > T e Missense Exon-13 A511S Possibly Tolerated (0.11) NA
Damaging (0.80)
M-144 GAS6 c.629C > T e Missense Exon-7 A210V Possibly Deleterious (0.01) NA
Damaging (0.87)
M-145 GAS6 c.497G > A e Missense Exon-6 G166D Probably Deleterious (0.03) NA
Damaging (0.92)
M-146 GAS6 c.922C > T e Nonsense Exon-9 R308* e e NA
M-147 GAS6 c.993G > T e Missense Exon-1 E331D Probably Deleterious (0.00) NA
Damaging (0.97)
M-148 GAS6 c.590-3C > T e Splice Mutation X197_splice e e NA
M-149 GAS6 c.1653G > A e Splice Mutation Q551 ¼ e e NA
M-150 GAS6 c.893G > A e Missense Exon-13 G298D Probably Deleterious (0.00) NA
Damaging (1.00)
M-151 GAS6 c.1818G > T e Missense Exon-9 E606D Benign (0.00) Tolerated (0.23) NA
(continued on next page)
 Table-2 (continued )

Dilara Fatma Akin and D. Ozkan

No Gene Nucleotide Accession Number Alteration type Localization AA position Clinical significance
alteration
Poly-Phen2 (score) SIFFT (score) COSMIC/FATHMM
prediction

M-152 GAS6 c.1638G > T e Missense Exon-14 K546N Possibly Tolerated (0.05) NA
Damaging (0.74)
M-153 GAS6 c.902T > C e Missense Exon-9 F301S Possibly Deleterious (0.02) NA
Damaging (0.53)

€
M-154 GAS6 c.412G > T e Missense Exon-5 G138C Probably Deleterious (0.00) NA
Damaging (0.97)
M-155 GAS6 c.1667C > T e Missense Exon14 A556V Possibly Tolerated (0.17) NA
Damaging (0.84)
M-156 GAS6 c.1163C > G e Missense Exon-11 A388G Benign (0.00) Tolerated (0.17) NA
M-157 GAS6 c.1735G > A e Missense Exon-14 V579I Benign (0.00) Tolerated (0.44) NA
M-158 GAS6 c.1622A > T e Missense Exon-13 D541V Possibly Deleterious (0.07) NA
Damaging (0.84)
M-159 GAS6 c.1804G > T e Missense Exon-14 A602S Benign (0.00) Tolerated (0.63) NA
M-160 GAS6 c.782A > G e Missense Exon-8 H261R Probably Deleterious (0.02) NA
Damaging (0.92)
M-161 GAS6 c.1714G > A e Missense Exon-14 G572S Benign (0.05) Tolerated (0.61) NA
M-162 GAS6 c.1592G > A e Missense Exon-13 R531H Benign (0.00) Tolerated (0.49) NA
M-163 GAS6 c.1466G > A e Missense Exon-12 S489N Benign (0.00) Tolerated (0.93) NA
M-164 GAS6 c.793C > T e Missense Exon-8 R265C Probably Deleterious (0.00) NA
Damaging (1.00)
M-165 GAS6 c.1481G > A e Missense Exon-13 R494Q Benign (0.01) Tolerated (0.52) NA
M-166 GAS6 c.1249T > C e Missense Exon-11 Y417H Benign (0.05) Tolerated (0.34) NA
M-167 PROS1 c.592G > T COSV62398338 Missense Exon-6 D198Y Possibly Deleterious (0.01) Pathogenic (score 0.86)
Damaging (0.91)
M-168 PROS1 c.719C > A COSV62397516 Missense Exon-7 S240Y e e Neutral (score 0.06)
318

M-169 PROS1 c.487G > A COSV62397576 Missense Exon-6 D163N Benign (0.02) Tolerated (0.17) Pathogenic (score 0.73)
M-170 PROS1 c.1682G > A COSV67776000 Missense Exon-14 R561Q Benign (0.03) Tolerated (0.22) Neutral (score 0.05)
M-171 PROS1 c.1220T > C COSV67776063 Missense Exon-11 I407T Probably Deleterious (0.00) Pathogenic (score 0.98)
Damaging (1.00)
M-172 PROS1 c.688G > A COSV62398184 Missense Exon-7 E230K e e Neutral (score 0.13)
M-173 PROS1 c.733G > T COSV67777321 Missense Exon-8 D245Y Probably Deleterious (0.00) Pathogenic (score 0.97)
Damaging (1.00)
M-174 PROS1 c.373C > T COSV62399770 Missense Exon-5 P125S Probably Deleterious (0.00) Pathogenic (score 0.96)
Damaging (0.99)

Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

M-175 PROS1 c.1493-1G > T Splice Region X498_splice e e
M-176 PROS1 c.892G > T COSV67776276 Nonsense Exon-9 E298* e e Pathogenic (score 0.89)
M-177 PROS1 c.178G > A e Missense E60K Probably Deleterious (0.00)
Damaging (0.99)
M-178 PROS1 c.895T > A e Missense Exon-9 L299I Possibly Tolerated (0.34) e
Damaging (0.54)
M-179 PROS1 c.272C > A COSV62398346 Missense Exon-4 S91Y Possibly Deleterious (0.04) Pathogenic (score 0.71)
Damaging (0.78)
M-180 PROS1 c.1498G > A COSV67777614 Missense Exon-13 V500I Benign (0.00) Tolerated (0.49) Neutral (score 0.01)
M-181 PROS1 c.1592A > G COSV67777614 Missense Exon-13 N531S Benign (0.04) Tolerated (0.42) Neutral (score 0.01)
M-182 PROS1 c.1327A > G COSV67775748 Missense Exon-12 N443D Probably Deleterious (0.00) Pathogenic (score 0.97)
Damaging (1.00)
M-183 PROS1 c.602-1G > T e Splice Region X201_splice e e
M-184 PROS1 c.361T > C COSV62398790 Missense Exon-5 C121R Probably Deleterious (0.00) Pathogenic (score 0.96)
Damaging (1.00)
M-185 PROS1 c.908C > T COSV67776359 Missense Exon-9 A303V Possibly Deleterious (0.00) Pathogenic (score 0.93)
Damaging (0.61)
M-186 PROS1 c.1480C > A COSV67774724 Missense Exon-12 H494N Benign (0.00) Tolerated (1.00) Neutral (score 0.02)
M-187 PROS1 c.97dup COSV62398363 FS ins Exon-2 S33Ffs*6 e e e
M-188 PROS1 c.850G > A COSV67774343 Missense Exon-9 V284I Benign (0.03) Tolerated (1.00) Neutral (score 0.15)
M-189 PROS1 c.810G > T COSV67774118 Missense Exon-8 K270N Deleterious (0.00) Neutral (score 0.24)
 Dilara Fatma Akin and D. Ozkan
Possibly
Damaging (0.52)
M-190 PROS1 c.454G > T COSV62397587 Nonsense Exon-5 E152* e e Neutral (score 0.28)
M-191 PROS1 c.1371G > T COSV67774347 Missense Exon-12 K457N Benign (0.00) Tolerated (1.00) Neutral (score 0.43)
M-192 PROS1 c.1352G > A COSV67774613 Missense Exon-12 R451Q Probably Deleterious (0.00) Pathogenic (score 0.97)
Damaging (1.00)
M-193 PROS1 c.508G > T COSV62398186 Missense Exon-6 G170C Probably Deleterious (0.00) Pathogenic (score 0.90)
Damaging (1.00)
M-194 PROS1 c.1229C > A e Missense Exon-11 P410H Possibly Deleterious (0.01) e

€
Damaging (0.57)
M-195 PROS1 c.1168G > T e Nonsense Exon-11 E390* e e e
M-196 PROS1 c.1822G > A e Missense Exon-14 D608N Benign (0.26) Tolerated (0.28) e
M-197 PROS1 c.1795G > T e Missense Exon-14 D599Y Probably Deleterious (0.00) e
Damaging (0.95)
M-198 PROS1 c.1033G > A Missense Exon-10 D345N Benign (0.28) Tolerated (0.31) e
M-199 PROS1 c.1708G > A e Missense Exon-14 D570N Benign (0.01) Tolerated (0.42) e
M-200 PROS1 c.181G > T e Missense Exon-14 E61* e e e
M-201 PROS1 c.1255G > T e Missense Exon-11 G419* e e e
M-202 PROS1 c.2022G > T e Missense Exon-15 K674N Benign (0.00) Tolerated (0.20) e
M-203 PROS1 c.1951C > A e Missense Exon-15 L651M Probably Deleterious (0.00) e
Damaging (1.00)
M-204 PROS1 c.1502C > T e Missense Exon-13 S501F Benign (0.37) Deleterious (0.04) e
M-205 PROS1 c.1295G > A COSV67774805 Missense Exon-11 R432Q Benign (0.03) Tolerated (0.62) e
M-206 PROS1 c.850-1G > A e Missense e X284_splice e e e
M-207 PROS1 c.1562G > A e Missense Exon-13 G521D Probably Deleterious (0.00) e
Damaging (0.96)
M-208 PROS1 c.497A > G e Missense Exon-6 N166S Benign (0.09) Tolerated (0.59) e
M-209 PROS1 c.1921G > T e Nonsense Exon-15 E641* e e e
M-210 PROS1 c.299C > T COSV62399923 Missense Exon-4 A100V Benign (0.02) Tolerated (0.90) Neutral (score 0.07)
M-211 PROS1 c.259G > T COSV62398453 Missense Exon-3 V87F Possibly Deleterious (0.03) Pathogenic (score 0.78)
319

Damaging (0.57)
M-212 PROS1 c.458del e FS del e K153Sfs*6 e e e
M-213 PROS1 c.76þ2T > C e Splice Region e X26_splice e e e
M-214 PROS1 c.832G > A COSV67777402 Missense Exon-8 D278N Possibly Deleterious (0.06) Pathogenic (score 0.95)
Damaging (0.86)
M-215 PROS1 c.1336C > G e Missense Exon-12 L446V Probably Deleterious (0.02) e
Damaging (0.99)
M-216 PROS1 c.1597G > A e Missense Exon-13 V533M Possibly Deleterious (0.02) e
Damaging (0.82)

Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

M-21 PROS1 c.1525A > C e Missense Exon-13 N509H Possibly Tolerated (0.01) e
Damaging (0.82)
M-218 PROS1 c.1024G > A e Missense Exon-10 E342K Probably Tolerated (0.15) e
Damaging (1.00)
M-219 PROS1 c.557G > A COSV62398792 Missense Exon-6 C186Y Probably Deleterious (0.02) Pathogenic (score 0.94)
Damaging (0.99)
M-220 PROS1 c.1012A > T e Missense Exon-10 I338L Benign (0.02) Tolerated (0.57) e
M-221 PROS1 c.1385G > T e Missense Exon-12 G462V Probably Deleterious (0.01) e
Damaging (0.94)
M-222 PROS1 c.478G > T e Nonsense Exon-6 E160* e e e
M-223 PROS1 c.169G > T e Nonsense Exon-2 E57* e e e
M-224 PROS1 c.779C > A e Missense Exon-8 P260H Probably Deleterious (0.01) e
Damaging (0.94)
M-225 PROS1 c.966-1G > T e Missense e X322_splice e e e
M-226 PROS1 c.1864C > A e Missense Exon-14 L622I Benign (0.01) Tolerated (0.41) e
M-227 PROS1 c.1828G > A e Missense Exon-14 A610T Benign (0.17) Tolerated (0.47) e
M-228 PROS1 c.79T > C e Splice Region Exon-2 L27 ¼ e e e
M-229 PROS1 c.1365G > C e Missense Exon-12 L455F Possibly Tolerated (0.01) e
Damaging (0.80)

M: Mutation; N/A: Not Applicable; AA: amino acid.

 €
Dilara Fatma Akin and D. Ozkan Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

Fig. 1. TAM receptors and their ligands. The TAM receptors are TYRO3; AXL and MerTK. The TAM ligands are Gas6 and Protein S (ProS).

Fig. 2. (A) Distribution of mutations in MerTK, AXL, TYRO3, CD47, GAS6, and PROS1 genes in TCGA UCEC cohort from cBioPortal. Percentages of overall mutations for each gene are
given on the left (B) Schematic representation of domain architecture of the MerTK, AXL, TYRO3, CD47, GAS6, and PROS1 proteins and mutations detected in TCGA UCEC cohort.
Human MerTK is a polypeptide comprising 999 amino acids. Human AXL is a polypeptide comprising 894 amino acids. Human TYRO3 is a polypeptide comprising 890 amino acids.
Human CD47 is a polypeptide comprising 323 amino acids. Human PROS1 is a polypeptide comprising 676 amino acids. Human GAS6 is a polypeptide comprising 678 amino acids.

320
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Dilara Fatma Akin and D. Ozkan
Fig. 3. (A) Comparative analysis of m-RNA expression values of mutant and wild type individuals in MerTK, AXL, TYRO3, CD47, GAS6, and PROS1genes (p < 0.05) (B) Comparison of
KaplaneMeier survival curves of the high and low expressions of MerTK, AXL, TYRO3, CD47, GAS6, and PROS1 in TCGA UCEC cohort (p < 0.05). Red line indicates the high
expressions of m-RNA; Green line indicates the low expressions of m-RNA.
(p-value 0.003, 0.004 and 0.02, respectively). The frequency of
somatic mutations detected in this gene in UCEC cohort was 8%.
PROS1 analysis
In this study, a total of 65 mutations were detected in the
PROS1. There were 55 missense mutations, 9 nonsense mutations,
5 splice regions, and 2 frame-shift mutations. The frequency of
somatic mutations detected in the gene in the UCEC cohort was
9%. PROS1 has 3 major domains: Gla domain, EGF-like calcium-
binding domain, SHBG domain. The nonsense mutations p. E57*
and p. E61* and the frame-shift mutation p. S33Ffs*6 may have
effects that alter the termination code and reading frame at the
very early stage of the 676 a. a long product. The p. X201_splice
mutation on the EGF-Like calcium binding domain and the p.
X322_splice mutation on the laminin G1 region in the SHBG
domain are located in the bases of the splicing region, which is
100% conserved between species in the evolutionary process.
These mutations lead to the formation of non-functional short
transcripts. It can lead to differences in m-RNA expression. PROS1;
AXL, PROS1; MerTK1, PROS1; TYRO3, PROS1; CD47, PROS1; All
associations with GAS6 mutations were found to be statistically
significant (p < 0.001).
Results of functional impact analysis of detected mutations
According to the results of the pathogenicity prediction analysis
of the Poly-Phen2 and SIFT Database programs, of the 191 missense
mutations detected in present study, 81 missense mutations were
classified as pathogenic in both programs. Because the pathogenic
scores of these mutations are close to 1 and are “affected,” they
were determined to be potentially pathogenic and predicted to
have disease-causing properties. The pathogenic mutations are
listed in detail in Table-2. Furthermore, 22 mutations are classified
as pathogenic in 3 different databases.
321
Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324
Gene expression profile and survival analysis results
Our analysis of the gene expression profile of MerTK, AXL, TYRO3,
CD47, GAS6 and PROS1 in UCEC cohort (n:174) and 91 healthy tissues
revealed that the expression levels of MerTK, AXL, GAS6 and PROS1
were statistically significantly lower in the patient group (p < 0.01)
(Fig.-3A). Among the target genes, only the expression level of CD47
was higher in the patient group than in the healthy group. Survival
analysis according to low and high gene expression profiles proved
not to be statistically significant in results (Fig. 3B). In analysis to
determine the effect of genetic mutations on m-RNA levels, patients
were divided into mutant and wild-type groups for the genes we
studied (MerTK, AXL, TYRO3, CD47, GAS6, and PROS1). When the
expression levels of these groups were compared, no statistically
significant difference was found between the groups (Fig.-4).
The profile of immune cell infiltration correlated with MERTK, AXL,
TYRO3, CD47, GAS6, and PROS1
When we analyzed the correlation between the m-RNA
expression level of target genes and the profile of immune infil-
tration in the UCEC cohort. The correlation between the expression
of target genes and immune infiltration in the UCEC cohort was
determined using data uploaded to the database TIMER. The
expression levels AXL and GAS6 were weakly correlated with
macrophage infiltration in UCEC cohort samples compared with
healthy samples (respectively R ¼ 0.283; R ¼ 0.217). On the other
hand, there was no correlation between PROS1 expression and
macrophage infiltration in the UCEC cohort (R ¼ 0.049). The
expression levels of MerTK, TYRO3, and CD47 were negatively
correlated with macrophage infiltration in the UCEC cohort
(respectively R ¼ 0.147, R ¼ 0.145, R ¼ 0.106) (Fig.-5).
Discussion
Endometrial carcinoma is viewed from different angles, but the
underlying pathogenic mechanisms are still relatively unknown,
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Dilara Fatma Akin and D. Ozkan Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324

Fig. 4. Comparative analysis of the tissue-specific differential expression MerTK, AXL, TYRO3, CD47, GAS6, and PROS1 genes in TCGA UCEC cohort using TIMER 2 program (p-value
Cutoff is 0.05; Log2 fold change).

and each study performed is important to identify the source of sequencing results of 509 patients diagnosed with UCEC in TCGA
new diagnostic, prognostic, and therapeutic targets. In present datasets. Furthermore to comprehensive mutation profiling study,
study, the mutational profiles of target genes MerTK, AXL, TYRO3, analyzes were performed using bioinformatics tools to determine
CD47, GAS6, and PROS1 were determined in detail in the genome the impact of detected mutations on m-RNA expression levels and

Fig. 5. Correlation between the MerTK, AXL, TYRO3, CD47, GAS6, and PROS1 expressions and immune infiltration (macrophage) in UCEC cohort using TIMER algorithm.

322
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Dilara Fatma Akin and D. Ozkan
survival. In the UCEC cohort consisting of 509 patients, a total of
229 mutations were detected in 7 genes (192 missense mutations,
17 nonsense mutations, 10 splice regions, 7 frame shift mutations, 3
fusion gene mutations) the most frequently mutated gene was AXL
and the least mutated gene was CD47. MerTK is a member of the
TAM receptor subfamily of receptor tyrosine kinases and plays a
role in phagocytic clearance of apoptotic cells. The catalytic domain
of tyrosine kinase, located in amino acid region 579e892 of MerTK,
contains various protein features such as ATP binding sites, proton
acceptor sites, phosphoserine and phosphotyrosine domains
[6,9,16,18,30]. Eleven missense mutations identified in this study in
this domain (p.I592N, p. K622N, p.M621I, p. E632D, p. R687Q, p.
K702E, p.M78I, p. T795M, p. Y810C, p. L814I, p. A868S) have path-
ogenic character and may alter their respective binding sites.
Furthermore, the frame shift alteration p. G58Sfs*4 in exon 2 and
the nonsense mutations p. R727*, p. R732*, p. E770* can cause the
formation of a stop codon in the early phase of the polypeptide. Its
oncogenic nature, arising from mutations in AXL, is manifested by
activation of signaling pathways related to proliferation, migration,
inhibition of apoptosis, and resistance to therapy [30]. The structure
of the AXL-GAS6 complex reveals that the first and second Ig-like
domains of AXL form a major and minor contact, respectively,
only with the first laminin G-like domain of GAS6. This binding
prevents any direct AXL/AXL OR GAS6/GAS6 contact [18,31]. In this
domain, LG1 and LG2 domains are formed by exons 2e5. In present
study, 6 (p.R71W, p. P168S, p. R48W, p. V213Sfs*37,p.G49*,p.P130S)
mutations were detected on these exons that we hypothesise can
alter the contact points. Again, the FNIII domain encoded by exon
6e9 provides the essential feature of AXL for its adhesion function.
The pathogenic mutations (p.H292Pfs*47, p. D382G, p. V309M, p.
S318T) may initiate transition to EMT by uncontrolled adhesion. It
was found that AXL can undergo an extracellular division event in
exon 11 near the transmembrane domain by protease, generating a
soluble fragment [27,31,32]. The nonsense mutation p. S442*
detected in this region in this study may cause the production of a
truncated protein that prevents the production of soluble AXL.
Because a type I transmembrane receptor, the enzymatic kinase
domain of AXL extends across exons 13e20 at its intracellular C-
terminus [31,32]. Our study group carries 9 missense mutations
(p.R507W, p. Y726H, p. R800W, p. R767H, p. R704H, p. L775M, p.
P621H, p. L680P, p. L600F) in this domain. We believe that muta-
tions causing this amino acid change may lead to dysfunctional
kinase activity.
TYRO3 mutations have been identified in cancer, but insufficient
functional studies have been performed to understand the poten-
tial significance of these mutations. TYRO3 consists of two
fibronectin-type domains III and two immunoglobulin like do-
mains in the extracellular part, a transmembrane part, and a kinase
domain in the cytoplasm [6,9,18,33]. The p. G527 ¼ and p. V761
mutations in the splicing region, especially in the kinase domain,
detected in this study may cause different transcripts.
The best studied TAM ligands, GAS6 and PROS1, form a bridge
between exposed phosphatidylserine externalized on the surface of
apoptotic cells and macrophage receptors TAM to trigger effer-
ocytosis [29,34]. GAS6/TAM disrupted ligandereceptor relationship
plays an important role in the development of many cancers,
including AML, ALL, schwannoma, glioma, thyroid cancer, ovarian
cancer, lung cancer, gastric cancer, prostate cancer, renal cell car-
cinoma, breast cancer, and melanoma [29,35]. The SHBG domain
consists of two globular laminin G-like regions (LG1 and LG2 re-
gion). Studies have reported that the SHBG domain must be present
for GAS6 to bind to its receptors [13,29,36]. In present study, a total
of 10 misense mutations in the SHBG domain were detected, 3 of
which we predicted to be pathogenic features (p.E331D, p. L421M,
and p. G642D). The p. G572S mutation discovered in the LG-2
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Taiwanese Journal of Obstetrics & Gynecology 62 (2023) 311e324
domain is located at the G572 phosphorylation site required for
binding to the receptors and is pathogenic in nature, which may
cause the phosphorylation site to lose its function. There is a
nonsense mutation p. R308* that can cause the truncated protein
we discovered in the LG1 region. This is located in the SHBG
domain, which is known to be the major binding region that
crosslinks with the immunoglobulin 1 and 2 regions of the GAS6
receptor [36]. Again, mutation of the splice region p.
Q551 ¼ prevents LG2 domain formation and may result in tran-
scripts with nonfunctional LG2 domains. It has been shown that the
EGF-like domain of GAS6 may play a role in stabilizing an active
conformation of GAS6 or modulating its activity [36,37]. In this
study, p. A210V and p. H261R were found to have 2 missense mu-
tations of pathogenic character in this domain, and it can be pre-
dicted that the protein to be formed may have possible
conformational deformations. The p. X197_splice mutation is
located on the splice site, and it may be that this mutation alters the
splice region if the protein has different transcript lengths.
CD47 generates a ‘do not eat me’ signal toward the immune
system that allows cells to escape the immune system [38]. This
signal is realized through the activation of a series of signaling
pathways triggered by binding to SIRPa [38,39]. Many studies have
found high expression of CD47 in most cancer cells. In this case, and
considering that CD47 generates the “do not eat me” signal, the idea
arose that tumor cells use this signal to escape the immune system.
CD47 is a transmembrane protein consisting of 5 transmembrane
domains, short intracytoplasmic C-terminal and N-terminal extra-
cellular immunoglobulin variants (19e127 a. a.) [28,38,39]. The p.
E47D, p. V54I, p. L91S, and p. I127V mutations detected in the UCEC
cohort in study are located in the domain encoding the extracel-
lular domain. This domain is a cell surface marker of CD47 that
binds SIRPa and inhibits phagocytosis by macrophages. Although
m-RNA expression level of CD47 was shown to be higher in tumor
tissue, a correlation to overall survival was not confirmed
throughout in UCEC cohort. Studies have reported that genetic al-
terations in the PROS1 have a negative effect on protein S function.
Protein S (PS) deficiency is most commonly caused by missense and
nonsense mutations, splice region mutations, insertions, and de-
letions. This region, known as the Gla domain, consists of modified
Gla residues at the N-terminus of the protein and has a high binding
affinity for membranes, such as in phosphatidylserine-bearing
activated platelets or apoptotic cells [13,40,41]. The nonsense mu-
tations p. E57* and p. E61*, which can cause the polypeptide to
terminate at a very early stage in this domain, were discovered in
present study group and are capable of forming a nonfunctional
truncated protein with no binding affinity. Because of the p.
X201_splice mutation in the splice acceptor region of exon 7, it is
highly likely that loss of the existing splice acceptor sequence re-
sults in non-functional transcripts and in-frame skipping of exon 7.
It has been reported that p. C121R, p. C186Y in the EGF domain that
we discovered in the UCEC cohort disrupted disulfide bond for-
mation and caused destabilization of PS. Similarly, APC cofactor
function was reported to be pathogenic by PS and decreased by 50%
in the presence of the p. D245Y mutation.
TYRO3, AXL, and MerTK have been reported to be ectopic or
overexpressed in numerous cancers, including leukemias, mela-
nomas, breast, lung, colon, liver, stomach, kidney, ovarian, and
brain cancers [18,32,33,35,37]. There is a few study in the literature
that states that AXL and GAS6 in particular are overexpressed in
endometrial cancer [42]. However, there is no study showing the
level of m-RNA expression and the functional effect of other TAM
receptors and ligands. The AXL receptor is overexpressed within
the tumor tissue of various cancers (i.e. breast cancer, renal cell
carcinoma, glioblastoma multiforme, ovarian cancer, pancreatic
cancer, and esophageal cancer), and high AXL expression was
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Dilara Fatma Akin and D. Ozkan
associated with a shorter overall survival and more advanced tu-
mor stage [27,30,31]. However, in this study, contrary to the liter-
ature, AXL gene expression was found to be low in EC. In present
study, it was found that the m-RNA expression of TAM receptors,
PROS1 and GAS6 ligands in the UCEC cohort with EC was lower than
that in the healthy group, while CD47 expression was statistically
significantly higher than that in the healthy group. Notably, CD47 is
overexpressed in various cancers and can bind directly to SIRPa,
which is mainly found in macrophages. The binding of CD47-SIRPa
sends a “do not eat me” signal that allows cancer cells to evade the
immune system [36,37,40]. Therefore, we believe that targeting the
phagocytosis checkpoint of CD47-SIRPa ax for therapeutic purposes
in EC may be an approach. Infiltration of macrophages in solid tu-
mors is associated with poor prognosis and is known to be related
to chemotherapy resistance in most cancers. It is known that the
pro-tumour effect of M2-like macrophages can be impaired by
blocking efferocytosis as a result of TAM receptor inhibition for
therapeutic purposes in other solid cancers. However, the results of
in-silico analysis showed that a limited or weak correlation was
observed between macrophage infiltration and the expression level
of TAM receptor and its ligands in EC.
Conclucion
We consider TAM receptors and ligands as important molecular
targets for the treatment of endometrial cancer. With this study, we
encourage researchers to validate the pathogenic mutations
described in this study and their effects in the genes encoding the
TAM receptor and their specific ligands in independent studies to
achieve the main goal of improved risk assessment at EC.
Funding
No funding was received.
Ethical approval and ethical standards
The data used in our study were obtained from public database
TCGA, therefore, ethical approval was not required.
Availability of data and materials
The datasets generated and analyzed during the present study
are available in TCGA database (https://www.cancer.gov/tcga), The
cbio cancer genomics portal (http://www.cbioportal.org/).
Declaration of competing interest
No potential conflict of interest was reported by the author(s).
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