Oncotarget, 2021, Vol. 12, (No. 3), pp: 145-159
www.oncotarget.com
Research Paper
High mRNA expression of LY6 gene family is associated with
overall survival outcome in pancreatic ductal adenocarcinoma
Eric Russ1, Krithika Bhuvaneshwar2, Guisong Wang3,4, Benjamin Jin1,4, Michele M.
Gage3,5, Subha Madhavan2, Yuriy Gusev2 and Geeta Upadhyay1,3
1
Department of Pathology, Uniformed Services University, Bethesda, MD, USA
2
Innovation Center for Biomedical Informatics, Georgetown University Medical Center, Washington DC, USA
3
Murtha Cancer Center/Research Program, Department of Surgery, Uniformed Services University of the Health Sciences,
Bethesda, MD, USA
4
The Henry M. Jackson Foundation for the Advancement of Military Medicine Inc, Bethesda, MD, USA
5
Walter Reed Navy Military Medical Center, Department of Surgery, Uniformed Services University, Bethesda, MD, USA
Correspondence to: Geeta Upadhyay, email: geeta.upadhyay@usuhs.edu
Keywords: LY6 genes; pancreatic cancer; immune cells; survival outcome
Received: November 21, 2020
Accepted: January 19, 2021
Published: February 02, 2021
Copyright: © 2021 Russ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.
ABSTRACT
Pancreatic cancer ranks one of the worst in overall survival outcome with a 5
year survival rate being less than 10%. Pancreatic cancer faces unique challenges
in its diagnosis and treatment, such as the lack of clinically validated biomarkers
and the immensely immunosuppressive tumor microenvironment. Recently, the LY6
gene family has received increasing attention for its multi-faceted roles in cancer
development, stem cell maintenance, immunomodulation, and association with more
aggressive and hard-to-treat cancers. A detailed study of mRNA expression of LY6
gene family and its association with overall survival (OS) outcome in pancreatic
cancers is lacking. We used publicly available clinical datasets to analyze the mRNA
expression of a set of LY6 genes and its effect on OS outcome in the context of the
tumor microenvironment and immunomodulation. We used web-based tools KaplanMeier Plotter, cBioPortal, Oncomine and R-programming to analyze copy number
alterations, mRNA expression and its association with OS outcome in pancreatic
cancer. These analyses demonstrated that high expression of LY6 genes is associated
with OS and disease free survival (DFS) outcome. High expression of LY6 genes
and their association with OS outcome is dependent on the composition of tumor
microenvironment. Considering that LY6 proteins are anchored to the outer cell
membrane or secreted, making them readily accessible, these findings highlight the
potential of LY6 family members in the future of pancreatic cancer diagnosis and
treatment.
for more than 90% of all pancreatic cancer diagnoses
[1, 2]. The poor prognosis of PDAC is due to several
challenges, especially the lack of manifestation screening
biomarkers, leading to most pancreatic cancer diagnoses
being made in the later, more aggressive stages.
New biomarkers and therapeutic targets of PDAC
are urgently needed. Currently, carbohydrate antigen (CA)
19-9 is the sole clinically approved serum biomarker for
pancreatic cancer [3]. CA-19-9 is only used for disease
monitoring due to a lack of specificity and sensitivity
INTRODUCTION
Pancreatic cancer is a high-risk malignant neoplasm
with a 5-year survival rate of less than 10%. The number
of new cases and deaths of pancreatic cancer worldwide in
2018 was 458,918 and 432,242, respectively. In 2020, the
projected number of incidences and deaths of pancreatic
cancer patients in the United States is 57,600 and 47,050,
respectively. Pancreatic ductal adenocarcinoma (PDAC)
is the most common type of pancreatic cancer, accounting
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for the early diagnosis of asymptomatic cases. PDAC
tends to have strong stem cell-like properties, causing
pancreatic cancer to be relatively resistant to traditional
chemotherapeutic strategies [4]. Mechanistically,
mutations in the KRAS gene has been well described in
PDAC, but this advanced knowledge has not resulted
in therapeutic innovations due to multiple factors [1, 2].
KRAS protein is relatively small and lacks deep binding
pockets, making it difficult to target with inhibitory drugs.
KRAS is an intracellular protein and cannot be targeted
with immunotherapy options such as antibodies or CAR-T
cells. Because of these limitations, KRAS has been viewed
as a difficult protein to develop targeted therapies [5, 6].
PDAC is presented with a highly immunosuppressive
tumor microenvironment, leading to challenges in
immunotherapy treatments such as the administration of
checkpoint inhibitors [7–9]. Thus, there is a critical need
for novel biomarkers relevant to therapeutic outcomes and
novel therapeutic targets in PDAC.
Recently, the human lymphocyte antigen-6 (LY6)
gene family has received increasing attention for its multifaceted roles in cancer development, stem cell maintenance,
immunomodulation, and association with more aggressive
and hard-to-treat cancers [2, 10, 11]. The LY6 gene family
is located on chromosomes 6, 8, 11, and 19. The LY6
family members in human chromosome 8 include PSCA,
LY6K, SLURP1, LYPD2, SLURP2, LY6D, GML, LY6E,
LY6L, LY6H, and GPIHBP1, mapped at 8q24.3 locus
[10]. Somatic amplification of 8q has been suggested to
be one of the most prevalent copy number gains in cancer
[12–14]. LY6D, LY6E, LY6H, and LY6K have increased
mRNA expression in tumor tissues of ovarian, colorectal,
gastric, breast, lung, bladder, brain, cervical, esophageal,
head and neck, and pancreatic cancer compared to adjacent
normal tissues. The increased mRNA expression of LY6D,
LY6E, LY6H and LY6K is associated with poor outcome
in ovarian, colorectal, gastric, breast, lung, bladder or
brain and CNS [2]. Increased levels of LY6A/E (Sca-1)
has been reported to promote breast tumorigenesis via
disruption of TGF-β signaling [15]. However, whether LY6
family members can be a biomarker for PDAC diagnosis,
prognosis, and therapy remains elusive.
In this report, we analyzed the mRNA expression
profile of 30 LY6 genes located on chromosomes 6,
8, 11 and 19 using the public The Cancer Genome
Atlas (TCGA) dataset of 177 PDAC patients and their
association with overall survival outcome. We used
publicly available online tools Kaplan-Meier Plotter (KM
Plotter), cBioPortal, Oncomine and survMisc R package
[16–18]. Herein, we demonstrate differential associations
of LY6 family members with patient survival outcome to
discuss the potential influence of our identified proteins
on the tumor microenvironment, immunomodulation, and
stem cell maintenance. Although the function of many LY6
family members is yet to be established, several of our
identified proteins have known roles in tumorigenesis and
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may be suitable candidate biomarkers for the diagnosis,
prognosis, and treatment of PDAC.
RESULTS
High expression of LY6 mRNA was associated
with overall survival outcome in pancreatic
ductal carcinoma
We used Kaplan-Meier (KM) plotter web tool
(https://kmplot.com) to see if LY6 gene expression was
significantly associated with overall survival (OS) in
pancreatic ductal adenocarcinoma (PDAC) [18]. We
found 21 out of 30 queried genes were significantly
associated with OS outcome in PDAC. We found that
high mRNA expression of 17 LY6 genes - PSCA,
SLURP1, LYPD2, LY6D, GML, Ly6E and LY6L
on chromosome 8; LYPD4, PLAUR, LYPD5 on
chromosome 19; PATE1, PATE2, PATE3 and CD59 on
chromosome 11and LY6G6C, LY6G6D and LY6G6F
on chromosome 6 to be significantly associated with
poor OS outcome. The hazard ratios for these genes
ranged from 1.68 to 2.99 indicating that patients with
high mRNA expression of genes had approximately
1.6 to 3 times the risk of death compared to patients
with low mRNA expression. We found high mRNA
expression of 4 LY6 genes - LY6H on chromosome
8; PINLYP on chromosome 19; and LY6G5C and
LY6G5B on chromosome 6 were significantly associated
with good OS outcome (Supplementary Table 1 and
Supplementary Figure 1).
We explored the association between RNA-seq
data from Pancreatic Adenocarcinoma from TCGA
with disease free survival (DFS) outcome using
R-programming. We found that 16 out of the 30 LY6
genes were significantly associated with DFS outcome
in PDAC. We found that high mRNA expression of 12
LY6 genes - PSCA, SLURP1, LYPD2, LY6D, GML and
LY6E on chromosome 8; LYPD3, PLAUR, LYPD5 on
chromosome 19; CD59 on chromosome 11 and LY6G6C
on chromosome 6 to be significantly associated with poor
DFS outcome. We found that high mRNA expression
of 4 LY6 genes - LY6H and GPIHBP1 on chromosome
8; and LY6G5C and LY6G5B on chromosome 6 were
significantly associated with good OS outcome (Figure 1).
We found that 13 LY6 genes were commonly
associated with both OS and DFS outcome. We found
high expression of 10 LY6 genes - PSCA, SLURP1,
LYPD2, LY6D, GML, LY6E on chromosome 8; PLAUR
and LYPD5 on chromosome 19; CD59 on chromosome
11 and LY6G6C on chromosome 6 were commonly
associated with poor OS and DFS outcomes. We found
high expression of 3 LY6 genes LY6H on chromosome
8; LY6G5C and LY6G5B on chromosome 6 were
commonly associated with good OS and DFS outcomes
(Supplementary Table 1 and Figure 1).
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This feature allowed us to observe the OS outcome in
patient samples with enriched or decreased cellular
content of PDAC. Mesenchymal stem cells (MSCs) in
tumor microenvironment have shown to be responsible
for increased tumor metastasis and immune tolerance of
tumors [19–21]. We studied how the LY6 genes affected
OS outcome in PDAC patients based on the MSCs
status in the tumor samples. We found high mRNA
expression of LY6D, SLURP1, CD59, PSCA, PATE2,
LY6G6F, LYPD5, LY6E, PATE1, LYPD2, LY6G6D,
PATE3, LYPD4, and GML was significantly associated
with poor OS outcome only in the MSC enriched patient
High expression of LY6 mRNA expression and
their association with overall survival outcome
was dependent on cell type content of the tumors
Tumor microenvironment is composed of various
cell types. The enrichment of specific cellular contents
in a tumor microenvironment may play an important
role as to how the tumor will progress or respond
to therapeutic interventions. To estimate if inherent
cellular content plays a role in the association of LY6
gene expression with the OS outcome, we used the
restricted analysis feature of Kaplan-Meier Plotter tool.
Figure 1: Disease free survival (DFS) outcome for 30 LY6 genes was analyzed using the RNA-seq data using the TCGA
data. The clinical and expression data were accessed through the cgdsr package, R programming. The optimal cutoff for mRNA expression
was determined using the method implemented in the survMisc R package. High mRNA expression of 15 genes associated with DFS
outcome of PDAC. Note: KM plot for GML gene expression has only 6 patients in high expression, however this was included based on
p value < 0.05.
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population. High expression of LY6G6C and GML was
associated with poor OS outcome in the MSC enriched
population. High expression of LY6G6C and GML was
associated with good OS outcome in the MSC decreased
population. LY6G5C was associated with good OS
outcome only in the MSC enriched population. Ly6G5B
was associated with good OS outcome independent of
MSC status. Ly6H, PINLYP was associated with good
OS outcome only in MSC enriched population. TEX101
was associated with good OS outcome only in MSC
decreased population (Table 1 and Supplementary
Figure 2).
Regulatory T cells (Treg cells) play important
roles in suppressing immune responses in the tumor
microenvironment. They can inhibit cytotoxic T-cell
lymphocytes (CTL) from attacking the cancer cells or
infiltrating the tumor microenvironment. We observed
that high expression of LY6D, SLURP1, PSCA,
LY6G6C, LY6E, LYPD5, LY6G6D, PATE2, and PATE1
were significantly associated with poor OS outcome
independent of the Treg status. High expression of
LY6G5C and LY6G5B were associated good OS outcome
independent of Treg status. CD59, LY6G6F, and PATE3
were associated with poor OS outcome only in Treg
decreased population. LYPD2 and PLAUR were only
associated with poor OS outcome only in Treg enriched
population. (Table 2 and Supplementary Figure 3).
CD8 positive T-cells are major defense against
cancer. Tumors with infiltrated CD8 positive T-cells are
termed as inflamed or hot tumors [22]. High expression
of PSCA, LY6D, SLURP1, PATE2, LYPD5, PATE1,
and LY6G6F was associated with poor OS outcome
independent of CD8 positive T-cells status. High
expression of LY6G6C and LY6G5B was associated
with good OS outcome independent of CD8 positive
T-cells status. High expression of PLAUR, LY6E, PATE3,
LYPD2, and CD177 was associated with poor OS outcome
only in CD8 enriched population. High expression of
LY6H was associated with good OS outcome only in
CD8 enriched population. High expression of LY6G6C,
LY6G6D, CD59, and LYPD4 was associated with poor
OS outcome only in CD8 decreased population. High
expression of LYPD3 was associated with poor OS
outcome in CD8 enriched and with good OS outcome in
CD8 decreased population (Table 3 and Supplementary
Figure 4).
Macrophages play an important role in pancreatic
beta cell function, pancreatic tissue homeostasis and
pancreatic cancer [23, 24]. High expression of LY6D,
CD59, and SLURP1 was associated with poor OS
outcome and LY6G5B and LY6G5C was associated with
good OS outcome independent of macrophage status.
High expression of PATE2 was associated with poor OS
outcome in macrophage enriched population. SPACA4,
LYPD4, LYPD3, and LY6L was associated with good OS
outcome in macrophages enriched population and with
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poor OS outcome in macrophages decreased population.
High expression of LY6H and LY6K was associated
with good OS outcome only in macrophages decreased
population. High expression of PSCA, TEX101, CD177,
LY6G6D, PATE1, LY6E, LY6G6F, LYPD2, PATE3,
LYPD5, GML and LY6G6C was associated with poor OS
outcome in macrophages decreased population (Table 4
and Supplementary Figure 5).
Natural killer T-cell (NKT cells) have the
characteristics of natural killer (NK) cells and T-cells
[25, 26]. High expression of LY6D, SLURP1,
LYPD5, PSCA, CD59, PATE2, LY6G6F and LY6E
was significantly associated with poor OS outcome
independent of NKT status. High expression of LY6G5C,
LY6H, and LY6G5B was significantly associated with
good OS outcome independent of NKT population. High
expression of LYPD2, PLAUR, LY6G6C, and CD177 was
significantly associated with poor OS outcome only in the
NKT enriched population. High expression of PATE4 was
associated with good OS outcome in NKT enriched and
poor OS outcome in NKT decreased population. High
expression of LYPD4, PATE3, PATE1, and LY6G6D was
associated with poor OS outcome only in NKT decreased
patient population (Table 5 and Supplementary Figure 6).
CD4 positive T-cells are helper memory T-cells
which can activate cytotoxic T-cells, natural killer
T-cells, B-cells and macrophages to activate immune
responses [27, 28]. High expression of LY6D, LY6E,
LY6L, LYPD4, LYPD5, and SLURP1 was significantly
associated with poor OS outcome independent of CD4+
memory T-cell status. High expression of LY6G5B was
strongly associated with good OS outcome independent
of CD4+ memory T-cell status. High expression of PATE1
was associated with good OS outcome in CD4+ enriched
and poor OS outcome in CD4+ decreased population.
High expression of PATE3, LYPD2, GML, PSCA, PATE2,
LY6G6D, CD59, and LY6G6C was associated with poor
OS outcome only in the CD4+decraesed population. High
expression of LY6H, and LY6G5C was associated with
good OS outcome in CD4+ decreased population (Table 6
and Supplementary Figure 7).
Presence of B-cells have been demonstrated
to correlate with good prognosis in many different
cancers [29–31]. Interestingly, LY6D has been shown
to be involved in B-cell differentiation [32, 33]. High
expression of LY6D, LY6E, PSCA, SLURP1, LYPD5,
and PATE2 was strongly associated with poor outcome
independent of B-cell status. High expression of LY6G5B
and LY6G5C was associated with good OS outcome
independent of B-cell status. High expression of PLAUR,
LYPD4, LY6G6C, LYPD2, CD59, LY6G6D, LY6L, GML,
PATE3, and PATE1 was associated with poor OS outcome
only in B-cell decreased population. High expression
of LY6H was associated with good OS outcome only in
B-cell decreased population. (Table 7 and Supplementary
Figure 8).
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hosted at Oncomine. LY6D, LY6E, PLAUR, PSCA, CD59
and LYPD3 mRNA expression was significantly increased
in the PDAC tumor tissues than normal adjacent tissues (p
< 0.01) (Figure 3).
LY6 DNA were amplified in pancreatic ductal
carcinoma
LY6 genes have been reported to be upregulated
in multiple cancers [11, 34]. To test whether LY6 gene
family members are amplified in PDAC, we assessed
copy number variation data including DNA amplification
and deep deletions from TCGA dataset (n = 177) and
Pancreatic Cancer UTSW dataset (n = 109) hosted on
cBioPortal tool [35]. We observed that LY6 genes located
on the chromosome 8q24.3, PSCA, LY6K, SLURP1,
LYPD2, LY6D, GML, LY6E, LY6H, and GPIHBP1 were
co-amplified in most 9 to 28% of PDAC cases in TCGA
and UTSW datasets. Interestingly, the cluster of LY6
genes which expressed on same genetic location were coamplified in the same patients. The deep deletions in LY6
genes were only observed in few cases (Figure 2).
DISCUSSION
The first step towards research and development
of pancreatic cancer treatment will be to identify and
define the novel tumor-specific biomarkers. Mutations
in the oncogenic KRAS gene occur in over 90% of
patients and are viewed as driving force of pancreatic
cancer [5]. However, a history of detailed knowledge
in the KRAS mechanistic pathway has not yet led to a
clinical breakthrough in the treatment of PDAC [5, 6].
With a five-year survival rate of less than 10%, there is an
urgent need for innovative treatment strategies. Compared
to other solid malignancies, challenges in pancreatic
cancer include the immensely immunosuppressive
tumor microenvironment, in addition to the presence
of a dense desmoplastic barrier, which limits the
diffusion of therapeutic drugs and the infiltration of
immunotherapy-based anti-tumor immune cells
[5–8]. An increased understanding of the key molecular
pathways unique to pancreatic cancer which contribute
to its immunosuppressive and stem cell-like properties
LY6 mRNA expression is upregulated in cancer
compared to normal tissues
The differential expression of LY6 genes between
the tumor tissue and the normal tissue will promote
the understanding of their potentials as prognosis and
therapeutic biomarkers. We evaluated 30 LY6 genes in
the Pei dataset of pancreatic tumors versus normal tissues
Figure 2: Oncoprint of copy number variation of 30 LY6 genes in TCGA and UTSW dataset using cBioPortal. Each
column represents a patient/sample. DNA amplification in red and DNA deep deletion in blue are depicted in the indicated genes.
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is required to develop novel and successful therapeutic
strategies against pancreatic cancer. Herein, we analyze
the expression of LY6 gene family and its association with
OS outcome in clinical samples of PDAC.
PDAC population enriched for Treg, CD8, macrophages,
NKT, B-cells and CD4+ immune cells. However, a single
pattern of association did not emerge for each of the LY6
genes, suggesting the LY6 genes may be differentially
regulated. In contrast to other LY6 genes, high expression
for CD59 was strongly associated with lower OS in
PDAC population decreased for CD8, Treg, macrophages,
NKT, B-cells and CD4+ immune cells. This observation
suggests that CD59 is associated with tumor environments
which present with lower immune cell infiltrates [37]. The
overexpression of CD59 in pancreatic cancer has major
consequences on the tumor microenvironment and was
previously shown to be required for stem cell evasion
of complement surveillance, a biological mechanism for
eliminating cancer stem cells in epithelial cancer [37].
We observed that increased mRNA of SLURP1
was associated with lower OS outcome in pancreatic
cancer. This observation was in agreement with public
data from The Human protein Atlas data which showed
that high expression of mRNA for SLURP1 is associated
with lower OS. This observation, however was in conflict
with a previously reported finding that high expression of
SLURP1 protein is associated with higher OS outcome
in pancreatic cancer [38]. Further studies are required to
understand the role of SLURP1 mRNA and protein in
pancreatic cancer and its association with OS outcome in
PDAC. In vivo evidence indicates that SLURP1 is a major
component of maintaining immune privilege through
inhibiting leukocytic binding and infiltration in a corneal
model, suggesting that SLURP1 can also serve as a potent
LY6 gene expression in the context of tumor
microenvironment
Mesenchymal stems cell (MSC) enriched pancreatic
tumors
MSC enriched tumors showed the strongest
association between high expression of LY6D/SLURP1/
PSCA/CD59 and low overall survival outcome in PDAC.
PDAC tumors with low MSC did not show significant
association with high LY6 gene expression to OS outcome.
This observation suggests that LY6 gene expression
and its association with OS outcome is specifically
relevant in the presence of MSCs in the pancreatic tumor
microenvironment. MSCs are instrumental in providing
the immunosuppressive tumor microenvironment. They
can suppress CD4 and CD8 positive T-cells. MSCs can
secrete various growth factors which can regulate gene
expression directly on cancer cells [36]. It remains to
be seen if the LY6 gene expression is associated with a
direct immunosuppressive environment due to presence
of MSCs.
Immune cell enriched pancreatic tumors
We observed that high expression of many LY6
genes were associated significantly with lower OS in
Figure 3: Oncomine tool showed significant increase expression of LY6 genes in pancreatic tumor vs normal tissues
in Pei dataset comparing normal vs tumor PDAC tissue. (1) Normal Pancreas (16 cases); (2) Pancreatic Carcinoma (36 cases).
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Table 1: High expression of LY6 genes and its association with overall survival outcome (OS) in
TCGA dataset of PDAC patient population based on mesenchymal stem cells (MSC) status, as
observed by using KM plotter tool
Gene
MSC enriched population
MSC decreased population
OS
p value
FDR
OS
p value
FDR
100%
LY6D
Poor
0.00002
1%
NS
0.11
SLURP1
Poor
0.00002
1%
NS
0.18
100%
PSCA
Poor
0.00005
1%
NS
0.33
100%
LY6G5C
Good
0.00005
2%
NS
0.16
100%
CD59
Poor
0.00007
2%
NS
0.22
100%
LY6G6F
Poor
0.00037
10%
NS
0.39
100%
PATE1
Poor
0.00061
20%
NA
NA
NA
PATE2
Poor
0.00130
50%
NS
0.21
100%
LY6G6D
Poor
0.00160
10%
NS
0.17
100%
LY6E
Poor
0.00290
50%
NS
0.26
100%
LY6G5B
Good
0.00300
50%
Good
0.041
50%
LY6G6C
Poor
0.00410
20%
Good
0.046
50%
LYPD5
Poor
0.00480
50%
NS
0.13
100%
LYPD2
Poor
0.00520
50%
NS
0.14
100%
PATE3
Poor
0.00640
50%
NS
0.59
100%
100%
LY6H
Good
0.00670
50%
NS
0.091
PINLYP
Good
0.00680
50%
NS
0.077
100%
LYPD4
Poor
0.02100
50%
NS
0.15
100%
LY6L
Poor
0.02200
50%
Good
0.036
50%
GML
Poor
0.03700
50%
NS
0.33
100%
TEX101
NS
0.12000
100%
Good
0.033
50%
LY6 gene expression with NS association in both categories are not displayed. The table is arranged in increasing p value for MSC enriched population.
For the original KM plots, sample sizes in MSCs enriched and decreased population and other details, please refer to Supplementary Figure 2. NS: Not
Significant, p < 0.05; OS: Overall Survival, FDR: False Discovery Rate, HR: Hazard Ratio.
inhibitor of immune activity [39, 40]. However this has not
been tested in tumor models, it is plausible that SLURP1
can contribute to pancreatic cancer’s immunosuppressive
tumor microenvironment and suppress anti-tumor immune
responses.
castration-resistant properties in prostate cancer [44].
Together, our analysis and previous research suggests
that LY6D expression may indicate a more aggressive
pancreatic cancer phenotype and it would therefore be
beneficial to explore the precise mechanism of action for
LY6D to determine its therapeutic potential.
LY6E, a GPI-anchored member of the LY6 family,
has recently been implicated as a driver of tumorigenesis
and stem cell maintenance through inhibiting expression
of the tumor suppressor PTEN and inducing the
upregulation of the HIF-1 pathway [45, 46]. Notably,
inhibition of LY6E with siRNA was shown to restore
PTEN expression, induce G1-S phase cell cycle arrest,
and increase apoptosis in gastric cancer, suggesting that
LY6E’s inhibition may be enough to cause anti-tumor
effects in some cancers [46]. On a clinical level, high
LY6E expression correlates with poor overall patient
survival in various malignant tumors such as those of
gastric, breast, head and neck, lung, bladder, brain, and
skin origin [11, 45]. Importantly, in pancreatic cancer
specifically, LY6E was suggested to be a marker for
cancer cells with stem cell properties and was used in
addition to the stem cell markers TACSTD1 and CD44
LY6 proteins and maintenance of proliferation
and stem cell-like properties
LY6D, LY6E, PSCA, and PLAUR, known
markers tumorigenesis and cancer cell maintenance,
were significantly associated with lower overall survival
outcome in our pancreatic cancer analysis [10, 11].
LY6D is a GPI-anchored member of the LY6 family
with a recently established association with aggressive
cancers and poor patient outcome [10, 11]. Under normal
physiological conditions, LY6D is most commonly used
as a marker of early B cell lineage; however, in response
to genotoxic stressors such as radiation and chemotherapy,
LY6D expression is upregulated in numerous cancer
types and is suggested to contribute to distant metastasis
in breast cancer [41–43]. LY6D serves as a marker of
luminal progenitors with bi-lineage capacity and intrinsic
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Table 2: High expression of LY6 genes and its association with overall survival outcome (OS) in
TCGA dataset of PDAC patient population based on T-regulatory cells (Treg) status, as observed
by using KM plotter tool
Gene
Treg enriched population
Treg decreased population
OS
p value
FDR
OS
p value
FDR
LY6D
Poor
0.00002
1%
Poor
0.00220
20%
SLURP1
Poor
0.00007
1%
Poor
0.00360
>50%
PSCA
Poor
0.00030
2%
Poor
0.01760
>50%
LY6G6C
Poor
0.00210
20%
Poor
0.00860
>50%
LYPD2
Poor
0.00220
20%
NS
0.09210
100%
LY6G5C
Good
0.00230
50%
Good
0.00030
5%
LY6G5B
Good
0.00290
10%
Good
0.00790
>50%
PLAUR
Poor
0.00390
50%
NS
0.20220
100%
>50%
LY6E
Poor
0.00810
50%
Poor
0.02210
LYPD5
Poor
0.01340
>50%
Poor
0.00770
50%
LY6G6D
Poor
0.02650
100%
Poor
0.04400
>50%
PATE2
Poor
0.02800
>50%
Poor
0.00200
50%
PATE1
Poor
0.03690
>50%
Poor
0.01290
>50%
>50%
LY6G6F
NS
0.05030
100%
Poor
0.00540
CD59
NS
0.07340
100%
Poor
0.00004
1%
PATE3
NS
0.16470
100%
Poor
0.02020
>50%
LY6 gene expression with NS association in both categories are not displayed. The table is arranged in increasing p value for Treg enriched population. For
the original KM plots, sample sizes in T-reg enriched and decreased population and other details, please refer to Supplementary Figure 3. NS: Not Significant,
p < 0.05; OS: Overall Survival, FDR: False Discovery Rate, HR: Hazard Ratio.
Table 3: High expression of LY6 genes and its association with overall survival outcome (OS) in TCGA dataset
of PDAC patient population based on CD8 positive (CD8+) status, as observed by using KM plotter tool
Gene
CD8+ enriched population
OS
p value
CD8+ decreased population
FDR
OS
p value
FDR
20%
LY6D
Poor
0.000074
1%
Poor
0.0016
SLURP1
Poor
0.0012
20%
Poor
0.0017
50%
PATE2
Poor
0.0013
20%
Poor
0.011
50%
LYPD5
Poor
0.0016
10%
Poor
0.035
50%
LY6G5C
Good
0.0022
10%
Good
0.0026
20%
PSCA
Poor
0.005
50%
Poor
0.0034
50%
LYPD3
Poor
0.0092
50%
Good
0.009
50%
PATE1
Poor
0.0095
50%
Poor
0.022
50%
PLAUR
Poor
0.01
50%
NS
0.21
100%
LY6G5B
Good
0.011
50%
Good
0.0049
50%
LY6E
Poor
0.015
50%
NS
0.082
100%
LY6H
Good
0.019
50%
NS
0.078
100%
LYPD2
Poor
0.02
50%
NS
0.097
100%
100%
CD177
Poor
0.023
50%
NS
0.27
LY6G6F
Poor
0.038
50%
Poor
0.0064
50%
LY6L
NS
0.049
50%
NS
0.13
100%
LY6G6C
NS
0.057
100%
Poor
0.015
50%
LY6G6D
NS
0.07
100%
Poor
0.014
50%
CD59
NS
0.1
100%
Poor
0.0014
20%
LYPD4
NS
0.21
100%
Poor
0.044
50%
LY6 gene expression with NS association in both categories are not displayed. The table is arranged in increasing p value for CD8+ enriched population. For
the original KM plots, sample sizes in in CD8+ cells enriched and decreased population and other details, please refer to Supplementary Figure 4. NS: Not
Significant, p < 0.05; OS: Overall Survival, FDR: False Discovery Rate, HR: Hazard Ratio.
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Table 4: High expression of LY6 genes and its association with overall survival outcome (OS) in TCGA
dataset of PDAC patient population based on macrophages status, as observed by using KM plotter tool
Gene
Macrophages enriched population
OS
p value
Macrophages decreased population
FDR
OS
p value
FDR
LY6D
Poor
0.00017
2%
Poor
0.00001
1%
LY6G5B
Good
0.00090
10%%
Good
0.04400
50%
CD59
Poor
0.00890
50%
Poor
0.00030
5%
100%
PATE2
Poor
0.00970
50%
NS
0.10000
SLURP1
Poor
0.01700
50%
Poor
0.00000
1%
SPACA4
Good
0.01900
50%
Poor
0.02900
50%
LYPD4
Good
0.02100
50%
Poor
0.00350
50%
LY6G5C
Good
0.02700
50%
Good
0.00002
1%
LYPD3
Good
0.02900
50%
Poor
0.03500
50%
LY6L
Good
0.04100
50%
Poor
0.00330
20%
PLAUR
NS
0.06900
100%
Poor
0.00092
10%
LY6H
NS
0.07800
100%
Good
0.00070
10%
PSCA
NS
0.09500
100%
Poor
0.00000
1%
TEX101
NS
0.09600
100%
Poor
0.00990
50%
CD177
NS
0.12000
100%
Poor
0.00790
50%
LY6G6D
NS
0.12000
100%
Poor
0.01400
50%
PATE1
NS
0.13000
100%
Poor
0.01200
50%
LY6K
NS
0.14000
100%
Good
0.00470
50%
LY6E
NS
0.14000
100%
Poor
0.00014
1%
LY6G6F
NS
0.18000
100%
Poor
0.00140
20%
LYPD2
NS
0.27000
100%
Poor
0.00006
1%
50%
PATE3
NS
0.29000
100%
Poor
0.01200
LYPD5
NS
0.35000
100%
Poor
0.00001
1%
GML
NS
0.45000
100%
Poor
0.02800
50%
LY6G6C
NS
0.48000
100%
Poor
0.00018
2%
LY6 gene expression with NS association in both categories are not displayed. The table is arranged in increasing p value for macrophages enriched
population. For the original KM plots, sample sizes in macrophages enriched and decreased population and other details, please refer to Supplementary Figure
5. NS: Not Significant, p < 0.05; OS: Overall Survival, FDR: False Discovery Rate, HR: Hazard Ratio.
to establish a sorting technique to obtain clonal colonyforming pancreatic cancer stem cells [47].
GPI-anchored LY6 family member PSCA is most
commonly attributed to prostate stem cells and prostate
cancer; however, its overexpression in pancreatic cancer
and limited expression in normal pancreatic cells is also well
recognized [48, 49]. PSCA can be a target of immunotherapy
strategies such as anti-PSCA antibody and anti-PSCA
CAR-T cell therapy, with a clinical trial currently underway
for the latter [6]. However, PSCA’s function in pancreatic
cancer is still unknown and requires further research.
expression data for these genes was found to be relatively
high in the patients with gene copy number amplifications,
suggesting that an amplified copy number for LY6
genes on chromosome 8 increases LY6 gene expression.
Understanding what causes both an amplification of LY6
gene copy number and an overexpression of LY6 genes
on chromosome 8 may reveal important insight into the
molecular phenotype of this subset of pancreatic cancer
patients.
Gene copy number amplifications of LY6
proteins on chromosome 8
High expression of LY6G5B, LY6G5C, and
LY6G6C was associated with high OS outcome. These
LY6 genes are located among the MHC class III proteins
and are known as MHC-linked LY6 genes. Although the
precise functions of the LY6 family members LY6G5B,
LY6G5C, LY6G6C are unknown, they are suggested
to be located on filopodia and secreted proteins with
binding potential to the cell surface and may be involved
The “good” LY6 genes
LY6D, LY6E, LY6H, LY6K, PSCA, SLURP1,
LYPD2, and GPIHP1 had co-amplifications of their gene
copy numbers as seen on cBioPortal in TCGA dataset
While increased DNA copy number does not necessarily
indicate increased levels of protein expression, the mRNA
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Table 5: High expression of LY6 genes and its association with overall survival outcome (OS) in TCGA
dataset of PDAC patient population based on NKT cell status, as observed by using KM plotter tool
Gene
NKT enriched population
NKT decreased population
OS
p value
FDR
OS
p value
FDR
LY6D
Poor
0.00009
2%
Poor
0.00100
20%
SLURP1
Poor
0.00010
2%
Poor
0.00330
50%
LY6G5C
Good
0.00030
2%
Good
0.00490
50%
LYPD2
Poor
0.00035
5%
NS
0.26000
100%
LYPD5
Poor
0.00170
20%
Poor
0.04700
50%
PSCA
Poor
0.00410
20%
Poor
0.00260
50%
PATE4
Good
0.00700
50%
Poor
0.01100
50%
CD59
Poor
0.00750
50%
Poor
0.00310
20%
LY6H
Good
0.00850
50%
Good
0.00900
50%
PLAUR
Poor
0.00990
50%
NS
0.42000
100%
LY6K
Good
0.01100
50%
NS
0.40000
100%
LY6G5B
Good
0.01400
50%
Good
0.00340
50%
100%
LY6G6C
Poor
0.01500
50%
NS
0.17000
PATE2
Poor
0.01600
50%
Poor
0.01100
50%
CD177
Poor
0.01700
50%
NS
0.33000
100%
LY6G6F
Poor
0.01700
50%
Poor
0.00660
50%
LY6E
Poor
0.03400
50%
Poor
0.00700
50%
LYPD4
NS
0.06300
100%
Poor
0.01900
50%
PATE3
Good
0.06900
100%
Poor
0.03400
50%
PATE1
NS
0.08700
100%
Poor
0.00650
50%
LY6G6D
NS
0.29000
100%
Poor
0.00160
20%
LY6 gene expression with NS association in both categories are not displayed. The table is arranged in increasing p value for NKT enriched population.
For the original KM plots, sample sizes in NKT cells enriched and decreased population and other details, please refer to Supplementary Figure 6. NS: Not
Significant, p < 0.05; OS: Overall Survival, FDR: False Discovery Rate, HR: Hazard Ratio.
in cell signaling. Interestingly, potential ligands for
LY6G5C, LY6G6C were found to be present on K562
cells, an undifferentiated megakaryocyte cell line,
among a panel of cell lines, indicating a potential role in
hematopoietic cell differentiation [50, 51]. It is unclear
how these genes are associated with higher OS outcome.
It is likely that cell-cell interaction may play important
role in LY6 signaling.
interaction context. With further research, these findings
may lead to potential successful screening markers for
PDAC, as well as new, personalized targeted therapy or
guidance of current standard chemotherapy regimens
based on LY6 gene expression.
MATERIALS AND METHODS
Overall survival (OS) outcome analysis
Concluding remarks
The Kaplan-Meier (KM) plotter (https://kmplot.
com/analysis/) online web-based tools allows users
to observe the association of mRNA expression to
over survival outcome with or the context of tumor
microenvironment cellular composition [18]. For our
analysis, we selected PDAC dataset on 177 patients
among the list of pan cancer dataset (TCGA collection)
in the gene expression RNA sequencing (RNA-seq) tab
on the homepage of KM plotter. This dataset was used to
visualize the association of mRNA expression of the LY6
gene family with overall survival (OS) outcome in PDAC.
The number of patients in low and high expression of
indicated gene is displayed in the Supplementary Table 1.
To estimate if inherent cellular content plays a role in the
LY6 family proteins are either attached to the outer
cell surface through a GPI-anchor or are secreted into the
extracellular matrix, making them relatively accessible
for drug inhibition or immunotherapy targeting. In this
report, we sought to analyze the mRNA expression of
LY6 gene family and its association with overall survival
(OS) outcome in pancreatic cancer patients. We focused
our exploration on thirty LY6 family genes scattered on
various chromosomes [2, 10, 11]. We found that high
expression of sixteen LY6 family members significantly
associated with lower OS outcome and high expression
of four genes significantly associated with higher OS
outcome. Future research is required to translate our
DNA and mRNA analysis into a proteomic and molecular
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Table 6: High expression of LY6 genes and its association with overall survival outcome (OS) in TCGA dataset
of PDAC patient population based on CD4+ memory T-cell status, as observed by using KM plotter tool
Gene
CD4+ enriched population
CD4+ decreased population
OS
p value
FDR
OS
p value
LY6D
Poor
0.000048
1%
Poor
0.0001
FDR
5%
LY6E
Poor
0.0012
10%
Poor
0.0248
>50%
LY6L
Poor
0.0037
20%
Poor
0.0056
>50%
>50%
LYPD4
Poor
0.0038
20%
Poor
0.003
LY6G5B
Good
0.0062
>50%
Good
0.009
50%
LYPD5
Poor
0.0142
>50%
Poor
0.0044
50%
SLURP1
Poor
0.0328
>50%
Poor
0.000013
1%
PATE1
Good
0.0465
>50%
Poor
0.0007
10%
PATE3
NS
0.061
100%
Poor
0.0052
>50%
LYPD2
NS
0.0854
100%
Poor
0.0041
>50%
GML
NS
0.1008
100%
Poor
0.0112
>50%
PSCA
NS
0.1014
100%
Poor
0.000084
2%
PATE2
NS
0.1275
100%
Poor
0.0004
20%
LY6G6D
NS
0.1351
100%
Poor
0.0016
10%
CD59
NS
0.1934
100%
Poor
0.0006
10%
LY6H
NS
0.214
100%
Good
0.006
50%
LY6G5C
NS
0.277
100%
Good
0.00006
2%
LY6G6C
NS
0.4561
100%
Poor
0.0037
50%
LY6 gene expression with NS association in both categories are not displayed. The table is arranged in increasing p value for CD4+ memory T-cells enriched population. For the
original KM plots, sample sizes in CD4+ memory T-cell enriched and decreased population and other details, please refer to Supplementary Figure 7. NS: Not Significant, p <
0.05; OS: Overall Survival, FDR: False Discovery Rate, HR: Hazard Ratio.
Table 7: High expression of LY6 genes and its association with overall survival outcome (OS) in TCGA
dataset of PDAC patient population based on B-cell status, as observed by using KM plotter tool
Gene
B cell enriched population
B cell decreased population
OS
p value
FDR
OS
p value
LY6D
Poor
0.00003
1%
Poor
0.0009
FDR
10%
LY6E
Poor
0.006
10%
Poor
0.0078
>50%
PSCA
Poor
0.0152
100%
Poor
0.0005
5%
LY6G5B
Good
0.0213
>50%
Good
0.0017
10%
SLURP1
Poor
0.0242
>50%
Poor
0.000009
1%
LY6G5C
Good
0.0283
100%
Good
0.0001
5%
LYPD5
Poor
0.0355
>50%
Poor
0.003
50%
>50%
PLAUR
NS
0.0433
100%
Poor
0.0644
PATE2
Poor
0.0488
100%
Poor
0.0014
50%
LYPD4
NS
0.0525
20%
Poor
0.009
>50%
LY6G6C
NS
0.055
100%
Poor
0.0078
>50%
LYPD2
NS
0.0967
100%
Poor
0.0021
50%
CD59
NS
0.1004
100%
Poor
0.0000068
1%
LY6G6D
NS
0.1892
100%
Poor
0.0038
>50%
>50%
LY6L
NS
0.2433
20%
Poor
0.043
LY6H
NS
0.3599
100%
Good
0.0007
20%
GML
NS
0.3734
100%
Poor
0.0385
>50%
PATE3
NS
0.4032
100%
Poor
0.0261
>50%
PATE1
NS
0.6189
>50%
Poor
0.0039
50%
LY6 gene expression with NS association in both categories are not displayed. The table is arranged in increasing p value for B-cells enriched population. For the original KM
plots, sample sizes in B-cells enriched and decreased population and other details, please refer to Supplementary Figure 8. NS: Not Significant, p < 0.05; OS: Overall Survival,
FDR: False Discovery Rate, HR: Hazard Ratio.
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association of LY6 gene expression with the OS outcome,
we used the restricted analysis feature of the KM Plotter
tool. This feature allowed us to observe the OS outcome
in patient samples with enriched or decreased cellular
content which included mesenchymal stem cell, CD8+ T
cells, macrophages, NK T cells, CD4+ memory T cells,
regulatory T cells, and B cells. The number of patients in
the various subgroups for low expressing genes were in
the range of 43 to 127 and for high expressing genes were
51 to 134. The exact number of patients in each subgroup
can be found in Supplementary Figure 1 through 8, which
corresponds to the data presented in Supplementary
Table 1, Tables 2–7.
Author contributions
Conceptualization, G.U.; formal analysis,
methodology, and writing, E.R., K.B., G.W., B.J., Y.G.,
and G.U.; critical reading and writing, E.R., K.B., G.W.,
B.J., M.G., S.M., Y.G., and G.U. All authors have read and
agreed to the published version of the manuscript.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest. The
funders had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing
of the manuscript, or in the decision to publish the results.
The opinions expressed herein are those of the
authors and are not necessarily representative of those
of the government of the United States, the Uniformed
Services University, the Department of Defense, NIH, or
any other US federal agency.
Disease free survival (DFS) analysis
RNA-seq normalized gene expression data at z-score
level and the corresponding clinical data from the TCGA
Pancreatic Adenocarcinoma cohort were downloaded from
the cBioPortal (https://www.cbioportal.org/) [16, 17]. The
TCGA dataset contained data from 179 patients, of which
139 had disease free survival (DFS) status, including one
metastasis sample. Thus, 138 primary tumor samples
were used for DFS analysis. DFS analysis was performed
using the R statistical programming environment. We
used the cgdsr R package (https://CRAN.R-project.
org/package=cgdsr) in the R statistical programming
software to download and query the data [52]. For each
gene, the optimal cutoff into “low” and “high” expression
was determined by inbuilt algorithm in the survMisc R
package [53]. We used this data to explore the association
of the LY6 genes with DFS outcome. The survival analysis
and KM plots were done using the survMisc R package.
Then the significance of the association of each gene with
DFS outcome was reported using a log rank test with
p-value < 0.05.
FUNDING
This research has been supported by NIH
R01CA227694 and Uniformed Services University StartUp funds by Vice President of Research office, USUHS to
Geeta Upadhyay.
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