Introduction
Head and neck squamous cell carcinoma (HNSCC) includes a heterogeneous group of malignancies
that arise in the oral cavity, larynx, and pharynx.[1 ]
[2 ] HNSCC is the seventh most common cancer worldwide, accounting for over 800,000 new
cases every year.[3 ] The high prevalence of HNSCC in regions such as Southeast Asia and Australia is
associated with the use of specific carcinogen-containing products (tobacco smoking,
areca nut chewing, and alcohol consumption), whereas increasing rates of oropharyngeal
infection with HPV have contributed to the high prevalence of HNSCC in the United
States and Western Europe.[1 ] Despite multimodal therapeutic approaches and advances in diagnostics, treatment,
and surveillance, the 5-year progression-free survival (PFS) of HPV-negative patients
with locoregionally advanced disease is approximately 40 to 50% and survival rates
for recurrent/metastatic (R/M) disease have not significantly improved over the past
40 years.[4 ]
The poor prognosis of HNSCC is primarily attributed to a lack of appropriate screening,
late-stage diagnosis, and antineoplastic resistance of cancer stem cells (CSCs). CSCs
constitute a minor fraction (1–3%) of the cells in primary tumors. The CSC hypothesis
proposes that CSCs are subpopulations of tumor cells that are responsible for tumorigenesis,
tumor differentiation, tumor maintenance, and invasion. The pool of CSCs with inherent
characteristics of self-renewal and tumor heterogeneity seldom divides and remains
undifferentiated. This facilitates the regeneration of new CSC promoting the development
and perpetuation of several human malignancies. The characterization of CSC markers
serves to identify novel therapeutic targets in HNSCC and can help in prognostication.[5 ]
Several molecular protein biomarkers of HNSCC CSCs have been proposed, with CD44 being
the most extensively validated for prognostic significance.[6 ] CD44, a cell surface receptor for hyaluronic acid and matrix metalloproteinases
(MMPs) participates in intercellular interactions, cell adhesion, and cell migration.
Upregulation of CD44 in HNSCC has been associated with increased loco-regional recurrence,
metastasis, invasion, decreased overall survival, and increased resistance to radiotherapy.[7 ] We studied the significance of the CD44 gene and protein expression in HNSCC by
using the Cancer Genome Atlas (TCGA) dataset accessed through the University of Alabama
at Birmingham Cancer (UALCAN) portal.
Data Source and Methods
The data on gene and protein expression of CD44 and its association with demographic
features, clinicopathological characteristics, and survival outcomes were curated
from UALCAN. UALCAN portal is an interactive and user-friendly web-based portal that
enables researchers to access level 3 RNA-seq data from TCGA. It facilitates gene
expression and survival analyses on around 20,500 protein-coding genes in 33 tumor
types. It is built on PERL-CGI with high-quality graphics using javascript and Cascading
Style Sheets. It is used to analyze relative gene transcript levels between tumors
and normal samples, and the relationship between these levels and clinicopathological
parameters.[8 ] Genomics data from the TCGA project include comprehensive molecular features of
various cancer types. The large sample data of TCGA efficiently addresses the queries
raised on tumor heterogeneity.
The mRNA transcript levels of CD44 in HNSCC and the corresponding normal controls
in TCGA were reviewed using UALCAN. The clinicopathological parameters including age,
gender, race, individual cancer stages, tumor grade, human papillomavirus (HPV), TP53
mutant status, and survival outcome of patients with primary HNSCC in TCGA-HNSCC were
retrieved and grouped according to median CD44 mRNA expression. The CD44 protein expression
analysis was done using data from Clinical Proteomic Tumor Analysis Consortium (CPTAC)
and the International Cancer Proteogenome Consortium datasets available in UALCAN.
Results
Gene and Protein Expression of CD44 (The Cancer Genome Atlas and Clinical Proteomic
Tumor Analysis Consortium Datasets in University of Alabama at Birmingham Cancer)
The TCGA dataset includes 520 cases of HNSCC and 44 healthy controls. Upregulation
of median (IQR) CD44 gene expression in HNSCC tumor samples (279.2) was significantly greater compared
with normal subjects (148.7; p = 0.0001). The CPTAC dataset comprised 108 HNSCC samples and 71 healthy controls.
Upregulation of CD44 protein expression in HNSCC tumor samples (median: 0.003) compared
with normal subjects (median: –1.3); statistically significant (p = 0.0008; [Fig. 1 ]).
Fig. 1 Gene and protein expression of CD44.
Association between CD44 Expression and Demographic Features (Age, Gender, and Race)
Though HNSCC patients in the age group between 41 and 60 years (n = 236) exhibited upregulation (median 285.1) of CD44 compared with the other age
groups, it was not statistically significant. Similarly, females with HNSCC (N = 136, 26%) showed overexpression of the CD44 gene compared with males, but it was not statistically significant ([Fig. 2) ].
Fig. 2 Association of CD44 and demographic features ([a] age; [b] gender; [c] race).
The TCGA-HNSC dataset included 383 males (74%) and 136 females (26%). Among patients
with HNSCC, females (median: 291.2) show overexpression of the CD44 gene compared with males (median: 278), statistically not significant (p = 0.2). Asians (median: 362.4), Caucasians (median: 279.2), and African-Americans
(median: 270.6) show a significant overexpression of the CD44 gene compared with the normal samples (median: 148.7), statistically significant
(p = 0.0006; [Fig. 2 ]).
Effect of CD44 Expression on Clinicopathological Characteristics (Tumor Stage, Tumor
Grade, Human Papillomavirus Status, and TP53 Mutation Status)
Overexpression of CD44 gene in Stage 1 of HNSCC patients (Median: 321.2) compared with normal and other
stages of HNSCC was statistically significant (p = 0.0001). Grade 2 HNSCC tumors (median: 297.1) demonstrate an upregulation of CD44
compared with HNSCC patients with grade 1 (median: 253.7), and grade 3 (median: 285.1),
and grade 4 (Median: 132.5) tumors and normal subjects (median: 148.7). HNSCC patients
with N1 and N0 nodal status (median: 291; p = 0.0003 and 286.4; p = 0.0001, respectively) have significant CD44 upregulation compared with normal subjects
(median: 147.8; [Fig. 3 ]).
Fig. 3 Effect of CD44 expression on tumor stage, tumor grade, and nodal status.
HNSCC tumors with negative HPV status (median: 304.9) exhibit greater CD44 gene expression compared with patients with positive HPV status (median: 152.7) and
normal subjects (median: 147.8), statistically significant (p = 0.0001). TP53 mutant status positive HNSCC patients (median: 309.8) showed significant
CD44 upregulation compared with TP53 nonmutant HNSCC patients (median: 222.9; [Fig. 4 ])..
Fig. 4 Effect of CD44 expression on HPV and TP53 status.
Association between CD44 Expression and Head and Neck Squamous Cell Carcinoma Patient
Survival
High CD44 expression group had lower median survival compared with the low/medium
expression group, but the difference was statistically not significant (p = 0.49; Plot 1/Graph 1/[Fig. 1a–d ]). Grade 3 tumors with high CD44 expression had a lesser median survival rate compared
with Grade 1 tumors with low CD44 expression, statistically not significant (p = 0.065). Irrespective of the differential expression of CD44, Caucasians had a higher
median survival time compared with African Americans (p = 0.0019) and had a higher median survival time compared with females (p = 0.17; [Fig. 5 ]).
Fig. 5 Association of CD44 expression and HNSCC patient survival.
Discussion
CD44, a cell membrane molecule, is a multistructural and multifunctional glycoprotein.
It has a range of functions including cell adhesion (aggregation and migration), hyaluronan
(HA) degradation, lymphocyte activation, lymph node homing, cell motility, cell migration,
cell signaling (cell–cell and cell–matrix interactions), gene transcriptions, myelopoiesis,
lymphopoiesis, angiogenesis, and release of cytokines because of its varied structure
and distribution in the body tissues. The alternative splicing of variable exons of
CD44 results in variants, which are denoted as CD44v, and the isoform with no variable
exon in the mRNA as the standard isoform, CD44s.[9 ] In this study, we analyzed the expression patterns of the CD44 gene and its association with demographic features, clinicopathological characteristics,
and overall survival of patients with HNSCC by in-silico analysis.
CD44 is the major HA receptor, and CD44 bound to HA participates in tumor biological
activities including tumor progression, metastasis, and proliferation. CD44 is the
most frequently observed CSC marker in solid tumors. CD44 binding to HA regulated
CSC survival, self-renewal, maintenance, and chemoresistance.[10 ]
CD44 was first identified in lymphocytes. CD44s (standard form) is found in the lungs,
epidermis, nervous system, stomach, pancreas, intestines, kidneys, urinary bladder,
and cervix. CD44v (variants) are distributed in keratinocytes, lymphocytes, macrophages,
and epithelial cells of the stomach, bladder, and cervix.[9 ] The data curated from the human protein atlas show the tissue-specific distribution
of CD44 with high protein expression scores in the squamous epithelial cells of oral
mucosa and tonsils and glandular cells of the salivary gland.
The pan-cancer profile of CD44 gene expression in the Timer 2.0 platform shows significant upregulation of the CD44 gene in cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma
multiforme, HNSCC, kidney chromophobe, kidney renal clear cell carcinoma, kidney renal
papillary cell carcinoma, lung adenocarcinoma, pheochromocytoma and paraganglioma,
prostate adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, thyroid carcinoma,
and uterine corpus endometrial carcinoma.[11 ]
Various studies support the significant overexpression of the CD44 gene in human cancers. Wang et al reported that a high CD44 expression promotes lung
cancer cell metastasis in vitro and in vivo through activation of ERK–ZEB1 signaling.[12 ] Gupta et al studied the immunohistochemical expression of CD44 in esophageal squamous
cell carcinoma (ESCC) and its predisposing lesions (mild, moderate, and severe dysplasia
and esophagitis). CD44 expression was significantly higher in ESCC as compared with
dysplasia and esophagitis.[13 ] Using immunohistochemistry on tissue array, Zanjani et al evaluated CD44 expression
in 206 renal tumor samples. Upregulation of CD44 expression was associated with aggressive
behavior and poor prognosis in clear cell renal cell carcinoma (RCC) but not in papillary
and chromophobe subtypes of RCC.[14 ] Wang et al in a meta-analysis of colorectal carcinoma (CRC) that included a total
of 48 studies reported that the total CD44 isoform overexpression was significantly
correlated with worse overall survival of patients with CRC.[15 ] CD44 modulates the aggressive phenotype of prostate cancer cells, by regulating
the expression of PDK1 (pyruvate dehydrogenase) and PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4).[16 ] In the meta-analysis to ascertain the prognostic value of CD44 in RCC, high CD44
expression correlated with high Fuhrman grade, recurrence, microvascular invasion,
and poor prognosis.[17 ] A study on the splice variants of CD44 demonstrated the distinct role of CD44v3
and CD44v6 in the progression of bladder cancer.[18 ] CD44v9 contributes to increased resistance to chemotherapy- or radiation-induced
cell death in gastric carcinoma. CD44 also promotes epithelial-mesenchymal transition
(EMT) in many cancer types such as colon cancer, gastric cancer, pancreatic cancer,
prostate cancer, liver cancer, and glioma by upregulating mesenchymal markers and
downregulating epithelial markers.[9 ]
The data mined from the UALCAN platform show upregulation of CD44 gene and protein expression in HNSCC compared with normal subjects. Sawant et al
reported that serum CD44 concentration was found significantly high in patients with
primary oral squamous cell carcinoma (OSCC) (n = 64) as compared with healthy individuals (n = 16; p < 0.001) and also in patients whose disease locally recurred (n = 10) as compared with those did not recur (n = 35; p = 0.0026).[19 ] Reategui et al characterized CD44v3 in HNSCC cell lines by reverse transcription
polymerase chain reaction. The mean CD44v3 values for HNSCC tumors were elevated 4.5
times (0.43 ± 0.44) compared with the normal tissues (0.10 ± 0.11; p < 0.01).[20 ] In an immunohistochemical study conducted by Tamatani et al, CD44 and CD44v9 expressions
were strongly detected in all OSCC tissues compared with normal epithelial cells.[21 ] Similarly in another study, CD44v6 expression was detected in the membrane of tumor
cells in 94% of the OSCC cases (n = 60).[22 ] In a comparative study conducted in Africa, CD44 expression was evaluated in the
whole unstimulated saliva of patients with oral leukoplakia and OSCC by ELISA. A statistically
significant overexpression of CD44 was demonstrated in OSCC patients compared with
those with leukoplakia and healthy controls.[23 ] El-Gendi et al reported that papillary thyroid carcinoma (PTC; n = 30) showed higher CD44v6 expression than follicular thyroid carcinoma (n = 10).[24 ] On the contrary, downregulation of the CD44 gene was associated with a poor prognosis
of laryngeal carcinoma.[25 ]
Age and gender did not show any significant association with CD44 expression. Similar
results were reported in the studies conducted by Monteiro et al and Wagih et al.[22 ]
[23 ] Asian, Caucasian, and African-Americans showed significant overexpression of the
CD44 gene expression compared with normal samples. Similar to our study results, Lee
et al, observed that OSCC with positive mutant p53 expression displayed enhanced expression
of CD44 (p < 0.001).[26 ] A significant upregulation of the CD44 gene in HPV-negative OSCC is observed in the present study. This is supported by
Slavik et al who studied the prognostic significance of the mutual combination of
CD44, EGFR, and p16 in HNSCC. The worst prognosis was seen in CD44+ /p16− , EGFR+ /p16− , and EGFR+ /CD44+ groups and in the EGFR+ /CD44+ within the p16 negative cohort.[27 ]
Upregulation of the CD44 gene was seen in Stage I and Grade 2 irrespective of N1 or N0 nodal status. Poor
survival was exhibited by high CD44 expression. Various study findings are congruent
with the current study results. Mack and Gires observed that CD44s and CD44v6 were
significantly upregulated in moderately differentiated SCC compared with poorly differentiated
SCC and carcinoma in-situ .[28 ] In another study by Sawant et al, CD44v6 staining intensity was detected as significantly
high in recurrent OSCC as compared with primary tumors (p < 0.001), and it also correlated with poor survival (p < 0.001). Furthermore, in combination, patients with increased CD44 concentration
in serum and CD44v6 expression in tumors significantly correlated with local recurrence
(p < 0.001) and poor survival (p < 0.001).[19 ] Okuyama et al and Fonseca et al showed that high CD44 expression was associated
with cervical lymph node metastasis.[29 ]
[30 ] Tandon et al observed an upregulation of CD44 was detected in 48% of well-differentiated
OSCC followed by a reduced expression in moderately differentiated and poorly differentiated
OSCCs and the expression correlated with the tumor size (T) in 23% of cases and with
lymph node metastases (N) in 42% of cases (p ≤ 0.05). This suggests an association of CD44 with tumor aggressiveness and EMT related
to loss of cell adhesion in a subset of OSCC.[31 ] Ortiz et al reported that CD44 immunoexpression was a significant predictor of lymph
node metastasis.[32 ]
In the present study, patients with Grade 3 HNSCC and high CD44 expression had a poor
prognosis compared with patients with Grade 1 and Grade 2 tumors. de Moraes et al
found that the 5-year cancer-specific survival rates for the CD44-negative and CD44-positive
groups were 74 and 38%, respectively, although this difference did not reach statistical
significance (p = 0.052).[33 ] OSCC patients with CD44 immunoreactivity of >30% had 2.08-fold increased risk of
death compared with those with <30% CD44 immunoreactivity.[34 ] Boxberg et al demonstrated that CD44 overexpression within the tumor core region
and in lymph node metastases was identified as an independent prognostic factor for
poor overall, disease-specific, and disease-free survival in subsets of patients with
advanced OSCC.[35 ] Morand et al reported that CD44 expression was associated with increased DOI (p = 0.018) and worse disease-specific survival (p = 0.041). A 1 mm increase in DOI was correlated with 31.1% higher chance of metastasis.
They explained that tumors with greater DOI showed stronger CD44 expression suggestive
of EMT and CSC signaling enabling the tumor to invade and metastasize to the nearby
lymph node. This suggests the importance of CD44 expression at the invasive tumor
front in early OSCC and its relation with tumor invasion and regional metastasis.[36 ]
Cohen et al, in a prospective study, showed that universal CD44 gross tissue staining
and total protein (TP) levels ≥ 1 mg/mL demonstrated poorer PFS, with the latter also
affecting OS. Poorer survival was associated with soluble CD44 ≥ 5.33 ng/mL and TP
≥ 1 mg/mL. Higher solCD44 and protein were significantly correlated with disease progression
(solCD44: p = 0.008, protein: p = 0.003). Patients who died exhibited significantly higher solCD44 and protein as
compared with those who lived (solCD44: p = 0.016, protein: p = 0.001).[37 ] In a systematic review and meta-analysis including 15 studies conducted by Chen
et al, it was found that the total percentage of CD44 expression was 57.8%, with 49.3%
in oral cancer patients, 66.4% in the pharynx, and 54.7% in larynx cancer patients
expressing CD44. No significant correlation between clinical features and CD44 expression
was revealed for oral cancer patients, but CD44 was shown to be associated with advanced
T categories, worse N categories, higher tumor grades, and 5-year OS rates in patients
with laryngeal and pharyngolaryngeal cancer.[10 ]
The binding of CD44 to HA promotes the transcription of several MMPs and initiates
focal matrix degradation. The intracellular domain of CD44 regulates a few target
genes CD44, cyclin D1, MMP-9, HIF-2α, c-myc, and Twist1. Binding of cytoskeletal proteins
ankyrin and ERM (ezrin, radixin, and moesin) to the cytoplasmic tail of CD44 links
CD44 to actin-cytoskeleton with functions involved in the regulation of epithelial-mesenchymal
transition in head and neck carcinogenesis.[38 ]
Computational approaches have been beneficial for researchers as they provide access
to an exhaustive collection of data and facilitate analysis within a short time and
cost-effective way. As with any in-silico study, the current study also has limitations.
There is a lack of enough clinical data to analyze the association between CD44 and
HNSCC. The dataset selected had individuals from different ethnic groups which made
data analysis and interpretation to be more generic rather than specific. CD44 can
undergo isoform switching in tumor cells. The association of CD44 variants with the
clinicopathological characteristics of the study population is not addressed. However,
there are several in-silico studies done to identify novel genetic markers which can
be applied for population-based screening.[12 ]
[39 ]
[40 ]
[41 ]
[42 ]
