Review

MUC4 as a diagnostic marker

in cancer
Subhankar Chakraborty, Maneesh Jain, Aaron R Sasson & Surinder K Batra†
†University
of Nebraska Medical Center, Eppley Institute for Research in Cancer,
1. Introduction Department of Biochemistry and Molecular Biology, Department of Surgery, Omaha,
2. MUC4 in the diagnosis of NE 68198-5870, USA
cancers of the upper
aerodigestive tract Background: Mucins are high molecular mass glycoproteins whose role
3. MUC4 in the diagnosis of
in diagnosis, prognosis and therapy is being increasingly recognized
gastrointestinal malignancies owing to their altered expression in a variety of carcinomas. MUC4, a
membrane-bound mucin encoded by a gene located on chromosome
4. MUC4 in the diagnosis of
locus 3q29, is aberrantly expressed in several cancers including those of the
reproductive tract malignancies
bile duct, breast, colon, esophagus, ovary, lung, prostate, stomach and
5. Correlation of subcellular
pancreas. Objective: This review considers the potential use of the
distribution of MUC4 with
MUC4 expression pattern in the diagnosis and prognosis of various cancers.
clinicopathologic characteristics
Results/conclusion: MUC4 expression is a specific marker of epithelial tumors
6. Summary and its expression correlates positively with the degree of differentiation in
7. Expert opinion several cancers. Importantly, MUC4 has emerged as a specific marker of
dysplasia, being expressed in the earliest dysplastic lesions preceding several
malignancies, including lethal pancreatic cancer. The presence of MUC4-specific
antibodies in the serum and of the transcript in peripheral blood mononuclear
cells of cancer patients raises the possibility of it emerging as a new
diagnostic biomarker for bedside application in high-risk individuals and
those with established cancer.

Keywords: cancer, diagnosis, MUC4

Expert Opin. Med. Diagn. (2008) 2(8):891-910

1. Introduction

There has been a steady increase in the incidence of cancer worldwide in recent
years. According to estimates from the American Cancer Society, > 1.4 million
new cases of cancer were diagnosed in 2007 and an estimated 560,000 patients
died from cancer-related causes in the US alone in the same year. Although the
total number of cases has increased, mortality resulting from cancer has shown a
slight, albeit welcome, decline [1]. Part of this is attributable to the development
of more potent anticancer drugs. A key contributing factor is the improvement in
diagnostic modalities that have permitted the detection of tumors at an early,
localized and surgically resectable stage. Although imaging modalities continue to
be the prime diagnostic tools being used, their accuracy and sensitivity have
reached almost saturation limits. In such a scenario, tumor markers, molecules
that show either an altered expression or a compositional change in tumor cells
compared with healthy or inflamed tissues, have come into the limelight. They
are useful as screening tools to identify patients suspected of harboring occult
tumors for subsequent confirmation with other non-invasive and invasive
diagnostic tests. Mucins are key biomolecules whose expression is deregulated
in several cancers. This altered expression pattern has been widely examined for
possible diagnostic application in several cancers.
MUC4 is a membrane-bound mucin that is normally expressed by the
epithelial lining in several tissues, including the conjunctiva, most of the
aerodigestive tract and the endocervix. Recently, MUC4 has been shown to

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MUC4 as a diagnostic marker in cancer
MUC4-α
SP NTR
TRD
Figure 1. Schematic structure of MUC4 apomucin.
CT: Cytoplasmic tail; Cys: Cysteine-rich regions; EGF: Epidermal growth factor-like domains; GDPH: Glycine-Aspartate-Proline-Histidine cleavage site;
NTR: Non-tandem repeat domain containing three imperfect repeats of 123 amino acids; SP: N-terminal signal peptide; TM: Transmembrane domain;
TRD: Tandem repeat domain.
be aberrantly expressed in several malignancies and
growing evidence supports its role as a useful marker for
diagnosis and as a possible target for therapy. This review
focuses on the potential of MUC4 as a biomarker in the
diagnosis and/or prognosis of cancer.
1.1 Mucins and MUC4
Body passages, comprising the aerodigestive and urogenital
tracts, are exposed to a multitude of agents, with the poten-
tial for damaging the delicate mucosal linings that protect
these passages. Several mechanisms are used by the body in
creating a protective barrier to counter this threat, including
epithelial tight junctions, microbicidal enzyme-containing
body fluids and commensal flora harbored outside and
within the body. A key role in this process is also played by
a hydrophilic mucus layer, which covers, protects and lubri-
cates the luminal surfaces of hollow passages. The viscoelas-
tic properties of mucus are determined chiefly by the
presence of mucins, which are heavily glycosylated, high
molecular mass glycoproteins, synthesized and secreted by
epithelial goblet cells and submucosal glands. Mucins are
multifunctional biomolecules. As components of mucus,
they help trap both foreign particles and invading organ-
isms. Additional functions being discovered include a role in
cellular signaling, maintenance of cell integrity and, in the
case of malignant cells, regulation of metastasis and the
resistance to apoptosis [2].
There are essentially two major functional classes of
mucin-type glycoproteins: the secreted or soluble (MUC2,
MUC5AC, MUC5B, MUC6–MUC8 and MUC19) and
the membrane-bound (MUC1, MUC3, MUC4, MUC12,
MUC13, MUC15, MUC17 and MUC20) [3,4]. Membrane-
bound mucins contain a transmembrane domain that
anchors them to the plasma membrane, in addition to a
short cytoplasmic tail [5,6]. The structure and function of
mucins in normal physiology and disease have been reviewed
in several articles [6-9]. MUC genes have a relatively tissue-
specific expression. Malignant transformation is associated
with either their deregulated expression or an alteration in
the structure of their protein backbone (e.g., a variable
892 Expert Opin. Med. Diagn. (2008) 2(8)
MUC4-β
EGF TM
Cys GDPH
Cys CT
number of tandem repeats [VNTR] polymorphism) or
glycosylation pattern [10,11].
MUC4 is a membrane-bound mucin that was first cloned
from a human tracheobronchial cDNA library and a human
pancreatic cancer cell line. The human MUC4 gene has
been mapped to the chromosomal region 3q29 [12-14]. The
protein core is synthesized as a precursor polypeptide, which
is subsequently processed to yield the mature peptide back-
bone, termed the MUC4 apomucin. The amino terminus of
the apomucin consists of a 27-residue signal peptide,
followed by 3 imperfect repeats of a motif that is
126 – 130 amino acids long, and a 554-amino acid unique
sequence. However, the largest domain is the central tandem
repeat domain, comprising a 16-amino-acid-long perfect
tandem repeat. The C-terminal region of MUC4 is divided
into 12 domains, including three epidermal growth factor
(EGF)-like domains. MUC4 is synthesized as a single poly-
peptide, which is postulated to undergo proteolytic process-
ing to yield two subunits, MUC4α (an extracellular
mucin-type glycoprotein) and MUC4β (a membrane-
tethered subunit), which are believed to be non-covalently
associated on the cell surface (Figure 1). The MUC4 gene
encodes numerous alternatively spliced forms, some of which
are devoid of the mucin hallmark, the tandem repeat
array [15]. The biology, evolution, functions and potential
therapeutic role of MUC4 have been the subject of several
reviews [5,8,16,17]. This article focuses on the diagnostic and
prognostic potential of MUC4 in various cancers.
1.2 MUC4 antibodies
Several antibodies have been reported in the literature that
recognize epitopes on either MUC4α (the extracellular
mucin-like subunit) or MUC4β (Figure 1). Hanaoka et al.
developed a rabbit polyclonal antibody to a peptide
constituting the tandemly repeated sequence on MUC4α.
Analysis of surface MUC4 expression in eight lung
cancer cell lines by flow-sorting (with this antibody) with or
without previous sialidase treatment revealed that although
50% of the cell lines were positive following sialidase
treatment, none in the untreated group was positive.

Table 1. Antibodies to MUC4 and its homolog rat ASGP used in studies on human tissues.
Name of antibody Nature Epitope recognized
8G7 Monoclonal (MAb) MUC4α TR
Anti-MUC4 Ab Rabbit polyclonal (PAb) MUC4α TR
Anti-MUC4 Ab Rabbit PAb MUC4α TR
MUC4-CT Rabbit PAb MUC4 cytoplasmic tail
SG9 Rabbit PAb MUC4α TR
M4P Chicken PAb Synthetic MUC4 TR peptide
LuCF12 and LuC18.2 MAb MUC4α TR
4F12 MAb rASGP-2 (rat ASGP-2)
1B1 MAb rASGP-2
15H10 MAb rASGP-1
MAb: Monoclonal antibody; TR: Tandem repeat.
This suggests the possibility that the carbohydrate chains
attached to the tandem repeat region of MUC4 could pre-
vent recognition of the extracellular peptide epitope by the
antibody [18]. Other suggested mechanisms for the failure to
detect surface MUC4 include shedding of either the whole,
or the tandem repeat-containing portion of MUC4α from
the cell surface into the surroundings. We have developed
and characterized a monoclonal antibody, 8G7, that recognizes
an epitope within a 16-amino acid sequence from the tandem
repeat (TR) region of human MUC4 [19]. The antibody
recognized a band > 500 kDa in size on western blot and
its reactivity was specific for MUC4α as evidenced by non-
reactivity against purified MUC1 and strong reactivity to a
16 amino-acid MUC4 tandem repeat peptide. Epitope
mapping by competitive ELISA showed that the binding of
the antibody to MUC4 tandem repeat peptide was com-
pletely inhibited by the 16-amino acid immunizing peptide,
suggesting that it recognizes a peptide epitope on native
MUC4. When tested for immunoreactivity against tissues,
the antibody showed a strong cytoplasmic staining, consis-
tent with the accumulation of mucus-containing secretory
granules in the cytosol of malignant cells. Confocal analysis,
however, showed a strong membrane staining with moderate
cytoplasmic staining in the MUC4 overexpressing pancreatic
cancer cell line CD18/HPAF. A possible explanation for this
could be the difference in subcellular distribution of mucins
in tissues versus that seen in cancer cell lines maintained
in vitro. Lopez-Ferrer et al. reported and characterized a rabbit
polyclonal antibody that specifically recognized a 16-amino
acid sequence from the tandem repeat of MUC4 but did
not react with sequences from the tandem repeat regions of
MUC1 – MUC8 [20]. MUC4 is a homolog of ascites sialo-
glycoprotein (ASGP)-1 and ASGP-2, which form the rat
sialomucin complex (SMC) [21]. A monoclonal antibody
1G8, raised against the rat AGSGP-2 (homologous to
human MUC4β), was shown to recognize human MUC4β,
producing bands with molecular mass similar to the rat
Expert Opin. Med. Diagn. (2008) 2(8)
Chakraborty, Jain, Sasson & Batra
Immunizing peptide sequence Ref.
STGDTTPLPVTDTSSV [19]
TSSASTGHATPLPVTD [20]
PVTD-TSSVSTGHATSLPVTD-TSSV [18]
SGARFSYFLNSAEAL [34]
STGDTTPLPVTDTSSV [110]
– [33]
TSSVSTRHATSLPVTD [41]
First 53 amino acids of rASGP-2 [111]
Between amino acid 53 – 683 of rASGP-2 [111]
O-linked carbohydrates on mature rASGP-1 [111]
ASGP-2. The antibody recognizes a unique sequence in the
non-tandem repeat region of rat ASGP-2 between the
N-terminal amino acid residue 53 and the transmembrane
domain of ASGP-2 [22]. Surprisingly, the antibody also rec-
ognized a band in heart tissue and weaker bands in the
kidney, liver and skeletal muscle, tissues that are negative for
MUC4 at the transcript level. In immunoblots, the antibody
recognized a protein with a molecular mass similar to that
expected for MUC4β. When used to immunoprecipitate the
proteins in MUC4β transfected HCT11 cells, the antibody
recognized two bands of 250 and 140 kDa. Given that
MUC4β has a predicted molecular mass of 80 kDa, it was
suggested that these bands represent two differentially glyco-
sylated forms of MUC4β. In vitro translated MUC4β was
also detected by the 1G8 antibody, suggesting that it recog-
nizes the peptide rather than the oligosaccharide epitope [23].
A monoclonal antibody (15H10) recognizing the O-liked
oligosaccharides on rat ASGP-1 (homologous to human
MUC4α) also recognized human MUC4 in formalin-fixed
paraffin-embedded tissues from normal and squamous cell
carcinoma bearing upper aerodigestive tract tissues [24].
MUC4 is characterized by a high degree of polymorphism
in its central TR region [25], which makes the antibodies
directed against the tandem repeats unsuitable for quantita-
tive analysis. Hence, antibodies directed against a non-
glycosylated region of the MUC4 protein are a pressing
need, especially for quantitative studies of MUC4 mucin in
body fluids [26]. A list of antibodies against MUC4 and
ASGP, its rat homolog, is summarized in Table 1.
1.3 MUC4 expression in normal tissues and
inflammatory conditions
MUC4 is expressed at high levels by several normal epithelia
including those of the conjunctiva [27], cornea [28], the tear
film [29], colon [30], fetal lungs, surface epithelium of the
adult respiratory tract from the trachea to the collecting
ducts [31], inferior nasal turbinates [32], eustachian tube [33],
893
MUC4 as a diagnostic marker in cancer
the striated and excretory ducts of the parotid and subman-
dibular salivary glands [34] and the endocervix [35]. A moder-
ately high level of expression is observed in the healthy adult
lungs, testes [36] and prostate [37], whereas low MUC4 levels
occur in the healthy ileal mucosa [38]. An alteration in
MUC4 expression is also observed in several disease states.
Benign conditions characterized by an upregulation of
MUC4 (at both the transcript and protein levels) include
inflammatory middle ear effusions, nasal polyps and hyper-
trophied adenoids in the upper aerodigestive tract [32-34,39,40],
follicular adenoma of the thyroid gland and nodular
goiter [41], the rare cap polyposis of the colon (mRNA
only) [42], in the caecum and colon of dextran sodium
sulfate-treated mice with features of acute colitis (mRNA
only) [43] and ulcerative colitis [44]. Conversely, a decreased
expression of the apomucin mRNA is observed in a mouse
model of allergic conjunctivitis [45]. In addition to an
alteration in its expression, an allelic variant (an A585S
polymorphism in the Von Willebrand domain of MUC4)
of the apomucin has been reported to be significantly
overrepresented in patients with ulcerative colitis [46].
MUC4 has been demonstrated to be aberrantly
expressed in several malignancies and has now been widely
investigated as a possible diagnostic marker (summarized in
Tables 2 – 6). This article examines the potential of MUC4
as a diagnostic and/or prognostic marker in cancer. For the
sake of convenience, the various malignancies are divided
into three groups, those arising from the upper aerodigestive
tract, the rest of the gastrointestinal system and those
originating from the reproductive system.
2. MUC4 in the diagnosis of cancers of the
upper aerodigestive tract
According to the National Cancer Institute, the upper
aerodigestive tract comprises the combined organs and
tissues of the respiratory tract and the upper part of the
digestive tract (including the lips, tongue, major salivary
glands, gums and adjacent oral cavity, floor of the mouth,
tonsils, oropharynx, nasopharynx, hypopharynx, the nasal
cavity, accessory sinuses, the middle ear and the larynx) [47].
MUC4 is expressed throughout the normal upper aerodiges-
tive tract (UADT) mucosa with a specific staining in the
most superficial and the suprabasal layers. However, no MUC4
expression is detected in the basal layers of the stratified
epithelium [24]. Cancers of the UADT, which constitute
∼ 4% of all malignancies [48], show not only an alteration
in MUC4 expression but also a change in its distribution in
the stratified epithelium (summarized in Table 2).
2.1 MUC4 expression in squamous cell carcinoma of
the UADT
Squamous cell carcinomas (SCCs) constitute > 95% of all
UADT cancers. Nearly 85% of these cancers showed at
least some degree of MUC4 staining in one study [49].
894 Expert Opin. Med. Diagn. (2008) 2(8)
The malignant epithelium, and more importantly the dys-
plastic mucosa adjacent to the areas of SCC, showed a dif-
fuse pattern of MUC4 staining extending throughout the
entire thickness of the epithelium. More importantly, the
normal mucosa distant from the areas of SCC preserved its
characteristic suprabasal staining pattern. Eleven of the
12 cervical lymph node biopsies harboring a metastasis from
a primary SCC were also positive for MUC4. Although no
correlation was evident between MUC4 expression and
either tumor grade or patient characteristics, the MUC4-
expressing tumors did have a lower rate of recurrence fol-
lowing resection and afforded the patients a significant
survival advantage. This suggests that the presence of MUC4
might predict a more favorable prognosis in SCCs arising in
the UADT. Further, a diffuse MUC4 staining in the mucosa
adjacent to an area of dysplasia could be an early marker of
subsequent malignant transformation [24].
2.2 MUC4 expression in salivary gland tumors
Salivary gland cancers comprise nearly 3% of all the head
and neck cancers. MUC4 is highly expressed in both the
major (parotid, submandibular and sublingual) and minor
salivary glands. By in situ hybridization, Liu et al.
demonstrated MUC4 expression in the cells lining the
striated and major excretory ducts (in the parotid and
submandibular glands) and in those lining the serous acini
(in submandibular glands only). A similar pattern of MUC4
expression was also observed by immunohistochemistry.
Further, MUC4 was also detected in secretions from the
all of the three major salivary glands [34]. MUC4
immunostaining was also detected in all the benign
salivary gland neoplasms examined (six out of six) and in
most of the salivary gland carcinomas in one study [24].
Mucoepidermoid cancer (MEC) is the most common
type of salivary gland malignancy. It is also characterized by
an overexpression of MUC4. MECs are unique in that
they exhibit a broad range of aggressiveness, ranging
from indolent localized tumors to highly invasive neoplasms
prone to local recurrence and metastasis. They are composed
chiefly of four types of cells – epidermoid, glandular,
mucinous and intermediate. Although MUC4 is generally
considered to be a marker of low-grade tumors, it was
found to be overexpressed in both low- and high-grade
MECs (55% positivity in high-grade tumors versus 91% in
low-grade tumors) [24]. Significantly, patients whose tumors
were MUC4-positive had a higher survival rate and a lower
rate of recurrence following surgery. In a separate study,
however, Handra-Luca et al. found no correlation between
MUC4 positivity and prognosis [50].
In addition to the increased expression, an alteration in
the distribution of MUC4 was also observed in the MECs.
Compared with the normal salivary glands where MUC4
was chiefly localized to the apical membrane of epithelial
cells lining the excretory ducts, the staining in MECs was
more diverse. It ranged from an apical staining in glandular
 Table 2. Expression pattern of MUC4 and its correlation with clinicopathologic parameters in upper aerodigestive tract malignancies.

Cancer Detection Sites of MUC4 expression Association with Prevalence of MUC4 positivity Association with Ref.
method stage/differentiation prognosis

Barrett’s esophagus RT-PCR NA NA Relative transcript levels Normal ND [62]

(precursor of esophageal epithelium: 1.1;
esophageal BO without dysplasia – 0.62;
adenocarcinoma) High-grade intraepithelial neoplasia:
1.27; Adenocarcinoma: 1.8
Mucoepidermoid IHC Polarized glandular cells: apical ↓ Expression in 79% (n = 63) No [50]
carcinoma of the membrane; Non-polarized high-grade neoplasms
salivary glands epidermoid and clear cells:
entire membrane
Mucoepidermoid IHC Supranuclear cytoplasm and apical ↑ Expression observed Normal salivary glands – 86%; ↑ MUC4 expression [49]
carcinoma of the membrane of normal ductal and in the WD low-grade MEC – 95% associated with lower rate of
salivary glands malignant cells MECs recurrence, a longer DFI and
an overall better prognosis
Mucoepidermoid IHC 15H10 MAb: both serous and MUC4 expression 1G8 MAb: Low-grade MECs: 82%; MUC4 positivity correlates [107]
carcinoma of the mucinous acini and ducts in normal decreases with Intermediate grade: 67%; High grade: with reduced risk of death
salivary glands salivary glands; 1G8 MAb: weaker increasing tumor grade 18% 15H10 MAb: Low grade: 91%; (p = 0.05). MUC4-negative
staining in the normal glands but Intermediate grade: 100%; tumors were more likely to

Expert Opin. Med. Diagn. (2008) 2(8)

strong staining of the tumor; High grade: 55% recur (p = 0.03)
Both MAbs: strong staining in
normal endothelial cells
SCC of the UADT* IHC Normal mucosa adjacent to invasive No correlation with 12.3% UADT tumors (n = 154) Positive membrane staining [24]
cancer: membrane staining of both differentiation; → 10% cells positive for MUC4 correlates with
basal and suprabasal cells Normal ↑ MUC4 positivity in ↑ survival (p = 0.06) and
UADT mucosa: membrane staining advanced stage tumors reduced risk of recurrence
in the suprabasal layer only (79% in stage III versus (p = 0.03)
66% in stage II)

*Included tumors from the oral cavity (excluding lips) oropharynx, hypopharynx and the larynx.
BO: Barrett’s esophagus; DFI: Disease-free interval; IHC: Immunohistochemistry; MAb: Monoclonal antibody; MEC: Mucoepidermoid carcinoma; NA: Not applicable; ND: Not discussed; RT-PCR: Real-time polymerase
chain reaction; SCC: Squamous cell carcinoma; UADT: Upper aerodigestive tract; WD: Well differentiated.

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Chakraborty, Jain, Sasson & Batra
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Table 3. Expression pattern of MUC4 and its correlation with clinicopathologic parameters in pulmonary and pleural malignancies.

Cancer Detection Sites of MUC4 expression Association Prevalence of MUC4 positivity Association with prognosis Ref.
method with stage/
differentiation

Lung cancer Northern Highest expression in ND [55]

Lung cancer
(non-small cell type)
blotting adenocarcinoma
IHC Adenocarcinoma: predominantly
both in cytosol and membrane
SCC: central, WD portion of the
SCC and necrotic areas
Adenosquamous carcinoma:
predominantly in the
ND Adenocarcinoma – 81% (n = 187); ↑ MUC4 expression associated [53]
SCC – 78% (n = 88); Adenosquamous with longer survival in stages I
carcinoma – 75% (n = 8); Large cell and II adenocarcinoma
carcinoma – 60% (n = 60) (p = 0.11)
MUC4 as a diagnostic marker in cancer

adenocarcinoma portion
Lung cancer IHC Adenocarcinoma: both cytosolic Lower in Normal bronchotracheal epithelium – 100% DFI and survival significantly [109]
(non-bronchioloalveolar and membrane staining, with the stage 1A than (n = 185). No MUC4 expression in the normal higher in patients with high
type ≤ 3 cm) latter being more common in other stages alveolar epithelium and stroma MUC4 expression (≥ 25%
Adenocarcinoma: 4 + (0.5% cases), 3 + (7%), cancer cell positivitiy) compared
2 + (5.9%) and 1+/0 (86.5%); High with low MUC4 expression
MUC4-expressing cells (intensity ≥ 2 +) detected (≤ 25% positivity)
more frequently in areas of stromal invasion Longer survival in stage 1A cases
with low MUC4 expression
Lung cancer IHC, Apical membrane of normal No Adenocarcinoma – 54% (protein), 78% (mRNA); No [18]
northern bronchioles SCC – 67% (protein), 71.2% (mRNA)
blot Strong cytoplasmic staining in
tumor cells
Lung cancer ISH Normal respiratory mucosa: Higher in Expressed by preinvasive (↑ in basal cell No [31]

Expert Opin. Med. Diagn. (2008) 2(8)

(a) Diffuse cytoplasmic staining in
all layers except basal layer
(b) Moderate to strong in the ducts
of normal submucosal glands
(c) Undetectable in alveoli; CIS:
Similar distribution but weaker
intensity; SCC: Moderate to strong
expression in the central, WD
portion
Primary pleural ISH, IHC Primary adenocarcinoma: diffuse
malignant cytoplasmic staining, rarely
mesothelioma membranous
Adenocarcinoma secondarily
involving the pleura: MUC4
staining strongest just
underneath pleura
WD cells (n = 1) and mucus cell hyperplasia [n = 2]
than in squamous metaplasia [n = 10] and
dysplasia [n = 4]) lesions, CIS (n = 5) and i
nvasive SCC (n = 20)
Malignant mesothelioma (n = 39) and effusions ND [56]
(n = 2) – 0%; Normal and hyperplastic mesothelial
cells (n = 32) – 0%; Lung adenocarcinoma tissues
(n = 40) – 89%; Pleural effusions (n = 32) – 91%;
Normal type II pneumocytes (n = 7), pneumocyte
hyperplasia (n = 10) and atypical hyperplasia
(n = 7) adjacent to the adenocarcinoma were
also positive

CIS: Carcinoma-in situ; DFI: Disease-free interval; IHC: Immunohistochemistry; ISH: In situ hybridization; ND: Not discussed; SCC: Squamous cell carcinoma; WD: Well differentiated.
 Table 4. Expression pattern of MUC4 and its correlation with clinicopathologic parameters in gastrointestinal malignancies.

Cancer Detection Sites of MUC4 Association with Prevalence of MUC4 positivity Association with prognosis Ref.
method expression stage/differentiation

Signet-ring cell IHC ND ND Colorectal (n = 11) – 91%; Gastric ND [112]

carcinoma (gastric, (n = 21) – 57%; Breast (n = 6) primary
colorectal and tumors – 0% (iv) Metastatic colorectal (n = 2)
breast) [100%], gastric (n = 4) [100%] and breast (n = 3)
tumor [33%]
Intraductal papillary ISH ND No 70% – [86]
mucinous tumor of
the pancreas
Pancreatic cancer Microarray ND No > 5-fold ↑ in expression in PC compared with ND [113]
normal pancreas and CP
Pancreatic cancer IHC Cytoplasmic ND Pancreatic cancer (n = 71) – 77% ND [78]
and membrane Chronic pancreatitis (n = 18) – 22%
Pancreatic cancer IHC Cytoplasmic No Invasive ductal adenocarcinoma (n = 135) – 31.9% Survival in high MUC4/high MUC4 and [108]
and membrane Normal pancreas – 0% high p27-expressing IDC significantly
lower than low MUC4/low MUC4 and
low p27-expressing IDC
MUC4 expression in IDC was a
predictor of poor prognosis

Expert Opin. Med. Diagn. (2008) 2(8)

Pancreatic cancer RT-PCR, NA High levels in well PC tissues – 75% ND [76]
RNA slot and MD but absent in PC cell lines – 73%
blot PD cancer cells. CP (n = 10) – 0%
No correlation with Normal pancreas (n = 7) – 0%
tumor stage
Pancreatic cancer IHC Cytoplasm ND PanIN-1: 17%; PanIN-2: 36%; PanIN-3: 85%; ND [81]
PC: 89% Normal ducts (n = 246) – 0%; CP
(n = 22) and demonstrating signs of intense
inflammation – 60%; Atropic ducts filled with
inspissated secretions (n = 7) – 57%
Colon cancer Northern NA None Normal colon – 100% (n = 9); Colon cancer – 55% ND [30]
blot (n = 8), comparable or increased MUC4 expression

CP: Chronic pancreatitis; IHC: Immunohistochemistry; ISH: In situ hybridization; MD: Moderately differentiated; NA: Not applicable; ND: Not discussed; PC: Pancreatic cancer; PD: Poorly differentiated; RT-PCR: Real-time
polymerase chain reaction.

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Chakraborty, Jain, Sasson & Batra
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Table 5. Expression pattern of MUC4 and its correlation with clinicopathologic parameters in gynaecologic malignancies.

Cancer Detection Sites of MUC4 expression Association with Prevalence of MUC4 positivity Association Ref.
method stage/differentiation with prognosis

Cervical SCC IHC, ISH and Normal ectocervix: diffuse cytosolic
western blotting staining in the parabasal layer
Normal endocervix: cytosolic staining
in the normal columnar cells and
endocervical glands;
Cervical dysplasia: diffuse cytoplasmic
staining throughout the thickness of
Highest in high-grade
intraepithelial neoplasia
(CIN grade II and III)
CIN grade I – 89% (n = 19), NA [90]
75 ± 40% cells positive; CIN-II
and -III – 100% samples, with 87 and
85.5% cells positive, respectively
MUC4 as a diagnostic marker in cancer

the epithelium
Cervical cancer CGH, Microarray, NA NA Cervical cancer: 2.5 – 10.9-fold NA [89]
RT-PCR increase compared with healthy
cervical tissue
Mucinous ovarian ISH Basal and/or apical membrane in NA Benign mucinous cystadenomas – 81% ND [95]
tumors columnar mucin-secreting tumor cells (n = 11); Borderline mucinous
tumors – 80% (n = 10)
Ovarian cancer Northern blotting NA ↓ With increasing stage Serous tumors – 25% (n = 16) ↑ Expression
Mucinous tumors (n = 2) and correlates with
endometrioid (n = 1) cancers – 100% longer survival
Ovarian cancer IHC Higher in early stage (100% Stages I and II (n = 19) – 100% No [105]
samples) than late stage Stages III and IV (n = 43) – 88%
(88% positive) Mucinous tumors (n = 5) – 100%

Expert Opin. Med. Diagn. (2008) 2(8)

Ovarian/primary Microarray, IHC, OC/PPC: cytoplasmic and membrane ND OC/PPC (n = 122) – 96% ND
peritoneal serous RT-PCR staining of tumor cells in both solid DMPM (n = 30) – 3% (IHC)
carcinoma tumors and effusions; DMPM: rarely
(OC/PPC) positive
Uterine cancer IHC Cervical dysplasia: entire thickness Expression detected in Endocervical adenocarcinoma-in situ ND [10]
staining in dysplastic squamous WD adenocarcinomas. PD [n = 8] (AIS) – 38%; Endocervical
epithelium cancers were negative (n = 9) – 75% and; Endometrial
Normal ectocervical epithelium: adenocarcinomas (n = 8) – 44%;
parabasal staining Normal endocervical epithelium and
Normal endocervix: cytoplasm and benign endocervical lesions – 100%
luminal portion of membrane of glands
Endometrial adenocarcinoma: focal
staining

AIS: Adenocarcinoma-in situ; CGH: Comparative genomic hybridization; CIN: Cervical intraepithelial neoplasia; DMPM: Diffuse peritoneal malignant mesothelioma; IHC: Immunohistochemistry; ISH: In situ hybridization;
NA: Not applicable; ND: Not discussed; PD: Poorly differentiated; SCC: Squamous cell carcinoma; WD: Well differentiated.

Table 6. Expression pattern of MUC4 and its correlation with clinicopathologic parameters in other malignancies.
Cancer Detection Sites of MUC4
method expression
Breast cancer IHC ND
Prostate cancer IHC Diffuse embrane
and cytoplasmic
staining pattern
Prostate cancer IHC (TMA) ND
SCC of the skin Microarray NA
IHC: Immunohistochemistry; NA: Not applicable; ND: Not discussed; SCC: Squamous cell carcinoma; TMA: Tissue microarray.
cells to a pan-membranous staining in the intermediate,
clear and epidermoid cells [50]. Further, a decrease in MUC4
positivity with increasing grade (and hence a loss of
differentiation) was also observed in the tumors, being
91, 80 and 50% in low, intermediate and high-grade MECs,
respectively [50]. Another study investigating a possible
correlation of the mucin expression profile in MECs with
clinicopathologic characteristics found that 95% of the
MECs overexpressed MUC4. Specifically, it was detected in
the supranuclear cytoplasm and the apical membrane of all
four cell types (outlined earlier) [49]. Most significantly, a
high expression of MUC4 was more commonly observed in
low-grade tumors (p = 0.002), and the presence of MUC4
was associated with a lower recurrence following resection
(20% recurrence rate in MECs expressing MUC4 in > 50%
cells versus 66% in those with fewer MUC4-positive cells)
and a longer disease-free survival (96 months in MUC4
expressing MECs compared with 28 months in MUC4
non-expressing MECs) [49].
Thus, MUC4 overexpression in MECs of the salivary
gland, and significantly its positive correlation with tumor
grade and a favorable prognosis, suggest its possible utility as
a prognostic marker in this malignancy.
2.3 MUC4 in the diagnosis of lung cancer
Lung cancer is one of the most commonly diagnosed malig-
nancies in the US and Europe [51]. About 75% of all cases
are of the non-small cell type. These include histologic sub-
types such as squamous cell carcinoma, adenocarcinoma and
large-cell undifferentiated adenocarcinoma (Table 3). The
remaining 25% are of the small-cell variety. Histologically,
adenocarcinomas form glandular structures with mucin
secretion, whereas SCCs are characterized chiefly by
keratinization [31]. MUC4 is expressed in the normal adult
lungs and strongly overexpressed in non-small cell lung
Expert Opin. Med. Diagn. (2008) 2(8)
Chakraborty, Jain, Sasson & Batra
Association with Prevalence of MUC4 Association Ref.
stage/differentiation positivity with prognosis
More common in 95% in breast cancer None [98]
low-grade tumors
None (i) Prostate cancer- 26.3% ND [37]
positivity {1+(21%),
+(5.3%), no 3+ staining}
(ii) Adjacent normal/
BPH- 84.2 % positivity
{1+ (28.9%), 2+ (18.4%),
3+(36.8%)}
NA No expression in both ND [100]
normal (n = 40) and cancer
tissues (n = 120)
NA NA NA [114]
cancer tissues and lung cancer cell lines, but rarely in small
cell lung cancer (3 positive cases out of 12 in one study) [52].
Interestingly, a high level of MUC4 expression was more
characteristic of adenocarcinoma and adenosquamous
carcinoma than squamous cell carcinoma or large cell
carcinoma [53]. Further, patients with stages I and II
adenocarcinomas with higher levels of MUC4 reactivity
showed a trend towards increased survival compared with
those with lower MUC4 immunoreactivity. According to
another report, MUC4 was detected in 67% of the
adenocarcinomas at the protein level, whereas the transcript
was identified in 72% of the samples [18]. In a multivariate
analysis of 185 cases of non-bronchoalveolar type lung
adenocarcinomas < 3 cm, stage 1A patients whose tumors
showed a high MUC4 expression (defined as positive
staining in ≥ 25% of neoplastic cells) had a significantly
lower survival than those with low MUC4 expression.
Univariate analysis also revealed that high MUC4 expression
correlated with vascular invasion and a greater frequency of
stromal invasion [54]. An earlier report had also shown that
MUC4 was the second most abundant mucin gene (after
MUC1) expressed in lung adenocarcinomas [55]. An intense
overexpression of the MUC4 transcript was also reported in
pre-malignant lesions of the lungs, including basal cell and
mucous cell hyperplasia and squamous metaplasia with or
without accompanying dysplasia [18]. In a study by
Llinares et al. [56], MUC4 protein was expressed in 32 out
of 35 lung adenocarcinomas but in none of the 41 malignant
mesotheliomas or the 32 samples of benign mesothelial cells.
A distinction between primary pleural malignant mesothe-
lioma and pleural infiltration by a spreading lung cancer is
crucial owing to the association of the former with an
occupational exposure to asbestos. Further, the two condi-
tions often present an identical clinical picture and are
difficult to distinguish histologically [57]. MUC4 positivity
899
MUC4 as a diagnostic marker in cancer
was characterized by a diffuse cytoplasmic staining and, less
frequently, membrane staining. In pulmonary adenocarcinomas
with weak to moderate MUC4 staining, MUC4 expression
was more frequent in the cancer cells invading the pleura,
suggesting that MUC4 may play a role in tumor progression
in lung adenocarcinoma. In addition, MUC4-positive
cells were also detected in 8 of the 10 cases of lung cancer-
associated pleural effusions. However, none of the pleural
fluid samples from malignant mesothelioma showed any
expression of the mucin. From this study, MUC4 emerged
not just as a highly specific and sensitive marker for lung
adenocarcinoma, but its differential expression in adenocar-
cinoma-associated pleural effusions suggested that it could
be useful as a marker to detect carcinomatous cells in pleural
effusions. The pattern of MUC4 expression in lung cancer
cell lines supports its role as a regulator and marker of
differentiation, with higher levels being detected in the
well-differentiated cell lines [58]. This observation is
supported further by the finding in cultured airway epithe-
lial cells wherein the well-differentiated but not the poorly
differentiated cells expressed the MUC4 transcript when
analyzed by northern blotting [59].
Hanaoka et al. reported that anti-MUC4 immunoglobulin
was detectable in the sera of lung cancer patients. Anti-MUC4
antibody was detected in the sera of lung cancer as well as
control patients (normal, breast cancer, tuberculosis and
metastatic renal cell carcinoma of the lung). However, antibody
titers were significantly higher in the sera from lung cancer
patients compared with the control group. Further, when an upper
limit for normal was defined and designed to give 100%
specificity, 6 of the 21 patients with lung cancer and none of
the controls were positive. Further, although all the MUC4-
positive sera contained the IgM isotype of the anti-MUC4
antibody, three samples also contained an IgG isotype [18].
These observations raise the possibility that MUC4
immunostaining in lung biopsies could serve to identify
potentially malignant lesions. Further, the detection of anti-
MUC4 antibodies might be useful as a serum-based marker
for the diagnosis of pulmonary adenocarcinoma.
3. MUC4 in the diagnosis of gastrointestinal
malignancies
Cancers of the gastrointestinal (GI) tract are widely
prevalent around the world and second in overall incidence
to malignancies affecting the genital tract. An alteration
in the expression of several mucins has been demonstrated
in malignancies arising from the GI tract, including cancers
of the esophagus, colon, stomach and the pancreatobiliary
tract (Table 4).
3.1 MUC4 in esophageal cancer
The normal esophageal mucosa is lined by a non-keratinized
stratified squamous epithelium. MUC1 and MUC4 are the
chief mucins expressed by the stratified epithelium. However,
900 Expert Opin. Med. Diagn. (2008) 2(8)
in response to chronic damage from irritants such as
alcohol, acid and tobacco, this stratified epithelium can
undergo malignant transformation. The resulting esophageal
carcinoma is classified into chiefly two histologic types:
squamous cell carcinoma and adenocarcinoma. The latter
usually develops from a precursor metaplastic lesion known
as ‘Barrett’s esophagus’ (BO) (hence it is also sometimes
called Barrett’s adenocarcinoma).
An increase in MUC4 transcript expression was observed
in all esophageal SCCs in one study [60]. The strongest signal
was localized to the superficial, most well-differentiated part
of the transformed epithelium. However, the expression was
patchier in the areas of Barrett’s adenocarcinoma [60]. BO is
the result of an abnormal repair of epithelial lesions that
are induced in the esophageal mucosa by persistent
gastroesophageal reflux. It is characterized by metaplasia of
the normal stratified squamous epithelium to columnar
epithelium. An onset of high-grade intraepithelial neoplasia
(HGN) in the metaplastic epithelium of BO is at present
the only marker used in the surveillance of patients to
identify those at a high risk for the development of
esophageal adenocarcinoma. However, the efficiency of
this process is thwarted by sampling errors, often due to
the difficulty in identifying an area of HGN. This is
compounded by the absence of focal neoplastic changes in
inadequate biopsies. This has made the identification of
markers of early dysplastic change in BO patients a
pressing requirement [60]. Most secreted mucins are observed
to be upregulated in BO, whereas in adenocarcinoma the
reverse is true, with a trend towards a reduced expression of
mucin genes [61]. In one study, MUC4 mRNA expression
was observed to be reduced in BO [62]. Further, the expression
of MUC4 transcripts was significantly increased following
the onset of HGN in existing BO lesions. This has previ-
ously been attributed to the induction of MUC4 expression
by bile salts present in the refluxed gastric contents [63].
MUC4 acts as a ligand for the receptor tyrosine
kinase ErbB-2, which is also aberrantly expressed in the
membranes of the epithelial cells in BO and the associated
adenocarcinoma [64]. The MUC4–ErbB2 complex has been
suggested to play a role in the inhibition of apoptosis [65].
However, the areas of BO with HGN showed an increase
in apoptosis (as evidenced by the elevated Bax:Bcl2 ratio)
compared with those with BO alone, despite a higher
expression of MUC4 in the former. This suggests that
MUC4 plays only a minor role in the regulation of
apoptosis in esophageal cancer. As HGNs are at an elevated
risk for malignant transformation, markers that can uncover
these foci in existing Barrett’s esophagitis lesions could
prove to be extremely useful in identifying individuals at
an elevated risk for the development of esophageal
adenocarcinoma. As MUC4 overexpression was observed
following the onset of HGN, it could serve as a marker
of dysplasia in the esophagus, although further studies
are warranted.

3.2 MUC4 in hepatobiliary tract diseases
MUC4 expression in the hepatobiliary duct system has also
been the focus of intense research. Stone formation in the
intrahepatic bile duct, a condition commonly encountered
in Southeast Asian countries, is frequently associated with a
preceding history of recurrent and residual stones and
frequent attacks of acute cholangitis. Chronic proliferative
cholangitis, the sequel of recurrent cholangitis episodes,
is regarded as a risk factor for the development of
cholangiocarcinoma. Cholangiocarcinoma is the most
common primary malignancy of the bile ducts and the
second most prevalent liver cancer. About 5% of cholangio-
carcinomas have associated hepatolithiasis. In an analysis
of mucin mRNA expression in tissue sections from stone-
containing intrahepatic bile ducts and cholangiocarcinomas
by in situ hybridization, MUC4 was found to be weakly
expressed in only 1 of the 10 normal sections and
overexpressed in 4 out of 6 cases of cholangiocarcinoma.
Significantly, MUC4 mRNA was detected in 10 out of the
12 sections with hepatolithiasis including 5 with hyperplastic
bile duct cells and 5 of 6 with dysplastic cells. The MUC4
probe labeled the entire cytoplasm of the epithelial cells lining
the bile ducts and the peribiliary glands with moderate to
strong intensity in sections of both hepatolithiasis and cho-
langiocarcinoma, albeit with a more heterogenous expression
in the latter [66].
Intrahepatic cholangiocarcinoma (ICC) is an extremely
lethal malignancy with most patients presenting with large
tumors and evidence of regional lymph node, pulmonary
and/or bone metastasis at the time of diagnosis [67].
Intrahepatic cholangiocarcinomas of the mass-forming type
(ICC-MFs) are the commonest types of ICC’s and are
associated an extremely poor prognosis even after surgical
resection of the tumor. ICC-MF tumors have been reported
to express MUC2 while being uniformly negative for
MUC1 [68]. In a study to examine the relationship between
MUC4 expression in ICC-MF tissue sections and patient
prognosis, MUC4 was detected in the cytoplasm of 37%
of the ICC-MF tissues, while no expression was observed
in the normal bile duct cells. When patients were
divided based on the length of survival into short-term
(< 12 months) and long-term (> 12 months) survivors,
MUC4 expression was observed in 60% of the short-term
and 8.3% of the long-term survivors. Patients whose tumors
expressed MUC4 (taken as positive staining in > 5% of
the tumor cells) had a significantly lower survival rate
(10% at 1 year and 0% at 3 years) than those with MUC4-
negative tumors (67% 1-year survival rate and 37% 3-year
survival rate). Further, the expression of both MUC4 and its
interacting partner, ErbB2, resulted in the worst prognosis
following surgery, whereas tumors expressing neither protein
afforded the best survival. Together with tumor size,
intrahepatic and lymph node metastasis, MUC4 expression
emerged as an important risk factor that adversely affected
the outcome in patients with a mass-forming ICC [69].
Expert Opin. Med. Diagn. (2008) 2(8)
Chakraborty, Jain, Sasson & Batra
MUC4 has also been show to be aberrantly expressed in
extrahepatic bile duct carcinomas. Patients whose tumors
showed a high level of MUC4 expression (> 20% of tumor
cells stained) had a significantly lower survival than those
with lower MUC4 expression levels (< 20% of cancer cells
stained). MUC4 staining was observed in the cytoplasm of
the cancer cells. Together with high MUC1 expression,
histologic grade of the tumor, surgical margin involvement
and nodal metastasis, a high MUC4 expression was a
predictor of poor prognosis in these patients [70].
Thus, MUC4 could serve to identify patients at a high
risk of cholangiocarcinoma. Also, it may have a role as a
prognostic marker in both intrahepatic and extrahepatic
biliary cancer.
3.3 MUC4 in colon cancer
Colon cancer is the third most common cancer among both
the sexes in the US, with > 112,000 estimated new cases
and > 52,000 deaths in 2007 [1]. Several mucins are
known to be overexpressed in colon cancer, including MUC1
(correlates with a poor prognosis) and MUC5AC [11].
Ogata et al. first reported the overexpression of MUC4 in
colon cancer cell lines and tissues [30]. MUC4 mRNA levels
were either unchanged or increased compared with the
normal colonic tissue in over half of the colon cancers
(five out of nine) examined. However, no correlation was
observed between MUC4 expression and the clinicopatho-
logic parameters. Zhang et al., studying MUC4 expression
in frozen tissue sections from multiple malignancies, reported
that 50% of colon and pancreatic cancer tissues expressed
the mucin [71]. Specifically, the median staining intensity of
MUC4 in these two cancers was 4+ in > 80% of the tumor
cells, suggesting a strong upregulation of MUC4 expression
in these malignancies.
Colonic polyps, specifically of the hyperplastic type,
are potentially malignant lesions that are known to
progress to adenocarcinoma. These polyps are generally
classified into hyperplastic polyps and adenomas. Serrated
adenomas are a subset of hybrid polyps showing features of
both hyperplastic polyps and adenomas that have been
documented to develop into intramucosal carcinoma. The
idea that hyperplastic polyps could progress to serrated
adenomas through a separate histogenetic pathway was first
introduced by Biemer-Hüttmann et al. [72]. Later, they also
showed that, although MUC4 was expressed in the normal
colonic mucosa, its expression was reduced in hyperplastic
polyps and completely abolished in serrated adenomas.
Further, a mucin signature of MUC2+, MUC4- and
MUC5AC+ was found to be highly specific to identify serrated
adenomas. Given the malignant potential of this hybrid
polyp, this observation suggests that loss of MUC4 expression
in serrated adenomas could be useful as a marker to identify
patients with an elevated risk of developing colon cancer [72].
This observation assumes an even greater significance
given the occurrence of hyperplastic polyps and serrated
901
MUC4 as a diagnostic marker in cancer

A. B. C.

MUC4 staining intensity

Figure 2. Differential overexpression of MUC4 during progression of pancreatic cancer. MUC4 is not expressed by the normal
pancreas (A), although its expression is observed in the pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions (B) and reaches
maximum intensity in pancreatic adenocarcinoma (C) (Magnification: × 10). The sections were stained with the mouse monoclonal
antibody 8G7 directed against the tandem repeat domain of human MUC4.

adenomas as part of the familial polyposis syndrome,
known to carry an elevated risk for subsequent development
of adenocarcinoma.
3.4 MUC4 in pancreatic adenocarcinoma
Pancreatic cancer (or pancreatic adenocarcinoma) is
the fourth most common cause of cancer-related deaths in
the United States, with > 37,000 new cases estimated
in 2007 [1]. It is an extremely lethal malignancy with a
5-year survival rate of < 5%. The poor survival is
attributed chiefly to an early development of micro
metastasis, the late onset of symptoms (except in cancer
affecting the pancreatic head) and an extreme resistance
to chemotherapy [73]. Measurement of carbohydrate antigen
(CA19 – 9) in serum is at present the most common
test used in the diagnosis and follow-up of pancreatic
cancer patients [74].
Several studies have demonstrated the aberrant overexpres-
sion of MUC4 in pancreatic cancer (PC) and suggested
that it could be a potential diagnostic marker. Balague et al.
first reported that MUC4 mRNA was overexpressed in pan-
creatic cancer but undetectable in normal pancreatic tissues
and in chronic pancreatitis [75]. MUC4 expression has since
been examined in both pancreatic cancer tissues and cell
lines. It was noted that although MUC4 was aberrantly
overexpressed in most pancreatic cancers (12/16 samples in
one study), it was uniformly absent in chronic pancreatitis
(CP) and the normal pancreas [76]. MUC4 expression was
also detected in a significant number of fine needle aspirates
(91%) from PC patients [77]. Further, in combination with
the non-expression of clusterin-β (a secreted glycoprotein),
MUC4 helped to distinguish reliably inflamed pancreatic
ductal epithelial cells from malignant ones [77]. MUC4 has
also been examined both alone and in combination with
other potential markers for its ability to differentiate PC
from CP. In one study, positive MUC4 immunostaining
of pancreatic tissue sections alone had a sensitivity and
specificity of 77 and 78%, respectively. When examined
in combination with immunostaining for other potential
markers of PC, a positive staining for either MUC4 or
p53 had the highest diagnostic sensitivity (96%). However,
the specificity was 73% compared with 78% for MUC4
alone. When immunoreactivity for both MUC4 and p53
was taken as the diagnostic criterion, the sensitivity fell
to 39% but specificity increased to 100% [78]. MUC4 also
emerged as an independent poor prognostic factor in a
multivariate analysis of 135 cases of invasive ductal
carcinoma of the pancreas [79]. The survival of 21 patients
with high MUC4 expression (> 20% MUC4-positive
neoplastic cells) was significantly worse than that of the
114 with low MUC4 expression.
The development of infiltrating adenocarcinoma of the
pancreas has been suggested to progress through an inter-
mediate preinvasive stage termed ‘pancreatic intraepithelial
neoplasia’ (PanIN) [80]. A progressive increase in MUC4
expression with the progression of PanIN lesions was
observed: from no detectable expression in the normal
pancreas, through an increasing expression in progressively
higher grades of PanIN, to the highest levels in well-
differentiated pancreatic cancer (Figure 2). MUC4 expres-
sion, however, decreased in poorly differentiated cancer [81].
Park et al. reported a similar observation, adding that
although MUC4 expression was detected as early as PanIN-1,
the strongest expression was observed in the PanIN-3 stage
of dysplasia [82].
Intraductal papillary-mucinous neoplasm (IPMN) of
the pancreas is a rare disease characterized by proliferation of
the pancreatic ductal epithelium with resultant mucin hyper
secretion. Importantly, it has the potential to progress to
invasive adenocarcinoma. IPMNs of the villus dark cell type
are chiefly characterized by MUC5AC+ and MUC2+,
whereas those of the papillary clear cell type are MUC5AC+

902 Expert Opin. Med. Diagn. (2008) 2(8)


and MUC2- [83-85]. IPMN adenomas showing dysplasia
were observed to be strongly positive for MUC2, MUC5AC
and, to a lesser extent, MUC4 [86,87]. A similar pattern
of mucin expression was also observed in colloid carcinomas
associated with IPMNs. On the other hand, invasive ductal
adenocarcinomas associated with IPMNs showed a loss of
MUC2 and gain of MUC1 and were associated with greater
metastatic potential. The altered mucin expression in IPMNs
of the adenoma type and the associated colloid carcinomas
has been suggested to contribute to their better prognosis
compared with conventional ductal adenocarcinoma.
Thus, MUC4, which is not detected in the normal
pancreas but appears de novo in the earliest preinvasive
stage of PC pathogenesis, could be an extremely useful
marker in patients who are at an elevated risk for developing
PC, including those with hereditary pancreatitis and
long-standing chronic pancreatitis. Further, we have detected
MUC4 transcripts in peripheral blood mononuclear cells
of pancreatic cancer patients but not in acute pancreatitis
or normal individuals (manuscript under preparation),
suggesting its possible role as a blood-based diagnostic
marker for PC.
4. MUC4 in the diagnosis of reproductive
tract malignancies
Although significant attention has focused on the role of
MUC4 in malignancies of the aerodigestive tract, it also
appears to play a role in cancers arising from the
reproductive system, chiefly, cervical and ovarian cancer in
females and prostate cancer in males (Table 5). An analysis
of MUC4 expression in the normal female reproductive
tract revealed that the mucin is expressed only in the lower
segment, specifically in the epithelium lining the ectocervix,
endocervix and, patchily, in the vagina. Further, on
following MUC4 expression in the normal endometrium
during the menstrual cycle, the highest expression was
detected just before the mid-cycle peak, when estrogen
levels are unopposed by progesterone, followed by a drop
thereafter, suggesting a possible hormonal regulation of
MUC4 expression in the endometrium [35].
4.1 MUC4 in cervical squamous cell carcinoma
Cancer of the cervix is the second most common
malignancy affecting women the world over, second only
to breast cancer in its incidence [1]. The squamocolumnar
junction (SCJ) between the ectocervix and the endocervix is
the usual site for the origin of the malignant transforma-
tion [88]. Using microarray analysis, Narayan et al. found
that MUC4 was one among 22 genes significantly
upregulated in cervical cancer tissues compared with the
healthy cervix [89]. An analysis of the MUC4 expression
profile in the normal, metaplastic and dysplastic cervical
epithelium revealed that whereas the apomucin is highly
expressed in the healthy endocervix, and patchily in the
Expert Opin. Med. Diagn. (2008) 2(8)
Chakraborty, Jain, Sasson & Batra
ectocervix, its expression was significantly upregulated
following the onset of squamous dysplasia at the SCJ [89,90].
Further, although the highest MUC4 expression was
observed in high-grade dysplasia (cervical intraepithelial
neoplasia or CIN grade II and III), it was detectable as early
as in CIN-I (75 ± 40% cells positive). A strong and diffuse
MUC4 immunostaining was observed throughout the
cytoplasm of the dysplastic cells and throughout the entire
thickness of the stratified epithelium. By contrast, the
normal endocervical epithelium showed MUC4 immuno-
staining only in the most superficial columnar epithelial layer
while the normal and metaplastic ectocervical epithelia had
a parabasal pattern of MUC4 staining. This suggests that
the presence of a diffuse MUC4 staining of the epithelium
at the SCJ could be a possible marker for dysplastic
changes in women at a high risk for developing cervical
cancer. Baker et al. further observed that although MUC4
was strongly expressed on the luminal surface of endocervi-
cal glands in benign cervical pathologies (tubal metaplasia,
micro glandular hyperplasia), and in endocervical dysplasia
and adenocarcinoma, its expression was negative in most
(five out of eight) samples of cervical adenocarcinoma-in situ
(AIS) [10]. Given that it is often extremely difficult to
distinguish AIS histologically from benign cervical
conditions, especially tubal metaplasia, the absence of MUC4
expression in the former could aid in distinguishing this
lesion from benign cervical pathologies.
Thus, evaluating MUC4 expression in cervical biopsies
has the potential to be useful as an early marker for cervical
dysplasia. Further, the absence of MUC4 expression could
also be used to distinguish high-grade cervical dysplasia from
other MUC4-expressing benign cervical pathologies [90].
4.2 MUC4 in endometrial carcinoma
Endometrial carcinoma is the most common type of uterine
cancer, with nearly 39,000 new cases estimated in 2007 [1].
Although the mortality from endometrial carcinoma has
declined in general, it continues to be higher in African-
American women than in Caucasians. Immunohistochemical
analysis of four mucin genes (MUC2, MUC4, MUC5AC
and MUC6) in normal, hyperplastic and malignant endo-
metrial tissues revealed that MUC4 was the predominant
mucin expressed by the normal endometrial epithelium
(36% positivity). MUC4 expression was reduced in the
proliferative and secretory phases of the endometrial cycle
(13 and 26% positive, respectively) but significantly increased
in simple endometrial hyperplasia (29% of cases positive),
with the most intense expression noted in tissues from
endometrial adenocarcinoma (77% of samples positive) [91].
Other studies reported a similar finding, albeit with a lower
incidence of MUC4 positivity in endometrial carcinoma
(45%). However, MUC4 staining did not correlate with
clinicopathological parameters [10,92]. This suggests that the
role of MUC4 in the progression and diagnosis of endometrial
carcinoma needs to be explored further.
903
MUC4 as a diagnostic marker in cancer
4.3 MUC4 in ovarian cancer
Ovarian cancer accounts for nearly 4% of all cancers in
women and is the eighth-leading cause of cancer-associated
deaths in the US [1]. It also has the dubious distinction of
being the most lethal cancer of the female reproductive
tract, chiefly owing to the advanced stage at which it is first
diagnosed [93]. The lack of early symptoms means that the
malignancy has often spread beyond the ovary when it is
first identified. One of the earliest reports of MUC4 over-
expression in ovarian cancer was a study by Giuntoli et al.,
wherein they reported that MUC4 was one of the mucin
genes whose expression was significantly increased in ovarian
cancer [94]. A key observation was the absence of MUC4
expression in a transformed but non-malignant ovarian epi-
thelial cell line, suggesting that its expression is related
specifically to the onset of malignant change in the
epithelial cells. It was also observed that the expression of
MUC4 was significantly decreased in late stage ovarian
cancer. Significantly, an increase in MUC4 expression
correlated with an improvement in patient survival. However,
the prognosis of ovarian cancer patients is also influenced
significantly by the histology. Based on the cell of origin,
malignant ovarian neoplasms are classified primarily into
surface-epithelial stromal tumors, germ cell and sex cord
stromal tumors. About 15% of ovarian tumors are also
classified as borderline. These are very similar in their
epidemiology to invasive tumors but occur more commonly
in younger women and have a more favorable outlook [66].
Histologically, the cells in borderline tumors present with
either a gastric, intestinal or an endocervical phenotype.
MUC4 mRNA was observed to be highly expressed by the
borderline ovarian tumors with an endocervical pheno-
type [95]. This subtype, although uncommon, carries a sig-
nificantly better chance of survival. Also, the MUC4-expressing
cells in these neoplasms were observed to coexist with either
intestinal-type goblet cells (MUC2+) or gastric-type epithelia
(MUC5AC+ or MUC6+). This is significant as borderline
ovarian tumors exhibiting an intestinal phenotype have an
extremely poor prognosis. Thus, it appears that while MUC4
expression alone is unlikely to provide significant prognostic
information, when combined with other mucins it could
help to identify high- or low-risk tumors and stratify patients
for treatment purposes [95]. The identification of tumors at
a localized and non-metastatic stage has been suggested as
the key to improving the poor prognosis in ovarian cancer
patients. In a study comparing the expression profile of
MUC4, MUC1 and MUC16 in ovarian cancer, it was
observed that MUC4 was significantly overexpressed in all
of the early stage tumors and a significant number of
advanced stage tumors (88%). Further, a combination of
MUC4 and MUC16 positivity identified 100% of the
late stage tumors (with 100% specificity). Significantly,
MUC4 but not MUC1 or MUC16 showed 100%
specificity for mucinous ovarian tumors. However, MUC4
expression did not correlate with patient survival.
904 Expert Opin. Med. Diagn. (2008) 2(8)
Thus, MUC4 expression appears to be upregulated in
the early stages of ovarian cancer pathogenesis, with a
high expression being maintained in advanced stage
tumors. Further, it appears to be a highly specific marker
for mucinous ovarian tumors. This suggests that it could
be useful in the diagnosis of early stage mucinous
ovarian tumors [94].
Diffuse malignant peritoneal mesothelioma (DMPM) is a
relatively rare yet aggressive cancer that originates from the
native mesothelial cells of the peritoneal cavity. Ovarian
cancer (OC) and its closely related and morphologically
indistinguishable counterpart primary peritoneal serous
carcinoma (PPC), are both thought to develop either from
the peritoneal mesothelium or from the ovarian surface
epithelium. Both DMPM and OC/PPC diffusely involve
the peritoneum, forming solid nodules and producing
ascites. In addition to their similar clinical presentation,
both OC/PPC and DMPM can be almost identical mor-
phologically. Further, both tumors share a similar immunos-
taining profile, which reflects their common histogenesis [96].
In a global gene expression analysis to identify differentially
expressed genes between OC/PPC and DMPM, MUC4
emerged as one of the 121 genes overexpressed in the for-
mer group [96]. In a follow-up study to examine the sites of
MUC4 expression in OC/PPC and its prognostic significance,
the same group reported that MUC4 was detected in carci-
noma cells present in OC/PPC-associated peritoneal effusion
in 141 of 142 cases examined [97]. Reactive mesothelial cells
present in 72 malignant and 10 reactive peritoneal effusions
examined were, however, uniformly negative. Additionally,
MUC4 expression was significantly higher in carcinomatous
cells present in the OC/PPC associated effusions than
in either the primary carcinomas or the solid metastases.
However, except for a higher expression in tumors from
older (> 60 year) patients, MUC4 expression did not correlate
with survival or any other clinicopathological parameters in
OC/PPC. These reports seem to suggest that MUC4 is a
highly specific marker to distinguish OC/PPC from both
benign (reactive) and malignant mesothelial cells.
4.4 MUC4 in breast cancer
Much effort is being made to identify markers with clinical
significance in breast cancer. Several of these studies have
reported an altered expression of mucins in breast cancer
tissues. An immunohistochemical study on tissues from
1447 cases of invasive breast cancer revealed that most
(91%) of the tissues were positive for MUC1 and MUC3.
Fewer tumors expressed MUC5AC (37%) and MUC2 (8%).
Significantly, MUC4 was expressed in nearly 95% of
the cases studied (Table 6). However, apart from a weak
correlation with tumor grade, no other correlation between
MUC4 expression and clinicopathological parameters was
observed [98]. MUC4 has also been reported previously in
breast milk [23]. Further, MUC4 expression has also been
suggested to be responsible for the resistance of breast cancer

cells to the anticancer drug Herceptin possibly by the mech-
anism of steric hindrance preventing access of Herceptin to
its target epitope on the HER-2 receptor [99].
4.5 MUC4 in prostate cancer
Prostate cancer is the most common cancer among men in
the US, with African-Americans having an incidence
twice as high as that of other racial and ethnic groups [1].
Although it is not clear whether screening helps to reduce
mortality, prostate-specific antigen (PSA) and digital rectal
examination (DRE) are two methods being employed at
present to screen men > 50 years of age. Using the anti-
MUC4 TR antibody 8G7, we observed that MUC4 expression
was significantly decreased in prostate cancer tissues compared
with either the normal prostate or areas of benign prostatic
hypertrophy (BPH) [37]. Out of the 38 tumor samples exam-
ined, a mere 26% had MUC4 staining, the intensity ranging
from faint to moderate. In contrast, nearly 85% of the adjacent
normal areas or those showing features of BPH were moderate
to strongly positive for MUC4 (Table 6). On the other
hand, MUC1, another membrane-bound mucin, was
detected in ∼ 54% of prostate cancer and ∼ 90% of the
adjacent normal/BPH areas. The staining pattern for both
mucins in this study was rather diffuse, staining both the
membrane and cytoplasm. However, it did not correlate
with either the cell type or tumor stage [37]. Using a different
antibody, Cozzi et al., however, reported no MUC4 expression
in either the normal or prostate cancer tissues [100]. This
suggests that the role of MUC4 in prostate cancer needs
further elucidation.
5. Correlation of subcellular distribution of
MUC4 with clinicopathologic characteristics
The subcellular localization of several molecules is known to
be altered in malignant cells compared with their normal
counterparts. For example, a cytoplasmic expression of
MUC1 in immunohistochemical analysis of invasive breast
carcinoma tissue sections correlated with positive lymph
node status in the patients [101]. Low cytosolic MUC1
expression also emerged as a predictor of good prognosis in
stage III ovarian cancer, whereas a low expression of MUC1
in the apical membrane of the neoplastic cells was
associated with early stage and a good outcome for patients
with invasive ovarian tumors [102]. In colorectal cancer,
the 5-year survival rate was significantly lower in cases
showing positive MUC1 staining in the circumferential
membrane and/or cytoplasm than those with staining only
in the apical membrane or those showing no staining
at all [103]. Hence, it is of interest to examine whether a
correlation exists between the subcellular location of MUC4
and clinicopathologic parameters. This could be especially
useful in predicting the outcome of patients diagnosed with
highly invasive and/or metastatic cancers, such as ovarian,
gastric and pancreatic adenocarcinoma. However, no
Expert Opin. Med. Diagn. (2008) 2(8)
Chakraborty, Jain, Sasson & Batra
correlation has been reported thus far with either patient
outlook or tumor characteristics. In the developing rat
foregut, MUC4 expression occurs initially on the surface of
the epithelial cells (lining the esophagus), followed by its
appearance in intracellular granules [104]. In malignant cells
on the other hand, MUC4 is expressed both in the cytosol
and in the apical membrane. Llinares et al. reported that
although cytoplasmic and membrane staining were both
frequently observed in lung adenocarcinomas, in some cases
the staining was limited entirely to the cytoplasm [56]. In
ovarian cancer, MUC4 expression was observed in both the
cell membrane and the cytosol in serous, mucinous and
clear-cell subtypes, whereas endometrioid tumors showed a
distinct apical and basal staining pattern with only a faint
cytoplasmic staining [105]. Eighty-six per cent of normal
salivary gland specimens were positive for MUC4 expression
in the supranuclear cytoplasmic region of their ducts.
However, in salivary gland MECs, both the cell membrane
and cytosol of all types of neoplastic cells were stained.
This difference in subcellular staining pattern of
membrane-bound mucins is believed to be due to
differences in post-translational processing of the mucin core
proteins leading to altered glycosylation patterns in
the neoplastic cells [49]. The normal ectocervix shows a
diffuse MUC4 staining limited to the cytosol in the cells
of the parabasal layer of the stratified squamous epithelium
in contrast to a stronger and diffuse cytoplasmic staining
throughout the entire thickness of the stratified epithelium
in dysplastic cervical epithelia [90]. Cytosolic MUC4
staining was also observed in both the columnar and goblet
cells of the normal colon, with the lower two-thirds of
the crypts showing the most intense staining. However,
MUC4 expression was nearly completely lost in serrated
adenomas and significantly reduced in hyperplastic polyps,
whereas the intensity, intracellular localization and crypt
distribution in tubular and tubulovillous adenomas were
identical to those observed in the normal colon [72].
Lung cancer tissues stained with a rabbit polyclonal
antibody to MUC4 showed a strong and diffuse
cytoplasmic MUC4 staining, whereas only weak MUC4
immunoreactivity restricted to the apical region of the
bronchioles was observed in the normal lung [106].
Thus, there is clearly an alteration in MUC4 distribution
with the onset of transformation in a normal epithelium.
Future studies to address the significance of the
subcellular distribution of MUC4 in relation to patient
survival, response to chemotherapy and presence
of distant metastasis would be extremely useful to
establish MUC4’s role as a prognostic marker in
neoplastic progression.
6. Summary
MUC4 expression is a specific marker of epithelial tumors
and its expression correlates positively with the degree of
905
MUC4 as a diagnostic marker in cancer
differentiation in several cancers. Most significantly, MUC4
has emerged as a specific marker of dysplasia, being
expressed in the earliest dysplastic lesions preceding several
malignancies, including esophageal, pancreatic and cervical
carcinoma. Also, the detection of MUC4-specific antibodies
in the serum and of the transcript in mononuclear cells of
cancer patients raises the possibility of it emerging as a new
diagnostic biomarker for bedside application in high-risk
individuals and those with established cancer.
7. Expert opinion
The preceding discussion has established that the detection
of MUC4 could serve as a new diagnostic marker in tissue
sections as well as body fluids. The use of MUC4 expression
as a yardstick to distinguish benign, dysplastic or malignant
lesions arising from a given site offers several advantages. For
example, it has a restricted expression in specific segments of
the gastrointestinal tract, which circumvents the possibility
of contamination from mucins present in secretions from
nearby portions of the gut. This is crucial as one of the
concerns in studies investigating the role of mucins as tumor
markers using homogenized tissues is contamination from
adjacent normal areas. In Barrett’s esophagus for instance, it
has been suggested that the detection of MUC2, MUC5AC
and MUC6 in the metaplastic epithelium could be the
result of reflux of the gastric mucus into the esophagus.
MUC4, however, is not expressed by the normal gastric
epithelium. Hence, its detection offers a greater specificity
for newly diagnosed esophageal lesions.
MUC4 is expressed de novo in several tissues during the
process of carcinogenesis, including the pancreas and the
bile duct epithelial cells. This prevents possible overlap
with endogenous MUC4 in diagnostic assays. The
availability of specific antibodies has also greatly helped
accelerate research into developing MUC4-based diagnostic
assays. Current imaging modalities, the cornerstone of
cancer diagnosis, can identify only macroscopic changes.
However, the microscopic and molecular alterations that
precede gross changes by a significant lag period can be
identified only by using molecular techniques. Here, tumor
markers come into prominence as tools to identify changes
that occur in tissues that appear histologically normal.
The de novo expression or the overexpression of MUC4 is
detectable in the early pre-malignant stages of several
lethal cancers including pancreatic, esophageal and cervical
carcinoma. As these cancers often present symptoms
only at an advanced stage, the emergence of MUC4 as a
hallmark of dysplasia could provide us with a tool to screen
and follow-up high-risk cases. We are currently engaged in
examining the pattern of MUC4 mRNA expression in the
pancreas and in PBMCs in a mouse model of spontaneous
906 Expert Opin. Med. Diagn. (2008) 2(8)
pancreatic adenocarcinoma (unpublished data) [80]. These
studies will elucidate the role of MUC4 in the early stages
of pancreatic cancer pathogenesis and explore the possibility
that detection of its mRNA in PBMCs might be a marker
of PanIN lesions in the pancreas.
Despite its over- or underexpression in cancer,
MUC4 alone is unlikely to be able to identify all cases
of a particular malignancy correctly. This is chiefly
due to the role of mucins (including MUC4) in inflamma-
tion and modulation of the immune response [39,43,46].
Distinguishing these reactive conditions from malignancy
is still a challenge, and one that will possibly require a
panel of markers to achieve the highest sensitivity and
specificity in diagnosis.
Finally, the role of MUC4 as a prognostic marker remains
to be examined further in larger studies. Although it
appears to be more highly expressed in well-differentiated
cancers, patients whose tumors express MUC4 appear
to fare better or worse in an organ-specific pattern.
For example, MUC4 expression is an indicator of a
good prognosis in SCC of the UADT [24], MECs of
the salivary gland [107] and in ovarian cancer [94]. On the
other hand, in intrahepatic cholangiocarcinomas, extrahepatic
bile duct tumors, invasive ductal adenocarcinomas and
small-sized lung adenocarcinomas, MUC4 expression was
associated with reduced survival and/or a poor
prognosis [69,70,108,109]. Further studies are needed to clarify
the ability of MUC4 to predict the outcome in cancer
patients as different techniques and antibodies were used in
these studies.
Although MUC4 is elevated in several malignancies at
tissue level, whether circulating MUC4 is present in the
serum is still an open question. It would be of interest to
examine whether elevation of serum MUC4 occurs in malig-
nancies like ovarian and pancreatic cancer that express high
levels of the mucin in the malignant cells. Further, evidence
of elevated MUC4 in various malignancies has raised
the possibility that it might serve as a novel target for
anti-cancer therapy using specific antibodies.
Acknowledgments
The authors thank K Berger and S Deb for editorial
assistance on this article. The authors on this work were
supported by the grants from the National Institutes of
Health (UO1 CA111294, RO1 CA 133774, RO1 CA
131944 and RO1 CA78590).
Declaration of interest
The authors have no conflict of interest to declare and no
fee has been received for preparation of the manuscript.

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Affiliation
Subhankar Chakraborty MD,
Maneesh Jain PhD, Aaron R Sasson MD &
Surinder K Batra† PhD
†Author for correspondence
University of Nebraska Medical Center,
Eppley Institute for Research in Cancer,
Department of Biochemistry and
Molecular Biology,
984525 Nebraska Medical Center,
Omaha, NE 68198-5870, USA
Tel: +1 402 559 5455; Fax: +1 402 559 6650;
E-mail: sbatra@unmc.edu