Expert Opinion in Medical Diagnostics (2008)

8/8/2019 Expert Opinion in Medical Diagnostics (2008)

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Review

10.1517/17530050802234194 2008 Informa UK Ltd ISSN 1753-0059 891

All rights reserved: reproduction in whole or in part not permitted

MUC4 as a diagnostic markerin cancerSubhankar Chakraborty, Maneesh Jain, Aaron R Sasson & Surinder K Batra

University of Nebraska Medical Center, Eppley Institute for Research in Cancer,Department of Biochemistry and Molecular Biology, Department of Surgery, Omaha,

NE 68198-5870, USA

Background: Mucins are high molecular mass glycoproteins whose role

in diagnosis, prognosis and therapy is being increasingly recognized

owing to their altered expression in a variety of carcinomas. MUC4, a

membrane-bound mucin encoded by a gene located on chromosome

locus 3q29, is aberrantly expressed in several cancers including those of the

bile duct, breast, colon, esophagus, ovary, lung, prostate, stomach and

pancreas. Objective: This review considers the potential use of the

MUC4 expression pattern in the diagnosis and prognosis of various cancers.

Results/conclusion: MUC4 expression is a specific marker of epithelial tumors

and its expression correlates positively with the degree of differentiation in

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 recentyears. According to estimates from the American Cancer Society, > 1.4 millionnew cases of cancer were diagnosed in 2007 and an estimated 560,000 patientsdied from cancer-related causes in the US alone in the same year. Although thetotal number of cases has increased, mortality resulting from cancer has shown aslight, albeit welcome, decline [1]. Part of this is attributable to the developmentof more potent anticancer drugs. A key contributing factor is the improvement indiagnostic modalities that have permitted the detection of tumors at an early,localized and surgically resectable stage. Although imaging modalities continue tobe the prime diagnostic tools being used, their accuracy and sensitivity havereached almost saturation limits. In such a scenario, tumor markers, moleculesthat show either an altered expression or a compositional change in tumor cellscompared with healthy or inflamed tissues, have come into the limelight. Theyare useful as screening tools to identify patients suspected of harboring occulttumors for subsequent confirmation with other non-invasive and invasivediagnostic tests. Mucins are key biomolecules whose expression is deregulatedin several cancers. This altered expression pattern has been widely examined forpossible diagnostic application in several cancers.

MUC4 is a membrane-bound mucin that is normally expressed by theepithelial lining in several tissues, including the conjunctiva, most of theaerodigestive tract and the endocervix. Recently, MUC4 has been shown to

1. Introduction

2. MUC4 in the diagnosis of

cancers of the upper

aerodigestive tract


gastrointestinal malignancies


reproductive tract malignancies

5. Correlation of subcellular

distribution of MUC4 with

clinicopathologic characteristics

6. Summary

7. Expert opinion


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MUC4 as a diagnostic marker in cancer

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

be aberrantly expressed in several malignancies andgrowing evidence supports its role as a useful marker fordiagnosis and as a possible target for therapy. This reviewfocuses on the potential of MUC4 as a biomarker in thediagnosis and/or prognosis of cancer.

1.1 Mucins and MUC4

Body passages, comprising the aerodigestive and urogenitaltracts, are exposed to a multitude of agents, with the poten-tial for damaging the delicate mucosal linings that protectthese passages. Several mechanisms are used by the body increating a protective barrier to counter this threat, includingepithelial tight junctions, microbicidal enzyme-containingbody fluids and commensal flora harbored outside andwithin the body. A key role in this process is also played bya 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 thepresence of mucins, which are heavily glycosylated, highmolecular mass glycoproteins, synthesized and secreted byepithelial goblet cells and submucosal glands. Mucins aremultifunctional biomolecules. As components of mucus,they help trap both foreign particles and invading organ-isms. Additional functions being discovered include a role incellular signaling, maintenance of cell integrity and, in thecase of malignant cells, regulation of metastasis and theresistance to apoptosis [2].

There are essentially two major functional classes ofmucin-type glycoproteins: the secreted or soluble (MUC2,

MUC5AC, MUC5B, MUC6MUC8 and MUC19) andthe membrane-bound (MUC1, MUC3, MUC4, MUC12,MUC13, MUC15, MUC17 and MUC20) [3,4]. Membrane-bound mucins contain a transmembrane domain thatanchors them to the plasma membrane, in addition to ashort cytoplasmic tail [5,6]. The structure and function ofmucins in normal physiology and disease have been reviewedin several articles [6-9]. MUC genes have a relatively tissue-specific expression. Malignant transformation is associated with either their deregulated expression or an alteration inthe structure of their protein backbone (e.g., a variable

number of tandem repeats [VNTR] polymorphism) orglycosylation pattern [10,11].

MUC4 is a membrane-bound mucin that was first clonedfrom a human tracheobronchial cDNA library and a humanpancreatic cancer cell line. The human MUC4 gene hasbeen mapped to the chromosomal region 3q29 [12-14]. Theprotein core is synthesized as a precursor polypeptide, whichis subsequently processed to yield the mature peptide back-bone, termed the MUC4 apomucin. The amino terminus ofthe apomucin consists of a 27-residue signal peptide,followed by 3 imperfect repeats of a motif that is126 130 amino acids long, and a 554-amino acid uniquesequence. However, the largest domain is the central tandemrepeat domain, comprising a 16-amino-acid-long perfecttandem repeat. The C-terminal region of MUC4 is dividedinto 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, MUC4 (an extracellularmucin-type glycoprotein) and MUC4 (a membrane-tethered subunit), which are believed to be non-covalentlyassociated on the cell surface (Figure 1). The MUC4 geneencodes numerous alternatively spliced forms, some of whichare devoid of the mucin hallmark, the tandem repeatarray [15]. The biology, evolution, functions and potentialtherapeutic role of MUC4 have been the subject of severalreviews [5,8,16,17]. This article focuses on the diagnostic andprognostic potential of MUC4 in various cancers.

1.2 MUC4 antibodiesSeveral antibodies have been reported in the literature thatrecognize epitopes on either MUC4 (the extracellularmucin-like subunit) or MUC4 (Figure 1). Hanaoka et al.developed a rabbit polyclonal antibody to a peptideconstituting the tandemly repeated sequence on MUC4. Analysis of surface MUC4 expression in eight lungcancer cell lines by flow-sorting (with this antibody) with or without previous sialidase treatment revealed that although50% of the cell lines were positive following sialidasetreatment, none in the untreated group was positive.

SPGDPH

EGF

MUC4- MUC4-

TRD CysTM

CT

NTR

Cys

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.


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Chakraborty, Jain, Sasson & Batra

Expert Opin. Med. Diagn. (2008) 2(8) 893

This suggests the possibility that the carbohydrate chainsattached to the tandem repeat region of MUC4 could pre-vent recognition of the extracellular peptide epitope by theantibody [18]. Other suggested mechanisms for the failure todetect surface MUC4 include shedding of either the whole,or the tandem repeat-containing portion of MUC4 fromthe cell surface into the surroundings. We have developedand characterized a monoclonal antibody, 8G7, that recognizesan epitope within a 16-amino acid sequence from the tandemrepeat (TR) region of human MUC4 [19]. The antibodyrecognized a band > 500 kDa in size on western blot andits reactivity was specific for MUC4 as evidenced by non-reactivity against purified MUC1 and strong reactivity to a

16 amino-acid MUC4 tandem repeat peptide. Epitopemapping by competitive ELISA showed that the binding ofthe 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 nativeMUC4. When tested for immunoreactivity against tissues,the antibody showed a strong cytoplasmic staining, consis-tent with the accumulation of mucus-containing secretorygranules in the cytosol of malignant cells. Confocal analysis,however, showed a strong membrane staining with moderatecytoplasmic staining in the MUC4 overexpressing pancreaticcancer cell line CD18/HPAF. A possible explanation for thiscould be the difference in subcellular distribution of mucins

in tissues versus that seen in cancer cell lines maintainedin vitro. Lopez-Ferrer et al. reported and characterized a rabbitpolyclonal antibody that specifically recognized a 16-aminoacid sequence from the tandem repeat of MUC4 but didnot react with sequences from the tandem repeat regions ofMUC1 MUC8 [20]. MUC4 is a homolog of ascites sialo-glycoprotein (ASGP)-1 and ASGP-2, which form the ratsialomucin complex (SMC) [21]. A monoclonal antibody1G8, raised against the rat AGSGP-2 (homologous tohuman MUC4), was shown to recognize human MUC4,producing bands with molecular mass similar to the rat

ASGP-2. The antibody recognizes a unique sequence in thenon-tandem repeat region of rat ASGP-2 between theN-terminal amino acid residue 53 and the transmembranedomain of ASGP-2 [22]. Surprisingly, the antibody also rec-ognized a band in heart tissue and weaker bands in thekidney, liver and skeletal muscle, tissues that are negative forMUC4at the transcript level. In immunoblots, the antibodyrecognized a protein with a molecular mass similar to thatexpected for MUC4. When used to immunoprecipitate theproteins in MUC4 transfected HCT11 cells, the antibodyrecognized two bands of 250 and 140 kDa. Given thatMUC4 has a predicted molecular mass of 80 kDa, it wassuggested that these bands represent two differentially glyco-

sylated forms of MUC4. In vitro translated MUC4 wasalso 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-likedoligosaccharides on rat ASGP-1 (homologous to humanMUC4) also recognized human MUC4 in formalin-fixedparaffin-embedded tissues from normal and squamous cellcarcinoma bearing upper aerodigestive tract tissues [24].MUC4 is characterized by a high degree of polymorphismin its central TR region [25], which makes the antibodiesdirected 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 inbody 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 epitheliaincluding those of the conjunctiva [27], cornea [28], the tearfilm [29], colon [30], fetal lungs, surface epithelium of theadult respiratory tract from the trachea to the collectingducts [31], inferior nasal turbinates [32], eustachian tube [33],

Table 1. Antibodies to MUC4 and its homolog rat ASGP used in studies on human tissues.

Name of antibody Nature Epitope recognized Immunizing peptide sequence Ref.

8G7 Monoclonal (MAb) MUC4 TR STGDTTPLPVTDTSSV [19]

Anti-MUC4 Ab Rabbit polyclonal (PAb) MUC4 TR TSSASTGHATPLPVTD [20]

Anti-MUC4 Ab Rabbit PAb MUC4 TR PVTD-TSSVSTGHATSLPVTD-TSSV [18]MUC4-CT Rabbit PAb MUC4 cytoplasmic tail SGARFSYFLNSAEAL [34]

SG9 Rabbit PAb MUC4 TR STGDTTPLPVTDTSSV [110]

M4P Chicken PAb Synthetic MUC4 TR peptide [33]

LuCF12 and LuC18.2 MAb MUC4 TR TSSVSTRHATSLPVTD [41]

4F12 MAb rASGP-2 (rat ASGP-2) First 53 amino acids of rASGP-2 [111]

1B1 MAb rASGP-2 Between amino acid 53 683 of rASGP-2 [111]

15H10 MAb rASGP-1 O-linked carbohydrates on mature rASGP-1 [111]

MAb: Monoclonal antibody; TR: Tandem repeat.


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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 adultlungs, testes [36] and prostate [37], whereas low MUC4 levelsoccur in the healthy ileal mucosa [38]. An alteration inMUC4 expression is also observed in several disease states.

Benign conditions characterized by an upregulation ofMUC4 (at both the transcript and protein levels) includeinflammatory 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 nodulargoiter [41], the rare cap polyposis of the colon (mRNAonly) [42], in the caecum and colon of dextran sodiumsulfate-treated mice with features of acute colitis (mRNAonly) [43] and ulcerative colitis [44]. Conversely, a decreasedexpression of the apomucin mRNA is observed in a mousemodel of allergic conjunctivitis [45]. In addition to analteration in its expression, an allelic variant (an A585Spolymorphism in the Von Willebrand domain of MUC4)of the apomucin has been reported to be significantlyoverrepresented in patients with ulcerative colitis [46].

MUC4 has been demonstrated to be aberrantlyexpressed in several malignancies and has now been widelyinvestigated as a possible diagnostic marker (summarized inTables 2 6). This article examines the potential of MUC4as a diagnostic and/or prognostic marker in cancer. For thesake of convenience, the various malignancies are dividedinto three groups, those arising from the upper aerodigestivetract, the rest of the gastrointestinal system and thoseoriginating from the reproductive system.

2. MUC4 in the diagnosis of cancers of theupper aerodigestive tract

According to the National Cancer Institute, the upperaerodigestive tract comprises the combined organs andtissues of the respiratory tract and the upper part of thedigestive tract (including the lips, tongue, major salivaryglands, gums and adjacent oral cavity, floor of the mouth,tonsils, oropharynx, nasopharynx, hypopharynx, the nasalcavity, 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 themost superficial and the suprabasal layers. However, no MUC4

expression is detected in the basal layers of the stratifiedepithelium [24]. Cancers of the UADT, which constitute 4% of all malignancies [48], show not only an alterationin MUC4 expression but also a change in its distribution inthe stratified epithelium (summarized in Table 2).

2.1 MUC4 expression in squamous cell carcinoma of

the UADT

Squamous cell carcinomas (SCCs) constitute > 95% of allUADT cancers. Nearly 85% of these cancers showed atleast some degree of MUC4 staining in one study [49].

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 theentire thickness of the epithelium. More importantly, thenormal mucosa distant from the areas of SCC preserved itscharacteristic suprabasal staining pattern. Eleven of the

12 cervical lymph node biopsies harboring a metastasis froma primary SCC were also positive for MUC4. Although nocorrelation was evident between MUC4 expression andeither tumor grade or patient characteristics, the MUC4-expressing tumors did have a lower rate of recurrence fol-lowing resection and afforded the patients a significantsurvival advantage. This suggests that the presence of MUC4might predict a more favorable prognosis in SCCs arising inthe UADT. Further, a diffuse MUC4 staining in the mucosaadjacent to an area of dysplasia could be an early marker ofsubsequent malignant transformation [24].

2.2 MUC4 expression in salivary gland tumors

Salivary gland cancers comprise nearly 3% of all the headand neck cancers. MUC4 is highly expressed in both themajor (parotid, submandibular and sublingual) and minorsalivary glands. By in situ hybridization, Liu et al.demonstrated MUC4 expression in the cells lining thestriated and major excretory ducts (in the parotid andsubmandibular glands) and in those lining the serous acini(in submandibular glands only). A similar pattern of MUC4expression was also observed by immunohistochemistry.Further, MUC4 was also detected in secretions from theall of the three major salivary glands [34]. MUC4immunostaining was also detected in all the benign

salivary gland neoplasms examined (six out of six) and inmost 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 byan overexpression of MUC4. MECs are unique in thatthey exhibit a broad range of aggressiveness, rangingfrom indolent localized tumors to highly invasive neoplasmsprone to local recurrence and metastasis. They are composedchiefly of four types of cells epidermoid, glandular,mucinous and intermediate. Although MUC4 is generallyconsidered to be a marker of low-grade tumors, it wasfound to be overexpressed in both low- and high-gradeMECs (55% positivity in high-grade tumors versus 91% in

low-grade tumors) [24]. Significantly, patients whose tumorswere MUC4-positive had a higher survival rate and a lowerrate of recurrence following surgery. In a separate study,however, Handra-Luca et al. found no correlation betweenMUC4 positivity and prognosis [50].

In addition to the increased expression, an alteration inthe 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 epithelialcells lining the excretory ducts, the staining in MECs wasmore diverse. It ranged from an apical staining in glandular


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Table4.

ExpressionpatternofMUC4anditscorrelationwithclinicopathologicparametersingastrointestinalmalignancies.

Cancer

Detection

method

SitesofMUC4

expression

Associationwith

stage/differentiation

PrevalenceofMUC4positivity

Associationwithprogno

sis

Ref.

Signet-ringcell

carcinoma(gastric,

colorectaland

breast)

IHC

ND

ND

Colorectal(n=11)91%;Gastric

(n=21)57%;Breast(n=6)primary

tumors0%(

iv)Metastaticcolorectal(n=2)

[100%],gastric(n=4)[100%]andbr

east(n=3)

tumor[33%]

ND

[112]

Intraductalpapillary

mucinoustumorof

thepancreas

ISH

ND

No

70%

[86]

Pancreaticcancer

Microarray

ND

No

>5-foldinexpressioninPCcomparedwith

normalpancreasandCP

ND

[113]

Pancreaticcancer

IHC

Cytoplasmic

andmembrane

ND

Pancreaticcancer(n=71)77%

Chronicpancreatitis(n=18)22%

ND

[78]

Pancreaticcancer

IHC

Cytoplasmic

andmembrane

No

Invasiveductaladenocarcinoma(n=1

35)31.9%

Normalpancreas0%

SurvivalinhighMUC4/highMUC4and

highp27-expressingIDCsignificantly

lowerthanlowMUC4/low

MUC4and

lowp27-expressingIDC

MUC4expressioninIDCw

asa

predictorofpoorprognosis

[108]

Pancreaticcancer

RT-PCR,

RNAslot

blot

NA

Highlevelsinwell

andMDbutabsentin

PDcancercells.

Nocorrelationwith

tumorstage

PCtissues75%

PCcelllines73%

CP(n=10)0%

Normalpancreas(n=7)0%

ND

[76]

Pancreaticcancer

IHC

Cytoplasm

ND

PanIN-1:17%;PanIN-2:36%;PanIN-3:85%;

PC:89%Normalducts(n=246)0%

;CP

(n=22)anddemonstratingsignsofin

tense

inflammation60%;Atropicductsfilledwith

inspissatedsecretions(n=7)57%

ND

[81]

Coloncancer

Northern

blot

NA

None

Normalcolon100%(n=9);Coloncancer55%

(n=8),comparableorincreasedMUC

4expression

ND

[30]

CP:Chronicpancreatitis;IHC:Immunohistochemistry;ISH:Insituhybridization;MD:Moderatelydifferentiated;NA:Notapplicable;ND:Notdisc

ussed;PC:Pancreaticcancer;PD:Poorlydifferentiated;RT-PCR:Real-time

polymerasechainreaction.


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cells to a pan-membranous staining in the intermediate,clear and epidermoid cells [50]. Further, a decrease in MUC4positivity with increasing grade (and hence a loss ofdifferentiation) was also observed in the tumors, being91, 80 and 50% in low, intermediate and high-grade MECs,respectively [50]. Another study investigating a possiblecorrelation of the mucin expression profile in MECs withclinicopathologic characteristics found that 95% of theMECs overexpressed MUC4. Specifically, it was detected inthe supranuclear cytoplasm and the apical membrane of allfour cell types (outlined earlier) [49]. Most significantly, a

high expression of MUC4 was more commonly observed inlow-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 MUC4expressing MECs compared with 28 months in MUC4non-expressing MECs) [49].

Thus, MUC4 overexpression in MECs of the salivarygland, and significantly its positive correlation with tumorgrade and a favorable prognosis, suggest its possible utility asa prognostic marker in this malignancy.

2.3 MUC4 in the diagnosis of lung cancerLung cancer is one of the most commonly diagnosed malig-nancies in the US and Europe [51]. About 75% of all casesare of the non-small cell type. These include histologic sub-types such as squamous cell carcinoma, adenocarcinoma andlarge-cell undifferentiated adenocarcinoma (Table 3). Theremaining 25% are of the small-cell variety. Histologically,adenocarcinomas form glandular structures with mucinsecretion, whereas SCCs are characterized chiefly bykeratinization [31]. MUC4 is expressed in the normal adultlungs and strongly overexpressed in non-small cell lung

cancer tissues and lung cancer cell lines, but rarely in smallcell lung cancer (3 positive cases out of 12 in one study) [52].Interestingly, a high level of MUC4 expression was morecharacteristic of adenocarcinoma and adenosquamouscarcinoma than squamous cell carcinoma or large cellcarcinoma [53]. Further, patients with stages I and IIadenocarcinomas with higher levels of MUC4 reactivityshowed a trend towards increased survival compared withthose with lower MUC4 immunoreactivity. According toanother report, MUC4 was detected in 67% of theadenocarcinomas at the protein level, whereas the transcript

was identified in 72% of the samples [18]. In a multivariateanalysis of 185 cases of non-bronchoalveolar type lungadenocarcinomas < 3 cm, stage 1A patients whose tumorsshowed a high MUC4 expression (defined as positivestaining in 25% of neoplastic cells) had a significantlylower survival than those with low MUC4 expression.Univariate analysis also revealed that high MUC4 expressioncorrelated with vascular invasion and a greater frequency ofstromal invasion [54]. An earlier report had also shown thatMUC4 was the second most abundant mucin gene (afterMUC1) expressed in lung adenocarcinomas [55]. An intenseoverexpression of the MUC4transcript was also reported inpre-malignant lesions of the lungs, including basal cell and

mucous cell hyperplasia and squamous metaplasia with or without accompanying dysplasia [18]. In a study byLlinares et al. [56], MUC4 protein was expressed in 32 outof 35 lung adenocarcinomas but in none of the 41 malignantmesotheliomas or the 32 samples of benign mesothelial cells. A distinction between primary pleural malignant mesothe-lioma and pleural infiltration by a spreading lung cancer iscrucial owing to the association of the former with anoccupational exposure to asbestos. Further, the two condi-tions often present an identical clinical picture and aredifficult to distinguish histologically [57]. MUC4 positivity

Table 6. Expression pattern of MUC4 and its correlation with clinicopathologic parameters in other malignancies.

Cancer Detection

method

Sites of MUC4

expression

Association with

stage/differentiation

Prevalence of MUC4

positivity

Association

with prognosis

Ref.

Breast cancer IHC ND More common inlow-grade tumors

95% in breast cancer None [98]

Prostate cancer IHC Diffuse embraneand cytoplasmicstaining pattern

None (i) Prostate cancer- 26.3%positivity {1+(21%),+(5.3%), no 3+ staining}(ii) Adjacent normal/BPH- 84.2 % positivity{1+ (28.9%), 2+ (18.4%),3+(36.8%)}

ND [37]

Prostate cancer IHC (TMA) ND NA No expression in bothnormal (n = 40) and cancertissues (n = 120)

ND [100]

SCC of the skin Microarray NA NA NA NA [114]

IHC: Immunohistochemistry; NA: Not applicable; ND: Not discussed; SCC: Squamous cell carcinoma; TMA: Tissue microarray.


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was characterized by a diffuse cytoplasmic staining and, lessfrequently, membrane staining. In pulmonary adenocarcinomaswith 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 progressionin 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 pleuralfluid samples from malignant mesothelioma showed anyexpression of the mucin. From this study, MUC4 emergednot just as a highly specific and sensitive marker for lungadenocarcinoma, but its differential expression in adenocar-cinoma-associated pleural effusions suggested that it couldbe useful as a marker to detect carcinomatous cells in pleuraleffusions. The pattern of MUC4 expression in lung cancercell lines supports its role as a regulator and marker ofdifferentiation, with higher levels being detected in the well-differentiated cell lines [58]. This observation issupported further by the finding in cultured airway epithe-lial cells wherein the well-differentiated but not the poorlydifferentiated cells expressed the MUC4 transcript whenanalyzed by northern blotting [59].

Hanaoka et al. reported that anti-MUC4 immunoglobulinwas detectable in the sera of lung cancer patients. Anti-MUC4antibody was detected in the sera of lung cancer as well ascontrol patients (normal, breast cancer, tuberculosis andmetastatic renal cell carcinoma of the lung). However, antibodytiters were significantly higher in the sera from lung cancerpatients compared with the control group. Further, when an upperlimit 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-MUC4antibody, three samples also contained an IgG isotype [18].

These observations raise the possibility that MUC4immunostaining in lung biopsies could serve to identifypotentially malignant lesions. Further, the detection of anti-MUC4 antibodies might be useful as a serum-based markerfor the diagnosis of pulmonary adenocarcinoma.

3. MUC4 in the diagnosis of gastrointestinalmalignancies

Cancers of the gastrointestinal (GI) tract are widely

prevalent around the world and second in overall incidenceto malignancies affecting the genital tract. An alterationin the expression of several mucins has been demonstratedin malignancies arising from the GI tract, including cancersof the esophagus, colon, stomach and the pancreatobiliarytract (Table 4).

3.1 MUC4 in esophageal cancer

The normal esophageal mucosa is lined by a non-keratinizedstratified squamous epithelium. MUC1 and MUC4 are thechief mucins expressed by the stratified epithelium. However,

in response to chronic damage from irritants such asalcohol, acid and tobacco, this stratified epithelium canundergo malignant transformation. The resulting esophagealcarcinoma is classified into chiefly two histologic types:squamous cell carcinoma and adenocarcinoma. The latterusually develops from a precursor metaplastic lesion known

as Barretts esophagus (BO) (hence it is also sometimescalled Barretts adenocarcinoma).

An increase inMUC4 transcript expression was observedin all esophageal SCCs in one study [60]. The strongest signalwas localized to the superficial, most well-differentiated partof the transformed epithelium. However, the expression waspatchier in the areas of Barretts adenocarcinoma [60]. BO isthe result of an abnormal repair of epithelial lesions thatare induced in the esophageal mucosa by persistentgastroesophageal reflux. It is characterized by metaplasia ofthe normal stratified squamous epithelium to columnarepithelium. An onset of high-grade intraepithelial neoplasia(HGN) in the metaplastic epithelium of BO is at presentthe only marker used in the surveillance of patients toidentify those at a high risk for the development ofesophageal adenocarcinoma. However, the efficiency ofthis process is thwarted by sampling errors, often due tothe difficulty in identifying an area of HGN. This iscompounded by the absence of focal neoplastic changes ininadequate biopsies. This has made the identification ofmarkers of early dysplastic change in BO patients apressing requirement [60]. Most secreted mucins are observedto be upregulated in BO, whereas in adenocarcinoma thereverse is true, with a trend towards a reduced expression ofmucin genes [61]. In one study, MUC4 mRNA expression

was observed to be reduced in BO [62]. Further, the expressionofMUC4 transcripts was significantly increased followingthe onset of HGN in existing BO lesions. This has previ-ously been attributed to the induction of MUC4 expressionby bile salts present in the refluxed gastric contents [63].MUC4 acts as a ligand for the receptor tyrosinekinase ErbB-2, which is also aberrantly expressed in themembranes of the epithelial cells in BO and the associatedadenocarcinoma [64]. The MUC4ErbB2 complex has beensuggested to play a role in the inhibition of apoptosis [65].However, the areas of BO with HGN showed an increasein 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 thatMUC4 plays only a minor role in the regulation ofapoptosis in esophageal cancer. As HGNs are at an elevatedrisk for malignant transformation, markers that can uncoverthese foci in existing Barretts esophagitis lesions couldprove to be extremely useful in identifying individuals atan elevated risk for the development of esophagealadenocarcinoma. As MUC4 overexpression was observedfollowing the onset of HGN, it could serve as a markerof dysplasia in the esophagus, although further studiesare warranted.


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3.2 MUC4 in hepatobiliary tract diseases

MUC4 expression in the hepatobiliary duct system has alsobeen the focus of intense research. Stone formation in theintrahepatic bile duct, a condition commonly encounteredin Southeast Asian countries, is frequently associated with apreceding history of recurrent and residual stones and

frequent attacks of acute cholangitis. Chronic proliferativecholangitis, the sequel of recurrent cholangitis episodes,is regarded as a risk factor for the development ofcholangiocarcinoma. Cholangiocarcinoma is the mostcommon primary malignancy of the bile ducts and thesecond most prevalent liver cancer. About 5% of cholangio-carcinomas have associated hepatolithiasis. In an analysisof mucin mRNA expression in tissue sections from stone-containing intrahepatic bile ducts and cholangiocarcinomasby in situ hybridization, MUC4 was found to be weaklyexpressed in only 1 of the 10 normal sections andoverexpressed in 4 out of 6 cases of cholangiocarcinoma.Significantly, MUC4 mRNA was detected in 10 out of the12 sections with hepatolithiasis including 5 with hyperplasticbile duct cells and 5 of 6 with dysplastic cells. The MUC4probe labeled the entire cytoplasm of the epithelial cells liningthe bile ducts and the peribiliary glands with moderate tostrong intensity in sections of both hepatolithiasis and cho-langiocarcinoma, albeit with a more heterogenous expressionin the latter [66].

Intrahepatic cholangiocarcinoma (ICC) is an extremelylethal malignancy with most patients presenting with largetumors and evidence of regional lymph node, pulmonaryand/or bone metastasis at the time of diagnosis [67].Intrahepatic cholangiocarcinomas of the mass-forming type

(ICC-MFs) are the commonest types of ICCs and areassociated an extremely poor prognosis even after surgicalresection of the tumor. ICC-MF tumors have been reportedto express MUC2 while being uniformly negative forMUC1 [68]. In a study to examine the relationship betweenMUC4 expression in ICC-MF tissue sections and patientprognosis, MUC4 was detected in the cytoplasm of 37%of the ICC-MF tissues, while no expression was observedin the normal bile duct cells. When patients weredivided 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-termand 8.3% of the long-term survivors. Patients whose tumors

expressed MUC4 (taken as positive staining in > 5% ofthe 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-yearsurvival rate). Further, the expression of both MUC4 and itsinteracting partner, ErbB2, resulted in the worst prognosisfollowing surgery, whereas tumors expressing neither proteinafforded the best survival. Together with tumor size,intrahepatic and lymph node metastasis, MUC4 expressionemerged as an important risk factor that adversely affectedthe outcome in patients with a mass-forming ICC [69].

MUC4 has also been show to be aberrantly expressed inextrahepatic bile duct carcinomas. Patients whose tumorsshowed a high level of MUC4 expression (> 20% of tumorcells stained) had a significantly lower survival than those with lower MUC4 expression levels (< 20% of cancer cellsstained). MUC4 staining was observed in the cytoplasm of

the cancer cells. Together with high MUC1 expression,histologic grade of the tumor, surgical margin involvementand nodal metastasis, a high MUC4 expression was apredictor of poor prognosis in these patients [70].

Thus, MUC4 could serve to identify patients at a highrisk of cholangiocarcinoma. Also, it may have a role as aprognostic marker in both intrahepatic and extrahepaticbiliary cancer.

3.3 MUC4 in colon cancer

Colon cancer is the third most common cancer among boththe sexes in the US, with > 112,000 estimated new casesand > 52,000 deaths in 2007 [1]. Several mucins areknown 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 incolon cancer cell lines and tissues [30]. MUC4mRNA levels were either unchanged or increased compared with thenormal colonic tissue in over half of the colon cancers(five out of nine) examined. However, no correlation wasobserved between MUC4 expression and the clinicopatho-logic parameters. Zhang et al., studying MUC4 expressionin frozen tissue sections from multiple malignancies, reportedthat 50% of colon and pancreatic cancer tissues expressedthe mucin [71]. Specifically, the median staining intensity of

MUC4 in these two cancers was 4+ in > 80% of the tumorcells, suggesting a strong upregulation of MUC4 expressionin these malignancies.

Colonic polyps, specifically of the hyperplastic type,are potentially malignant lesions that are known toprogress to adenocarcinoma. These polyps are generallyclassified into hyperplastic polyps and adenomas. Serratedadenomas are a subset of hybrid polyps showing features ofboth hyperplastic polyps and adenomas that have beendocumented to develop into intramucosal carcinoma. Theidea that hyperplastic polyps could progress to serratedadenomas through a separate histogenetic pathway was firstintroduced by Biemer-Httmann et al. [72]. Later, they also

showed that, although MUC4 was expressed in the normalcolonic mucosa, its expression was reduced in hyperplasticpolyps and completely abolished in serrated adenomas.Further, a mucin signature of MUC2+, MUC4- andMUC5AC+was found to be highly specific to identify serratedadenomas. Given the malignant potential of this hybridpolyp, this observation suggests that loss of MUC4 expressionin serrated adenomas could be useful as a marker to identifypatients with an elevated risk of developing colon cancer [72].This observation assumes an even greater significancegiven the occurrence of hyperplastic polyps and serrated


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adenomas as part of the familial polyposis syndrome,known to carry an elevated risk for subsequent developmentof adenocarcinoma.

3.4 MUC4 in pancreatic adenocarcinoma

Pancreatic cancer (or pancreatic adenocarcinoma) isthe fourth most common cause of cancer-related deaths inthe United States, with > 37,000 new cases estimatedin 2007 [1]. It is an extremely lethal malignancy with a5-year survival rate of < 5%. The poor survival isattributed chiefly to an early development of micrometastasis, the late onset of symptoms (except in cancer

affecting the pancreatic head) and an extreme resistanceto chemotherapy [73]. Measurement of carbohydrate antigen(CA19 9) in serum is at present the most commontest used in the diagnosis and follow-up of pancreaticcancer patients [74].

Several studies have demonstrated the aberrant overexpres-sion of MUC4 in pancreatic cancer (PC) and suggestedthat it could be a potential diagnostic marker. Balague et al.first reported that MUC4mRNA was overexpressed in pan-creatic cancer but undetectable in normal pancreatic tissuesand in chronic pancreatitis [75]. MUC4 expression has sincebeen examined in both pancreatic cancer tissues and celllines. It was noted that although MUC4 was aberrantly

overexpressed in most pancreatic cancers (12/16 samples inone study), it was uniformly absent in chronic pancreatitis(CP) and the normal pancreas [76]. MUC4 expression wasalso detected in a significant number of fine needle aspirates(91%) from PC patients [77]. Further, in combination withthe non-expression of clusterin- (a secreted glycoprotein),MUC4 helped to distinguish reliably inflamed pancreaticductal epithelial cells from malignant ones [77]. MUC4 hasalso been examined both alone and in combination withother potential markers for its ability to differentiate PCfrom CP. In one study, positive MUC4 immunostaining

of pancreatic tissue sections alone had a sensitivity andspecificity of 77 and 78%, respectively. When examinedin combination with immunostaining for other potentialmarkers of PC, a positive staining for either MUC4 orp53 had the highest diagnostic sensitivity (96%). However,the specificity was 73% compared with 78% for MUC4alone. When immunoreactivity for both MUC4 and p53 was taken as the diagnostic criterion, the sensitivity fellto 39% but specificity increased to 100% [78]. MUC4 alsoemerged as an independent poor prognostic factor in amultivariate analysis of 135 cases of invasive ductalcarcinoma of the pancreas [79]. The survival of 21 patients

with high MUC4 expression (> 20% MUC4-positiveneoplastic cells) was significantly worse than that of the114 with low MUC4 expression.

The development of infiltrating adenocarcinoma of thepancreas has been suggested to progress through an inter-mediate preinvasive stage termed pancreatic intraepithelialneoplasia (PanIN) [80]. A progressive increase in MUC4expression with the progression of PanIN lesions wasobserved: from no detectable expression in the normalpancreas, through an increasing expression in progressivelyhigher 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 thatalthough MUC4 expression was detected as early as PanIN-1,the strongest expression was observed in the PanIN-3 stageof dysplasia [82].

Intraductal papillary-mucinous neoplasm (IPMN) ofthe pancreas is a rare disease characterized by proliferation ofthe pancreatic ductal epithelium with resultant mucin hypersecretion. Importantly, it has the potential to progress toinvasive adenocarcinoma. IPMNs of the villus dark cell typeare chiefly characterized by MUC5AC+ and MUC2+,whereas those of the papillary clear cell type are MUC5AC+

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 reachesmaximum intensity in pancreatic adenocarcinoma (C) (Magnification: 10). The sections were stained with the mouse monoclonalantibody 8G7 directed against the tandem repeat domain of human MUC4.


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and MUC2- [83-85]. IPMN adenomas showing dysplasiawere observed to be strongly positive for MUC2, MUC5ACand, to a lesser extent, MUC4 [86,87]. A similar patternof mucin expression was also observed in colloid carcinomasassociated with IPMNs. On the other hand, invasive ductaladenocarcinomas associated with IPMNs showed a loss of

MUC2 and gain of MUC1 and were associated with greatermetastatic potential. The altered mucin expression in IPMNsof the adenoma type and the associated colloid carcinomashas been suggested to contribute to their better prognosiscompared with conventional ductal adenocarcinoma.

Thus, MUC4, which is not detected in the normalpancreas but appears de novo in the earliest preinvasivestage of PC pathogenesis, could be an extremely usefulmarker in patients who are at an elevated risk for developingPC, including those with hereditary pancreatitis andlong-standing chronic pancreatitis. Further, we have detectedMUC4 transcripts in peripheral blood mononuclear cellsof pancreatic cancer patients but not in acute pancreatitisor normal individuals (manuscript under preparation),suggesting its possible role as a blood-based diagnosticmarker for PC.

4. MUC4 in the diagnosis of reproductivetract malignancies

Although significant attention has focused on the role ofMUC4 in malignancies of the aerodigestive tract, it alsoappears to play a role in cancers arising from thereproductive system, chiefly, cervical and ovarian cancer infemales and prostate cancer in males (Table 5). An analysis

of MUC4 expression in the normal female reproductivetract revealed that the mucin is expressed only in the lowersegment, specifically in the epithelium lining the ectocervix,endocervix and, patchily, in the vagina. Further, onfollowing MUC4 expression in the normal endometriumduring the menstrual cycle, the highest expression wasdetected just before the mid-cycle peak, when estrogenlevels are unopposed by progesterone, followed by a dropthereafter, suggesting a possible hormonal regulation ofMUC4expression 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 onlyto breast cancer in its incidence [1]. The squamocolumnarjunction (SCJ) between the ectocervix and the endocervix isthe usual site for the origin of the malignant transforma-tion [88]. Using microarray analysis, Narayan et al. foundthat MUC4 was one among 22 genes significantlyupregulated in cervical cancer tissues compared with thehealthy cervix [89]. An analysis of the MUC4 expressionprofile in the normal, metaplastic and dysplastic cervicalepithelium revealed that whereas the apomucin is highlyexpressed in the healthy endocervix, and patchily in the

ectocervix, its expression was significantly upregulatedfollowing the onset of squamous dysplasia at the SCJ [89,90].Further, although the highest MUC4 expression wasobserved in high-grade dysplasia (cervical intraepithelialneoplasia or CIN grade II and III), it was detectable as earlyas in CIN-I (75 40% cells positive). A strong and diffuse

MUC4 immunostaining was observed throughout thecytoplasm of the dysplastic cells and throughout the entirethickness of the stratified epithelium. By contrast, thenormal endocervical epithelium showed MUC4 immuno-staining only in the most superficial columnar epithelial layer while the normal and metaplastic ectocervical epithelia hada parabasal pattern of MUC4 staining. This suggests thatthe presence of a diffuse MUC4 staining of the epitheliumat the SCJ could be a possible marker for dysplasticchanges in women at a high risk for developing cervicalcancer. Baker et al. further observed that although MUC4was strongly expressed on the luminal surface of endocervi-cal glands in benign cervical pathologies (tubal metaplasia,micro glandular hyperplasia), and in endocervical dysplasiaand 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 todistinguish AIS histologically from benign cervicalconditions, especially tubal metaplasia, the absence of MUC4expression in the former could aid in distinguishing thislesion from benign cervical pathologies.

Thus, evaluating MUC4 expression in cervical biopsieshas the potential to be useful as an early marker for cervicaldysplasia. Further, the absence of MUC4 expression couldalso 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 uterinecancer, with nearly 39,000 new cases estimated in 2007 [1]. Although the mortality from endometrial carcinoma hasdeclined in general, it continues to be higher in African-American women than in Caucasians. Immunohistochemicalanalysis of four mucin genes (MUC2, MUC4, MUC5ACand MUC6) in normal, hyperplastic and malignant endo-metrial tissues revealed that MUC4 was the predominantmucin 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 increasedin simple endometrial hyperplasia (29% of cases positive), with the most intense expression noted in tissues fromendometrial adenocarcinoma (77% of samples positive) [91].Other studies reported a similar finding, albeit with a lowerincidence of MUC4 positivity in endometrial carcinoma(45%). However, MUC4 staining did not correlate withclinicopathological parameters [10,92]. This suggests that therole of MUC4 in the progression and diagnosis of endometrialcarcinoma needs to be explored further.


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4.3 MUC4 in ovarian cancer

Ovarian cancer accounts for nearly 4% of all cancers inwomen and is the eighth-leading cause of cancer-associateddeaths in the US [1]. It also has the dubious distinction ofbeing the most lethal cancer of the female reproductivetract, chiefly owing to the advanced stage at which it is first

diagnosed [93]. The lack of early symptoms means that themalignancy has often spread beyond the ovary when it isfirst identified. One of the earliest reports of MUC4 over-expression in ovarian cancer was a study by Giuntoli et al., wherein they reported thatMUC4 was one of the mucingenes whose expression was significantly increased in ovariancancer [94]. A key observation was the absence ofMUC4expression in a transformed but non-malignant ovarian epi-thelial cell line, suggesting that its expression is relatedspecifically to the onset of malignant change in theepithelial cells. It was also observed that the expression ofMUC4 was significantly decreased in late stage ovariancancer. Significantly, an increase in MUC4 expressioncorrelated with an improvement in patient survival. However,the prognosis of ovarian cancer patients is also influencedsignificantly by the histology. Based on the cell of origin,malignant ovarian neoplasms are classified primarily intosurface-epithelial stromal tumors, germ cell and sex cordstromal tumors. About 15% of ovarian tumors are alsoclassified as borderline. These are very similar in theirepidemiology to invasive tumors but occur more commonlyin younger women and have a more favorable outlook [66].Histologically, the cells in borderline tumors present witheither 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, theMUC4-expressingcells in these neoplasms were observed to coexist with eitherintestinal-type goblet cells (MUC2+) or gastric-type epithelia(MUC5AC+ or MUC6+). This is significant as borderlineovarian tumors exhibiting an intestinal phenotype have anextremely poor prognosis. Thus, it appears that while MUC4expression alone is unlikely to provide significant prognosticinformation, when combined with other mucins it couldhelp to identify high- or low-risk tumors and stratify patientsfor treatment purposes [95]. The identification of tumors ata localized and non-metastatic stage has been suggested as

the key to improving the poor prognosis in ovarian cancerpatients. In a study comparing the expression profile ofMUC4, MUC1 and MUC16 in ovarian cancer, it wasobserved that MUC4 was significantly overexpressed in allof the early stage tumors and a significant number ofadvanced stage tumors (88%). Further, a combination ofMUC4 and MUC16 positivity identified 100% of thelate stage tumors (with 100% specificity). Significantly,MUC4 but not MUC1 or MUC16 showed 100%specificity for mucinous ovarian tumors. However, MUC4expression did not correlate with patient survival.

Thus, MUC4 expression appears to be upregulated inthe early stages of ovarian cancer pathogenesis, with ahigh expression being maintained in advanced stagetumors. Further, it appears to be a highly specific markerfor mucinous ovarian tumors. This suggests that it couldbe 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 thenative mesothelial cells of the peritoneal cavity. Ovariancancer (OC) and its closely related and morphologicallyindistinguishable counterpart primary peritoneal serouscarcinoma (PPC), are both thought to develop either fromthe peritoneal mesothelium or from the ovarian surfaceepithelium. Both DMPM and OC/PPC diffusely involvethe peritoneum, forming solid nodules and producingascites. 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 differentiallyexpressed genes between OC/PPC and DMPM, MUC4emerged as one of the 121 genes overexpressed in the for-mer group [96]. In a follow-up study to examine the sites ofMUC4 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 effusionin 141 of 142 cases examined [97]. Reactive mesothelial cellspresent in 72 malignant and 10 reactive peritoneal effusionsexamined were, however, uniformly negative. Additionally,MUC4 expression was significantly higher in carcinomatous

cells present in the OC/PPC associated effusions thanin either the primary carcinomas or the solid metastases.However, except for a higher expression in tumors fromolder (> 60 year) patients, MUC4 expression did not correlatewith survival or any other clinicopathological parameters inOC/PPC. These reports seem to suggest that MUC4 is ahighly specific marker to distinguish OC/PPC from bothbenign (reactive) and malignant mesothelial cells.

4.4 MUC4 in breast cancer

Much effort is being made to identify markers with clinicalsignificance in breast cancer. Several of these studies havereported an altered expression of mucins in breast cancer

tissues. An immunohistochemical study on tissues from1447 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% ofthe cases studied (Table 6). However, apart from a weakcorrelation with tumor grade, no other correlation betweenMUC4 expression and clinicopathological parameters wasobserved [98]. MUC4 has also been reported previously inbreast milk [23]. Further, MUC4 expression has also beensuggested to be responsible for the resistance of breast cancer


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cells to the anticancer drug Herceptin possibly by the mech-anism of steric hindrance preventing access of Herceptin toits 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 incidencetwice as high as that of other racial and ethnic groups [1]. Although it is not clear whether screening helps to reducemortality, prostate-specific antigen (PSA) and digital rectalexamination (DRE) are two methods being employed atpresent to screen men > 50 years of age. Using the anti-MUC4 TR antibody 8G7, we observed that MUC4 expressionwas significantly decreased in prostate cancer tissues comparedwith either the normal prostate or areas of benign prostatichypertrophy (BPH) [37]. Out of the 38 tumor samples exam-ined, a mere 26% had MUC4 staining, the intensity rangingfrom faint to moderate. In contrast, nearly 85% of the adjacentnormal areas or those showing features of BPH were moderateto strongly positive for MUC4 (Table 6). On the otherhand, MUC1, another membrane-bound mucin, wasdetected in 54% of prostate cancer and 90% of theadjacent normal/BPH areas. The staining pattern for bothmucins in this study was rather diffuse, staining both themembrane and cytoplasm. However, it did not correlatewith either the cell type or tumor stage [37]. Using a differentantibody, Cozzi et al., however, reported no MUC4 expressionin either the normal or prostate cancer tissues [100]. Thissuggests that the role of MUC4 in prostate cancer needsfurther elucidation.

5. Correlation of subcellular distribution ofMUC4 with clinicopathologic characteristics

The subcellular localization of several molecules is known tobe altered in malignant cells compared with their normalcounterparts. For example, a cytoplasmic expression ofMUC1 in immunohistochemical analysis of invasive breastcarcinoma tissue sections correlated with positive lymphnode status in the patients [101]. Low cytosolic MUC1expression also emerged as a predictor of good prognosis instage III ovarian cancer, whereas a low expression of MUC1in the apical membrane of the neoplastic cells wasassociated 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 casesshowing positive MUC1 staining in the circumferentialmembrane and/or cytoplasm than those with staining onlyin the apical membrane or those showing no stainingat all [103]. Hence, it is of interest to examine whether acorrelation exists between the subcellular location of MUC4and clinicopathologic parameters. This could be especiallyuseful in predicting the outcome of patients diagnosed withhighly invasive and/or metastatic cancers, such as ovarian,gastric and pancreatic adenocarcinoma. However, no

correlation has been reported thus far with either patientoutlook or tumor characteristics. In the developing ratforegut, MUC4 expression occurs initially on the surface ofthe epithelial cells (lining the esophagus), followed by itsappearance in intracellular granules [104]. In malignant cellson the other hand, MUC4 is expressed both in the cytosol

and in the apical membrane. Llinares et al. reported thatalthough cytoplasmic and membrane staining were bothfrequently observed in lung adenocarcinomas, in some casesthe staining was limited entirely to the cytoplasm [56]. Inovarian cancer, MUC4 expression was observed in both thecell membrane and the cytosol in serous, mucinous andclear-cell subtypes, whereas endometrioid tumors showed adistinct apical and basal staining pattern with only a faintcytoplasmic staining [105]. Eighty-six per cent of normalsalivary gland specimens were positive for MUC4 expressionin the supranuclear cytoplasmic region of their ducts.However, in salivary gland MECs, both the cell membraneand cytosol of all types of neoplastic cells were stained.This difference in subcellular staining pattern ofmembrane-bound mucins is believed to be due todifferences in post-translational processing of the mucin coreproteins leading to altered glycosylation patterns inthe neoplastic cells [49]. The normal ectocervix shows adiffuse MUC4 staining limited to the cytosol in the cellsof the parabasal layer of the stratified squamous epitheliumin contrast to a stronger and diffuse cytoplasmic stainingthroughout the entire thickness of the stratified epitheliumin dysplastic cervical epithelia [90]. Cytosolic MUC4staining was also observed in both the columnar and gobletcells 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 serratedadenomas and significantly reduced in hyperplastic polyps, whereas the intensity, intracellular localization and cryptdistribution in tubular and tubulovillous adenomas wereidentical to those observed in the normal colon [72].Lung cancer tissues stained with a rabbit polyclonalantibody to MUC4 showed a strong and diffusecytoplasmic MUC4 staining, whereas only weak MUC4immunoreactivity restricted to the apical region of thebronchioles 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 thesubcellular distribution of MUC4 in relation to patientsurvival, response to chemotherapy and presenceof distant metastasis would be extremely useful toestablish MUC4s role as a prognostic marker inneoplastic progression.

6. Summary

MUC4 expression is a specific marker of epithelial tumorsand its expression correlates positively with the degree of


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differentiation in several cancers. Most significantly, MUC4has emerged as a specific marker of dysplasia, beingexpressed in the earliest dysplastic lesions preceding severalmalignancies, including esophageal, pancreatic and cervicalcarcinoma. Also, the detection of MUC4-specific antibodiesin the serum and of the transcript in mononuclear cells of

cancer patients raises the possibility of it emerging as a newdiagnostic biomarker for bedside application in high-riskindividuals and those with established cancer.

7. Expert opinion

The preceding discussion has established that the detectionof MUC4 could serve as a new diagnostic marker in tissuesections as well as body fluids. The use of MUC4 expressionas a yardstick to distinguish benign, dysplastic or malignantlesions arising from a given site offers several advantages. Forexample, it has a restricted expression in specific segments ofthe gastrointestinal tract, which circumvents the possibilityof contamination from mucins present in secretions fromnearby portions of the gut. This is crucial as one of theconcerns in studies investigating the role of mucins as tumormarkers using homogenized tissues is contamination fromadjacent normal areas. In Barretts esophagus for instance, ithas been suggested that the detection of MUC2, MUC5ACand MUC6 in the metaplastic epithelium could be theresult of reflux of the gastric mucus into the esophagus.MUC4, however, is not expressed by the normal gastricepithelium. Hence, its detection offers a greater specificityfor newly diagnosed esophageal lesions.

MUC4 is expressed de novo in several tissues during the

process of carcinogenesis, including the pancreas and thebile duct epithelial cells. This prevents possible overlap with endogenous MUC4 in diagnostic assays. Theavailability of specific antibodies has also greatly helpedaccelerate research into developing MUC4-based diagnosticassays. Current imaging modalities, the cornerstone ofcancer diagnosis, can identify only macroscopic changes.However, the microscopic and molecular alterations thatprecede gross changes by a significant lag period can beidentified only by using molecular techniques. Here, tumormarkers come into prominence as tools to identify changesthat 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 severallethal cancers including pancreatic, esophageal and cervicalcarcinoma. As these cancers often present symptomsonly at an advanced stage, the emergence of MUC4 as ahallmark of dysplasia could provide us with a tool to screenand follow-up high-risk cases. We are currently engaged inexamining the pattern ofMUC4 mRNA expression in thepancreas and in PBMCs in a mouse model of spontaneous

pancreatic adenocarcinoma (unpublished data) [80]. Thesestudies will elucidate the role of MUC4 in the early stagesof pancreatic cancer pathogenesis and explore the possibilitythat detection of its mRNA in PBMCs might be a markerof PanIN lesions in the pancreas.

Despite its over- or underexpression in cancer,

MUC4 alone is unlikely to be able to identify all casesof a particular malignancy correctly. This is chieflydue to the role of mucins (including MUC4) in inflamma-tion and modulation of the immune response [39,43,46].Distinguishing these reactive conditions from malignancyis still a challenge, and one that will possibly require apanel of markers to achieve the highest sensitivity andspecificity in diagnosis.

Finally, the role of MUC4 as a prognostic marker remainsto be examined further in larger studies. Although itappears to be more highly expressed in well-differentiatedcancers, patients whose tumors express MUC4 appearto fare better or worse in an organ-specific pattern.For example, MUC4 expression is an indicator of agood prognosis in SCC of the UADT [24], MECs ofthe salivary gland [107] and in ovarian cancer [94]. On theother hand, in intrahepatic cholangiocarcinomas, extrahepaticbile duct tumors, invasive ductal adenocarcinomas andsmall-sized lung adenocarcinomas, MUC4 expression wasassociated with reduced survival and/or a poorprognosis [69,70,108,109]. Further studies are needed to clarifythe ability of MUC4 to predict the outcome in cancerpatients as different techniques and antibodies were used inthese studies.

Although MUC4 is elevated in several malignancies at

tissue level, whether circulating MUC4 is present in theserum is still an open question. It would be of interest toexamine whether elevation of serum MUC4 occurs in malig-nancies like ovarian and pancreatic cancer that express highlevels of the mucin in the malignant cells. Further, evidenceof elevated MUC4 in various malignancies has raisedthe possibility that it might serve as a novel target foranti-cancer therapy using specific antibodies.

Acknowledgments

The authors thank K Berger and S Deb for editorialassistance on this article. The authors on this work were

supported by the grants from the National Institutes ofHealth (UO1 CA111294, RO1 CA 133774, RO1 CA131944 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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